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Chromosomal heteromorphisms are interindividual variation of chromosomes without phenotypic consequences and are detected most frequently in 1q12, 9q12, 16q11.2, yq12 and in the short arms of acrocentric chromosomes (13, 14, 15, 21 and 22). In some cases, the enlarged acrocentric p arms of p arm can mask a cryptic translocation and therefore a partial trisomy . Here, we report a moroccan 1-year - old girl addressed for suspicion of trisomy 21 having an extremely enlarged p arm in one of her chromosomes 22 . Her length was 78 cm (25 centile), weight 10 kg (25 centile) and head circumference 46 cm (50 centile). She had no psychomotor delay but she had mild dysmorphism including round face, up slanting palpebral fissures, epicanthus, small nose, low - set ears and high arched palate [figure 1]; echocardiography was normal . The index propositus having a down - syndrome like facies cytogenetics studies were performed on metaphase chromosome preparations obtained from phytohemagglutinin stimulated lymphocyte cultures according to standard procedures . Chromosome analysis was carried out applying rhg banding at a 400 band level as previously reported and according to the international system for human cytogenetic: nomenclature iscn 2009 . Nor staining was realized using standard protocols, as well . Fluorescence in situ hybridization (fish) was carried out following respectively manufacturer's instructions and/or as previously reported . 30 - 19002) encompassing two genes dscr4 and dscr8 with 3 markers d21s259, d21s341, d21s342 in 21q22.13-q22.2 (down - syndrome critical region) and a whole chromosome paint (wcp) 21 probe (cytocell cat n lpp21r). Besides a microdissection derived home brew probe specific for all acrocentric short arms and denominated as midi54 and a the centromere - specific probe d14z1/d22z1, which at the same time hybridizes to centromeres of chromosome 14 and 22 (kreatech). Cytogenetics studies were performed on metaphase chromosome preparations obtained from phytohemagglutinin stimulated lymphocyte cultures according to standard procedures . Chromosome analysis was carried out applying rhg banding at a 400 band level as previously reported and according to the international system for human cytogenetic: nomenclature iscn 2009 . Nor staining was realized using standard protocols, as well . Fluorescence in situ hybridization (fish) was carried out following respectively manufacturer's instructions and/or as previously reported . 30 - 19002) encompassing two genes dscr4 and dscr8 with 3 markers d21s259, d21s341, d21s342 in 21q22.13-q22.2 (down - syndrome critical region) and a whole chromosome paint (wcp) 21 probe (cytocell cat n lpp21r). Besides a microdissection derived home brew probe specific for all acrocentric short arms and denominated as midi54 and a the centromere - specific probe d14z1/d22z1, which at the same time hybridizes to centromeres of chromosome 14 and 22 (kreatech). Cytogenetics studies were performed on metaphase chromosome preparations obtained from phytohemagglutinin stimulated lymphocyte cultures according to standard procedures . Chromosome analysis was carried out applying rhg banding at a 400 band level as previously reported and according to the international system for human cytogenetic: nomenclature iscn 2009 . Nor staining was realized using standard protocols, as well . Fluorescence in situ hybridization (fish) was carried out following respectively manufacturer's instructions and/or as previously reported . 30 - 19002) encompassing two genes dscr4 and dscr8 with 3 markers d21s259, d21s341, d21s342 in 21q22.13-q22.2 (down - syndrome critical region) and a whole chromosome paint (wcp) 21 probe (cytocell cat n lpp21r). Besides a microdissection derived home brew probe specific for all acrocentric short arms and denominated as midi54 and a the centromere - specific probe d14z1/d22z1, which at the same time hybridizes to centromeres of chromosome 14 and 22 (kreatech). R banding analysis of the chromosomes from peripheral blood of the propositus revealed a normal karyotype apart from an enlarged short arm for one chromosome 22 inherited from the mother [figure 2]. Thus, the karyotype was reported as 46, xx, der (22) add (22)(p13) mat . Applying the nor staining results were quite variable and overall non - informative (data not shown). A partial r banding karyotype of the propositus (a) and her mother (b) showing enlarged short arm of one chromosome 22 a probe lsi 21 of the critical region for the down - syndrome critical region and a wcp 21 probe excluded the presence of a cryptic trisomy 21 [figure 3]. Fish using a probe for all acrocentric short arms characterized the additional material on the der (22) as heterochromatin derived from an acrocentric short arm [figure 4]. Therefore, according to cytogenetic and molecular cytogenetic characterization the karyotype was 46, xx,22ph+++ mat . Metaphases hybridized with down - syndrome critical region probe lsi 21 (a) and whole chromosome paint (wcp) for chromosome 21 (b). (a) (b) wcp 22 revealed a complete hybridization of the two long arms of chromosome 21 . Neither der 22 nor other chromosomes revealed signal for any of the two probes the short arm of the chromosome 22p++ on the right was completely stained by a probe specific for the acrocentric short arm (midi54) indicating its heterochromatic nature cytogenetic and fish investigation of the propositus excluded (cryptic) trisomy 21 as already reported in down - syndrome like syndrome phenotypes . Instead of clarifying the genetic reason of the clinical problems in this specific case unexpectedly a familial 22ph+++ was detected and further characterized . Uninformative results as seen after nor silver staining was reported by some authors as well, previously . Thus, nowadays fish analysis is more straightforward for the characterization of heteromorphisms in general and acrocentric short arm variants in special . This kind of molecular cytogenetic analysis also enables to distinguish heteromorphic variants from partial trisomies due to cryptic translocations . Most likely heterochromatin loss or gain are not causative for acquired or inherited disorders, even though such relations still are discussed . To the best of our knowledge, this special here reported kind of chromosome 22 short arm enlargement, leading ro ~1.5 larger size than the normal long arm fish should always be used to resolve similar situations involving cytogenetic aberrations within the short arms of acrocentric chromosomes . Further follow - up of the propositus may allow us to have more clinical information and making some correlations between this heteromorphism and/or other genetic changes below the cytogenetic resolution and the phenotype of the patient.
Pulmonary arterial hypertension (pah) is a progressive disease that can cause right ventricular (rv) dilatation and dysfunction as a result of increased pulmonary vascular resistance (pvr) and pulmonary arterial (pa) pressure.1)2) echocardiographic evidence of rv systolic dysfunction and clinical features of right - sided heart failure have been regarded as the main determinants of morbidity and mortality in patients with pah.3)4)5) despite the obvious clinical importance of assessing rv systolic function, the estimation of rv function can be challenging.6)7) because of their invasiveness and/or high - cost, right heart catheterization (rhc) and cardiac magnetic resonance imaging (cmr) are impractical for frequent serial assessment . Although transthoracic 2-dimensional echocardiography (2de) provides important information about rv anatomy, function and hemodynamic status, the complex geometry of the rv makes accurate assessment by conventional 2de difficult.8) moreover, conventional 2de measures including velocity and displacement based analyses can be affected by translational motion of the heart and respiratory variation.9) two - dimensional strain echocardiography has been introduced to assess regional and global rv function in various disease categories including pah.6)10)11)12) because longitudinal shortening contributes to overall rv systolic function, rv longitudinal strain (rvls) assessed by strain echocardiography can give direct information about regional and global rv mechanics . However, there are relatively small numbers of validation data in rv strain measurement . Thus, we compared rvls with functional capacity and invasive hemodynamic data by rhc in patients with primary pah . We retrospectively studied consecutive adult patients (> 18 years of age) with world health organization (who) group 1 pah evaluated from february 2000 to december 2007 in the pah clinic of cleveland clinic . All patients fulfilled the contemporary diagnostic criteria,13) and they had regular outpatient follow - ups in this institution . Patients with left ventricular (lv) systolic dysfunction, significant valvular heart disease, chronic thromboembolic disease, and pulmonary parenchymal diseases were excluded in this study . We included and analyzed echocardiographic data without change of medication during the time interval between echocardiography and rhc . Patients without baseline echocardiographic examination or rhc in this institution, with poor echocardiographic images unable to analyze, atrial fibrillation, or medication change during time interval of two examinations were excluded . Conventional 2de examinations were performed according to the guidelines and standard recommendations of the american society of echocardiography and were recorded digitally.14) in our institution, all echocardiographic images are stored digitally into echocardiographic database with digital imaging and communications in medicine standard format . Echocardiographic images were reviewed by sonographers unaware of the clinical, laboratory, and hemodynamic information . Rv fractional area change (rvfac) was calculated from the apical 4-chamber view using the percentage change in areas of the end - diastolic and end - systolic areas of the rv . The tricuspid annular plane systolic excursion (tapse) was recorded with m - mode echocardiography parallel to the lateral rv wall and across the tricuspid annular plane and measured as the distance of systolic movement of the rv annulus in the longitudinal direction.8) rv tei index was defined as the ratio of isovolumic relaxation time (ivrt) and isovolumic contraction time (ivct) divided by ejection time (et) of rv.8) rv free wall thickness was measured using m mode from the subcostal view at the peak of the r wave at the level of the tricuspid valve chordae tendineae . Rv tei index was defined as the ratio of ivrt and ivct divided by et of rv.8) pa systolic pressure was estimated from the maximal continuous - wave doppler velocity of the tricuspid regurgitation (tr) jet imaged from multiple planes plus estimated central venous pressure calculated by the diameter of inferior vena cava and distensibility.8) an average of three measurements was used for all analyses . Rv strain and strain rate were analyzed off - line with velocity vector imaging (vvi) software (axius, siemens medical solutions, mountain view, ca, usa). After manual tracing of the endocardial border of the rv (about 10 to 16 points) over one frame, the endocardial borders were automatically tracked throughout the cardiac cycle . Myocardial velocity is derived as the ratio between frame - to - frame displacement of the speckles and the time interval.12) global longitudinal strain and time - to peak strain were calculated . Negative strain values indicate tissue shortening and a smaller value (that is, higher absolute value) indicates better rv systolic function . Global longitudinal strain of rv (rvlsglobal) was calculated by the average of six segmental values of the lateral wall and interventricular septum . Global longitudinal strain of rv free wall (rvlsfw) was measured by the averages of three segmental values (base, mid and apex) of the lateral wall . Hemodynamic data included the pa systolic pressure (spap), mean pa pressure (mpap), pulmonary capillary wedge pressure (pcwp), right atrium and rv pressures, and cardiac output . Pvr was calculated using the formula (mpap - pcwp)/cardiac output and reported in wood units . A 6-minute walking distance was performed according to the american thoracic society protocol.15) total distance walked, expressed in meters, was recorded . Brain natriuretic peptide (bnp) was measured by triage immunoassay reagents (biosite, san diego, ca, usa) formatted for beckman coulter instrumentation (brea, ca, usa). Longitudinal strain measurements for each patient were performed offline in a blinded manner by two independent observers . Each observer performed two independent measurements of regional and global rv strain values with vvi on same echocardiographic images in a randomly selected 10 patients . The data were analyzed using spss version 20.0 (spss inc ., chicago, il, usa) and medcalc version 12.4 (medcalc software, mariakerke, belgium). Linear regression analysis was performed to evaluate the relationship between rvls and other functional or invasive hemodynamic variables . A paired student t test was applied to evaluate the difference of basal and follow - up values . In the variables without normal distribution, wilcoxon signed rank test was used to evaluate the difference . To assess the overall differences on rvlsglobal between groups without versus with change of treatment (i.e. Treatment initiation or intensification), applied a linear mixed effects model with unstructured covariance for random effects was . Effects of groups were tested as a factor, time after initial echocardiographic assessment as a covariate, as well as timegroup interactions . Variability was tested with 2-way analysis of variance with calculation of intra- and inter - observer standard error or measurement (semintra and seminter). Semintra expresses the random error by a single observe, while seminter is an indicator of the mean variation between different observers.16) all tests were two - sided and p value<0.05 was considered statistically significant . We retrospectively studied consecutive adult patients (> 18 years of age) with world health organization (who) group 1 pah evaluated from february 2000 to december 2007 in the pah clinic of cleveland clinic . All patients fulfilled the contemporary diagnostic criteria,13) and they had regular outpatient follow - ups in this institution . Patients with left ventricular (lv) systolic dysfunction, significant valvular heart disease, chronic thromboembolic disease, and pulmonary parenchymal diseases were excluded in this study . We included and analyzed echocardiographic data without change of medication during the time interval between echocardiography and rhc . Patients without baseline echocardiographic examination or rhc in this institution, with poor echocardiographic images unable to analyze, atrial fibrillation, or medication change during time interval of two examinations were excluded . Conventional 2de examinations were performed according to the guidelines and standard recommendations of the american society of echocardiography and were recorded digitally.14) in our institution, all echocardiographic images are stored digitally into echocardiographic database with digital imaging and communications in medicine standard format . Echocardiographic images were reviewed by sonographers unaware of the clinical, laboratory, and hemodynamic information . Rv fractional area change (rvfac) was calculated from the apical 4-chamber view using the percentage change in areas of the end - diastolic and end - systolic areas of the rv . The tricuspid annular plane systolic excursion (tapse) was recorded with m - mode echocardiography parallel to the lateral rv wall and across the tricuspid annular plane and measured as the distance of systolic movement of the rv annulus in the longitudinal direction.8) rv tei index was defined as the ratio of isovolumic relaxation time (ivrt) and isovolumic contraction time (ivct) divided by ejection time (et) of rv.8) rv free wall thickness was measured using m mode from the subcostal view at the peak of the r wave at the level of the tricuspid valve chordae tendineae . Rv tei index was defined as the ratio of ivrt and ivct divided by et of rv.8) pa systolic pressure was estimated from the maximal continuous - wave doppler velocity of the tricuspid regurgitation (tr) jet imaged from multiple planes plus estimated central venous pressure calculated by the diameter of inferior vena cava and distensibility.8) an average of three measurements was used for all analyses . Rv strain and strain rate were analyzed off - line with velocity vector imaging (vvi) software (axius, siemens medical solutions, mountain view, ca, usa). After manual tracing of the endocardial border of the rv (about 10 to 16 points) over one frame, the endocardial borders were automatically tracked throughout the cardiac cycle . Myocardial velocity is derived as the ratio between frame - to - frame displacement of the speckles and the time interval.12) global longitudinal strain and time - to peak strain were calculated . Negative strain values indicate tissue shortening and a smaller value (that is, higher absolute value) indicates better rv systolic function . Global longitudinal strain of rv (rvlsglobal) was calculated by the average of six segmental values of the lateral wall and interventricular septum . Global longitudinal strain of rv free wall (rvlsfw) was measured by the averages of three segmental values (base, mid and apex) of the lateral wall . Hemodynamic data included the pa systolic pressure (spap), mean pa pressure (mpap), pulmonary capillary wedge pressure (pcwp), right atrium and rv pressures, and cardiac output . Pvr was calculated using the formula (mpap - pcwp)/cardiac output and reported in wood units . A 6-minute walking distance was performed according to the american thoracic society protocol.15) total distance walked, expressed in meters, was recorded . Brain natriuretic peptide (bnp) was measured by triage immunoassay reagents (biosite, san diego, ca, usa) formatted for beckman coulter instrumentation (brea, ca, usa). Longitudinal strain measurements for each patient were performed offline in a blinded manner by two independent observers . Each observer performed two independent measurements of regional and global rv strain values with vvi on same echocardiographic images in a randomly selected 10 patients . The data were analyzed using spss version 20.0 (spss inc ., chicago, il, usa) and medcalc version 12.4 (medcalc software, mariakerke, belgium). Linear regression analysis was performed to evaluate the relationship between rvls and other functional or invasive hemodynamic variables . A paired student t test was applied to evaluate the difference of basal and follow - up values . In the variables without normal distribution, wilcoxon signed rank test was used to evaluate the difference . To assess the overall differences on rvlsglobal between groups without versus with change of treatment (i.e. Treatment initiation or intensification), applied a linear mixed effects model with unstructured covariance for random effects was . Effects of groups were tested as a factor, time after initial echocardiographic assessment as a covariate, as well as timegroup interactions . Variability was tested with 2-way analysis of variance with calculation of intra- and inter - observer standard error or measurement (semintra and seminter). Semintra expresses the random error by a single observe, while seminter is an indicator of the mean variation between different observers.16) all tests were two - sided and p value<0.05 was considered statistically significant . A total 34 patients (29 females, mean age 4513 years old) with who group i pah {dana point group i: 26 idiopathic, 5 familial, and 3 associated with other diseases (2 with connective tissue disease and 1 with systemic to pulmonary shunt)} were included in this study . Their mean duration of disease was 2331 months (range 0 - 94 months) and the majority of patients were who functional class ii (29%) and iii (62%). Their mean time interval between echocardiography and rhc was 5 days (range: 0 - 30 days). Twenty patients had been diagnosed as pah in this institution and the remaining 16 patients were transferred from other hospitals . Eight patients were taking a single vasodilator medication {1 on a phosphodiesterase-5 inhibitor (pde5i), 3 on an oral endothelin receptor antagonists (era), and 4 on prostaglandins} and 8 patients were taking a combination of vasodilators (3 on era and prostaglandins, 3 on pde5i and prostaglandins, and 2 on pde5i, era, and prostaglandins). The baseline clinical, laboratory, echocardiographic, and hemodynamic data (table 1) were typical for a pah population, with normal lv systolic function, elevated pa pressure, rv enlargement, and rv systolic dysfunction . In patients with advanced clinical symptoms (nyha functional class iii / iv) had significantly decreased rvlsglobal (-17.16.1 vs. -11.43.1%, p=0.01). Rvlsfw showed lower in the patients with advanced symptoms without statistical significance (-18.37.1 vs. -13.44.4%, p=0.08, fig . Rvlsglobal showed good correlations with 6-minute walking distance (6mwd, r=-0.54, p<0.01) and logarithmic transformation of bnp concentration (logbnp, r=0.65, p<0.01, fig . 2a and c). Also, rvlsfw demonstrated significant correlations with 6mwd (r=-0.57, p<0.01) and logbnp (r=0.65, p<0.01, fig . Rvlsglobal had significant correlations with hemodynamic parameters including mpap (r=0.35, p<0.05), cardiac index (r=0.50, p<0.01, and pvr (r=-0.45, p=0.01, fig . Rvlsfw showed similar correlation with cardiac index (r=-0.47, p<0.01) and pvr (r=0.45, p=0.01). However, it did not have a significant correlation with mpap (r=0.31, p=0.08, fig . Follow - up rhc data were available in 25 patients with median time interval of two rhcs was 33 months (inter - quartile range=21 - 57 months). During the period, they were treated with additional specific pulmonary vasodilator therapy (19 with prostaglandin therapy, 3 with pde5i, 1 with era, and 2 with combination of era and pde5i). The follow - up clinical, echocardiographic and hemodynamic data are listed in table 2 . With specific pulmonary vasodilator treatment, clinical symptoms were improved, 6mwds were increased and bnp levels were decreased . Follow - up rvlsglobal value also showed significant correlation with follow - up 6mwd (r=-0.44, p=0.04) whereas rvlsfw showed insignificant correlation (r=-0.40, p=0.06). As baseline logbnp, follow - up logbnp revealed significant correlations with rvlsglobal (r=0.75, p<0.01) and rvlsfw (r=0.68, p<0.01). There were significant correlations between the change of mpap and rvlsglobal (r=0.45, p<0.01) and rvlsfw (r=0.43, p<0.01, fig . The change of pvr also had significant correlations with change of rvlsglobal (r=0.40, p=0.01) and rvlsfw (r=0.31, p<0.05, fig . However, 5 patients had a worsening of 6mwd . In patients with worsening of 6mwd, rvlsglobal showed no significant change (from -13.53.8% to -13.94.7%, p=0.86). In patients with improvement of 6mwd, rvlsglobal improved from -12.75.8% to -16.05.2% (p=0.03). Interestingly, the rvfac (a " conventional " marker of rv function) was significantly changed in both patient groups (in patients with worsening of 6mwd: from 204% to 264%, p=0.02; in patients with improvement in 6mwd: from 217% to 299%, p<0.01). In our cohort, all patients underwent echocardiographic follow - up studies early (within 1 to 6 months, median 95 days) and late (> 1 year, median 772 days). There were 6 patients without change of treatment after initial echocardiography and 28 patients with either treatment initiation or intensification (i.e., increase in dose or addition of another agent). 5 . There was an improvement of rvlsglobal during follow - up in patients with change of medication (p=0.03 for the difference in slopes). Interestingly, while rvlsglobal was overall higher in the group without the change of medication (p=0.04), there was no additional change during follow - up . A total 34 patients (29 females, mean age 4513 years old) with who group i pah {dana point group i: 26 idiopathic, 5 familial, and 3 associated with other diseases (2 with connective tissue disease and 1 with systemic to pulmonary shunt)} were included in this study . Their mean duration of disease was 2331 months (range 0 - 94 months) and the majority of patients were who functional class ii (29%) and iii (62%). Their mean time interval between echocardiography and rhc was 5 days (range: 0 - 30 days). Twenty patients had been diagnosed as pah in this institution and the remaining 16 patients were transferred from other hospitals . Eight patients were taking a single vasodilator medication {1 on a phosphodiesterase-5 inhibitor (pde5i), 3 on an oral endothelin receptor antagonists (era), and 4 on prostaglandins} and 8 patients were taking a combination of vasodilators (3 on era and prostaglandins, 3 on pde5i and prostaglandins, and 2 on pde5i, era, and prostaglandins). The baseline clinical, laboratory, echocardiographic, and hemodynamic data (table 1) were typical for a pah population, with normal lv systolic function, elevated pa pressure, rv enlargement, and rv systolic dysfunction . Rvlsglobal was -13.35.0% and rvlsfw was -15.15.8% . In patients with advanced clinical symptoms (nyha functional class iii / iv) had significantly decreased rvlsglobal (-17.16.1 vs. -11.43.1%, p=0.01). Rvlsfw showed lower in the patients with advanced symptoms without statistical significance (-18.37.1 vs. -13.44.4%, p=0.08, fig . Rvlsglobal showed good correlations with 6-minute walking distance (6mwd, r=-0.54, p<0.01) and logarithmic transformation of bnp concentration (logbnp, r=0.65, p<0.01, fig . 2a and c). Also, rvlsfw demonstrated significant correlations with 6mwd (r=-0.57, p<0.01) and logbnp (r=0.65, p<0.01, fig . Rvlsglobal had significant correlations with hemodynamic parameters including mpap (r=0.35, p<0.05), cardiac index (r=0.50, p<0.01, and pvr (r=-0.45, p=0.01, fig . Rvlsfw showed similar correlation with cardiac index (r=-0.47, p<0.01) and pvr (r=0.45, p=0.01). However, it did not have a significant correlation with mpap (r=0.31, p=0.08, fig . Follow - up rhc data were available in 25 patients with median time interval of two rhcs was 33 months (inter - quartile range=21 - 57 months). During the period, they were treated with additional specific pulmonary vasodilator therapy (19 with prostaglandin therapy, 3 with pde5i, 1 with era, and 2 with combination of era and pde5i). The follow - up clinical, echocardiographic and hemodynamic data are listed in table 2 . With specific pulmonary vasodilator treatment, clinical symptoms were improved, 6mwds were increased and bnp levels were decreased . Rv systolic function was improved, and mpap and pvr were decreased . Follow - up rvlsglobal value also showed significant correlation with follow - up 6mwd (r=-0.44, p=0.04) whereas rvlsfw showed insignificant correlation (r=-0.40, p=0.06). As baseline logbnp, follow - up logbnp revealed significant correlations with rvlsglobal (r=0.75, p<0.01) and rvlsfw (r=0.68, p<0.01). There were significant correlations between the change of mpap and rvlsglobal (r=0.45, p<0.01) and rvlsfw (r=0.43, p<0.01, fig . The change of pvr also had significant correlations with change of rvlsglobal (r=0.40, p=0.01) and rvlsfw (r=0.31, p<0.05, fig . However, 5 patients had a worsening of 6mwd . In patients with worsening of 6mwd, rvlsglobal showed no significant change (from -13.53.8% to -13.94.7%, p=0.86). In patients with improvement of 6mwd, interestingly, the rvfac (a " conventional " marker of rv function) was significantly changed in both patient groups (in patients with worsening of 6mwd: from 204% to 264%, p=0.02; in patients with improvement in 6mwd: from 217% to 299%, p<0.01). In our cohort, all patients underwent echocardiographic follow - up studies early (within 1 to 6 months, median 95 days) and late (> 1 year, median 772 days). There were 6 patients without change of treatment after initial echocardiography and 28 patients with either treatment initiation or intensification (i.e., increase in dose or addition of another agent). 5 . There was an improvement of rvlsglobal during follow - up in patients with change of medication (p=0.03 for the difference in slopes). Interestingly, while rvlsglobal was overall higher in the group without the change of medication (p=0.04), there was no additional change during follow - up ., echocardiographic follow - up data showed that changes in rvls reflect changes in treatment during follow - up . This is relevant since, to our knowledge, there are no studies documenting the changes in rvls during pulmonary hypertension treatment . These findings imply that, while single measurement of rvls is prognostically relevant, it may be also necessary to track changes in treatment to appreciate its impact, and improve prognostication . Finally, when compared to previous studies,17)18) in this manuscript we correlate rvls with measured, rather than estimated, rvsp and pulmonary vascular resistance . This finding is also relevant since a recent study19) showed that echocardiographic estimation may not be always accurate . Although rv systolic dysfunction is an important prognostic factor in pah patients, rv assessment can be challenging.20) rhc gives important pressure- and flow - derived parameters in the diagnosis and management of pah patients.21) however, frequent serial measurement is invasive and impractical.22) cmr is a useful imaging method providing morphological and functional information of the rv.23)24) however, it is expensive, time consuming, and not readily available in most institutions, and may not be performed in some patients . It provides several doppler - derived parameters about right heart hemodynamics and some parameters; tapse or presence of pericardial effusion, are known as prognostic factors in pah patients.20) however, the assessment of rv function parameters by standard 2de often remains insufficient because of measurement variability intrinsic to the parameters measured.19) two - dimensional strain echocardiography is a new method providing relatively easy and angle - independent quantification of myocardial deformation on 2de images.12) because the predominant orientation of muscle fiber in the rv is in the longitudinal plane, longitudinal motion of rv is a major determinant of rv systolic function, especially in patients with pah.7) strain echocardiography can measure rvls, and rvls can give direct information about regional and global rv mechanics . Rv dysfunction is a well - known factor of functional impairment in pah patients.25) presently, patients with advanced symptoms had more decreased rvls . Our result is similar to the previously reported studies showing pah patients with worse rv longitudinal peak systolic strain had worse functional class and lower rv systolic function.12)26) patients with poor exercise capacity (6mwd<332 m) have poor prognosis.20)27) rvls significantly correlated with functional capacity assessed by 6mwd at baseline and follow - up in this study . After adjustment of age, gender, body mass index, cardiac index and mpap, rvlsglobal (standardized =-0.63, p<0.01) and rvlsrvfw (standardized =-0.59, p<0.01) showed significant association with baseline 6mwd . Increased pvr, as consequences of pathological changes in the pulmonary arteries, results in rv pressure overload and rv systolic dysfunction in pah.25) chronic rv pressure overload directly affect rv longitudinal systolic function and result in impairment of rvls.17) pirat et al.12) reported significantly impaired global and regional rv systolic function in 57 patients with pah compared with age - matched normal controls . Sachdev et al.6) documented significantly decreased rv longitudinal peak systolic strain of rv free wall (-155%) in a cohort of 80 pah patients . They showed that patients with impaired rv free wall strain (> -12.5%) had higher mpap and pvr . In our study, mean rvlsglobal was -13.25.0% and rvlsfw was -15.15.8%, and these values are similarly decreased as in the study of sachdev et al.6) we showed significant correlations of baseline rvls with functional and hemodynamic parameters . Recent development of pharmaceutical therapies has resulted in improved hemodynamics, exercise capacity, and prognosis in pah patients.28) the effect of specific pulmonary vasodilator therapy on rvls and their relationship with invasive hemodynamic parameters was also investigated . Moreover, there are several reports regarding how impaired rvls can be used as a prognostic markers in pah patients.6)18)26) these results suggest that rvls can be a useful non - invasive indicator in the serial assessment of rv systolic function in pah patients . In this retrospective and observational study, major limitations include the relatively small numbers of pah patients and the number of pah patients who did not have repeat right heart catheterization . Bias may have been introduced from patient selection . This result suggests that the assessment of rvls may be useful to track clinical changes in pah patients . We analyzed rvls with stored images, and the analysis may have been affected by image quality and echocardiographic machines . Secondly, we used a vendor - independent platform to measure rvls with echocardiographic images studied by different echocardiographic machines . However, using a vendor - independent platform from different echocardiographic machines can be an option in the comparison of rvls in serial echocardiographic examinations, especially in the assessment of treatment effects . Echocardiographic parameters and cardiac catheterization were not performed simultaneously . The decision for a second catheterization nonetheless, a prospective study with large numbers of patients using the same echocardiographic machine will be needed to confirm the correlations and the clinical impact of this measurement . Rvls calculated by strain echocardiography correlates with functional capacity and invasive hemodynamic parameters in patients with pah . The increase of rvls corresponds with the decrease of mpap and pvr with specific pulmonary vasodilator therapy . Decrease of mpap and pvr as a result of treatment was associated with improvement of rvls . In this retrospective and observational study, major limitations include the relatively small numbers of pah patients and the number of pah patients who did not have repeat right heart catheterization . This result suggests that the assessment of rvls may be useful to track clinical changes in pah patients . We analyzed rvls with stored images, and the analysis may have been affected by image quality and echocardiographic machines . Secondly, we used a vendor - independent platform to measure rvls with echocardiographic images studied by different echocardiographic machines . However, using a vendor - independent platform from different echocardiographic machines can be an option in the comparison of rvls in serial echocardiographic examinations, especially in the assessment of treatment effects . Echocardiographic parameters and cardiac catheterization were not performed simultaneously . The decision for a second catheterization nonetheless, a prospective study with large numbers of patients using the same echocardiographic machine will be needed to confirm the correlations and the clinical impact of this measurement . Rvls calculated by strain echocardiography correlates with functional capacity and invasive hemodynamic parameters in patients with pah . The increase of rvls corresponds with the decrease of mpap and pvr with specific pulmonary vasodilator therapy . Decrease of mpap and pvr as a result of treatment was associated with improvement of rvls.
This was a multicenter, community - based, retrospective observational study of patients with pns, ranging from 8 to 20 mm in diameter, presenting to 18 geographically representative outpatient pulmonary clinics across the united states . The study was approved at 15 sites by a central institutional review board and at three sites by local institutional review board approval . Four hundred forty sites were identified based on investigator databases and claims data from a large insurance carrier whose coverage population was representative of the overall us population . Of these, 77 sites expressed interest in participating, and 48 sites went on to sign confidentiality agreements . Of these, 17 did not request additional information, leaving 31 sites undergoing qualification review . Eighteen outpatient pulmonary clinics were chosen to participate based on the following criteria: (1) management of patients with pns, (2) availability of medical records, and (3) ability to perform data abstraction . In addition, investigators targeted enrollment of geographically diverse patients to limit the potential bias associated with differences in practice patterns and to account for variation in disease prevalence (eg, endemic mycoses) that could alter management decisions . Patients were identified by querying databases (eg, billing and scheduling systems) using five international classification of diseases, ninth revision, clinical modification codes for pn (793.1, 786.6, 518.89, 519.8, 519.9) to ensure homogeneity in patient identification and inclusion . Manual chart abstraction was then used to identify those who met the criteria . To minimize selection bias, the sites were not permitted to use additional codes during database query to identify patients . To ensure a systematic sample, inclusion criteria included age 40 years and 89 years at the time of nodule finding, presentation to a pulmonologist, nodule size 8 to 20 mm, and definitive diagnosis ascertained by tissue diagnosis or radiographic follow - up for 2 years . Exclusion criteria included chest ct scan performed> 60 days prior to the initial visit, prior diagnosis of any cancer within 2 years of nodule detection, or incomplete chart data . Patients were categorized into three groups by the most invasive procedure performed during management, as follows: surveillance (serial imaging), biopsy (ct scan - guided transthoracic needle aspiration [ttna] or bronchoscopy), or surgery (including mediastinoscopy, video - assisted thorascopic surgery, and/or thoracotomy). Clinical data were abstracted retrospectively by designated study staff into an electronic data capture system from initial consultation through establishment of a definitive diagnosis (ie, pathology results) or a minimum 2-year follow - up . Data included patient demographic and clinical characteristics, pn characteristics, imaging tests, invasive testing, and surgery . Pet scan reports were reviewed and abstracted where available for a subset of patients . To adjudicate a pet scan report as positive or negative, an algorithm was developed that prioritized the following components of the report from highest to lowest: final radiology impression, description of findings, and standard uptake values (suvs) (e - fig 1). Pet scanning was defined as negative if the report included any of the following statements: no evidence of malignancy, no 18f - fluorodeoxyglucose uptake or hypermetabolic activity, or an suv of <2.5 . A positive pet scan was defined as a report that included any of the following statements: concern or suggestion of malignancy, findings that described increased 18f - fluorodeoxyglucose uptake or hypermetabolic activity, or an suv 2.5 . In the 27 cases in which the findings were discordant with the final impression, programmed edit checks were built into the electronic data capture system and at the conclusion of chart abstraction, each site provided access to a random 10% sample of deidentified patient records for review . Two previously developed and validated models were used to estimate the pca in each patient . Receiver operating characteristic curves and the area under the curve were generated with 95% cis . The pca was calculated for each patient and categorized into three groups (<5%, 5% to <65%, and 65%). Or analysis of variance tests were used to compare subgroups, and p values <.05 were considered significant . A nodule was classified as benign based on confirmed benign pathology or the absence of radiographic change as determined by the managing physician during surveillance for at least 2 years . Multivariate logistic regression was performed to identify factors associated with the use of an invasive diagnostic procedure . All statistical analyses were performed using sas / stat, version 9.3 (sas institute inc). Four hundred forty sites were identified based on investigator databases and claims data from a large insurance carrier whose coverage population was representative of the overall us population . Of these, 77 sites expressed interest in participating, and 48 sites went on to sign confidentiality agreements . Of these, 17 did not request additional information, leaving 31 sites undergoing qualification review . Eighteen outpatient pulmonary clinics were chosen to participate based on the following criteria: (1) management of patients with pns, (2) availability of medical records, and (3) ability to perform data abstraction . In addition, investigators targeted enrollment of geographically diverse patients to limit the potential bias associated with differences in practice patterns and to account for variation in disease prevalence (eg, endemic mycoses) that could alter management decisions . Patients were identified by querying databases (eg, billing and scheduling systems) using five international classification of diseases, ninth revision, clinical modification codes for pn (793.1, 786.6, 518.89, 519.8, 519.9) to ensure homogeneity in patient identification and inclusion . Manual chart abstraction was then used to identify those who met the criteria . To minimize selection bias, the sites were not permitted to use additional codes during database query to identify patients . To ensure a systematic sample, inclusion criteria included age 40 years and 89 years at the time of nodule finding, presentation to a pulmonologist, nodule size 8 to 20 mm, and definitive diagnosis ascertained by tissue diagnosis or radiographic follow - up for 2 years . Exclusion criteria included chest ct scan performed> 60 days prior to the initial visit, prior diagnosis of any cancer within 2 years of nodule detection, or incomplete chart data . Patients were categorized into three groups by the most invasive procedure performed during management, as follows: surveillance (serial imaging), biopsy (ct scan - guided transthoracic needle aspiration [ttna] or bronchoscopy), or surgery (including mediastinoscopy, video - assisted thorascopic surgery, and/or thoracotomy). Clinical data were abstracted retrospectively by designated study staff into an electronic data capture system from initial consultation through establishment of a definitive diagnosis (ie, pathology results) or a minimum 2-year follow - up . Data included patient demographic and clinical characteristics, pn characteristics, imaging tests, invasive testing, and surgery . Pet scan reports were reviewed and abstracted where available for a subset of patients . To adjudicate a pet scan report as positive or negative, an algorithm was developed that prioritized the following components of the report from highest to lowest: final radiology impression, description of findings, and standard uptake values (suvs) (e - fig 1). Pet scanning was defined as negative if the report included any of the following statements: no evidence of malignancy, no 18f - fluorodeoxyglucose uptake or hypermetabolic activity, or an suv of <2.5 . A positive pet scan was defined as a report that included any of the following statements: concern or suggestion of malignancy, findings that described increased 18f - fluorodeoxyglucose uptake or hypermetabolic activity, or an suv 2.5 . In the 27 cases in which the findings were discordant with the final impression, adjudication was performed by two independent pulmonologists and agreement was reached in all cases . Programmed edit checks were built into the electronic data capture system and at the conclusion of chart abstraction, each site provided access to a random 10% sample of deidentified patient records for review . Two previously developed and validated models were used to estimate the pca in each patient . Model accuracy was determined by comparing the pca with the final diagnosis . Receiver operating characteristic curves and the area under the curve were generated with 95% cis . The pca was calculated for each patient and categorized into three groups (<5%, 5% to <65%, and 65%). Or analysis of variance tests were used to compare subgroups, and p values <.05 were considered significant . A nodule was classified as benign based on confirmed benign pathology or the absence of radiographic change as determined by the managing physician during surveillance for at least 2 years . Multivariate logistic regression was performed to identify factors associated with the use of an invasive diagnostic procedure . All statistical analyses were performed using sas / stat, version 9.3 (sas institute inc). Eighteen sites were chosen based on geographic location and ability to meet the inclusion criteria (e - fig 2). Most of the exclusions were based on nodule size (<8 mm or> 20 mm) (n = 1,314), missing clinical information (n = 424), or insufficient follow - up time (n = 303). There was some variation at both the site level and regionally in the numbers of patients excluded because of missing data (e - tables 1, 2). Inclusion and exclusion . Eval = evaluation . Of the 377 patients included, 283 (75%) had a nodule that was benign, and 94 (25%) had a malignant nodule (table 1). The average age was 64.5 years, although patients with malignancy were slightly older (p <.02). Those with cancer were more likely to be current or former smokers (p <.0001). Although the number of pack - years smoked was significantly higher in those with malignancy (45 vs 27, p <.0001), a substantial proportion of those with benign nodules smoked (67%). Malignant nodules were larger than benign nodules (p <.0001), although there did not appear to be a reliable cutoff above which malignancy could be predicted . Twelve percent of nodules 11 mm in size were malignant, whereas 40% of nodules between 16 and 20 mm were malignant . Other cancer diagnoses include spindle cell, mesothelioma, and metastatic cancer . Diagnostic outcome by nodule size (n = 377). Current smokers (or = 3.29; 95% ci, 1.36 - 7.98) and patients with larger nodules (16 - 20 mm) were more likely (or = 4.77; 95% ci, 2.95 - 7.72) to have a procedure (biopsy or surgery) performed . There was no difference in receipt of procedure based on age, family history of cancer, or geographic location (table 2). Factors associated with the decision to pursue invasive procedures in the management of pns pn = pulmonary nodule . To further understand nodule management decisions, we used two prediction models to determine the pca for each individual in the study . These models had very good accuracy in this cohort of patients, with an area under the curve of 0.77 (95% ci, 0.72 - 0.81) and 0.74 (95% ci, 0.69 - 0.80), respectively (e - fig 3). Because the results did not vary significantly between models, we report the results from the veteran affairs model here and results from the mayo model in e - table 3 . Table 3 lists the pretest probability for cancer by three ranges as defined by the chest guidelines for pn management: low risk (pca <0.05), intermediate risk (pca 0.05 to <0.65), and high risk (> 0.65). The average pca for all patients as calculated by the model was 0.33, which is only slightly higher than the cancer prevalence of 25% in this population . Thirty - six patients had nodules that had a low risk of malignancy, and all were benign . Slightly more than one - half were followed by serial imaging alone (n = 20, 56%). However, despite a pca of <0.05, 10 (28%) underwent biopsy, and six (17%) had surgical resection . The majority of nodules (n = 300) fell into the intermediate - risk category, and 75% of these were benign . Forty - one patients (11%) had high - risk nodules, and 23 (58%) were benign . The rate of surgical resection was similar among the three groups (17%, 21%, 17%, respectively; p = .69). Alternative cut points for the low - risk category were investigated . All nodules with a pca of <0.10 were found to be benign, whereas 98% of those with a pca of <0.15 were benign (table 4). (%) or mean sd . See table 3 for expansion of abbreviations . Nearly one - half of all patients (46%, n = 175) were followed with serial imaging only . The median number of scans performed was three (range, one to seven). Although the majority (62%) had three to five follow - up imaging studies, 4% underwent seven repeat scans . All nodules in this group were benign by resolution or 2 years of radiographic stability . Biopsy (ttna or bronchoscopy) was performed as the most invasive procedure in 125 patients (33%) and was positive for malignancy in 44 (35%). In more than one - half (n = 71, 57%), a specific benign diagnosis was obtained, whereas 10 (8%) were nondiagnostic and subsequently followed for 2 years . A total of 77 patients (20%) had surgery as their most invasive test . Prior to surgery, 19 patients (25%) underwent a ttna or bronchoscopy; 11 had a nondiagnostic biopsy (seven malignant, four benign at surgery), and eight had a benign biopsy result (three malignant, five benign at surgery). Overall, malignancy was diagnosed in 65% (n = 50; 95% ci, 53.2%-75.5%) of those who received surgery, whereas 35% (n = 27; 95% ci, 24.5%-46.8%) underwent surgery for benign disease . A pet scan was performed in 141 patients (37%). Overall, pet scan use increased with the intensity of testing (surveillance 24%, biopsy 42%, surgery 60%, p <.0001). Results were available for 78% (n = 110) of those who underwent pet scanning (fig 3). The overall accuracy of pet scanning was 74%, with a false - positive (fp) rate of 39% and a false - negative (fn) rate of 9% . In 8- to 11-mm nodules with pet scan data (n = 38), the fn rate was 10% and the fp rate was 36% . Nodules measuring> 11 to 15 mm (n = 48) had fn and fp rates of 9% and 36%, respectively . The fn rate for pet scans in nodules> 15 mm (n = 24) was 8%, and the fp rate was 55% . Pet scan use grouped based on most invasive procedure . A, surveillance . (n = 175), 42 (24%) underwent pet scanning, for whom 31 reports were available . Seven (23%) had pet scan positive results and were followed with serial imaging . Among the 125 patients who underwent biopsy as the most invasive procedure, 53 (42%) underwent pet scan prior to the biopsy . Twenty - nine (69%) were pet scan positive and 13 (31%) were negative, yet all went on to biopsy . In the 77 patients who underwent surgery, 46 (60%) underwent a preoperative pet scan, for which 37 reports were available . Thirty - one (84%) had pet scan positive results . Among the 19 patients who underwent preoperative biopsy, this study describing the management of intermediate pns referred to community pulmonologists has several important findings . First, a quarter of patients referred for intermediate - sized pn evaluation were found to have cancer, emphasizing the importance of the diagnostic evaluation . Third, despite guideline recommendations for surveillance of pns with <5% pca, 44% of low - risk patients underwent one or more invasive procedures for a benign nodule . Finally, the rate of surgical resection for nodules with various pretest probabilities of malignancy was similar . This suggests that pulmonologists do not routinely consider pca, are unaware that guidelines exist for nodule management, or do not to follow them . To our knowledge, this is the first study that documents the prevalence of cancer (25%) in patients with intermediate - sized nodules who are referred to community - based pulmonologists . This finding has implications for managing pns in the community; because the risk of cancer is substantial, patients should be managed based on their individualized risk of malignancy . We found that a high proportion of patients who underwent surgery had benign disease without variation when stratified by the pretest probability of cancer in the nodule . The rates of resection for benign disease varied from 9% to 23% in screening trials and surgical series . Guidelines recommend surgery if the pretest probability of cancer is high (pca> 65%) because a negative biopsy result would not dissuade the physician from pursuing a definitive diagnosis and a positive biopsy result would lead to surgery anyway . The risk models we evaluated were very good at identifying those patients who did not have cancer (100% nodules with <5% pca were benign), yet surgical resection was performed at rates similar to those with a high probability of cancer . Similarly, there was no difference in the proportion of patients undergoing surveillance when stratified by pca . This suggests that pulmonologists did not readily follow the guidelines because they were unaware of them, did not believe them useful, or overestimated the pca . This study does not allow us to explore the reasons why pulmonologists aggressively manage some nodules that turn out to be benign . There are several possible explanations as to what may have led to biopsy or surgery, and they include being misled by pet scan results, patient preferences for diagnostic certainty, an overestimation of the pca, or incentives for procedure referral . Further research is needed to determine the exact drivers of physician decision - making in pn management . Although physicians did not appear to use pca in making decisions about surgery or surveillance, there was a trend toward an increasing use of biopsy as the pca increased (p = .07). This finding suggests that pulmonologists may use clinical intuition and experience to distinguish benign from malignant disease, although, admittedly, data on provider years in practice and the proportion who were board certified were not available . A significant proportion of patients in this study with benign nodules were older and had significant smoking histories and larger nodules, all which typically suggest malignancy and may have led physicians to overestimate pca . In the setting of a positive or ambiguous pet scan interpretation, it is possible that the pulmonologists felt compelled to confirm the abnormal findings with a definitive tissue diagnosis . The fp rate for pet scans in this study was 39%, as compared with the 5% to 28% rate reported in the literature . Conversely, in some instances, pet scan results did not seem to be taken into account when making management decisions . For instance, in the surveillance - only group, eight patients with positive pet scans did not undergo further diagnostic procedures . Similarly, in the biopsy group, 13 patients with negative pet scan results still underwent an invasive procedure . The guidelines advocate a patient - centered approach to decision - making after patients are informed of the pca . Thus, some patients may request surgery despite a low pca because of anxiety about the unknown and an unwillingness to continue with surveillance . The cornerstone of management for pns has been calculating pca and acting on those calculations . This study provides evidence that physicians may not use pca to guide decision - making and also suggests that an alternative low - risk - category pca higher than the current chest guidelines would shift more patients into the surveillance management strategy without missing malignancy . Although the exact reason for management decisions cannot be elicited, it is clear that there is wide variation in the management of pns by pulmonologists . The goal for nodule management is to efficiently detect lung cancer while decreasing the number of unnecessary invasive procedures . A number of radiographic improvements, novel tests, and biomarkers have been developed with the potential to help determine the likelihood of malignancy in a nodule . The use of semiautomated volumetric software to measure the diameter and volume doubling time of a nodule between scans has been used successfully in screening trials to improve the sensitivity, specificity, and negative predictive value of ct scans . Similarly, volumetric measurements added to traditional nodule malignancy prediction models have also been shown to improve the classification of malignancy from 60% to 80%, with better performance than that of the traditional model . Identification of aberrations in airway epithelial cell gene expression, circulating tumor antibodies, plasma proteomics, exhaled breath analysis, and microrna are all being studied actively for use in diagnosing lung cancer . These novel tests have the potential to further refine nodule management and alleviate the ambiguity of current management strategies . First, the retrospective design precluded a complete understanding of the rationale for test ordering . However this design did allow for a definitive final diagnosis to be established with biopsy, surgery, or 2 years of radiographic surveillance . Second, incomplete pet scan data coupled with difficulties in scan interpretation resulted in variations in the definition of a positive pet scan . To overcome this limitation finally, excluding patients who had had a ct scan> 60 days prior to initial consultation may have selectively excluded some patients with low probability nodules . The findings are generalizable because of the large sample size, geographic distribution, and multicenter design . The study also focuses on a size range of nodules (8 - 20 mm) that most often presents a diagnostic dilemma for physicians . Nodules smaller than 8 mm are followed with surveillance because the pca is low, pet scans do not reliably characterize small nodules, and biopsy of small nodules is difficult; by contrast, nodules> 20 mm are likely to undergo biopsy or surgery because the pca is high . As opposed to other published series, this study allowed for all three management strategies to be studied in detail . In conclusion, one in four patients referred with a nodule to a community pulmonologist has cancer, making management decisions critical to patient outcomes . To spare patients unnecessary testing, use of a higher pca to define a nodule as low risk seems reasonable . There is a wide variation in how nodules are managed and the choice of management may be influenced by a variety of factors in addition to pretest probability . Future research should focus on what influences decision - making in pn management so that interventions may be developed to promote proper guideline adherence and avoidance of unnecessary invasive procedures.
Migraine is a chronic disease of the brain that enhances morbidity with complains like photophobia, headache attacks, phonophobia, and nausea . In the second edition of the headache classification by the international headache society, migraine is among the primary headaches and migraine without aura, migraine with aura, migraine cursors in childhood, retinal migraine, and migraine complications are grouped as potential migraines . According to the results of the turkish headache database, the number of female patients is 4 times that of male patients . There were 84% of the adults who were migraine without aura patients, and 14% who were migraine with aura patients . Although most of these patients have been found to have comorbidity with other diseases, eye diseases were excluded from those studies; however, photophobia is reported to be the most frequent accompanying symptoms . The vision system is reported to play a role, especially in migraine with aura headaches . One of 5 patients with a headache is directed to the department of eye diseases, mostly because almost all migraine with aura patients have visual auras . The relationship between migraine and refractive errors has been studied for more than 100 years; however, conflicting results have emerged from those studies [79]. In this study, we compared the auto - refractometric values of migraine patients with and without visual aura, with those patients without migraine complaints . We prospectively compared the patients who consulted the neurology policlinic between june 2013 and march 2014 with a headache complaint and diagnosed with migraine in accordance with the ichd-2 criterion with the individuals of the same gender and age group with no headache complaint . This study adhered to the tenets of the declaration of helsinki and was carried out with the approval of the institution . The patients were given full detailed eye examinations and findings were noted . Out of these patients, those over age 18 years who had 20/20 uncorrected vision and who did not have ocular pathology in their examination were included in the study . The patients were divided into 3 groups: group 1: 86 eyes of 43 migraine patients without aura; group 2: 38 eyes of 19 migraine patients with aura; and group 3: 60 eyes of 30 patients without headache . The same ophthalmologist examined the patients in the period with no headache . The corrected and uncorrected vision of right eye and left and bilateral eye of all the patients were individually measured . Those patients whose uncorrected vision did not have 20/20 value for both eyes separately and bilaterally were excluded from the study . The 3-dimensional vision and eye movements of the patients were evaluated by the worth 4-point test and those patients who failed this test were excluded from the study . Biomicroscopic and fundus examinations of the patients were carried out and the intraocular pressure values were measured with goldmann applanation tonometry; those patients with ocular pathology were excluded from the study . Before the eye examination, following the eye examinations, 45 minutes after the patients were given 1% cyclopentolate hcl drips 3 times with 5 minutes intervals, their auto - refractometry values were recorded again . The intended purpose of using the cyclopentolate with mydriatic and cycloplegic effect was that with the cycloplegic effect, ciliary muscles contraction was hindered and refractivity of the lenses increases through cambering; in other words, accommodation was hindered . Thus, we aimed to provide more accurate refractivity values for the eye structures . In the study, spherical equivalents (se) and cycloplegic spherical equivalents (cse) from the auto - refractometrical values of the patients were used . Spherical equivalent is equal to the sum of the sphere value and half the cylindrical value, which were measured by the auto - refractometer device . Data were analyzed using spss (statistical package for social sciences) for windows 17.0 software . For quantitative data to compare the groups with normal distribution of parameters, we prospectively compared the patients who consulted the neurology policlinic between june 2013 and march 2014 with a headache complaint and diagnosed with migraine in accordance with the ichd-2 criterion with the individuals of the same gender and age group with no headache complaint . This study adhered to the tenets of the declaration of helsinki and was carried out with the approval of the institution . The patients were given full detailed eye examinations and findings were noted . Out of these patients, those over age 18 years who had 20/20 uncorrected vision and who did not have ocular pathology in their examination were included in the study . The patients were divided into 3 groups: group 1: 86 eyes of 43 migraine patients without aura; group 2: 38 eyes of 19 migraine patients with aura; and group 3: 60 eyes of 30 patients without headache . The corrected and uncorrected vision of right eye and left and bilateral eye of all the patients were individually measured . Those patients whose uncorrected vision did not have 20/20 value for both eyes separately and bilaterally were excluded from the study . The 3-dimensional vision and eye movements of the patients were evaluated by the worth 4-point test and those patients who failed this test were excluded from the study . Biomicroscopic and fundus examinations of the patients were carried out and the intraocular pressure values were measured with goldmann applanation tonometry; those patients with ocular pathology were excluded from the study . Before the eye examination, 45 minutes after the patients were given 1% cyclopentolate hcl drips 3 times with 5 minutes intervals, their auto - refractometry values were recorded again . The intended purpose of using the cyclopentolate with mydriatic and cycloplegic effect was that with the cycloplegic effect, ciliary muscles contraction was hindered and refractivity of the lenses increases through cambering; in other words, accommodation was hindered . Thus, we aimed to provide more accurate refractivity values for the eye structures . In the study, spherical equivalents (se) and cycloplegic spherical equivalents (cse) from the auto - refractometrical values of the patients were used . Spherical equivalent is equal to the sum of the sphere value and half the cylindrical value, which were measured by the auto - refractometer device . Data were analyzed using spss (statistical package for social sciences) for windows 17.0 software . For quantitative data to compare the groups with normal distribution of parameters, forty - three (69%) out of the total of 62 patients had migraine without aura and 19 of them (31%) had aura migraine . Age, gender distribution, and auto - refractometrical values of the migraine and control patients are given in table 1 . Although the cycloplegic spherical equivalents results of the right eyes were not statistically significant, statistical significance was found in the tukey test between the migraine patients with aura and the control group (p=0.05). The right and left eyes of the patients were evaluated together and a significant correlation was found between the groups . It was also found through the tukey test that the source of significance was patients with aura (p=0.010). When the refractive value differences between both eyes were evaluated, no significant difference was found in se and cse measurements (p=0.411 and 0.536). To evaluate the impact of cycloplegia in patients, se and cse values were obtained and differences between these values were evaluated . In the post hoc tukey test, it was found that the change in group 2 patients was significantly lower than the change in group 3 patients (p=0.024). The differences between cse and se in all eyes in both groups are given in table 3 . Ninety - nine percent of migraine patients with aura had visual symptoms . It was observed that since visual auras such as photopsy, scintillating scotoma, and hemianosmic changes in the foreground, especially in migraine with aura, the potential ocular problems in migraine pathogenesis have to be taken into account and the patients consulting with headaches were referred to ophthalmologists . We give eye examination to the migraine patients referred to our policlinics from the department of neurology and evaluated their examination findings . There are studies stating that migraine - related findings and refraction defects can trigger migraine . It was reported that migraine may impact the eyelids, conjunctiva, iris, pupil, optical nerve, retina, and extraocular muscles . The relationship between the refractive errors and migraine has been researched since the beginning of the 20 century . Although these studies especially emphasized that anisometropia and astigmatism were the triggers of migraine, the value of their results were limited because they were not controlled studies and no statistical evaluation was made [79]. Emphasized that blurry vision generated by astigmatism could create a hyperexcitability on the visual cortex and this could be a triggering factor for migraine . When the migraine patients in our study were compared with the same age and gender groups, the spherical equivalents were found to be the same, but the cycloplegic spherical equivalents were significantly lower . When the migraine patients are divided into groups with and without aura and evaluated this way, while se values of the patient group with aura were similar to the control group, their cse values were significantly lower . Compatible with this finding, it was found that the changes in migraine patients with aura between the measurements before and after eye drops (1% cyclopentolate hcl) were significantly lower . Similar to our study, there are studies demonstrating that the refractive errors were not different in migraine patients and it was also suggested in these studies that binocular vision was important . However, even though we found no significant difference in refractive values between the 2 eyes in our study, all the patients involved in the study had 3-dimensional and uncorrected 20/20 vision . The fact that the cycloplegic refraction values, especially in patients with aura, were found to be lower in comparison to healthy individuals could be an indication that these patients did not make sufficient accommodation . Since these patients could have blurry vision and difficulty focusing, it should be noted that this could be the trigger of a headache . On the other hand, no anisocoria was found in any of our patients; however, we failed to measure the pupil diameters since we did not have a pupillometer . It is possible that lack of accommodation in addition to isochoric and mydriatic pupils may create an attack with the increase in the amount of light entering the retina . It was found in the previous studies that visual stimulus triggered a migraine in more than 1/3 of the patients and that the migraines triggered by light decreased in patients who were given colored lenses during the migraine attack . Bright light was a triggering factor in 87% of the migraine patients with aura, and this rate was 76% in migraine patients without aura . The general characteristic of this condition is that with the third cranial nerve the most affected, it is accompanied by ocular cranial nerve palsy together with the migraine - like headaches in the form of attacks . In these situations, in which the pathophysiology is not fully understood in 75% of the patients, there was contrast increase and thickening in the mri - scanned nerves during the attacks, and it was thought that there might be an accompanying migraine or independent neuropathy . In another study, in a spect - scanned nerve area, a low level of blood flow was detected and it was thought that a temporary ischemic attack might follow . In addition, ciliary ganglion cells or migraine - related elongated mydriasis might have occurred as a result of temporary dysfunctioning of postganglionic and parasympathetic fibers during a migraine attack . The researchers who coined the term sillier ganglioplegic for this particular situation stated that this might be another result of ophthalmoplegic migraine pathogenesis . According to this hypothesis, the cause of ophthalmoplegic migraine may also cause elongated mydriasis by clinging to the parasympathetic fibers . In the same study, it was stated that, in theory, the pain stemming from the sillier ganglions may find its way to the ophthalmic region of the trigeminal nerve and may create a bilateral headache . Based on this study, it is possible to conclude that innervation deterioration in the sillier ganglions into the sillier muscle in these patients may cause insufficient accommodation . However, it was not possible to evaluate these hypotheses in our study since there were insufficient numbers of migraine patients with aura; they were evaluated during the remission period and no mri tests were carried out . We are of the opinion that the main causes of these conflicting results are misdiagnosis of headaches, changes in the diseases classification due to the new diagnostic techniques, evaluation of all the migraine patients together, and the lump - sum evaluation of all the eye impairments and visual levels . To keep our study free of such handicaps, we only evaluated the patient with uncorrected 20/20 vision, dividing them into groups with and without aura . We found that the cycloplegic spherical equivalent values of our patients with aura were lower than in control patients . However, given the fact that our findings were obtained from the eye examinations of the patients during the remission period, it is not possible to determine whether migraine with aura is the trigger or the result of those attacks . However, the number of migraine patients with aura is less than those without aura and it is very difficult to detect by the patient s ophthalmologist during the attack period . In order to be more conclusive about this particular issue, the neurologist and ophthalmologist should be in communication and there is a need for more comprehensive studies with larger number of participants.
Radiofrequency ablation of the slow pathway is considered to be the gold standard treatment for patients with atrioventricular nodal reentrant tachycardia (avnrt). Pulmonary artery agenesis is a rare anomaly that may occur during embryological development of the heart.1 this agenesis may be accompanied by a complete or partial absence of the lung . In the great majority of the cases, the diagnosis is usually made at or soon after birth and it can be associated with multiple anomalies . An otherwise normal heart may be displaced into the right hemithorax by unilateral pulmonary agenesis.2 catheter ablation of avnrt in the setting of dextrocardia is potentially challenging . There have been a few reports of successful slow pathway ablation in patients with dextrocardia, but usually at the expense of prolonged procedure and fluoroscopy times.37 to our knowledge, slow pathway ablation of a patient with dextrocardia due to pulmonary agenesis has not been described to date . A 35-year - old woman was admitted to our cardiology clinic with complaints of palpitation . She had a 7-year history of paroxysmal palpitations . The tachycardia, at a rate of 170200 bpm, was associated with dyspnea and chest tightness and lasted for up to 60 minutes at a time . Her physical examination revealed no breathing sound on the right and a normal breathing sound on the left hemithorax, while the heart s sound was heard from the right hemithorax . The chest radiogram incidentally showed a rudimentary, opacified right hemithorax with mediastinal shift and herniation of the contralateral lung . Except for dextroposition of the heart, a 12-lead resting electrocardiogram (ecg) showed sinus rhythm at a rate of 64 bpm, a positive qrs complex in avr, a positive p wave in avr and a biphasic p wave in avl, and a prominent r wave in v1 with undetermined horizontal axis (figure 1). The clinical tachycardia was a regular, narrow qrs tachycardia at a rate of 170 bpm, during which the p wave was indiscernible (figure 2). Computed tomography showed a complete absence of the right lung with dextroposition of the heart (figure 3). Electrophysiological study was performed after obtaining written informed consent and discontinuation of all drugs for five half - lives . The right atrium and the right ventricle were imaged on left lateral and anteroposterior views by contrast injection through inferior vena cava to reveal the cardiac anatomy (figure 4). Then, a 4 mm - tipped ablation catheter (cordis webster, baldwin park, ca, usa) was advanced through the 6f sheath into the right atrium and a four - pole fixed - curve diagnostic catheter was placed on the bundle of his . During the electrophysiological study, a narrow qrs tachycardia with a cycle length of 270 ms was reproducibly induced by atrial premature stimulation with an extrastimulus (figure 5a). The ventriculoatrial interval, measured from the onset of ventricular activation on the surface ecg to the earliest deflection of the atrial activation in the his bundle electrogram, was <60 ms, and the diagnosis of typical slow fast avnrt was confirmed . His bundle records were taken with an electroanatomi - cal approach . Then, slow pathway potentials were recorded (figure 5b). Fluoroscopic views of the catheters during the ablation and mapping of the coronary sinus ostium are shown in figures 6 and 7 . During ablation, the radiofrequency energy was adjusted to obtain a catheter tip temperature between 50c and 60c . The current was applied for 60 seconds after junctional tachycardia was observed during ablation (figure 8). After the ablation, there was no residual slow pathway conduction and the clinical arrhythmia was not inducible without or with infusion of isoproterenol . The total procedure time was 30 minutes with a total radiofrequency delivery time of 8 minutes and fluoroscopy time of only 6 minutes . No complication occurred after the procedure and the patient was free of symptoms at 1-year follow - up . Dextrocardia or complex cardiac anatomy of the heart may be challenging to electrophysiologists during catheter ablation procedures . There have been only a few case reports of catheter ablation of supraventricular tachyarrhythmias in patients with dextrocardia.37 pulmonary agenesis can be localized to a single lobe and it can affect an entire lung or, in rare cases, both lungs.1 although a majority of patients with unilateral agenesis die soon after birth or in early childhood, in some extreme cases, patients can survive up to adulthood, like our patient . The most common and familiar form is the mirror image of normal or mirror - image dextrocardia, in which the migration of the apex of the l - bulboventricular loop is into the right hemithorax, as expected . The anterior posterior relationship of the various parts of the heart is normal, but their right - to - left orientation is reversed . The second most common type is dextroversion, in which the heart appears to be rotated into the right hemithorax relative to its normal position . 3) the third type of dextrocardia is dextroposition, in which an otherwise normal heart is displaced or shifted into the right hemithorax by other extracardiac causes such as agenesis or fibrosis of the right lung . Our case was a type 3 dextrocardia . To recognize the ecg findings of dextrocardia necessitates a clear understanding of the electrical axis . Global negativity in lead i, a positively deflected qrs complex in avr, and right axis deviation and reverse r wave progression in precordial leads are the mean ecg findings in dextrocardia . The finding of a positively deflected qrs complex in avr and a negatively deflected ors complex in lead i may lead to misdiagnosis . The most common cause for this finding is reversed electrode placement of arm leads (left and right). During a standard catheter ablation procedure, the catheter is targeted to the ablation region based on typical local electrogram characteristics (slow - pathway potentials) and anatomical landmarks . In contrast to patients with normal anatomy, localization of the slow pathway and stabilization of the catheter are more difficult in patients with dextrocardia . Therefore, the time spent in the catheterization laboratory and the fluoroscopy time are markedly longer than in cases with normal hearts . Our case illustrates the role of imaging by contrast injection in a patient with dextrocardia . The identification of the accurate anatomy using imaging modalities such as computerized tomography and magnetic resonance imaging and three - dimensional image reconstruction using mapping systems may be useful not only to help the electrophysiologist, enabling a safe and successful catheter ablation procedure, but also to understand the complex anatomical structures and to guide optimal catheter access . However, these modalities are quite expensive and are not easily accessible in every unit . Before the ablation procedure, we easily localized the coronary sinus ostium and his bundle position . As such, we did not need an additional catheter in this ablation procedure, but an additional catheter might further facilitate the procedure . We are presenting this pulmonary agenesis case in view of its atypical presentation in adulthood, which is exceptionally rare . As demonstrated in this case, revealing cardiac anatomy by contrast injection or other imaging modalities may shorten the procedure time and decrease the complication rates.
T cells and b cells derived from the lymphoid lineage belong to the adaptive immune system . Function via production of effector cytokines after differentiation and activation . In comparison to the cytotoxic feature of both adaptive cd8 t cells and innate cnk cells, the helper feature of cd4 th cells was considered to be a unique characteristic of the adaptive system acquired during evolution . However, over the past few years several groundbreaking works on a novel member of the innate immune system, the innate lymphoid cell (ilc), have dramatically changed our knowledge about the composition of the innate lymphoid lineage and led us to reconsider the relationship between innate and adaptive lymphoid lineages in the context of evolution . Like other lymphocytes, ilcs also develop from the common lymphoid progenitors (clps) found in fetal liver and adult bone marrow . They were not discovered and classified as a new lymphocyte family until recently, partly due to their distinct enrichment in nonlymphoid tissues such as mucosal tissues, skin, and adipose tissues, with scarce distribution in lymphoid tissues . Their lack of any known lineage surface markers may also contribute to their belated discovery . In actuality, scientists noticed certain subsets of ilcs such as lymphoid tissue inducers (ltis) as early as the 1990s [3, 4], but it was not until three independent reports on type 2 cytokine producing innate lymphoid cells (ilc2s) in 2010 [57] that people began to recognize the possible existence of an innate population with a helper feature mirroring adaptive th cells . The nomenclature of innate lymphoid cells (ilcs) was then formed based on the existence of helper lymphocyte in the innate arm of the immune system . Similar to the classification of th cells, mature helper - like ilcs can be categorized into three groups based on their master regulator expression and signature effector cytokine production . Ilc2s, the innate counterpart of th2 cells, express high levels of gata-3 and are capable of producing type 2 cytokines such as il-5 and il-13 [57]. Ilc3s express rort and are capable of producing il-22 and il-17, similar to th17/th22 cells [810]. The ccr6 lineage includes lymphoid tissue inducer (lti) cells, while ccr6ilc3 can give rise to a special population of rort ilc3s that express the natural cytotoxicity receptor (ncr) nkp46 (encoded by ncr1) in mice and nkp44 (encoded by ncr2) in humans . Ccr6ilc3s can also express t - bet which drives further development of this lineage into the ncr stage [1113]. Finally, ilc1s are the innate counterpart of th1 cells . They are t - bet positive and better ifn- producers than cnk cells [14, 15]. Absence of rort and eomes expression in ilc1s distinguishes them from rort - expressing ncr ilc3 and eomes - expressing cnk cells . Initially, the nomenclature of ilcs included cnk cells, a notion many scientists still hold . But in this review, we limit ilcs to helper - like ilcs to distinguish these special innate lymphocytes from cnk cells . A defining function of ilcs is their production of a similar set of effector cytokines as those produced by cd4 th cells in the adaptive immune system . This feature of ilcs enables them to mount robust immune responses at the innate stage via acting on other immune or structure cells . Through production of il-5 and il-13, ilc2s may induce the first wave of eosinophil recruitment and stimulate epithelial and smooth muscle cells, during type 2 responses to helminth infection or allergen inoculation [17, 18]. Steady state production of il-22 by ilc3s is crucial for the homeostasis between host and commensals within the mucosal tissues . Upon infection, ilc3s are also the major innate source of il-22 after receiving il-23 stimulation [8, 10]. Ilc1 cells are relatively scarce in the gut but enriched in the liver in steady state [14, 20]. During early type 1 responses, ilc1s are better ifn- producers than cnk cells and they provide the initial protection in mice infected with t. gondii . Ilc2s and th2 cells may collaborate to mount robust type 2 immune responses during the effector phase . Some ilc2s express mhc class ii and thus are able to stimulate th2 cells to produce il-2, which in turn promotes ilc2 proliferation and cytokine production [21, 22]. However, possibly due to the lack or low level of costimulatory molecules cd80/cd86 on ilc3s, this type of antigen presentation functions through a suppressive mechanism to maintain the homeostasis of commensal specific th cell in the colon . Ilcs have additional functions, which may or may not be shared by th cells . For example, ilc2 can produce amphiregulin, which facilitates the repair and reorganization of damaged tissues after viral infection . For example, ilc2s are enriched in adipose tissues and contribute to the beiging of white adipose tissue through production of cytokines and/or methionine - enkephalin [26, 27]. Through il-22 and lymphotoxin production, ilc3s can induce intestinal epithelial cell expression of fucosyltransferase 2 (fut2) and thus regulate the epithelial fucosylation, which provides the metabolic substrates for commensals [28, 29]. The development, maturation, and maintenance of distinct ilc subsets are regulated by a set of specific transcriptional regulators, including t - bet, gata-3, and rort, similar to the regulation of effector th cell differentiation . A master regulator that determines th cell differentiation towards a particular subset also seems to direct the development of the related ilc subset . In addition, other factors such as ror, bcl11b, and ahr are involved in regulating the development and functions of ilc subsets . Mirroring their critical functions during th1, th2, and th17 differentiation, the master regulators t - bet, gata-3, and rort are also implicated in fate determination of ilc subsets . Furthermore, gata-3 is required for the maintenance and function of fully developed ilc2s [30, 31]. Deletion of gata3 in mature ilc2s results in dramatic diminution of il-5 and il-13 production followed by the rapid disappearance of these cells . Gata-3 is also expressed in ilc1 and ilc3 cells as well as in ilc progenitors, in which gata-3 has a critical function; we will discuss gata-3 function during early ilc development in detail in the progenitor section below . T - bet induction in ilc3s may be driven by notch signals, il-23 stimulation, and the microbiota . The gradient increment of t - bet levels directs further development of ccr6ilc3s into ncr ilc3s . Some cells may even turn off rort expression to become t - betncr ex - ilc3s . In addition, t - bet regulates ifn- production by the ncr ilc3s . Besides ccr6ilc3s accordingly, t - bet deficient mice lack ilc1s and ncr ilc3s but have normal ilc2s and ccr6 ilc3s . Ilc1s were previously confused with cnk cells as both express t - bet and are capable of producing ifn-. However, cnk cells express eomes while ilc1s do not . In addition, ilc1s may also express cell surface markers such as cd49a, cd160, and/or cd127, which are usually absent on cnk cells . Ror is highly expressed by ilc2s and is necessary for their development [32, 33]. Ror deficiency results in dramatic reduction of ilc2s but does not affect the development of other ilc subsets . Thus, mice reconstituted with ror deficient bone marrow are useful tools for studying ilc2 functions during immune responses . Bcl11b is a critical factor during early stages of t cell development [34, 35]. But during ilc development, bcl11b deficiency only blocks the development of ilc2s as shown by two independent studies published this year [3638]. Bcl11b is expressed as early as the common ilc progenitor stage and it may suppress the development of ilc3s . The mechanism of how bcl11b specifies ilc2 fate is yet to be determined . Unlike gata-3 function in mature ilc2s, bcl11b deletion does not affect the maintenance or function of mature ilc2s despite bcl11b being highly expressed in mature ilc2s . The aryl hydrocarbon receptor (ahr) is well known to be involved in th17 cell differentiation and function . Ahr is also expressed by both ccr6 and ccr6 ilc3s [40, 41]. In adult ahr deficient mice, ilc3 cell number in the gut is dramatically reduced, probably due to the defective accumulation and/or enhanced apoptosis of ilc3s . Ahr is also critical for the function of ilc3s by regulating il-22 production and the ahr effects on ilcs seem to be ilc3 specific . Ilcs, cnk cells, and t cells all develop from clps found in fetal liver and adult bone marrow . At late stages of t cell development, nave cd4 and cd8 t cells develop from the cd4cd8 double positive cells in the thymus whereas cd4 t effector th cells are differentiated from nave cd4 t cells in the periphery . Considering the functional similarity between mature ilcs and cd4 th cells thus, a hypothesis concerning ilc development is that, like t cells, there may be a common progenitor for all innate lymphocytes, including ilcs and cnk cells, after the clp stage . In a subsequent stage, a common ilc progenitor would be capable of giving rise to all ilcs, in parallel with the potential of nave cd4 t cells to become different th effector cells . This hypothesis is supported by the fact that certain gene deletions affect the development of all innate lymphocytes and/or all ilcs . Indeed, during the last year, we have witnessed a few breakthrough studies in identifying the common progenitors for ilcs [14, 42]. Id2 is required for the development of all ilc populations since its deficiency results in the loss of all ilcs . Using an id2 reporter mouse strain, a lineagecd127flt-3integrin47 population has been reported in a stage after clp and has lost the potential to become t, b, and cnk cells . Id2 expression within this population has been shown by expression of an id2 fluorescent reporter . Previously reported immature ilc2s in bone marrow are also id2; however, they can be excluded by cd25 staining . The multipotential capacity of these id2cd25 progenitors for all ilc subsets was also confirmed by in vitro single cell development assay . These lineagecd127flt-3integrin47id2cd25 progenitors are thus termed as common helper - like innate lymphoid progenitors (chilps). In addition to the critical role in maintenance and function of ilc2s, gata-3 is also crucial in the general development of all ilcs . It has been recently reported that gata3 deficiency prior to the clp stage affects the development of all ilcs in a cell intrinsic manner, indicating that gata-3 is a critical regulator for ilc development . In mice carrying a cre construct driven by the vav1 promoter to conditionally delete gata3 at the hematopoietic stem cell stage, dramatic defects in all il-7r-expressing ilcs in the periphery are noted . Moreover, these gata3 conditional - deficient mice do not generate lymph nodes or peyer's patches, consistent with the finding that the development of functional lti cells is defective at the fetal stage . They also succumb to citrobacter rodentium infection, consistent with another report showing that gata-3 mediates the development of ilc3s by using chimera mice with hematopoietic precursor cells from e12.513.5 gata3 embryos . The essential role of gata-3 during ilc development is consistent with its indispensable function during cd4 t cell development after the cd4cd8 stage in the thymus . Consistent with its critical role during ilc development, gata-3 expression levels in the common ilc progenitors, such as chilps, are comparable to that in ilc2s . Thus, gata-3 is likely a master regulator for the development of common ilc progenitors . Given that both id2 and gata-3 are highly expressed in ilc progenitors, it is reasonable to speculate that while id2 may direct the acquisition of the innate feature of ilcs similar to cnk cells gata-3 may play an indispensable role in directing the helper feature of ilcs similar to cd4 t cells . Because of this, gata-3 may distinguish the helper lineage from cytotoxic cnk lineage during innate cell development . Altogether, id2 and gata-3 coexpression during the common ilc progenitor stage may establish a special regulatory network that determines the innate and helper features of the ilcs . By analyzing a plzf fate mapping mouse strain, researchers found that the majority of mature ilcs have expressed plzf with the exception of the ccr6 ilc3 lineage . Plzf is hardly detectable in mature ilcs but is transiently expressed by a subset of the chilp population in fetal liver and adult bone marrow . These plzf progenitors are multipotential cells and are able to give rise to all ilcs except ccr6 lti cells . Furthermore, these plzf progenitors do not develop into t cells, b cells, or cnk cells . Plzf progenitors may develop after the chilp stage and have lost the potential to develop into lti lineage . The function of plzf during ilc development remains elusive particularly because plzf is only transiently expressed during the early development stage . Furthermore, although zbtb16 mice with plzf deficiency have altered ilc development especially for ilc2s, all the ilcs can still be detected in these mice . An obvious issue for these defined common ilc progenitors is that they are actually heterogeneous populations . Within the chilp cells, there should be some ccr6 ilc3 progenitors within the plzf population, which will never turn on plzf expression . Even within the plzf population, only a minority of the cells have the potential to become multiple ilcs during in vitro single cell development assay . It is likely that both id2 chilps and plzf common ilc progenitors contain a large fraction of partially committed ilc immediate progenitors . Common progenitor multipotent for all ilcs is still required . Meanwhile, in addition to fetal liver and adult bone marrow, the common ilc progenitors may also exist in other tissues, such as fetal intestine, as was shown in a recent study using arginase-1 reporter mice . This report suggests that we should not limit ourselves to study fetal liver and adult bone marrow cells to gain further knowledge on ilc progenitors . As mentioned above, many ilc progenitors found in fetal liver and adult bone marrow may have already committed partially to a specific ilc lineage . Indeed, bcl11b - expressing progenitors appear within the chilp population; yet these cells have already committed to the ilc2 fate . There are also abundant immature ilc2s, presumably the most immediate ilc2 precursors, present in the bone marrow; these cells express mature ilc2 markers sca-1 and cd25 . Plzf fate mapping analysis indicates that most immature ilc1s have expressed plzf and thus are derived from the plzf ilc progenitors rather than the cnk progenitors . A couple of other transcription factors, nfil3 [4851] and tox, have also been found to be involved in the development of both ilcs and cnk cells . Since their expression is detected earlier than id2 expression, which occurs at the chilp stage, the expression of these transcription factors may mark even earlier common progenitors for both ilcs and cnk cells, similar to cd4cd8 thymocytes during t cell development . However, the phenotypes of ilc development in mice deficient in either of these regulators are not as dramatic as those resulting from id2 or gata3 deficiency . The mechanisms through which nfil3 and tox function during ilc and cnk cell development require further investigation . Given the functional similarities between the innate ilcs and cnk cells and the adaptive cd4 and cd8 t cells, it is reasonable to propose that ilcs and cnk cells follow a similar developmental pathway to that of t cells . A clp may give rise to a common progenitor for all innate lymphocytes including ilcs and cnk cells, which subsequently gives rise to chilps . Distinct ilc subsets are further developed from the plzf chilp stage as the differentiation of th effectors from nave cd4 t cells . Thus, the development of innate lymphocyte subsets, including ilcs and cnk cells, to a certain extent, mirrors that of t cells (figure 1). As discussed above, more and more regulators are being found to be involved in the development of ilcs . Interestingly, most of these regulators are also involved with t cell development . However, distinct from t cell development, the regulatory functions of the various transcription factors during ilc development are not related to tcr - mediated thymic selection, which, to some extent, may explain why the deficiency of certain regulators has distinct effects on ilc and t cell development . Although deletion of either one of the newly identified genes, including nfil3, tox, or tcf7 [53, 54], results in defective ilc development to a various extent, none of these defects are as severe as that resulting from id2 or gata3 deficiency . Thus, it is possible that in the early progenitors of the ilc lineage, these regulators may function through a network designed for fine - tuning ilc development . Id2 and gata-3 might form the core complex, while other regulators are involved in tuning the optimal function of the core components . Id2 or gata3 deficiency dramatically affects ilc development, whereas deletion of other regulatory components results in various incomplete defects of ilc development . Nuclear factor interleukin-3 (nfil3; also known as e4-binding protein 4, e4bp4), a basic region leucine zipper transcription factor, is critical for cnk cell development . Based on this, it is possible that nfil3 is expressed and functions at a progenitor stage common to both ilcs and cnk cells . Nfil3 deficient mice showed dramatic defects during the transition from clp to chilp stage [48, 51]. However, ilc development is not completely abolished in nfil3 deficient mice; in the periphery, there are still substantial numbers of ilcs that have escaped the developmental block, although the population is too small to control infectious challenges . Nfil3 expression is induced by il-7 signaling, which is also critical for the development of ilcs . After development, nfil3 maintains a very high level of expression in mature ilcs, a level higher than that in cnk cells . Nfil3 is dispensable for ilc3 maintenance; however, its function in other mature ilcs remains unclear . Thymocyte selection - associated high - mobility group box protein (tox) is a member of the high - mobility group box superfamily . It contains a dna - binding domain and is required for the development of t [56, 57], nk, nkt, and lti cells . A new study this year showed that tox also has broad effects in ilc development . In tox deficient mice, the chilps as well as mature ilcs were severely reduced compared with these cells in wild type mice . There is an intrinsic defect in the notch signaling pathway in tox deficient chilps, which may explain the defect in ilc development in tox deficient mice . T cell factor 1 (tcf-1) is a critical transcription factor for t cell fate specification at the early development stage . Tcf7 (gene encode tcf-1) deficiency affects both ilcs [53, 54] and cnk cells . Similar as during t cell development, tcf-1 is regulated by notch signaling and may be involved in the induction of il-7r and gata-3 expression during ilc development [53, 54]. Based on the sequential expression of nfil3, tox, tcf-1, id2, and gata-3 and the knowledge we have concerning their relationships in different systems, it is likely that nfil3 expression initially increases after the clp stage, which in turn regulates the expression of tox and id2 . After id2 is turned on, the core assembly involved in directing the innate features of ilc development begins to function . Although gata-3 is expressed at low levels at the clp stage, its function during ilc development requires high expression levels . Tox is involved in the regulation of notch signaling pathway, which in turn regulates tcf-1 and gata-3 . Tcf-1 is then required for optimal expression of gata-3 . Upon increased expression of gata-3, the helper transcription factor assembly nfil3, tox, and tcf-1, in connection with the notch pathways, form a network to prepare for the upregulation of id2 and gata-3, which together form the executive regulatory network to direct lineage fate determination of ilcs, possibly with continued assistance from the initiation transcription factors such as tcf7, tox, and nfil3 (figure 2). Additional experiments are required to confirm this proposed regulatory network model and it is likely that additional regulators for ilc development may soon be discovered . Studies on the newly identified ilcs in recent years have enriched our knowledge on the innate lymphoid lineage development and have provided us with more evidence supporting the close relationship between the innate and adaptive immune systems during evolution . The classification of the ilc population was initially based on their capacity to produce a similar set of effector cytokines compared to th cells . Subsequent studies have revealed that the development of ilcs is also regulated by a similar set of key transcription factors required for t cell development . After the clp stage, innate lymphoid lineages and t cells lineages start to develop separately but the regulatory mechanisms may still be shared . Nfil3, tox, and tcf-1 expression at an early stage immediately after clp in the innate lymphoid lineage may mark the common progenitor for both ilcs and cnk cells . Ilc and cnk lineages are further separated by the induced expression of id2 and gata-3 in the common ilc progenitors . At later stages, additional regulators are upregulated to drive the distinct fates of different ilc subsets . In conclusion, the ilc cell fate is precisely regulated during development by a network of multiple serially expressed regulators, which may form a regulatory network during development and render the developed ilcs with both innate and helper features.
Thymic hyperplasia is defined as the enlargement of the thymus with a histologically normal cortical and medullary component . The hyperplastic gland can become very large; however, besides its larger size, the imaging appearance of the enlarged thymus is normal (1). No child cases of pericardial lipomatosis have been reported in the english literature . To the best of our knowledge, this is the first case report of a child with thymic hyperplasia simulating a fat containing mass accompanied by pericardial lipomatosis and right facial hemihypertrophy . An 8-year - old boy visited the outpatient clinic for evaluation of facial asymmetry and narrowing of the right external auditory canal . Upon physical examination, the patient showed right side facial enlargement, cranial protrusion, gingival hypertrophy, and soft tissue thickening of the external auditory canal . There was no evidence of asymmetry of the body trunk and limbs or any subcutaneous / cutaneous lesions noted . The patient denied any prior history of medical, surgical problems, or any family history of hemihyperplasia . A chest posteroanterior film identified a large mediastinal mass draping over the right cardiac shadow (fig . Chest ct revealed a large mass occupying the anterior mediastinum showing no mass effect or displacement of adjacent structures . It had a bilobular configuration with asymmetric mass - like enlargement of the right lobe which contained curvilinear or nodular areas of fatty component . Concomitant diffuse fat attenuation filling the pericardial space suggestive of pericardial lipomatosis was also noted . 1c - f). A fat containing benign mediastinal mass including thymolipoma or lipoblastoma was suspected . A malignant mass such as liposarcoma or malignant germ cell tumor was also included in the differential diagnoses . As it was difficult to determine whether the more predominantly enlarged right lobe was the only portion involved or both the right and left lobes were pathologic, mediastinal mri was performed to further determine the precise extent of the lesion . The mri results revealed a large anterior mediastinal mass that showed heterogeneous iso - signal intensity to the chest wall muscles on t1- and t2-weighted images and heterogeneous enhancement on fat saturated t1-weighted images . There were whorls or nodular areas of high signal intensity within the thymus on both t1- and t2-weighted images which showed suppression on fat saturated contrast enhanced t1-weighted sequence . The pericardial space was also filled with high signal intensity on both t1- and t2-weighted images and showed fat suppression on fat saturated contrast enhanced t1-wieghted images (fig . 1 g, h). For evaluation of facial asymmetry and narrowing of the right external auditory canal, contrast - enhanced ct of the paranasal sinuses was also performed . Paranasal sinus ct showed hypertrophy of the right side parotid gland, adenoid tissue, temporalis muscle, and sternocleidomastoid muscle . Moreover, mild narrowing of the right external auditory canal was noted due to soft tissue thickening along the posterior wall of the canal (fig . The operative findings revealed a large anterior mediastinal mass showing no invasion into the mediastinal structures and the parietal pleura . On gross specimen, the mass was yellowish with a lobulating contour confined within a thin fibrous capsule . The mass was about 17 13 3 cm in size and weighed 280 g (fig . The cut surface showed multifocal areas of light - yellow spots of fatty tissue scattered within the thymic mass . There was no evidence of hemorrhage or areas of cystic, necrotic changes (fig . Microscopically, the tumor showed normal distribution of the cortex, medulla and hassall's corpuscles . It had fibrous and myxoid stroma with focal areas of adipose tissue admixed in various proportions (fig . The postoperative course was uneventful and the patient was discharged to follow - up on an outpatient basis for further management of right facial hemihypertrophy . The patient was followed up for about a year and a half and has not shown any evidence of progressive course or cutaneous / subcutaneous lesions thus far . Thymic hyperplasia is defined as an enlarged thymus beyond the normal upper limit for any given patient age . Hence, the imaging appearance is similar to a normal thymus except for its larger size (1). Moreover, thymic hyperplasia shows homogeneous attenuation similar or slightly lower than the muscles on noncontrast ct and homogeneous enhancement of 20 - 30 hounsfield units after contrast enhancement . On mri, thymic hyperplasia show slightly brighter si than that of the muscles on both t1-weighted and t2-weighted images (1 - 3). Opposed - phase images may show diffuse decreased signal intensity (4). A normal thymus has a homogeneous appearance in childhood and begins to appear heterogeneous on imaging studies at about the age of thirty when the adipocytes become dominant . Hence, any condition in which the thymus appears heterogeneous in a child should raise suspicion of a pathologic condition (3). In our case, the enlarged thymus had a heterogeneous appearance with areas of fat admixed within the thymic tissue . It was an unusual finding for a child and a fat containing anterior mediastinal mass such as thymolipoma, lipoblastoma was to be considered . Malignant lesions such as malignant germ cell tumor or liposarcoma was also considered as a possible differential diagnosis . The mass was confirmed to be a hyperplastic thymus composed of normal thymic components and cellular organization suggestive of a true thymic hyperplasia on histologic examination . Although it is well known that the thymus can undergo hyperplastic changes after suppression from recent stressful conditions such as chemotherapy for neoplasm, grave's disease, corticosteroid therapy for cushing's disease, irradiation or thermal burns and some autoimmune diseases (systemic lupus erythematosus, hashimoto thyroiditis, addison's disease, acromegaly), our patient did not have any of these well known systemic stresses or conditions causing thymic hyperplasia (5). However, the patient did have facial hemi - hypertrophy on the right side . As it has been reported that the left lobe of the thymus is usually larger than the right, the predominant enlargement of the right lobe with more prominent fatty component on the right side shown in our case may be related to the underlying right side hypertrophic condition of this patient (2, 3). Complimentary to this finding, the patient also had diffuse pericardial lipomatosis, which is also an extremely rare finding with only a few cases reported in adult patients (6, 7). Pericardial lipomatosis, whether isolated or accompanied by other diseases, has not been reported in the pediatric population . The combination of thymic hyperplasia, pericardial lipomatosis, and facial hemihypertrophy seen in our case is also the first to be reported, both in children and adults . Although the patient did not undergo any genetic studies, considering that the patient has facial hemihypertrophy, thymic hyperplasia, and deep soft tissue lipomatosis, there is a possibility that the clinical and imaging features of this patient could be a spectrum of hemihyperplasia syndromes . Hyperplasia syndromes include a heterogeneous group of disorders with overgrowth of one or more limbs or body parts as a predominant finding and includes disease entities such as beckwith - wiedemann syndrome, proteus syndrome, hemihyperplasia multiple lipomatosis syndrome (hhml), cowden / bannayan - riley - ruvalcaba syndrome (brrs) among others . Beckwith - wiedemann syndrome is the most well known hemihyperplasia syndrome and is associated with anomalies such as macroglossia, abdominal wall defects, hypoglycemia, and increased risk of embryonal tumors . It is also well known because of its genotypic abnormalities of the distal region of chromosome arm 11p . Proteus syndrome is a rare, highly variable hamartomatous syndrome which is clinically diagnosed if the patient satisfies the necessary diagnostic criteria of disproportionate overgrowth, connective tissue nevi, dysregulated adipose tissue, and vascular malformation . Cutaneous lesions such as connective tissue nevi, tends to appear over time and may delay the diagnosis . Some report patient with proteus syndrome showing mutation of pten (phosphatase and tensin homologue deleted on chromosome 10) and glypican 3 (gpc3)gene, while others cast doubts over its involvement . Hhml syndrome is thought to be a distinct form of proteus syndrome which does not satisfy the diagnostic criteria and shows less severe hyperplasia of the limbs or body parts . Brrs is characterized by macrocephaly, mild mental retardation, cutaneous lipomas, or hemangiomas, while pigmented macules of the glans penis and the onset is noted at birth or shortly thereafter . Cowden syndrome is an adult - onset condition with mucocutaneous signs and increased risk of cancer (9, 10). Among the disease entities of the hemihyperplasia syndromes listed above, the latter three are those which are known to show both hyperplasia and lipomas as in our case . However, as the asymmetric overgrowth in our patient was not from birth and the patient did not show any sign of mental retardation, brrs may be excluded and the possible diagnosis could be narrowed down to proteus syndrome or hhml syndrome . Until now, our patient was followed up for about a year and a half and the most fitting diagnosis appears to be hhml . However, although the patient did not show cutaneous, epidermal nevi or capillary, venous, and lymphatic malformation, they may develop over time and show progressive enlargement . Hence, further clinical observation and imaging studies including a serial skeletal survey will be necessary for the diagnosis or exclusion of proteus syndrome . Unfortunately, genetic studies that may support the diagnosis of hemihyperplasia syndrome was not performed . Although the diagnosis and exclusion of several hyperplastic syndromes may be available with genetic studies, it may not be of any help in differentiating proteus syndrome from hhml since there are no molecular studies available at this moment . Until molecular mechanisms of the hemihyperplasia syndromes are further elucidated, thorough observation of any change in the clinical findings will remain essential for the diagnosis of patients . In conclusion, we have reviewed and discussed the imaging findings of a rare combination of thymic hyperplasia, pericardial lipomatosis and facial hemihypertrophy in a pediatric patient.
Infant and child mortality rates are regarded as indicators of the prevailing health conditions in a society; they measure the success of health programmes and policies aimed at their education . The world summit for children, held in 1990, instituted a package of objectives for implementation by the year 2000 . Among these objectives was the aim to reduce infant and under - five mortality by one third or to 50 and 70 deaths per 1000 births, respectively, whichever was less.1 this was reaffirmed at the 1994 international conference on population and development (icpd).2 the 1992 population policy of sudan announced the intention to reduce the mortality rates of children under five years of age to 60 deaths per 1000, from the level in 1992 by the year 2002.3 sudan is a developing country characterized by high levels of infant and child mortality . According to the 1993 fourth national population census, under - five mortality rate was 150 per 1000 live births and one out of ten infants died before reaching their first birth day . The safe mother survey (sms) which was conducted in 1999 showed that under - five mortality rate was 105 per 1000 live births and infant mortality rate was 69 per 1000 live births.4 the persistence of high infant and child mortality rates, call for the identification of the main causes of this phenomenon . The main objectives of this study were to determine the level of infant and child mortality rates in wad - medani and to identify the main causes . It is the second largest town in sudan and lies on the bank of the blue nile . According to the fourth national census, wad - medani has a population of 430,487 comprising 214,670 males and 215,817 females.5 its population is a mixture of different tribal groups of sudan, most of whom are government employees, traders, farmers, and casual laborers . The average health situation in wad - medani town is better than the national average and also better than any of the other towns in sudan.6 the data of this study came from a household survey conducted in september 2004 . A simple random sampling technique was used with anticipated population proportion estimated to yield 300 women who had at least one child under five years of age . A sample of 504 children under - five in 300 household were investigated using a questionnaire . This questionnaire consisted of two sets of data; the first set asked for the child's sex, birth weight, birth order, birth interval, immunization and child mortality, while the second set dealt with the mother's socio - economic status and other environmental variables . After the data were collected and coded, the statistical packet for social sciences (spss) were used to analyze the data . Was used to evaluate some associations between the dependent variable infant and child mortality and a set of independent variables . It is the second largest town in sudan and lies on the bank of the blue nile . According to the fourth national census, wad - medani has a population of 430,487 comprising 214,670 males and 215,817 females.5 its population is a mixture of different tribal groups of sudan, most of whom are government employees, traders, farmers, and casual laborers . The average health situation in wad - medani town is better than the national average and also better than any of the other towns in sudan.6 the data of this study came from a household survey conducted in september 2004 . A simple random sampling technique was used with anticipated population proportion estimated to yield 300 women who had at least one child under five years of age . A sample of 504 children under - five in 300 household were investigated using a questionnaire . This questionnaire consisted of two sets of data; the first set asked for the child's sex, birth weight, birth order, birth interval, immunization and child mortality, while the second set dealt with the mother's socio - economic status and other environmental variables . After the data were collected and coded, the statistical packet for social sciences (spss) were used to analyze the data . Was used to evaluate some associations between the dependent variable infant and child mortality and a set of independent variables . Table 1 shows that 20.6% of the children were below the age of one and 79.6% of them were between 1 and 5 years . The sex percentages of infants and children under five was 43.7% males and 56.3% females . The birth weight of the children was classified into three groups: under weight, normal weight and unknown weight . Those underweight were 26.2%, children with normal birth weight accounted for 62.7% and unknown birth weight were 11.1% . Demographic and biomedical characteristics of the children on the immunization status of the children, 65.1% of the children had been fully vaccinated, 27.8% partially immunized, while only 7.1% had no immunizations . The distribution of children by birth order is shown in table 1 . As can be seen from the table, 40.9% of the children were in the group (six and more) births, 32.1% in the group (four to five) births, 22.2% in the group (two- three) births and only 4.8% in the first group (first child). For 33.3% of the children, the birth interval was less than one year, for 46.1% it was 1 - 2 years and it was more than two years for 20.6% . The percentage of women with 4 - 8 years of education was 11.7%, 9 - 12 years was 58.7%, 13 - 16 years was 29% and 0.6 formed the last category with more than 16 years of education . The same table shows that 91% of the women were housewives, 7% were employed,1% had other occupations . It was found that 98.6% were married, 0.7% were widowed and 0.7% were divorced . Of the mothers, 96.3% used contraceptive measures, while 3.7% did not . Mother's educational, occupational and marital status table 3 shows the distribution of the sample households by the level of expenditure . It is obvious from the table that the majority of families (60.7%) belonged to the middle income group, whereas the low and high income groups formed 30.0% and 9.3% respectively . Family income and some selected community and environmental variables housing conditions and sanitation are known to be important determinants of the level of infant and child mortality . Table 3 shows the types of houses, and number of the rooms and latrines . Ninety - seven point 3 percent of the women reported that their houses were made of bricks, 36.3% reported their houses had two rooms, 31.3% three rooms, and 17.1% had four rooms while 15.3% had one room only . When asked about latrines, 63.3% of the same women said they had pit - latrines, while only 36.7% had siphon . Seventy - five families kept animals, 60% of whom said the animals were kept inside the house, while 40% kept them outside . It was found that 7.7% of the women's infants had died, while 92.3% of them are alive . For the age group 1 - 5 years, 6.7% of the children had died, while 93.3% of them were alive, yielding child mortality rate for that age group as 67 per 1000 live births . Infant and child mortality in this part, cross - tabulation for the most important variables relating to infant and under - five mortality are presented . In each case, a test of independence was performed using (chi -square) test of independence . The independent variables that were assumed to affect the mortality of infants and children under five significantly (dependent variable) were child order, child interval, child weight, child immunization, mothers education, family income, keeping of animals, latrines and the use of contraceptives . Table 5 shows that child order, child weight, interval between children, child immunization, the use of contraceptives, family income, mother's education and latrines were all significant at 1%, while only the keeping of animals was significant at 5% . This means that all these variables were strongly associated with the mortality of children under five years old and increased the risk of infant and child mortality . Table 1 shows that 20.6% of the children were below the age of one and 79.6% of them were between 1 and 5 years . The sex percentages of infants and children under five was 43.7% males and 56.3% females . The birth weight of the children was classified into three groups: under weight, normal weight and unknown weight . Those underweight were 26.2%, children with normal birth weight accounted for 62.7% and unknown birth weight were 11.1% . Demographic and biomedical characteristics of the children on the immunization status of the children, 65.1% of the children had been fully vaccinated, 27.8% partially immunized, while only 7.1% had no immunizations . The distribution of children by birth order is shown in table 1 . As can be seen from the table, 40.9% of the children were in the group (six and more) births, 32.1% in the group (four to five) births, 22.2% in the group (two- three) births and only 4.8% in the first group (first child). For 33.3% of the children, the birth interval was less than one year, for 46.1% it was 1 - 2 years and it was more than two years for 20.6% . The percentage of women with 4 - 8 years of education was 11.7%, 9 - 12 years was 58.7%, 13 - 16 years was 29% and 0.6 formed the last category with more than 16 years of education . The same table shows that 91% of the women were housewives, 7% were employed,1% had other occupations . It was found that 98.6% were married, 0.7% were widowed and 0.7% were divorced . Of the mothers, it is obvious from the table that the majority of families (60.7%) belonged to the middle income group, whereas the low and high income groups formed 30.0% and 9.3% respectively . Family income and some selected community and environmental variables housing conditions and sanitation are known to be important determinants of the level of infant and child mortality . Table 3 shows the types of houses, and number of the rooms and latrines . Ninety - seven point 3 percent of the women reported that their houses were made of bricks, 36.3% reported their houses had two rooms, 31.3% three rooms, and 17.1% had four rooms while 15.3% had one room only . When asked about latrines, 63.3% of the same women said they had pit - latrines, while only 36.7% had siphon . Seventy - five families kept animals, 60% of whom said the animals were kept inside the house, while 40% kept them outside . It was found that 7.7% of the women's infants had died, while 92.3% of them are alive . For the age group 1 - 5 years, 6.7% of the children had died, while 93.3% of them were alive, yielding child mortality rate for that age group as 67 per 1000 live births . In this part, cross - tabulation for the most important variables relating to infant and under - five mortality are presented . In each case, a test of independence was performed using (chi -square) test of independence . The independent variables that were assumed to affect the mortality of infants and children under five significantly (dependent variable) were child order, child interval, child weight, child immunization, mothers education, family income, keeping of animals, latrines and the use of contraceptives . Table 5 shows that child order, child weight, interval between children, child immunization, the use of contraceptives, family income, mother's education and latrines were all significant at 1%, while only the keeping of animals was significant at 5% . This means that all these variables were strongly associated with the mortality of children under five years old and increased the risk of infant and child mortality . Since the 1970s, the estimated annual number of deaths among children of less than 5 years has decreased by almost a third . This reduction has been very uneven and in some countries the rates of childhood mortality are increasing . In 1998, more than 50 countries including sudan still had childhood mortality rates of over 100 per 1000 live births.7 according to rutstein (1984), infant mortality rate (imr) is the number of infants dying in the first year of life per 1000 live birth, whereas child mortality rate (1 - 5) is the probability of dying between the first and fifth birthday expressed per 1000.8 the analysis of infant and child mortality in wad - medani reveals that infant mortality rate is 77 per 1000 live births . This figure is consistent with the findings of the 1999 safe mother survey (sms),7 in which the national level of infant mortality was estimated at 68 deaths per 1000 live births . Also the study estimated under - five mortality as 67 deaths per 1000 live births . The multiple indicators cluster survey (mics) which was conducted in the gezira state in 2002, showed an analogous level in which 59 out of 1000 children died before reaching the age of five.9 on examination of the determinants of infant and child mortality, the study found a strong association with child order . Many studies suggest that infant and child mortality increase with the increase of parity after the second birth . The higher the parity, the shorter the birth interval, and the shorter the birth interval the higher risk of dying for a child . The risk of dying is considerably higher for a child who has a sibling born within the preceding two years.1011 on the other hand, other studies suggest that infant and child mortality is generally higher for first born children, especially during the first year of life.12 the study showed that there is a significant association between birth interval and infant and under - five mortality . The length of the birth interval is a very important factor for the survival status of infants and children . If the length of the birth interval is short, the probability of dying is high . The probability of dying before age five for children born less than two years after a previous birth is more than double that for children born four or more years after a previous birth.13 moreover, short birth intervals have indirect effects through such factors as mother's depletion, premature birth and limited family resources.11 the birth weight of the child has been found to be statistically significant as a determinant of under - five mortality . In line with results found elsewhere, mortality decreased with the child's increase in weight at birth . The rates of mortality for children who were under weight at birth were higher than those who had normal weight at birth.14 further, the study showed a significant association between mother's education and infant and under - five mortality . Maternal education has been identified as one of the most important socioeconomic determinants of infant and child mortality . Many studies show that the higher the level of maternal education, the lower the infant and child mortality.15 caldwell (1981) provided three explanations for the phenomenon: better educated mothers become less fatalistic about their children's illnesses . They are more capable of manipulating available health facilities and personnel and they greatly change the traditional balance of familial relationships with a profound effect on child care . In addition to these, they are more likely to have had antenatal care, to have given birth with some medical attention, and to have taken their children at some point to see a physician.16 . Regarding the significant association between the use of contraception and child mortality, we found that 73% of the women had not used any contraceptive methods within the last twelve months because of lack or unavailability in many of the areas covered by the study . However, not all the children had been vaccinated (full immunization 65.1%), because of the fear of complications from immunization . Lack of latrines leads to high environmental contamination in houses and makes children more vulnerable to infectious diseases . For example, in an inter - american study, the incidence of diarrheal disease was lowest in households with pipe - borne drinking water and flush toilet facilities . Current access to proper toilet facilities was found to influence infant mortality, especially beyond the post - neonatal stage.12 considering the association between the keeping of animals and child mortality, it was found that most households kept animals inside their houses, which increased the hazards of infectious diseases . The conclusion of this study is that infant and under - five mortality in gezira state was still high and socioeconomic and environmental factors had a lot of influence on the health status of children in wad medani . Finally, this study recommends the expansion of immunization of children, and education for mothers in order to promote family planning and improve child health . Also further detailed research is needed to provide better explanations for the determinants of the higher rates of infant and child mortality in sudan.
Mastitis represents a major economic cost to dairy farmers with losses of up to 190 per case depending on severity and 60 per cow for the average milk supplier . The losses associated with mastitis include discarded milk, increased number of culled cows, cost of antibiotic treatment and reduced milk quality and price . Even though, it has been shown that factors such as genetic characteristics, impaired immune - function, feeding regimes and machine milking are related to mastitis, poor milking hygiene has been associated with increased somatic cell count (scc), reduced milk production and inferior milk quality . Machine milking may also be considered as a major cause of bacterial cross contamination from cow to cow . However, a good premilking hygiene routine can decrease the cow infection ratio by not only reducing udder bacterial contamination from the environment, but also reducing bacterial contamination from other infected animals . Newbould . Showed a positive relationship between bacteria presence on teats and new intramammary infection . Staphylococcus aureus is one of the major and more virulent pathogens that can cause mastitis infection and lactating cows can be considered one of the main reservoirs of this species . Moreover, staphylococcus aureus colonisation of teat skin increases the risk of staphylococcus aureus intrammmary infection . The aim of any teat cleaning routine is not only to reduce mastitis infection risk, but also to enhance milk quality . . Also showed that spore concentration in milk was highly correlated with spore concentration on teats; hence milk bacterial spores most likely originated from dirt and faeces attached to the teats at the time of milking . Many methods of pre - milking udder preparation are practiced by producers and overall, one of the most important aspects of pre - milking udder hygiene is udder dryness at the time of machine attachment (visser et al . Bacteria contaminated water can also increase milk bacterial counts (visser et al . 2007). A commonly used pre - milking teat preparation method involves washing teats by hand with water and drying teats with a paper towel just before the machine is attached . There is strong evidence that among all pre - milking procedures, wet cleaning treatment, followed by paper towel manual drying will result in the lowest bacterial counts: this practice is particularly effective in reducing milk bacterial contamination during the winter housing period . Similar seasonal bacterial infection trends linked to the pasturing / housing routine have been observed by hutchison et al . . As an alternative to washing and drying teats, many producers now dip teats pre - milking with various disinfectant products such as iodophor solution, iodine based gel, sodium hypoclorate, dodecyl benzene sulfonic acid (ddbsa), chlorine, chlorhexidine, phenolics and alcohol . Showed that both iodine based gel and 0.5% iodophor solution significantly reduced milk bacterial count and clinical mastitis occurrence compared to teat washing and drying with paper towels . However, showed that pre - milking disinfection with 0.25% iodine dip or phenolics solution did not reduce the incidence of clinical mastitis when compared to post - milking disinfection only . However, pankey et al . Reported that predipping reduced the rate of intrammamary infection with major mastitis pathogens such as staphylococcus aureus, streptococcus agalactiae and coliforms . In addition to iodine products, chorhexidine when used as a pre - dip, significantly decreased scc values in herds infected with mastitis . Furthermore, gibson et al . Concluded that a chlorine based dip followed by a dry wipe was an effective pre - milking treatment for controlling cow mastitis . The benefit of using some disinfectant products pre - milking in reducing new mastitis infection has been demonstrated . A study by roberson et al . Demonstrated that teat orifices colonised with staphylococcus aureus were 3.3 times more likely to have intramammary infection . Therefore, reducing the microbial count on teats prior to milking is an important step in the prevention of mastitis . The type of disinfectant product used as a pre - dip may have varying degrees of success in reducing the microbial count on teats . However, there is no knowledge on the effect of pre - milking disinfection using a range of newly - formulated teat disinfectants in reducing the microbial counts on teats . Thus, the objective of this study was to investigate the effectiveness of six different pre - milking teat preparation procedures on lowering the staphylococcal, streptococcal and coliform bacterial count on teat skin . Six pre - milking teat preparations were applied to spring calving holstein friesian cows during two herd management periods, while cows were housed (indoors) and while cows were grazed on pastures (outdoors). During the indoor period and for the previous three months, cows were housed in an easy - feed slatted house with matted cubicle beds and dressed with lime daily to maintain a dry bed . During the outdoor period and for the previous three months, cows were grazed under a rotational grazing system and moved every 24 hours to new pasture . Ten cows which were free from clinical mastitis infection were randomly chosen for each pre - milking preparation for a period of five days per treatment . The six pre - milking applications applied are identified as' washing and drying',' iodine',' chlorhexidine',' chlorine',' wipes' and' no preparation' . Description of pre - milking teat preparation treatments an iodine (2.0% w / v) teat disinfectant (1 - 4 mix) containing emollients was applied as a post - milking teat disinfectant to the following treatments,' iodine',' washing and drying',' no preparation' and disinfectant' wipes' using an in - parlour sprayer .' Chlorhexidine' teat foam and' chlorine' teat foam were used for post - milking disinfection, for the respective pre - milking treatments . The same foam products were used for post - milking disinfection, as it was considered more likely that where the foam products are used on farms as a pre - milking disinfectant, they would also be used for post - milking disinfection . The possibility of a teat skin reaction if different pre- and post - teat disinfectant products were used was also considered . Analysis of the bacterial counts on teats prior to teat preparation showed no effects of using different post milking disinfectant products . The' chlorine' teat foam product was prepared daily by mixing a foam active base and activator (50/50). The entire circumference of the teat was covered in teat foam by immersing each individual teat in the teat cup .' Iodine' was applied as a pre - milking disinfectant by spray, as this was considered the most common application method on irish farms . Approximately, 15 mls of iodine was applied to the teats of each cow when used as a pre and post milkinf disinfectant . Teats disinfected pre - milking were dried with individual disposable paper towels approximately 30 seconds after the disinfectant was applied and prior to milking . Cows were milked in a 20-unit, 80-degree side - by - side milking parlour and were milked in the morning at 07:30 h and in the afternoon at 15:30 h. cows were exposed to each pre - milking preparation for a period of five days, for each management period at both the morning and afternoon milking . On day four and five, all teats from each cow were swabbed using one sterile swab (cultiplast, lp italian spa, via carlo reale, 15/4, 20157, milano, italy) per cow before teat preparation and repeated after teat preparation at the morning milking (table 2). The sterile swab was rubbed across the teat orifice and down the side of each teat avoiding contact with the udder hair or cows flank at all times . A small number of teats (<7%) that were considered excessively soiled with faeces, and where swabbing of the teat skin was not possible, in addition, by omitting these soiled teats, the potential contamination of the agar plates was avoided . In those instances, there were no differences in the number of excluded teats between treatments or management periods . Swabbing procedure carried out before and after teat preparation for six pre - milking teat preparation procedures * = one swab per cow, * * = all repeated after teat preparation immediately after teat swabbing was completed, the swabs were placed in individual sterile bottles containing 5 mls of tryptic soy broth (bd . Bbl trypticase soy broth (soybean - casein digest broth). The broth was manufactured by becton, dickinson and company, sparks, md 21152 usa.38800 le pont - de - claix, france . The broth was prepared in 500 ml amounts and autoclaved at 121c for 15 minutes, and then distributed into 5 ml aliquots in a laminar flow cabinet . The sterile bottles containing the swabs were frozen (-20) awaiting laboratory analysis for the presence of staphylococcal, streptococcal, and coliforms . The swabs were subsequently plated on three separate selective agars: baird parker (staphylococcal), edwards (streptococcal), and macconkey (coliforms). Specific bacteria types within each category were not defined . Following incubation at 37c for 24 hours, microbial counts (cfu / ml) for each pathogen type were manually counted and assigned to one of six categories depending on the bacterial counts measured . (1 = no pathogen present, 2 = 1 to 10, 3 = 11 to 20, 4 => 20, 5 = numerous, 6 = infinite). The results were analysed by logistic regression using sas . Preliminary analysis of the results for before and after treatment was by fitting generalised logits for the multinomial response because, while the response was ordinal in nature, the data did not meet the assumptions of a proportional odds model . 1 = lower category count after treatment and 0 = same or greater category count after treatment . Standard maximum likelihood estimation of the regression could not proceed because of the technical condition of quasicomplete separation for the effects . An alternative strategy due to heinze and schemper was implemented using their sas macro code there were no differences in the levels of staphylococcal, streptococcal and coliform bacterial counts measured on teats (assigned to different teat preparations)' before' teat preparation . However, there was a significant reduction (p <0.001) in the levels of staphylococcal pathogens on teats after teat preparation with all treatments except the' no preparation' pre - milking treatment . The probability of a reduction in the staphylococcal counts tended to be higher (p <0.06) for cows managed outdoors compared too indoors . Treatment with' chlorine' teat foam resulted in a 30% reduction in staphylococcal counts when used on cows at pasture compared to its use on cows indoors, likewise' iodine' and' wipes' resulted in an increased staphylococcal count reduction of 18% and 20%, respectively, when used on cows at pasture (table 3). On the other hand' chlorhexidine' teat foam had a 20% greater reduction in bacterial counts when used on housed cows, compared to cows at pasture . However, the median reduction in the staphylococcal count for all treatments was 21% better for cows on pasture compared to its use on housed cows . The probability of a reduction in streptococcal bacterial counts in response to overall teat preparation was not significantly different between indoor and outdoor management periods . However, some pre - milking treatments resulted in greater reductions in counts when used on cows housed compared to when used on cows at pasture and vice versa . The treatment containing' iodine' resulted in a 26% greater reduction in streptococcal counts when used on cows indoors compared to when used on cows at pasture (table 3). Similarly, the' chlorhexidine' teat foam treatment had a 20% greater reduction when used indoors, compared to when used on cows outdoors . However, both the' chlorine' and disinfectant' wipes' teat preparation treatments resulted in a 30% greater reduction when used outdoors compared to when used on cows indoors . The reduction observed for' coliform' counts was low for both indoor and outdoor management periods (table 3). However, the probability of a greater response to teat preparation for' coliform' bacteria is more likely outdoors (or = 0.27; p <0.05) compared to indoors (table 4). Effect of pre - milking teat preparation treatment in reducing staphylococcal, streptococcal and coliform bacteria counts on teats at two time periods (indoor and outdoor) (% reduction) estimated odds ratios (or) and their 95% confidence intervals (ci) for the effect of pre - milking teat preparation and management period on staphylococcus spp . (str) and coliform (col) counts table 4 shows the association between pre - milking teat preparation procedure and management period on the probability of a reduction in the microbial levels of staphylococcal, streptococcal and coliform pathogens .' Washing and drying' had a higher probability (p <0.001) of reducing both the staphylococcal and streptococcal counts on teats compared to' no preparation', as would be expected . Both' chlorhexidine' (or = 4.46) and' wipe' treatments (or = 4.46) had an increased probability (p <0.01) of reducing the staphylococcal count on teats compared to' washing and drying' . Treatments with' chlorine' (or = 3.45) and' wipes' (3.45) had the highest probability (p <0.01) of reducing the streptococcal count compared to' washing and drying' . Both' iodine' (or = 1.24) and' chlorhexidine' (or = 1.65) also tended to have greater probability of reducing streptococcal counts compared to' washing and drying' .' Washing and drying' had a higher probability (p <0.05) of reducing the coliform count on teats compared to' no preparation' . Any of the remaining treatments did not enhance reduction in coliform numbers (p>0.05) compared to' washing and drying' . No differences in the microbial counts were observed on teats, regardless of the post - milking disinfectant products used at the previous milking . Therefore, using different products as a post disinfectant in this study did not influence the outcome of the study . In this study, the use of some disinfectant products for pre - milking teat preparation had beneficial effects on reducing the levels of staphylococcal and streptococcal pathogens on teat skin compared to' washing and drying' and' no preparation' . Where teat preparation is omitted, increased teat colonisation could be expected and this may result in new intramammary infection . This would concur with the findings of pankey et al . Who reported that pre - dipping can reduce the rate of intrammamary infection with major mastitis pathogens . Furthermore, gibson et al . Concluded that most pre - milking teat cleaning treatments reduce the teat total bacterial count, but that cleaning effectiveness was influenced by the type of disinfectant and the application methods . While commercially available disinfectant products may appear to use similar ingredients, the levels and strength of ingredients with additional emollients may influence the success of a product in reducing somatic cell count and improving teat condition over a longer period . When' iodine' was used as a pre - milking disinfectant, while it did not significantly reduce bacterial numbers, it was 2.3 times more likely to reduce staphylococcal and 1.24 times more likely to reduce streptococcal counts on teats compared to' washing and drying' or no preparation treatments . This result tends to agree with the findings of ingawa et al . Who demonstrated that iodine reduced the bacterial count on teats compared to washing and drying . The use of a 0.25% iodine solution pre - milking has also been shown by oliver et al . To reduce major pathogen intramammary infections resulting from streptooccus uberis and dysgalactiae by as much as 49% . However, including' iodine' as a pre - milking teat preparation treatment may have implications for milk residues as pre or post dipping with an iodine product can increase iodine levels in milk . Therefore, correct disinfectant concentration and drying after application with paper towels must be advised to reduce milk residues .' Chlorhexidine' teat foam which is a new product sold on the irish market was 4.46 times more likely to reduce the staphylococcal count on teats prior to milking when compared to washing and drying . This is of particular signifance to irish dairy farmers as staphylococcus aureus pathogens are to be found in 51% of irish bulk milk samples . Staphylococcus aureus colonisation on teat ends has been shown to increase the risk of intrammmary infection . Therefore, a reduction in staphylococcal numbers on teat ends may reduce the new infection rate on irish farms . Additionally, it has previously been demonstrated that a disinfectant product containing chlorhexidine, when used as a post disinfectant and when used as a pre - milking disinfectant over a long time period, reduced the somatic cell count . The' chlorine' teat foam product used in this study was 3.45 times likely to reduce the streptococcal count on teats prior to milking when compared to the' washing and drying' treatment . This is in agreement with gibson et al . Who showed that a similar chlorine based dip followed by a dry wipe was a most effective treatment for controlling cow mastitis and reducing milk contaminants . The positive effect observed with' wipes' in reducing both staphylococcal and streptococcal counts on teats may be due to the physical manipulation of teats combined with the disinfectant . Dry wiping without disinfectant has been shown to reduce the total bacterial count in bulk milk . However, the use of dry or wet towels by themselves did not have any significant effect on reducing coliform counts in milk . In this study, the reduction in the coliform count on teats with any of the teat preparation treatments used was low . This may be influenced by a low initial level of coliform pathogens present on teats prior to treatment . Teat washing combined with drying with individual paper towels reduced staphylococcal, streptococcal and coliform pathogens compared to' no preparation' and was particularly effective in reducing streptococcal counts when used on cows indoors compared to outdoors . This is in agreement with the findings of mckinnon et al . Who concluded that washing and drying teats prior to milking significantly reduced milk bacterial counts during the winter housing period, compared to the pasture summer period . However, the results of this study would indicate that the probability of a reduction in staphylococcal and streptococcal counts could be expected to be greater where a disinfectant is used pre - milking compared to' washing and drying' or' no preparation' treatments . This study shows that the use of some disinfectant products for pre - milking teat preparation can have beneficial effects on reducing the levels of staphylococcal and streptococcal pathogens on teat skin . Therefore, the possibility of bacterial transfer from cow to cow during milking could be expected to be reduced compared to many farm situations where' no preparation' is normally practiced . The study time period was not sufficient to come to any conclusions on the effect of premilking teat disinfection with regard to teat condition or somatic cell count . In conclusion, bacterial numbers, specifically staphylococcal and streptococcal numbers on cow teat surfaces, were significantly reduced when disinfection products were applied to teats . The use of wipes was particularly effective due to the physical wiping action in conjunction with the disinfectant application . While the practice of washing and drying did reduce bacterial numbers compared to not cleaning teats at all, it could not be considered to be as effective as cleaning with disinfectant products . Given the level of bacterial numbers on non - prepared teats and the reduction observed with chemical disinfectant, it may be advisable to include this process as part of the milking routine . However, pre - milking teat disinfection must be followed by teat drying using individual paper towels to minimise the possibility of chemical residues in milk.
The issue of who should have responsibility for pacs has been around for many years . In the early days of pacs in the 1990s, there were valid reasons supporting pacs management by the radiology department . In those days, pacs usually ran as standalone systems and were not widely used outside of the radiology department . Today, more compelling reasons support the treatment of pacs as a component of an enterprise strategy that appropriately falls under the chief information officer (cio) and the it organization . The cio is the executive who has responsibility for integrating information technology into the health care workplace . Over the past few years, the cio s role has become more complex as public policy has encouraged the adoption of the electronic medical record (emr). The total emr, including computerized provider order entry (cpoe) and clinical documentation, is the strategic goals for most health care cios in the usa . Achieving this goal pacs is only one of the pieces that must be considered in the context of how it fits into and contributes to the emr . Because it is the cio s responsibility to deliver the emr, it is appropriate that the selection, implementation, and operation of the system be under his or her authority . A second compelling reason is that pacs is no longer a radiology - only asset . Diagnostic images are part of clinical information that clinicians outside of radiology expect to have readily available when viewing the emr . Moreover, pacs technology is regularly used by many other areas, such as cardiology, anatomical pathology, ophthalmology, gastroenterology, and document image management . Many of the large emr vendors have taken pacs architecture and expanded it to incorporate the potential for any nontextual clinical information . As pacs technology becomes more pervasive in the organization, it must be centrally managed to avoid duplication of costs and maintain consistency of service . Another reason that pacs should be managed by the cio is the technical complexity of today s it environment . Health care organizations are moving away from an application - centric approach to an enterprise - wide approach in managing systems . This migration has been triggered by regulatory and economic requirements . Under the health insurance portability and accountability act (hipaa) security rule,1 health care organizations have a fiduciary responsibility to safeguard protected health information . Hipaa requirements are mirrored in the joint commission on accreditation of health care organizations information management standards, which are being updated for 2009.2 approaching these requirements on an application - by - application basis is too costly and too complex to ensure compliance . When any information system is managed outside of the it organization, it becomes difficult to ensure compliance, and the entire organization is at risk . Pacs requires more storage capacity than any other single application.3 pacs storage requirements will also increase more rapidly than other applications as more types of images are captured and stored . Despite the fact that data storage costs have been decreasing rapidly, storage is a significant cost element that requires careful management . Many organizations have begun to plan their storage requirements on an enterprise - wide basis rather than on an application - by - application basis . Organizations derive significant benefits by planning and managing data storage on an enterprise - wide basis, particularly in meeting system availability and data redundancy requirements . The final reason why pacs should be managed by the cio has to do with its importance to the emr . Pacs is a strategic component of the emr and must be sold to the organization in that way . The cio is more likely to get capital support for pacs than if the organization sees pacs as a departmental system . The cio and the it organization are structured to be service providers to the rest of the organization . They are more likely to have the resources necessary to support pacs and are better positioned to secure future funding . The issue of who should have responsibility for pacs has been around for many years . In the early days of pacs in the 1990s, there were valid reasons supporting pacs management by the radiology department . In those days, pacs usually ran as standalone systems and were not widely used outside of the radiology department . Today, more compelling reasons support the treatment of pacs as a component of an enterprise strategy that appropriately falls under the chief information officer (cio) and the it organization . The cio is the executive who has responsibility for integrating information technology into the health care workplace . Over the past few years, the cio s role has become more complex as public policy has encouraged the adoption of the electronic medical record (emr). The total emr, including computerized provider order entry (cpoe) and clinical documentation, is the strategic goals for most health care cios in the usa . Achieving this goal pacs is only one of the pieces that must be considered in the context of how it fits into and contributes to the emr . Because it is the cio s responsibility to deliver the emr, it is appropriate that the selection, implementation, and operation of the system be under his or her authority . A second compelling reason is that pacs is no longer a radiology - only asset . Diagnostic images are part of clinical information that clinicians outside of radiology expect to have readily available when viewing the emr . Moreover, pacs technology is regularly used by many other areas, such as cardiology, anatomical pathology, ophthalmology, gastroenterology, and document image management . Many of the large emr vendors have taken pacs architecture and expanded it to incorporate the potential for any nontextual clinical information . As pacs technology becomes more pervasive in the organization, it must be centrally managed to avoid duplication of costs and maintain consistency of service . Another reason that pacs should be managed by the cio is the technical complexity of today s it environment . Health care organizations are moving away from an application - centric approach to an enterprise - wide approach in managing systems . This migration has been triggered by regulatory and economic requirements . Under the health insurance portability and accountability act (hipaa) security rule,1 health care organizations have a fiduciary responsibility to safeguard protected health information . Hipaa requirements are mirrored in the joint commission on accreditation of health care organizations information management standards, which are being updated for 2009.2 approaching these requirements on an application - by - application basis is too costly and too complex to ensure compliance . When any information system is managed outside of the it organization, it becomes difficult to ensure compliance, and the entire organization is at risk . Pacs requires more storage capacity than any other single application.3 pacs storage requirements will also increase more rapidly than other applications as more types of images are captured and stored . Despite the fact that data storage costs have been decreasing rapidly, storage is a significant cost element that requires careful management . Many organizations have begun to plan their storage requirements on an enterprise - wide basis rather than on an application - by - application basis . Organizations derive significant benefits by planning and managing data storage on an enterprise - wide basis, particularly in meeting system availability and data redundancy requirements . The final reason why pacs should be managed by the cio has to do with its importance to the emr . Pacs is a strategic component of the emr and must be sold to the organization in that way . The cio is more likely to get capital support for pacs than if the organization sees pacs as a departmental system . The cio and the it organization are structured to be service providers to the rest of the organization . They are more likely to have the resources necessary to support pacs and are better positioned to secure future funding . Radiology is too large, too complex, too valuable, and too dependant on it to be treated as an ordinary it customer . Radiologists and technical staff are advanced users of complex information systems . The hospital it organization originated as billing systems under the control of the chief financial officer . While it has grown up, the organizational culture is still predominantly corporate lacking clinical expertise . Without domain expertise and local accountability to radiology system requirements frequently lack exception reporting in workflow or adequate support response times to ensure the clinical mission . An it organization without any accountability to radiology has a very hard time doing the necessary tailoring of technology to make it successful . The information systems in imaging are not generic systems; they require specialty knowledge and maintenance skills . It is a fulltime job that does not end when the go - live date passes . Radiology is crucial to the financial well being of a medical center . At a large academic medical center, such as northwestern memorial hospital, more than 20% of patients revenue from the technical component of imaging procedures can approach 2025% of net patient revenues of the institution . Revenue in excess of expenses subsidizes many other areas of the institution and provides for a state - of - the - art imaging environment . Maintaining these revenue streams in the face of decreasing reimbursement and sigma4 parlance, this means defining, measuring, analyzing, improving, and controlling the improvement of every process in the department . It needs to go beyond simple support but be an active participant in process redesign . Another challenge to an independent it organization structure is the allure of using a single vendor over best of breed solutions . No single system from a single vendor can provide all of the it functionality necessary for an imaging department of any significant size . Yet, central it cannot resist the appearing simplicity of synergies and lower costs from a single vendor at the expense of end user functionality and satisfaction . If a group is not cognizant of their users needs and how things really work in practice, it is difficult to differentiate vendors on factors other than cost . Although some of the processes found in medical imaging are common business activities, such as human resource and supply chain management, other processes, such as the integrating the health care enterprise (ihe) radiology integration profiles5 are unique and complex . Mastering the analysis of these processes requires in - depth knowledge of the imaging environment . It staff must be embedded in departmental operations, often arising from the rank - in - file . It is somewhat surprising that many enterprise it organizations do not use industry best practices in business continuity and fault tolerance . This can be understood in part because the vast majority of enterprise it systems are not defined as mission critical . What is the response time to a pacs failure in the operating room? Detailed fallback and what - if plans must be in place throughout the department . Executing these plans in a specific situation requires dedicated it resources with detailed knowledge of the environment and personnel . Understanding the appropriate response model is hard to appreciate for a corporate it group which is frequently based outside of the hospital . The only standard they are experienced with is the hl7 version 3 messaging interface standard . There is little knowledge of the digital imaging and communications in medicine (dicom) standard, the predominant standard in radiology . Lastly, radiology as a specialty has been a rapid adopter of disruptive technology such as multidetector computed tomography scanners . It takes a more diligent awareness of new technology and its impact upon architecture than traditional areas in the health care enterprise . Radiology is too large, too complex, too valuable, and too dependant on it to be treated as an ordinary it customer . Radiologists and technical staff are advanced users of complex information systems . The hospital it organization originated as billing systems under the control of the chief financial officer . While it has grown up, the organizational culture is still predominantly corporate lacking clinical expertise . Without domain expertise and local accountability to radiology system requirements frequently lack exception reporting in workflow or adequate support response times to ensure the clinical mission . An it organization without any accountability to radiology has a very hard time doing the necessary tailoring of technology to make it successful . The information systems in imaging are not generic systems; they require specialty knowledge and maintenance skills . It is a fulltime job that does not end when the go - live date passes . Radiology is crucial to the financial well being of a medical center . At a large academic medical center, such as northwestern memorial hospital, more than 20% of patients revenue from the technical component of imaging procedures can approach 2025% of net patient revenues of the institution . Revenue in excess of expenses subsidizes many other areas of the institution and provides for a state - of - the - art imaging environment . Maintaining these revenue streams in the face of decreasing reimbursement and sigma4 parlance, this means defining, measuring, analyzing, improving, and controlling the improvement of every process in the department . It needs to go beyond simple support but be an active participant in process redesign . Another challenge to an independent it organization structure is the allure of using a single vendor over best of breed solutions . No single system from a single vendor can provide all of the it functionality necessary for an imaging department of any significant size . Yet, central it cannot resist the appearing simplicity of synergies and lower costs from a single vendor at the expense of end user functionality and satisfaction . If a group is not cognizant of their users needs and how things really work in practice, it is difficult to differentiate vendors on factors other than cost . Although some of the processes found in medical imaging are common business activities, such as human resource and supply chain management, other processes, such as the integrating the health care enterprise (ihe) radiology integration profiles5 are unique and complex . Mastering the analysis of these processes requires in - depth knowledge of the imaging environment . It staff must be embedded in departmental operations, often arising from the rank - in - file . It is somewhat surprising that many enterprise it organizations do not use industry best practices in business continuity and fault tolerance . This can be understood in part because the vast majority of enterprise it systems are not defined as mission critical . What is the response time to a pacs failure in the operating room? Detailed fallback and what - if plans must be in place throughout the department . Executing these plans in a specific situation requires dedicated it resources with detailed knowledge of the environment and personnel . Understanding the appropriate response model is hard to appreciate for a corporate it group which is frequently based outside of the hospital . The only standard they are experienced with is the hl7 version 3 messaging interface standard . There is little knowledge of the digital imaging and communications in medicine (dicom) standard, the predominant standard in radiology . Lastly, radiology as a specialty has been a rapid adopter of disruptive technology such as multidetector computed tomography scanners . It takes a more diligent awareness of new technology and its impact upon architecture than traditional areas in the health care enterprise . Channin illustrate the traditional perspective of silos of care . From the perspective of the radiology department, he is absolutely right that the priorities of the radiology department, and it will probably never be the same . Coordinating the care of a patient among all of the diagnostic and treatment options in the most efficient and cost - effective manner must be the priority of our health care delivery system . It has been charged with delivering the emr, which focuses on the patient not the hospital department . The patient must be the priority, even if this means compromises elsewhere in the delivery system . What is best for the patient may not necessarily be the best or most efficient for individual departments . An it organization that is truly responsive to the needs of the organization will be embedded within each department and will have domain expertise . An enlightened cio is not threatened by it innovation within departments but will try to find ways to work with departments to develop solutions . In the end, however, everything must go back to the number one priority: the patient . Although it may be true that the cio has ultimate responsibility for any it activity within the institution, the role of the cio is clearly strategic not tactical . Glaser and williams wrote, the cio is a critical contributor to the development of the organization s strategy; a valued member of the c suite; a leader of and manager of a high - performance it staff; able to lead and support major change in organizational processes; an astute judge of the potential of new technologies; effective in managing the organization s it suppliers .... .6 no leader operating at that level, regardless of technical expertise and background, can hope to understand the detailed requirements and technology of the myriad clinical and support entities . The cio must lead in the support of standards, interoperability, compliance with policies, procedures, and regulations . He or she should supply intellectual and financial nourishment to let a garden of innovation grow . I contend that radiology understands the requirements and needs better than a centralized it organization . Health care providers need to view and manipulate images as well as be assisted by the work product of the radiologists . The radiology community has developed a number of technical frameworks that serve as an example of how clinically centric it can be developed and managed locally . Radiology can and does also serve as a technology exemplar for the other -ologies . Nowhere is this more evident than in the evolution of ihe . Similarly, within an institution, we can share our best practices and our infrastructure with our colleagues . If it makes sense for pathology images to be in pacs, great; if it makes sense for them to be in pathology, all the better . Standards and interoperability will push them where they need to be, just in time, for clinical decision support . Central it has no role in managing a department s evolution, and certainly making them evolve in lockstep would be disruptive . My argument that the technical complexity of radiology mandates local it ownership stands . That regulatory complexity is increasing is a fact of life with which every organizational unit must contend . The role of the cio is, again, to provide leadership and guidance and to monitor compliance . The vice president of safety and facilities does not come to our department to lecture on the chicago fire code . All staff shall be versed in fire response procedures (a joint commission requirement); we make it so . Senior management, including the cio, need to make prioritization decisions that leverage its resources wisely . Once those decisions on resources are made, however, only the department has the knowledge to make contracting decisions and plan, deploy, and manage the technology required to meet those metrics . We do not plumb our own water lines, generate our own electricity, or smith our locks . Specific detailed operations and complex devices, however, are not commodities and cannot be treated as such.
An increased man - made radiation exposure - risk from the use of high - dose imaging modalities such as computed tomography and angiographic suites is now being observed in many health - care centers with over 3.6 million diagnostic examinations performed annually worldwide . Interventional procedures are performed by cardiologists, radiologists, endovascular surgeons, operation theater staff, etc ., due to the well - known benefits in medicine . However, it is crucial for the referring clinician and the interventionalist (radiologist / cardiologist / clinician in the operation theater) to assess the potential benefit - risk ratio for various interventional procedures as some of the procedures involve a high radiation exposure due to prolonged fluoroscopic screening . All interventional cardiological procedures are invariably performed using dedicated fluoroscopy and angiography suites equipped with either image intensifier (ii)-based or flat panel detector (fpd)-based systems . The ii - based systems have been used for fluoroscopy for more than two decades . On the other hand, the fpd - based systems for medical imaging emerged in the 1990s initially for two - dimensional (2d) projection x - ray image, and subsequently for a real - time interventional angiography suites equipped with ii or fpd have the potential to impart high radiation doses to patients if optimization strategies are not well - implemented . Stringent optimization involves orientation of staff, consistent restriction of frame rates during image acquisition, using low dose settings, judicious use of magnification, etc . It is also necessary to understand the potential risks due to radiation from different interventional procedures . For this reason, it is necessary that one should be knowledgeable in the magnitude of radiation dose associated with each intervention . This can be achieved by measuring real - time doses using devices such as a dose area product (dap) meter . Most of the newer angiography machines are equipped with a dap meter fitted on the collimator assembly of the machine . Dap is particularly a useful method for assessing and comparing the radiation dose from screening procedures and acts as a surrogate for radiation risk . Entrance surface dose (esd) is also used for measuring radiation doses . From the dose descriptors, it is possible to estimate organ doses as well as effective doses for each procedure . Radiation doses from interventional procedures have been widely reported in literature, with more emphasis on doses from ii - based systems . However, there are only a few reports on radiation doses from fpd systems as it may be a transition period from ii to fpd for most of the interventional users . Some patient and phantom - based studies reported in literature state that doses from fpd are higher than ii systems . Few other studies report that radiation doses from fpd are lower than ii systems . In comparing conventional and digital systems, hence, it is not clear whether fpd imparts lower radiation dose than ii and whether there would still be a need to further optimize doses in the newer fpd systems . The purpose of this article is to review and compare radiation doses from ii and fpd - based systems in interventional cardiology in a tertiary referral center that has introduced fpd system recently . It is anticipated that this information will be useful for those performing cardiological interventions and for those who are on a transition from ii to fpd . Cardiovascular interventions were performed using two dedicated catheterization labs, each equipped with philips allura fd10 flat panel system (netherlands). The dose area product (dap) values for coronary angiography (cag, n = 222) and percutaneous transluminal coronary angioplasty (ptca, n = 75) procedures performed during a one year period 20122013 were prospectively recorded using a built - in calibrated dap meter (transmission ionization chamber). The ptca procedure was invariably performed by a senior interventionalist assisted by two junior cardiologists, while the cag was performed either by the senior interventionalist or by junior cardiologists . For a similar comparison of clinical protocols adopted in the institution, dap values from cag and ptca performed using philips integris h3000 and h5000 ii - based systems (netherlands) reported earlier all the x - ray systems were on periodic qa programs and conformed to the manufacturer's specifications . The ii and fpd systems had low-, normal-, and high - dose settings, respectively, for fluoroscopy . These machines incorporated a total filtration of 2.5 mmal with possibility of selecting spectral filters such as 0.1 mm, 0.2 mm, 0.4 mm cu for dose reduction . During the course of the study, low dose setting with 0.4 mm cu filter was invariably selected during fluoroscopy . In the earlier work using ii - based system, a 23-cm image intensifier format (iif) was used during fluoroscopic screening in cag and ptca procedures for tracing the path of the catheter from the region of arterial puncture and to the screening of the cardiac valve region . A 17-cm iif was used for the oblique, caudal, cranial, and lateral projections delineating the coronary anatomy . In the fpd system, 25 cm fluoro format was used during screening and 20 cm was used for other projections to delineate coronary anatomy . A transition from ii to fpd system for a catheterization lab would require adequate justification in terms of radiation dose, image quality, maintenance, and investment . It has been reported that the fpds designed specifically for fluoroscopic purposes provide superior image quality and dose efficiency compared to the ii systems, except at the lowest fluoroscopic dose levels . Prieto et al ., reports that even after upgrading to the fpd from ii, significant increase in patient doses were observed though the fluoroscopic time and number of images remaining the same in both cases during the initial transition period . As only a few studies on radiation doses are available for fpd systems; more work is required on optimization strategies in the fpd systems . Table 1 shows the dap values and fluoroscopic time duration for ii and fpd systems from the referral center where the study was conducted . The dap values for ii - based systems represented in table 1 is from the use of optimized protocol as reported in the previous published article from the same referral center where the study was conducted . Prior to optimization in ii systems, the doses were above 50%; however, it was possible to optimize dose by halving the entrance dose ratesby selecting 0.4 mm cu filtration (generally recruited in pediatric protocols) during fluoroscopy . Selection of 0.4 mm cu filtration did not suffer significant loss of image quality; however, tube potentials were increased from 80 kvp to 103 kvp during fluoroscopy . In the fpd system, tube potentials ranged from 90 kvp110 kvp when 0.4 mm cu filtration was selected with low dose settings during fluoroscopy . Having adhered to the same optimization strategies in both systems, doses were similar owing to the fact that further optimization is warranted in fpd system . Reported dap values of 31 and 33 gycm from cag with the use of ii and fpd system, respectively . They have also reported that the total dap from fluoroscopy and cine for ii and fpd are not significantly different and the image quality from fpd is better than ii in cine mode with no difference in the imaging performance in fluoroscopy mode . Fluoroscopic time and dose area product values from ii and flat panel detector (fpd)-based systems from cardiological interventions tables 2 and 3 shows dap values for cag and ptca procedures from various studies in literature . The arithmetic mean dap and fluoroscopic time duration using ii system as reported in literature was 39 gycm for 6.6 min and 61.2 gycm for 17 min for cag and ptca respectively [table 2]. With the use of fpd, mean dap and fluoroscopic time duration were 28.4 gycm for 7 min and 61 gycm for 18.05 min for cag and ptca, respectively [table 3]. From tables 2 and 3, radiation doses from fpd were significantly low for cag but were similar for ptca when compared to ii systems . It should be noted that time duration for cag and ptca was not available for some reported studies in tables 2 and 3 . Wide variation of doses is observed from these studies which may be attributed to the angiographic system used, time duration of the procedure and work environment . Doses of the order of 492 gycm were recorded in ptca procedure from fpd system which was higher than the ii systems . Chida et al ., conducted studies from various ii and fpd system and the average entrance doses of cine angiography and fluoroscopy in fpd systems were not significantly different . Though fpds possess good detective quantum efficiency, they did not inherently reduce the radiation dose . Jensen et al ., observed that patient doses from fpd were lower than ii systems; however, the eye lens doses for radiologists were higher in fpd than in ii due to the use of high filtration and recruitment of high tube potentials . In our study, high tube potentials were recruited when low dose settings involving high filtration were selected, which may have the potential to increase staff doses . Radiation doses from cardiological interventions performed using image intensifier - based systems radiation doses from cardiological interventions performed using flat panel detector - based systems it is prudent to adopt stringent optimization measures in fpd as the dose may be higher than the ii systems as reported by prieto et al . And dose reduction is possible in either ii or fpd systems . During the initial stages of our study, the doses from fpd were similar to the ii system though high filtration was used . Reports that it is possible to achieve mean dap of 6.2 and 10.4 gycm for cag and ptca procedures performed using ii system . They further report that the reduction of doses was by influencing the quality of fellowship training, consistent restriction to mean values of 171 frames per cag, 165 frames per ptca, low - level fluoroscopy, training in the use of fluoroscopy - free blind positioning to the region of interest, restrictions to achieve lower ii entrance dose for adequate image quality . From tables 1 and 3, the mean dap for cag from fpd were higher than those reported by kuon et al . Tsapaki et al ., reported the doses from fpd were increased by 35% compared to ii when fluoroscopy levels were changed from low to high mode; they also recorded a minimum dap value of 6.1 and 14.3 gycm for cag and ptca, respectively, with the use of low dose fluoroscopy settings in fpd . Dekker et al ., reported that the new generation fpds incorporated with good image processing and noise reduction techniques resulted in reducing patient doses by 43% without compromising image quality and staff doses by 50% during electrophysiological interventions . Though fpd has reduced entrance dose rates, it does not automatically reduce radiation doses in clinical practice . Further work is necessary to study the possibilities of dose reduction in fpd so as to be implemented in the clinical set up . The patient dose and image quality in any newer modality needs to be permanently monitored and transition from ii to fpd requires careful attention . This is a preliminary study as the institution where the study was conducted recently moved from ii to fpd - based systems . Though radiation doses for cardiological interventions from fpd were similar to the ii - based system achieved after optimization, the advantages of fpd in terms of good image uniformity, improved patient imaging accessibility due to smaller size, absence of geometric distortion / veiling glare or vignetting make the fpd superior to the ii systems . To achieve improved patient dose reduction, it is advisable to strictly adhere to low dose protocols with high filtration in fpd systems . In addition, more attention for staff doses is warranted especially for interventionalists when this stringent patient dose reduction is employed . It is recommended to follow stringent dose reduction strategies right from the period of initial installation when there is a transition from ii to fpd systems . Further studies are required to develop dose optimization in fpd, though use of high filtration is already in place.
The norbeck social support questionnaire (nssq) [1, 2] is a network - based social support inventory . That is, unlike global support measures which assess overall perception of how supported one feels, the nssq asks participants to take detailed stock or inventory of (a) how many supportive network members they have and (b) how much affection, affirmation, and aid each network member provides . The strength of a network - based inventory is that it allows testing of multiple social support hypotheses, which are impossible to test using global measures of social support . Moving from general to specific, one can investigate the effects of total functional support (affect, affirmation, and aid) from the entire network, each of the three types of support from the entire network, total functional support from each network relationship (e.g., total functional support from spouse, friends, etc . ), or each of the three types of support from each network relationship (e.g., affect from spouse). Despite the advantages of being a network based inventory, house and kahn argued that the nssq scoring system both creates extraneous variance and is a source of measurement error . They noted that network size varies considerably, because participants can nominate up to 24 network members . Thus, when participants' support ratings of network members are summed, nssq support scores may be confounded from extraneous variance from network size, and this is especially problematic when using total network scores versus specific network relationship scores . That is, because more network members implies more support, support scores from the entire network most heavily reflect both support ratings and number of supporters listed . Recognizing this problem, some investigators use averaged scores (support ratings divided by network size) to remove the influence of network size variability . However, though effective in removing the effects of network size variability, as fully detailed below, norbeck discouraged this practice, cautioning that averaging can unduly lower scores of some participants with large networks . Though this may be true, to date, the effects of averaging on support scores as network size increases have not been investigated . Moreover, because extraneous variance due to network size variability in raw scores increases measurement error, averaged scores continue to be used . In fact, 23% of nssq - based studies published since norbeck's caution in 1995 report averaged scores . The purpose of this paper is twofold: first, using three different data sets, we evaluate norbeck's concern by investigating whether averaged support scores do indeed decrease as network size increases . Second, we evaluate the statistical efficiency of averaged versus raw scores by comparing the powers of their correlations with a reasonable criterion variable . The conceptual basis for the nssq [1, 2] is kahn's definition of social support, and it thus includes measures of functional support (affect, affirmation, and aid), network size, and network relationships . For this reason, the nssq was commended in house and kahn's classic work on social support concepts and measures, where they urged investigators to consider all three of these aspects of support, because network size is a necessary condition and hence a partial determinant (page 85) of network relationships and the types of support given . Due to its comprehensive scope and extensive ongoing psychometric evaluation [1, 2, 4, 68], it is one of the most widely used social support measures in nursing research . In fact, since its inception in 1981, the nssq has been used in over 250 studies published in peer - reviewed journals, and its use increases each year . One reason for its widespread use is that unlike other network - based support inventories, the nssq is completed by the participant without input from an interviewer . This makes it ideal for use in large - scale studies such as mailed surveys . Because of this self - report feature, the nssq requires a unique layout . Specifically, participants are first asked to list from 1 to 24 network members who provide personal support for you or who are important to you and then specify their relationship (spouse, parent, friend, etc . ). After completing the network list, they are instructed to successively turn the half pages and rate each listed network member (04) on six functional support questions measuring three types of support: affect, affirmation, and aid (see table 1). Normative data (n = 1,067) shows that the average network size is 10.9 members, but the high standard deviation of this average (5.9) reveals the considerable variability between reported network sizes . This is because participants' network size is dependent on many factors, including how influenced participants are by the presence of 24 spaces as well as the size of their immediate and extended family . For example, family (other than spouse) is the most often listed relationship, so a participant with two living parents, a spouse, and four children may list up to seven immediate family members plus supportive siblings, friends, neighbors, and so forth . In contrast, a participant with deceased parents, a spouse, and two children will only have three possible immediate family members to list . Because support ratings for each network member are summed, support scores (range = 0576) vary greatly due to network size alone . Thus, the above participant with seven immediate family members functionally inflates his / her support score . In fact, in the three samples [911] used in the present study, network size was very highly correlated with affect scores (.95, .94, and .95, resp .) And affirmation scores (.92, .90, and .92, resp .) And a bit less with aid scores (.81, .82, and .82, resp . ). It is likely that aid's lower correlations with network size are the result of more participants giving some network members aid ratings of 0 than giving 0's for affect or affirmation ratings . When this happens, the participant has effectively dropped that person from their network, and thus reduced the influence of network size on that support score . This happens most often with aid, because some participants list network members who may like (affect) and agree with (affirmation) them but be unable to provide tangible help (aid) such as children, elderly parents, and peripheral network members . In fact, in the present study's third sample, where data were entered at this level of specificity (i.e., affect, affirmation, and aid scores from each network member), it was determined that for aid scores, 33% of participants gave a 0 aid rating to at least 1 network member, and 20% of participants gave more than 1 network member a 0 aid rating . In contrast, only 9% of participants gave a 0 affect rating, and 19% gave a 0 affirmation rating . Few participants gave more than one network member a 0 rating for affect or affirmation questions . In summary, though nssq support scores are meant to measure quantity of support, they have two determinants of variability: support ratings and network size . Therefore, raw support scores cannot be taken at face value but should be viewed as support ratings relative to network size . For this reason, many investigators remove the influence of network size variability by averaging nssq scores, that is, dividing the support score by the network size . Averaging is not a problem when participants rate all network members uniformly highly or lowly . For example, participant a lists 7 highly supportive network members, and participant b lists 14 equally supportive members . B's score (305) is higher than a's (154) only because she listed more network members . However, a's and b's averaged support scores (a (154/7 = 22) and b (305/14 = 21.79)) reflect their support quality relative to their respective network sizes . This is because only their network sizes varied; their support ratings were consistently high . All of their network members uniformly liked them (affect), agreed with them (affirmation), and could help them (aid), but this equality among network members is not typical . In a typical nssq network, only a few supporters give large amounts of all three types of support and the others contribute in varying degrees, and this reflects reality . That is, some network members make one feel loved and/or are good confidants but cannot offer tangible support and vice versa . This pattern is typified by participant c: like b, participant c has a relatively large network (14), but unlike b, her network members' ratings were more varied . C rated 7 network members highly on most support questions but varied the ratings of the other 7 network members giving some high and some low ratings for some types of support . Though both a and c each have 7 highly supportive network members and c has 7 additional network members giving some support, c's averaged score (277/14 = 19.79) is 2.21 points lower than a's (154/7 = 22). Because of variations in her ratings, unlike b, c's numerator (support score) did not keep up with her denominator (network size). Of course, it is possible that this deflation of averaged scores happens at all network sizes . In fact, if averaging lowered scores consistently for all participants, then lowered scores due to averaging would be the norm and would result in true regressions to the mean . Thus, as is the case with uniformly high or low ratings, if all participants vary their support ratings averaging is again not a problem . Norbeck was concerned, however, because participants' support ratings show increased variability with increased network size . That is, the more people one has in his / her network (denominator), the more room for variability of ratings (numerator) and the more chance that one's score will be unduly lowered by averaging if the numerator does not keep pace with the denominator . Indeed, we saw previously that raw support scores increase as network size increases as evidenced by the high positive correlations . Thus, the question is whether averaging results in a statistically significant lowering of (averaged) scores as network size increases . The potential for this is greatest for total network scores and less so for source - specific scores due to the smaller denominators (one relationship category). Nevertheless, though norbeck's concern about the effects of averaging may be warranted, one must also be aware of the effects of using support scores which contain variability due to network size . For example, if high support is related to low stress but support scores are reflective of support as well as network size, it is likely that the size of the relation between support and stress will be reduced . This is due to measurement error or the extraneous variance present in support scores resulting from network size variability . Thus, the risk of making a type 2 error (failing to detect a real effect) may be greater when using raw support scores, and the deleterious effects of averaging, if any, must be weighed against their beneficial effects in terms of explained variance in the criterion variable . Is there a statistically significant negative correlation between averaged total functional support scores (entire network) and number in network? In addition, are there statistically significant negative correlations between averaged affect, affirmation, and aid scores (entire network) and number in network? Does using averaged total functional support scores provide a measure less infected with extraneous variance and thus produce a more efficient measure than raw total functional support scores? Do averaged total functional support scores yield higher powers than raw scores produced under the same conditions? Similarly, do averaged affect, affirmation, and aid scores provide an analysis with a higher power than respective analyses with raw scores? With institutional review board approval, a secondary analysis was conducted on data from three different samples [911] of women who were mothers attending college for their first postsecondary school degree . The same data collection protocol was used in all three studies: participation was invited during a brief in - class presentation of the study, and participants completed the self - administered surveys on their own time and anonymously returned surveys in postage - paid envelopes addressed to the first author . Response rates were high (66%, 45%, and 57%, resp . ). Along with the nssq, participants completed the perceived multiple role stress scale and role involvement questionnaires as well as a demographic data sheet . Table 2 shows that these samples' ((n = 157); (n = 263); (n = 189)) parametric properties are consistent with norbeck's normative data for community dwelling adult females (n = 1,067). Though norbeck gives no information concerning network size distribution, the majority (75%) of participants in the present study's samples listed 14 network members, and 50% listed 10 - 11 members . All samples' total functional support scores were statistically significantly nonnormal, showing positive skews (5.78, 3.32, and 5.60, resp . ), and all except sample two showed statistically significantly positive kurtoses (3.5 and 4.54). These findings are consistent with findings concerning normality throughout the nssq literature . That is, due to high network size, some participants have very high support scores . Research questions 1 and 2.using pasw 18 with a.05 alpha level, a bivariate pearson correlation was computed to determine if there was a statistically significant negative correlation between averaged total functional support scores (summed affect, affirmation, and aid scores from the entire network divided by total number in network) and number in the network . In addition, separate correlations were computed to determine if there were statistically significant negative correlations between averaged affect scores, averaged affirmation scores, and averaged aid scores from the entire network and network number . Because the effect of network size is removed from averaged scores, a significant decrease in averaged scores as network number increases (i.e., a significant negative correlation between averaged scores and network number) would support the claim that averaging scores indeed unduly lowers support scores as network size increases . Using pasw 18 with a.05 alpha level, a bivariate pearson correlation was computed to determine if there was a statistically significant negative correlation between averaged total functional support scores (summed affect, affirmation, and aid scores from the entire network divided by total number in network) and number in the network . In addition, separate correlations were computed to determine if there were statistically significant negative correlations between averaged affect scores, averaged affirmation scores, and averaged aid scores from the entire network and network number . Because the effect of network size is removed from averaged scores, a significant decrease in averaged scores as network number increases (i.e., a significant negative correlation between averaged scores and network number) would support the claim that averaging scores indeed unduly lowers support scores as network size increases . Research questions 3 and 4.in order to answer these research questions, pmrs was used as the criterion variable . Pmrs is the amount of role stress experienced by women who are both mothers and students . It has been hypothesized that increased social support is related to decreased levels of pmrs [911, 13]. Using the same software and criteria as above, we tested the correlations between pmrs and both raw and averaged total functional support scores and affect, affirmation, and aid scores.using g * power 3, we then computed the power of each of the tests given the respective sample sizes, correlations with pmrs of both the various raw and the averaged scores, and = .05 . Analogous to the power of a microscope, tests with higher powers can detect finer differences (and thus avoid more type ii errors) than less powerful tests conducted under the same level of statistical rigor . Although there is no established standard for minimally acceptable levels of power, cohen suggested that power should be at least .80 . That is, there is an 80% chance of finding a real, significant effect . Given that averaged scores retain the same information about support quantity as do raw scores but remove the variance associated with differences in network sizes, we expected higher powers among averaged scores than among raw scores . In order to answer these research questions, pmrs is the amount of role stress experienced by women who are both mothers and students . It has been hypothesized that increased social support is related to decreased levels of pmrs [911, 13]. Using the same software and criteria as above, we tested the correlations between pmrs and both raw and averaged total functional support scores and affect, affirmation, and aid scores . Using g * power 3, we then computed the power of each of the tests given the respective sample sizes, correlations with pmrs of both the various raw and the averaged scores, and = .05 . Analogous to the power of a microscope, tests with higher powers can detect finer differences (and thus avoid more type ii errors) than less powerful tests conducted under the same level of statistical rigor . Although there is no established standard for minimally acceptable levels of power, cohen suggested that power should be at least .80 . That is, there is an 80% chance of finding a real, significant effect . Given that averaged scores retain the same information about support quantity as do raw scores but remove the variance associated with differences in network sizes, we expected higher powers among averaged scores than among raw scores . Research questions 1 and 2results are presented in table 3 . In all three samples, there are no statistically significant decreases in averaged total functional support scores as network size increases . Nor are there statistically significant decreases in averaged affect or affirmation support scores as network size increases . Thus, averaged total functional support scores and averaged affect and affirmation scores do not unduly lower scores as one's network size increases.however, this is not true for averaged aid scores . In all three samples, there are statistically significant decreases in averaged aid support scores as network size increases . These results are most likely due to the aforementioned high percentage of participants who rated some network members 0 (none at all) for one or both of the aid questions . When participants scored a network member as providing 0 aid, it was most often for network members mentioned later in the network list . That is, it appears that participants begin completing the list of network members by nominating their closest supporters followed by more peripheral supporters . Thus, with the exception of young children and elderly parents, these close supporters are likely able to offer more tangible help than the others . When rating members as providing 0 aid, participants already reduced the influence of network size, and averaging penalized them further, because the denominator was not adjusted to account for this . Averaging does indeed unduly lower aid scores of participants as network number increases . Results are presented in table 3 . In all three samples, there are no statistically significant decreases in averaged total functional support scores as network size increases . Nor are there statistically significant decreases in averaged affect or affirmation support scores as network size increases . Thus, averaged total functional support scores and averaged affect and affirmation scores do not unduly lower scores as one's network size increases . However, this is not true for averaged aid scores . In all three samples, there are statistically significant decreases in averaged aid support scores as network size increases . These results are most likely due to the aforementioned high percentage of participants who rated some network members 0 (none at all) for one or both of the aid questions . When participants scored a network member as providing 0 aid, it was most often for network members mentioned later in the network list . That is, it appears that participants begin completing the list of network members by nominating their closest supporters followed by more peripheral supporters . Thus, with the exception of young children and elderly parents, these close supporters are likely able to offer more tangible help than the others . When rating members as providing 0 aid, participants already reduced the influence of network size, and averaging penalized them further, because the denominator was not adjusted to account for this . Averaged total functional support scores, averaged affect scores, and averaged affirmation scores resulted in higher powers when correlated with pmrs than their respective raw scores . These results are most dramatic in samples one and three, where powers increased .50 (.47 to .97) and .66 (.13 to .79), respectively . Though sample two's results do not show as marked an improvement, gains in power did considerably improve their probabilities by .24 to .35 points . It should be noted that all else being equal larger sample sizes will yield higher power; sample two had 74 more participants than sample one and 106 more participants than sample three . Moreover, we found no statistically significant lowering of averaged scores, as network size increased when investigating research questions 1 and 2 above . Therefore, we recommend using averaged total functional support scores and averaged affect and affirmation scores.however, averaged aid scores did not perform as well as raw aid scores . The gains in power were modest (.15 and .20) for samples one and three, while power actually decreased in sample two by .19 . Thus, in light of results of research questions 1 and 2 showing the statistically significant lowering of averaged aid scores as network size increases and the equivocal effect on power of a test correlation, use of averaged aid scores is not recommended . Averaged total functional support scores, averaged affect scores, and averaged affirmation scores resulted in higher powers when correlated with pmrs than their respective raw scores . These results are most dramatic in samples one and three, where powers increased .50 (.47 to .97) and .66 (.13 to .79), respectively . Though sample two's results do not show as marked an improvement, gains in power did considerably improve their probabilities by .24 to .35 points . It should be noted that all else being equal larger sample sizes will yield higher power; sample two had 74 more participants than sample one and 106 more participants than sample three . Moreover, we found no statistically significant lowering of averaged scores, as network size increased when investigating research questions 1 and 2 above . Therefore, we recommend using averaged total functional support scores and averaged affect and affirmation scores . However, averaged aid scores did not perform as well as raw aid scores . The gains in power were modest (.15 and .20) for samples one and three, while power actually decreased in sample two by .19 . Thus, in light of results of research questions 1 and 2 showing the statistically significant lowering of averaged aid scores as network size increases and the equivocal effect on power of a test correlation, use of averaged aid scores is not recommended . Averaging reduces the influence of varied network size, but norbeck was concerned that if support ratings decrease (lower numerators) as network size increases (higher denominators), averaging may unduly lower scores as network size increases . It was found that averaging does not significantly lower total functional support scores or affect and affirmation scores as network size increases . Furthermore, these averaged scores improve analyses by decreasing measurement error as demonstrated by an increase in power . Use of these averaged scores is acceptable, given the underlying nature of the data and the improvements in power . However, norbeck's concern about averaging is well founded regarding averaged aid scores . Because network size's influence on aid scores was already reduced by participants' ratings of 0 (none at all) for some network members, averaged aid scores unduly penalize participants as network size increases . Moreover, reduction in measurement error improved only slightly in two samples, and measurement error actually increased in one sample.
Molecular dynamics simulations have been used as a powerful tool to study lipid membranes but are limited by the length and time scales of the probing systems . In particular, the lateral diffusion rate of a lipid in a membrane, although varying among different experimental techniques, is in the range 1010 cm s. this means that it will take a lipid about hundreds of nanoseconds to microseconds time scale to cover a 1 nm area . Furthermore, lipids are observed to move collectively with their neighbors, and therefore, the rate at which a lipid swaps position with its neighbor is even slower . This poses a serious problem when we simulate lipid bilayers with multiple components, since the lateral organization of different components requires extensive simulation to attain equilibrium and common accessible simulation time scales may provide configurations ensemble biased toward the initial conditions . Temperature replica exchange molecular dynamics (t - remd) is one of the methods that has been widely used to accelerate equilibration in simulations and achieved numerous success in protein folding . However, its application to lipid bilayers is rare since the number of replicas scales with the degrees of freedom (dofs) of the whole system, and lipid membranes usually have many more dofs than the protein systems, making it computationally prohibitive to use t - remd to study lipid bilayers . One promising method to get around the poor scalability of t - remd with system size is replica exchange with solute tempering (rest), which was initially proposed by berne and co - workers . Rest is a specific variation of a generalized hamiltonian replica exchange method . By changing the solute solute and solute solvent interactions in the system although rest has been demonstrated to sample the conformational ensemble of the alanine dipeptide successfully, its efficiency in folding larger proteins remains unclear, which impedes a wide adoption of the method . In this work, we show that rest can be used as an efficient way to accelerate lateral equilibration in a mixed lipid bilayer . We applied constant pressure rest to a 1,2-dipalmitoyl - sn - glycero-3-phosphocholine (dppc) bilayer with 50 mol% cholesterol (chol). Constant pressure in rest simulation is used because volume expansion at high temperature increases lipids lateral diffusion . By carefully choosing the tempering solute, we managed to simulate the system with only 12 rest replicas, which compares favorably to the requirement for 100 replicas with t - remd at the same replica exchange rate . The relative diffusion rate between molecules in rest is, on average, an order of magnitude faster than the rate in standard md simulation . We also show that the lateral radial distribution function between all molecular pair types (chol chol, chol - dppc, dppc finally, we use rest to obtain the gibbs free energy profiles between different molecular pair types from the corresponding lateral radial distribution functions . Let us start with a brief review of replica exchange with solute tempering (rest). Rest can be derived from a more general form of replica exchange called hamiltonian replica exchange . In hamiltonian replica exchange, replica m is simulated with potential energy em, at temperature tm (the corresponding inverse temperature will be referred as m, where m = 1/kbtm and kb is the boltzmann constant) and constant pressure pm . In an isothermal isobaric ensemble, the probability of configuration xm with volume vm in replica m is1where zm is the corresponding partition function . The exchange between replica m and n can be treated as the change from state i to state f in the generalized ensemble,2and we use t(i f) to denote the transition probability for i f. applying the detailed balance condition3gives the ratio of the transition probabilities4where5 if the replicas are simulated at the same temperature t0 and pressure p0 but a different potential energy, eq 5 can be further reduced to6 the detailed balance condition guarantees the boltzmann sampling of all replicas when sufficiently long simulations are performed . In rest, each replica is simulated at the same temperature (t0) and pressure (p0) but a different potential energy function . We can divide the system potential energy into three terms:7where each term, in order, represents solute solute, solute solvent, solvent solvent interactions . For replica m, we scale its potential energy according to8 following from eq 1, the boltzmann distribution for replica m becomes9equation 9 shows that, thermodynamically, we can interpret the solute of the system as if it is simulated at the scaled potential (m/0)ess at temperature t0, or at the original potential ess at an effective temperature tm . In each rest setup, we should always ensure that there is one replica simulated with tm = t0 . We will refer t0 also as the target temperature because it is the temperature of interested for the studied system . Replicas simulated with tm t0 are used for the sole purpose of enhancing sampling at t0 and configurations obtained from these additional replicas do not represent any thermodynamic ensembles that have experimental counterparts . First, the definition of solute and solvent in a system is not absolute . The solute can generally present a part of the system whose sampling we want to accelerate, while the solvent is defined as the rest of the system . Second, the potential function of replica m can be of a form different from eq 8 . Rest is just a specific form of hamiltonian replica exchange, and there is no restriction in the form of the potential energy function used in each replica in hamiltonian replica exchange . The advantage of using eq 8 is that there is a physical interpretation associated with it, but this is not required . Therefore, the choice of the prefactor in front of esw is not unique . We choose (m/0) for the ease of implementation, as suggested by terakawa et al . Following eq 6, the exchange ratio between replica m and n is determined by10it is clear from the above equation that the exchange ratio is independent of solvent solvent interactions (eww). Therefore, one can enhance the solute dynamics and simultaneously reduce the number of tempered degrees of freedom, which results in a reduction of the number of required replicas . We want to point out that in our implementation of rest we use eq 6 to calculate exchange rate rather than eq 10 for practical reasons . Specifically, the total potential energy is easily available from the simulation code . In the following text, we describe how we scale the potential energy function according to eq 8, using separate force field parameters for each replica . In common molecular dynamics force fields, the potential function consists of11where the first two terms are coulomb and lennard - jones interactions and the third and the following terms are bonded interactions which define the bond length potential, the bond angle potential, the torsion potential, and so forth . The scaling of the potential in replica m descried in eq 8 can be done as follows: (1) for the bonded interactions in the solute, scale the spring constants by (m/0), (2) scale the charges in solute by (m/0), and (3) scale ij by m/0 if both i and j are in the solute, and by (m/0) if only i or j is in the solute . We scale ij directly because it can be applied to any combination rule . By these the system we studied consisted of 144 dppcs, 144 cholesterols (chol), and 14k water molecules . Each monolayer in the bilayer was built independently by randomly placing 72 dppcs and 72 chols on 12 12 planar grids . We define the z axis as the bilayer normal and refer the values of z> 0 nm and z <0 nm as the upper and lower monolayers . Then, we equilibrated the system for 20 ns at 323 k and 1 atm . The resulting configuration was used as the initial structure for all replicas in rest and the standard md simulations . We note here that every replica in rest has the same starting configuration as in the md simulation . In rest, we chose dppc molecules as the solute and treated cholesterols and waters together as solvent . The explanation for this choice is provided in the results section . In total, 12 replicas were used and a 25% exchange rate was achieved between the neighboring replicas . Each replica was simulated at 323 k (t0), while the effective temperatures of dppc were set at 323, 341, 360, 380, 400, 421, 445, 471, 500, 531, 564, and 600 k (tm). Each replica in rest was simulated for 60 ns, while the md simulation was conducted for 400 ns . We implemented hamiltonian replica exchange in gromacs 4.5.7 software package to conduct rest . The default hamiltonian replica exchange in gromacs is done through thermodynamic integration, and it suffers from a great performance loss if the potential function involving a large portion of the system is altered . Systems were simulated under periodic boundary conditions, at constant temperature and pressure . For temperature coupling, dppc and chol molecules we note here that in rest, each replica is simulated at 323 k and the heating of solute is done by reducing the solute rahman barostat at 1 atm was used and the pressure in the plane of the bilayer was coupled separately from the pressure normal to the bilayer . The temperature and pressure time constants of coupling were 0.1 and 2.0 ps, respectively . The spc / e model was used for water and the 43a1-s3 force field was used for dppc and chol . Settle was used to constrain water molecules and lincs was used to constrain all other bond lengths in the system . The sixth - order particle mesh ewald (pme) method was used for electrostatic interactions with a fourier spacing of 0.15 nm . The real space coulomb interactions and pair - list calculations were set to 1.0 nm . A 1.0/1.6 nm twin - range cutoff scheme was used for vdw interactions and the pair - list was updated every 10 steps . To study how molecules diffuse relative to one another, we calculated the relative diffusion coefficient dij between each pair of molecules i, j. the relative diffusion removes the contribution from collective motions of molecules to the diffusion and should give a better estimate of how fast the system samples various lateral configurations . We define the mean squared relative displacement between molecule i, j in a time interval t as1213where ri(t) is the position of molecule i at time t and the bracket means an average over different starting times t. then we linearly fit rij2(t) as a function of t for larger t . Dij was assigned as /4 (two - dimensions) the slope of the curve . We note here that in rest, the relative diffusion rate is an average over temperatures (due to the exchange among replicas, replicas jump in the temperature ladder); however, it should still provide a meaningful description of how fast the simulations sample bilayer lateral configurations in general . As mentioned in the methods session, the choice of solute for rest simulations is not absolute . The solute can generally be the part of the system whose dynamics we want to accelerate, while the solvent is the rest of the system . Since in this study we want to accelerate the dynamics of lipid bilayers, it is natural to choose both the dppc and chol as the solute . Before we conduct rest, it is always a good practice to test the system at the highest temperature that we want to simulate in rest . We found that when we simulated the dppc and chol at 600 k (tm), the chol moved out of the monolayers and formed a third layer sandwiched by the dppc bilayer . A snapshot of the system is shown in figure 1 . A possible explanation of this can be obtained by carefully examining chol molecular structures . Chol has a small hydrophilic alcohol headgroup and a bulky hydrophobic body . At low temperature, the hydrophilic interactions between the chol alcohol group and water favors aligning chol along the bilayer normal . When the effective temperature of chol increases, the entropic effect becomes dominant and chol will gain entropically by placing itself in the middle of the bilayer . On the other hand, dppc has a larger hydrophilic headgroup than chol; thus, even at 600 k, it still can anchor itself upright to form the bilayer . Snapshot of the system where both dopc and chol molecules are tempered at 600 k. water is colored blue . The oxygen atom in chol and the phosphate atom in dppc one way is to lower the highest temperature in rest to keep chol aligned with the bilayer normal . Another way is to use only dppc as solute and treat both chol and water as solvent . We want to note that we should be able to just conduct rest with both dppc and chol set to 600 k, even though chol moved out of the monolayers at this temperature . However, this would be very inefficient . Configurations similar to figure 1 obtained from high solute temperature replicas will have vanishingly small probabilities in the target temperature replica ensemble (t0 = 323 k) due to the detailed balance condition (eq 6). As a result neale et al . Has observed such phenomena in another hamiltonian replica exchange system . In this case, high temperature replicas will not enhance the sampling in the target temperature replica but consume computing time . In this work, we take the second approach in which only dppc is chosen as the tempered solute . Since this reduces the dofs in the solute, it further decreases the number of replicas required to span our temperature range . By choosing dppc as solute, we managed to heat dppc from 323 to 600 k with 12 replicas . The exchange rates between neighboring replicas are 25%, 22%, 24%, 26%, 27%, 22%, 23%, 22%, 22%, 24%, and 22% . In order to compare the efficiency between rest and t - remd, we estimate how many replicas we need in our system to conduct t - remd to maintain a 25% exchange rate between neighboring replicas . Figure 2 shows that if 12 replicas are used in t - remd, we can only cover the temperature ranging from 323 to 340 k. this would hardly accelerate the simulation as t - remd usually gains simulation efficiency by increasing the enthalpy barrier crossing rate in high temperature replicas . Comparatively, rest has a significant advantage over t - remd as we can heat the dppc to a much higher temperature with the same number of replicas . Number of replicas required to obtain a 25% exchange rate between neighboring replicas in rest and t - remd (temperature replica exchange). Below, we compare the sampling efficiency between rest and a single long md simulation . In our work, we ran rest for 60 ns and standard md for 400 ns . The equilibrium sampling of lipid bilayers, especially of bilayers with different components, depends on the ability to sample different lateral organizations . The faster the system can explore various lateral configurations, the quicker equilibrium will be reached . Therefore, the lateral diffusion coefficient plays a key role in determining the equilibrium rate . However, lipids usually move collectively in bilayers . This collective motion generally does not facilitate the sampling of various lateral configurations but contributes significantly to the lateral diffusion coefficient of each individual molecule . Therefore, we calculated the relative diffusion coefficient between every pairs of molecule as it removes the contribution from collective motion among lipids . Dppc) of relative diffusion coefficients are calculated from the md and rest simulations . It clearly indicates that in all molecular pair types, molecules in rest diffuse an order of magnitude faster than in standard md . Also, we observe that the diffusion between chol and chol is the fastest while the diffusion between dppc and dppc is the slowest . Dppc, on the other hand, has two acyl tails that are usually entangled with other dppc, which reduces the diffusion . It is also worth noting that even though we only increase the effective temperature of dppc, the diffusion of chol increases as well . By omitting the chol from the tempered solute, we further reduced dofs in the solute and therefore reduced the number of replicas required for the system . This also suggests that the efficiency of rest may be further optimized by carefully choosing the tempering solute, which is also pointed out in the original paper . We note that the total simulation time in rest (60 12 = 720 ns) is almost twice as much as the time in md (400 ns). However, considering an order of magnitude increase in the lipid diffusion, rest is quite efficient . Probability density of all pairwise relative diffusion coefficients between chol chol (c c), dppc chol (d c), and dppc dppc (d d) in md and rest simulations . The radial distribution function (rdf) quantitatively describes the lateral organization of molecules in a bilayer . We define the lateral distance between two molecules as the center of mass (com) distance between the molecules projected in the x y plane . As the coupling between separate monolayers is weak, the rdf calculated from the upper and lower monolayers should converge to the same distribution . Therefore, we can judge the convergence of a simulation by the rdf difference calculated from separate monolayers . Figure 4 and supporting information figures s1 and s2 show the rdfs among chol chol, chol the rdfs are calculated from separate monolayers (blue, upper monolayer; red, lower monolayer) from the rest and md simulations using different block sizes . In all cases chol rdfs (figure 4) for example, with a 10 ns block size, the rdfs in rest show reasonable convergence between separate monolayers (figure 4a c). However, this is not the case for the md simulation (figure 4d f). The rdf difference in md simulation in the last 10 ns block (figure 4f) is even larger than the difference in the first 10 ns block (figure 4d). We reason that as molecules diffuse slowly in the md simulation, the sampled lateral configurations are highly correlated in time; thus, a larger block size is required to obtain independent configurations . Therefore, we increased the time block size to 120 ns to calculate the rdf from the md simulation . Figure 4g i show that rdf converges better when the block size is increased . The converged rdf in md (figure 4i) has a very similar shape with the rdf obtained from rest (figure 4c). The blue / red line in each subplot represents the rdf calculated from the upper / lower monolayer, respectively . Supporting information figures s1 and s2 show the rdfs between chol - dppc and dppc dppc pairs, respectively . We observe that the rdf of dppc dppc does not converge as well as the rdf of chol chol or chol - dppc . Dppc pairs diffuse the slowest in both md and rest; therefore, we can expect that the rdf of dppc dppc takes the longest time to converge . To quantitative analyze the convergence rate, we define the rdf difference between separate monolayers (rdfdiff) as14where rdfu(i) and rdfl(i) are the value of the ith bin of rdfs from the upper and lower layer, respectively . Figure 5 shows the convergence of rdf as a function of time block size in rest and md simulations . Figure 5 shows that the rdf in rest converges an order of magnitude faster than in md . Difference between rdfs calculated from the upper and lower monolayers as a function of block size . Error bars are estimated from consecutive blocks with the same block size . In this session, we compare several bilayer structural properties calculated from the rest and md simulations . For rest, only the replica simulated with solute temperature at tm = 323 k is used for analysis, as it represents the system with the original hamiltonian . Normally, the area per lipid serves a good indicator on bilayer structural properties . However, since the bilayer we studied has both chol and dppc, we calculated the average area per molecule (aapm) instead . Aapm is defined as the projected area of the bilayer in the x y plane divided by the number of molecules in a monolayer . The aapm are 43.1 0.6 in rest and 42.8 0.4 in md . Another important bilayer property is the deuterium order parameter (scd) of the lipid acyl tails . The order parameter of a methylene at position i is defined as15where i is the angle between a c d vector of the ith methylene in an acyl chain and the normal of the bilayer (z axis). Our calculation suggests that rest and md simulations have similar bilayer properties, which is a good validation of the rest method . Deuterium order parameters \|scd\| of dppc palmitoyl chains at 323 k calculated from the rest and md simulations . Chol is well - known for its condensing effect on lipid bilayers composed of lipids with saturated acyl tails . It smooths the lipid liquid / gel phase transition to the lipid disordered / ordered phase transition . It is reported that the average value of \|scd\| for dppc at liquid ordered phase and disordered phase are 0.36 and 0.21, respectively.supporting information figure s3 shows the \|scd\| of dppc at different solute temperatures in rest . At high solute temperatures, the dppc acyl tails are more flexible and the \|scd\| indicates that the bilayer is in the liquid disordered phase . The range of sampled \|scd\| in rest suggests that rest works well even when the system experiences the liquid disordered / ordered phase transition . Based on the high resolution rdf obtained from rest, we calculate the total gibbs free energy g(r) and excess gibbs free energy g(r) profiles between chol chol, chol dppc, and dppc dppc . G(r) and g(r) are defined as16and17g(r) is the lateral radial distribution function (rdf), and r0 is the reference distance where we set g(r0) = 0 . G(r) is the contribution to the g(r) due to the jacobian or area effect in the two - dimensional space . This means that neither chol or dppc tends to aggregate at the 50% chol concentration . Chol gibbs free energy profile in dilute conditions and found that g(r) drops below zero in the range 1.0 <r <1.5 nm . Dppc g(r) is almost flat when r> 1.0 nm, suggesting a random distribution of dppc at large distance . Chol g(r), indicating preferential interacting locations for chol chol pairs . This supports some phenomenological models, such as the supperlattice model and umbrella model, which suggest long - range ordering for chol . However, the barriers between the free energy minimums are of the order of kt scale, suggesting that the ordering of cholesterols is sensitive to the temperature . As the derivative of g(r) is the mean force, the g(r) we obtained can be used as a reference for various coarse grained models for this system . Total free energy g(r) (solid line) and the excess free energy g(r) (dashed line) profiles between different molecular types as a function of the lateral molecular center of mass distance . A general guideline is to choose the part of the system for which we want to accelerate the dynamics as the solute . For example, if we study the lipid protein interactions and are interested in the affinity of different lipid components to the protein, we can temper the lipids to accelerate their diffusion . On the other hand, if we are interested in how the protein adapt its conformation to the bilayer environment, we can temper the protein instead . With a good choice of solute, rest can accelerate the dynamics of the system with fewer replicas (compare to t - remd). The sole purpose of the replicas with the scaled potentials (tm t0) is to sample configurations that are likely to occur in the target temperature ensemble (tm = t0). Therefore, if we scale the potential in such a way that the system samples configurations with low probability to populate at the target temperature ensemble (configurations such as figure 1, in our case), rest will be less efficient . However, no matter what the choice of the solute is, we always have one replica in rest that is simulated at the original unscaled potential . Choosing the solute that makes rest efficient may require an intuitive trial and error approach . In the past usually, coarse grained systems have fewer degrees of freedom than their atomic counterparts, which results in smoother free energy landscape and faster lipid diffusion . Another method, developed by tajkhorshid and co - workers, is a membrane mimetic model, which separates the lipid headgroup from its hydrophobic tails . This method facilitates headgroup diffusion while maintaining a hydrophobic core and has been used to study the insertion of peripheral proteins . Wang et al . Also developed a method based on the accelerated molecular dynamics (amd) method, which accelerates lipid diffusion by adding a boost potential to the original system . Rest provides an efficient way of accelerating the equilibrium of lipid bilayer systems while simulating at least one copy of an unperturbed potential and maintaining atomistic details . In this work, we applied replica exchange with solute tempering (rest) to a cholesterol dppc bilayer system . In rest, part of the system is chosen as solute and the solute solute and solute solvent interactions are scaled such that thermodynamically, the solute is effectively sampling at a different temperature . We found that choosing both cholesterols and dppcs as solute is not efficient because most of the cholesterols moved out of the monolayers to form a third layer at high temperature . Therefore, we chose to temper the dppc molecules only . Since the number of replicas for rest only scales with the degrees of freedom in the solute, we managed to use 12 replicas to sample dppc at temperature ranging from 323 to 600 k, which, otherwise, would require 100 replicas in the traditional temperature replica exchange molecular dynamics (t - remd). The relative diffusion coefficients between all molecular pair types (chol chol, chol dppc) in rest are, on average, an order of magnitude larger than in standard md simulation, indicating a better sampling of lateral structures in rest . Dppc, and dppc dppc radial distribution function (rdf) between separate monolayers in the rest and md simulations . Since the coupling between monolayers is weak, the rdf should converge to the same distribution from different monolayers . Our results show that the rdf converges much faster in the rest than in the md simulation . Bilayer structural properties such as average area per molecules and deuterium order parameters are similar between the rest and md simulations . Finally, we obtained the lateral free energy profile between different molecular types from rest, which could be used as a reference to coarse - grained models of the system . Chol lateral free energy has several local minima and shows a long - range ordering, but the free energy barriers between minima are on the kt scale, indicating the ordering may be sensitive to the temperature . While we see a significant advantage of using rest to accelerate lateral equilibrium in mixed lipid bilayers, we believe that rest, or more generally, hamiltonian replica exchange will have broader applications . For example, the preferential interactions between different lipids and membrane proteins can be studied by tempering the lipids while leaving the protein and solvent at the target temperature . Also, combined with umbrella sampling, rest can be used to accelerate relaxation on degrees of freedom orthogonal to the reaction coordinate, which is reported as a hurdle in free energy calculations for lipid membranes.
Bartter syndrome (bs) is a rare inherited renal tubular disorder characterized by renal salt wasting, hypokalemic metabolic alkalosis and normotensive hyperreninemic hyperaldosteronism1, 2). Although bs is a typical tubular disorder, there have been several case reports of patients who developed focal segmental glomerulosclerosis (fsgs), a glomerular lesion, during the course of bs3 - 7). Such cases support a possible link between the two diseases, and indicate that stimulation of the renin - angiotensin system (ras) and increased angiotensin ii in response to chronic glomerular hyperfiltration due to salt - losing tubulopathy may lead to secondary fsgs3, 4, 8). In addition to fsgs, several other factors have been reported to influence the renal survival of patients with bs, including recurrent episodes of dehydration during early infancy, chronic hypokalemia, long - term use of non - steroidal anti - inf lammatory drugs, nephrocalcinosis, and mutations in the bsnd gene9 - 11). However, there have been only a few case reports of patients with bs who underwent renal transplantation6, 12 - 15). Here, we present a case of a patient with bs who developed fsgs and subsequently underwent renal transplantation due to aggravation of his renal function . We also review previously reported cases of both diseases and cases of individuals with bs who underwent renal transplantation . There were no perinatal problems including polyhydramnios, and the family history was unremarkable . At age 3 months, the patient visited a hospital due to recurrent febrile episodes and poor weight gain . The patient was diagnosed as bs, and potassium supplement and indomethacin medication were started . However, his compliance to the drug was very poor . Detailed clinical and laboratory data from that time are unavailable . At age 7, proteinuria was incidentally detected upon routine follow - up urinalysis . A renal ultrasonography revealed diffusely increased parenchymal echogenicity without definite evidence of nephrocalcinosis or nephrolithiasis . At age 8, a renal biopsy was conducted at the hospital due to persistent proteinuria (24-hour urine protein excretion 3,840 mg / day, serum albumin 3.4 g / dl, and serum creatinine 1.2 mg / dl). His proteinuria did not respond to 4 weeks of oral prednisolone (2 mg / kg / day) treatment . At age 10, the patient's height was 123 cm (<3rd percentile), weight was 36 kg (90 - 97th percentile), and blood pressure was 117/69 mmhg at the time of admission . The laboratory examination revealed a serum creatinine level of 3.1 mg / dl, a sodium level of 138 mmol / l, a potassium level of 3.7 mmol / l, a chloride level of 103 mmol / l, a bicarbonate level of 30 meq / l, a magnesium level of 2.0 / ml / hr (normal range 1 - 2.5), an aldosterone level of 558 pg / ml (normal range 50 - 194), and a spot urine calcium (mg) to creatinine (mg) ratio of 0.02 . Additionally, the patient's bone age was 6 years 5 months upon admission . Genetic analysis revealed a homozygous p.trp(tgg)610stop(tga) mutation in exon 16 on the clcnkb gene . His renal function deteriorated progressively for one year thereafter, at which point hemodialysis was started . Subsequently, the patient underwent renal transplantation successfully from a deceased donor at age 16 . The post - transplantation course has been uneventful for more than 3 years, with complete disappearance of bs without recurrence of fsgs . Clinically, bs can be classified into two variants, antenatal / neonatal bs and classic bs according to the onset age . Genetically, bs can be classified into at least 5 subtypes according to underlying mutant genes, all of which are expressed in the tubular epithelial cells of the thick ascending limb of the loop of henle . Bs type i is caused by loss - of - function mutations of slc12a1 encoding the apical sodium - potassium - chloride cotransporter (nkcc2), while bs type ii is caused by loss - of - function mutations of kcnj1 encoding the apical inwardly - rectifying potassium channel (romk), bs type iii is caused by loss - of - function mutations of clcnkb encoding the basolateral chloride channel (clc - kb), bs type iv is caused by loss - of - function mutations of bsnd encoding barttin, and bs type v is caused by gain - of - function mutations of casr encoding the basolateral calcium sensing receptor (casr)2). Bs is an inherited tubular disorder; however, our patient developed fsgs, an acquired glomerular lesion, later in the disease course . There have been five similar case reports of bs complicated by fsgs3 - 7) (table 1). In addition, there is an additional report of a patient with classic bs who developed end - stage renal disease due to glomerulopathy12). However, this patient was excluded from the review because the renal pathology was described as' diffuse' glomerulosclerosis . As shown in table 1, patients with bs and fsgs show development of fsgs independently of gender, clinical variants of bs or genetic subtypes of bs . One patient had nephrocalcinosis, and three patients had a history of long - term non - steroidal anti - inflammatory drug treatment prior to the onset of proteinuria . Three patients had progressed to chronic renal failure, but the rate of progression was variable . One other patient revealed mildly decreased renal function one year after diagnosis with fsgs5). Normal renal function was maintained in the remaining two patients, and, interestingly, proteinuria resolved completely in one of these patients after one year treatment with potassium supplements, spironolactone, and indomethacin3). The renal pathologic findings were described in detail in three cases, two of which had perihilar type fsgs . The perihilar variant is common in patients with secondary forms of fsgs mediated by glomerular hyperfiltration or other adaptation after loss of renal mass8, 16). These findings suggest that fsgs complicated by bs may be a secondary lesion due to adaptive response to chronic hyperfiltration by chronic salt - loss and resultant chronic stimulation of the renin - angiotensin system . Several case reports of gitelman syndrome complicated by fsgs support this explanation17, 18). Case reports of patients with bs who underwent renal transplantation are also rare, and the causes of esrd include progression of fsgs or other causes including complication of long - term non - steroidal anti - inflammatory drug treatment6, 12 - 15). In one of the patients, pre - emptive bilateral native nephrectomies and renal transplantation were conducted prior to the onset of esrd due to severe, clinically brittle, neonatal bs15). In all of the cases, transplantation was successful and bs disappeared completely after transplantation as shown in our patient . In summary, bs may be complicated by fsgs due to adaptive response to chronic salt - losing nephropathy, and fsgs may cause esrd in some patients.
Exploiting the incretin effect [1, 2], glucagon - like peptide-1 (glp-1) analogues are primarily utilised for their glucose lowering abilities through stimulation of insulin in a glucose - dependent manner [3, 4]. Nevertheless, glp-1 analogues provide additional benefits which include the control of glucose excursions by attenuating the rate of gastric emptying, inhibition of glucagon secretion, and promotion of weight loss by augmenting the sensation of satiety [5, 6]. Most promisingly, glp-1 analogues promote the proliferation, survival, and neogenesis of pancreatic -cells, a facet not shared by existing antihyperglycaemic agents [79]. With such beneficial properties, glp-1 analogues are commonplace in the glycaemic control of patients with type 2 diabetes mellitus (t2 dm) and presently four drugs within this class are licensed for use in t2 dm: exenatide (byetta), liraglutide (victoza), lixisenatide (lyxumia), and prolonged - release exenatide (bydureon). As established by the national institute of health and care excellence (nice), glp-1 analogues are utilised as a third - line therapy when both first - line metformin and second - line sulfonylureas fail to provide adequate glycaemic control despite appropriate diet and exercise interventions . To date, several clinical trials have demonstrated their efficacious ability to induce weight loss and improve glycaemic control in type 2 diabetic patients [1424]. Nonetheless, within such studies, glp-1 analogues also fail in a cohort of individuals due to adverse effects associated with these agents (primary failure) or by not achieving the defined end - goal (secondary failure). Although the definition of primary failure is standardised between healthcare institutes, different healthcare systems adhere to differing end - goal definitions . Within the uk, nice define an individual who is said to have responded to therapy if their baseline weight and glycated haemoglobin (hba1c) have reduced by 3% and an 11 mmol / mol (1%), respectively, after six months of glp-1 analogue administration . If such criteria have been satisfied glp-1 analogue therapy is continued whereas if not the next appropriate therapy, typically insulin, is considered . Despite the required weight loss ensuring that glp-1 analogues are used cost - effectively, an 11 mmol / mol (1%) drop in hba1c is known to reduce the development of microvascular complications, such as diabetic retinopathy, nephropathy, and neuropathy, by 25% . However, limited information is currently available which delineates factors that predict whether individuals will respond or not to glp-1 analogues . At present, the few studies in this area of research have identified that baseline hba1c appears to be the strongest predictor of response to glp-1 analogues [2631]. Nevertheless, such studies are hindered by both small sample sizes and a lack of comparisons at multiple time points . Furthermore, identification of individuals unlikely to respond to glp-1 analogues early on would nullify the theoretical risk of exposing patients to a 6-month period of side - effects, ranging from nausea and vomiting to acute pancreatitis [1417], and the significant expense associated with glp-1 analogues without any clinical benefit . Therefore, the possibility of differentiating between responders and nonresponders to glp-1 analogues based upon patient characteristics and/or changes in anthropometric and metabolic parameters remains a topical subject amongst clinicians . Subsequently, the primary aim of our study was to determine whether we could identify, in a group of patients with t2 dm initiated on the glp-1 analogue exenatide, any predictors of response by identifying any differences between parameters in those that achieved the nice required reduction in hba1c (responders) and those that did not (nonresponders). Additionally, our secondary aim was to translate any identified predictors of glycaemic response into a quantifiable figure, whereby this figure could be used to distinguish between individuals who would then achieve the targeted hba1c reduction from those that would not . This retrospective observational study was registered and conducted at the countess of chester nhs foundation trust (coch), cheshire, united kingdom . Participants were identified following review of patients with diabetes mellitus who were initiated on a glp-1 analogue from 2008 to 2014 at the endocrinology department at the coch . Medical records for participants fulfilling the inclusion criteria were then accessed using the programme meditech (medical information technology, massachusetts). Data collected included the patient's age, gender, and the number of years diagnosed with t2 dm, concurrent diabetic medications, and the patient's weight, body mass index (bmi), and hba1c at baseline (date initiated on exenatide), 3 months, and 6 months after exenatide initiation . All variables explored in this study were included in the audit protocol, thereby conforming to the ethical standards and framework set by the coch . Patients were included if they met the following criteria: diagnosis of t2 dm made using the clinical criteria established by the nice, 18 years of age, initiated on exenatide, and required data available at baseline, 3 months, and 6 months after exenatide therapy . Exenatide was chosen as the sole glp-1 analogue under analysis due to it being the most prescribed glp-1 analogue at the coch and our experience with this agent in clinical practice . Additionally, prolonged - release exenatide was not considered due to the increased time period required for this drug to be efficacious; thus our defined time points would not necessarily reflect any glycaemic benefit . Patients were excluded if diagnosed with other subsets of diabetes mellitus (dm), such as type 1 diabetes mellitus (t1 dm) and gestational diabetes, exposure to previous glp-1 analogue treatment, prescribed glp-1 analogues that did not include exenatide, prescribed weight reducing medications alongside exenatide, and if complete data was not available at the aforementioned time points . Participants who satisfied the inclusion criteria were divided into two groups: responders and nonresponders . Responders refer to individuals whose baseline hba1c fell by the nice instigated 11 mmol / mol (1%) reduction after 6 months of exenatide use, whereas nonresponders categorise patients who failed to achieve this decrease . The additional 3% weight loss requirement also stated by nice was not included in the response definition, as the 25% reduction in microvascular risk associated with a 11 mmol / mol reduction in hba1c is, in our opinion, of greater clinical importance . Furthermore, we also categorised another group of participants as those with primary failure to exenatide treatment . This group included individuals that experienced side effects within the first two weeks of initiating exenatide resulting in the participant discontinuing further treatment . Results obtained from the study are presented as mean standard deviation (sd) or as percentages (%) for continuous and categorical variables, respectively . The significance of any differences between mean values obtained from both responders and nonresponders was determined using the independent student's t - test for continuous data and chi - square test or fisher's exact for categorical data; fisher's exact test was used if sample size within categories was less than 5 . To determine if any changes observed in weight, bmi, or hba1c over the three time points for the entire cohort, responder group, and nonresponder group were of statistical significance, repeated measures analysis of variance (rm - anova) was used . If statistical significance was observed, tukey's test was used to correct for multiple comparisons . In order to develop a model that could predict and define the relationship between statistically varying variables and attainment of the defined hba1c reduction, both linear regression and binary logistic regression analysis were used . Results were deemed statistically significant if the p value was <0.05 and all statistical analyses were conducted using the statistical software package graphpad prism 6 . 253 individuals were excluded on the basis of incomplete data (n = 206), prescribed a glp-1 analogue other than exenatide (n = 46; liraglutide = 31; lixisenatide = 7; prolonged - release exenatide = 8), or diagnosed with t1 dm (n = 1). This left 112 participants available for statistical analyses with 63 responding and 49 not responding to exenatide, as defined by a reduction 11 mmol / mol (1%) in hba1c from baseline to six months . None of the 112 participants reported any adverse effects and none reported any symptomatic hypoglycaemia . Initial comparisons of patient demographics between responders and nonresponders identified no significant differences in gender (percentage male), age of participants within the cohorts, and duration of t2 dm (table 1). Additionally, differences in medications prescribe alongside exenatide were similar between the two groups with the exception of the number prescribed exenatide with insulin . Nonresponders were shown to have a significantly greater proportion of participants on exenatide with insulin compared to responders (20.4 versus 3.17%, p = 0.0047). Weight for the entire cohort reduced from 113 17.9 kg at baseline to 109 18.1 kg at 3 months and 106 18.9 kg at 6 months (p <0.0001). Similarly, the bmi for the entire cohort reduced from 39.2 5.85 kg / m at baseline to 37.7 5.91 kg / m at 3 months and 36.8 6.31 kg / m at 6 months (p <0.0001). Hba1c across the study period reduced from a baseline value of 80.9 14.6 mmol / mol to 70.4 13.4 mmol / mol by 3 months and 67.8 14.9 mmol / mol at 6 months (p <0.0001). Analysis of these changes between time points showed it to be statistically significant (p <0.0001). For both responders and nonresponders, there was a significant reduction in both weight and bmi from baseline to six months (p <0.001) (table 2). Although responders had a higher baseline weight compared to nonresponders (114 18.6 versus 111 16.3 kg) (39.4 5.63 versus 38.4 5.7 kg / m), both groups achieved the same mean weight by 3 months (106 20.6 versus 106 16.8 kg) and responders had a lower bmi by 6 months compared to nonresponders (36.5 6.57 versus 37.1 6 kg / m) (table 2). Comparisons in the hba1c change within responders identified a reduction from 85.1 15.4 mmol / mol at baseline to 67.8 13.4 mmol / mol by 3 months and 60.7 12.2 mmol / mol by 6 months (p <0.001). However, nonresponders demonstrated an initial reduction in hba1c by 3 months (73.7 12.9 mmol / mol) from baseline (75.5 11.4 mmol / mol), yet by 6 months the hba1c had risen to a level greater than baseline (77 13.2 mmol / mol). Although the overall change in the nonresponders was shown to be significant (p = 0.028), following correction for multiple comparisons, it was observed that the only statistically significant difference was the elevation in hba1c from 3 months to 6 months (p = 0.032) (table 2). Analysis of weight between responders and nonresponders identified no significant differences between the two cohorts at baseline (114 18.6 versus 111 16.3 kg, p = 0.54), 3 months (109 19.1 versus 108 16.9 kg, p = 0.86), and 6 months (106 20.6 versus 106 16.8 kg, p = 0.83) (figure 1(a)). Similarly, any differences in the bmi between responders and nonresponders at baseline (39.4 5.63 versus 38.4 5.7 kg / m), 3 months (37.6 5.91 versus 37.7 5.97 kg / m), and 6 months (36.5 6.57 versus 37.1 6 kg / m) were also not significant with p values of 0.75, 0.92, and 0.61, respectively (figure 1(b)). However, the hba1c recorded for responders and nonresponders was shown to significantly differ at baseline (85.1 15.4 versus 75.5 11.4 mmol / mol, p <0.001), 3 months (67.8 13.4 versus 73.7 12.9 mmol / mol, p = 0.021), and 6 months (60.7 12.2 versus 77 13.2 mmol / mol, p <0.001) (figure 1(c)). Linear regression analysis identified a significant relationship between the baseline hba1c and changes in hba1c from baseline to 6 months (p <0.0001), with the x - intercept at 60.3 14 mmol / mol (figure 2). Therefore, individuals with a baseline hba1c <60.3 mmol / mol were not likely to respond, yet those with a baseline hba1c 60.3 mmol / mol were more likely to respond to exenatide therapy over the 6-month period . Additionally, those with a higher baseline hba1c experienced a greater relative and absolute reduction in their hba1c over the study period . No significant relationship was observed for any other variable (p> 0.05). Of the several variables included within the binary logistic regression, only baseline hba1c predicted response as defined by a reduction in 11 mmol / mol after 6 months of exenatide therapy (table 3). Our model demonstrates that individuals with a higher baseline hba1c have a 5% increased odds of being classed as responders to exenatide by 6 months (p = 0.004). Although our model identified exenatide with insulin therapy as increasing the odds of responding compared to exenatide alone, the wide confidence interval range makes this variable unlikely to predict response to exenatide therapy . In order to determine the change in hba1c required for response to exenatide therapy, we quantified the percentage difference in hba1c from baseline to 3 months against the proportion of participants responding and not responding to exenatide by 6 months (table 4). Comparisons within categories revealed that a reduction of 1520% in hba1c by 3 months compared to baseline was the first statistically significant difference that resulted in a greater proportion of responders compared to nonresponders (p = 0.033). Therefore, this would indicate that if the patient's baseline hba1c decreases within a range of 1520% (or more) by 3 months, they are more likely to achieve the 11 mmol / mol reduction in hba1c by 6 months . Furthermore, a reduction in the hba1c from baseline between 0 and 5% resulted in the first significant difference that resulted in a greater proportion of nonresponders compared to responders . Subsequently, those who have a reduction in hba1c between 0 and 5% (or less) by 3 months are more likely to not reach the 11 mmol / mol reduction in hba1c by 6 months . The clinical benefits of exenatide as a monotherapy or in combination with either existing oral antihyperglycaemic agents (ohas) and/or insulin have been extensively documented elsewhere [1417]. Our study reflects similar improvements as exemplified by the significant reduction observed overall in the mean hba1c, weight, and bmi across the entire cohort of participants . Therefore, our study reinforces the utilisation of exenatide as a glp-1 analogue in the management of t2 dm and additionally emphasises the efficacious properties of this glp-1 analogue with regard to improvements in both weight and glycaemic control . However, within the aforementioned studies there is a significant proportion of the initial cohort that either experience side - effects preventing them from continuing exenatide (primary failure) or fail on the basis of not achieving the defined end - point (secondary failure). Our study reflects this phenomenon as demonstrated by 18.8% of the initial cohort discontinuing therapy following primary failure due to intolerable nausea and vomiting . Additionally, of the participants that did complete 6 months of exenatide therapy, 43.8% failed to achieve the required 11 mmol / mol reduction in hba1c stipulated by nice . Such observations reflect those reported by the limited literature presently available with studies reporting primary failure rates within the range of 11.433.6%, in addition to secondary failure rates ranging from 39 to 61% [26, 27, 2931]. Although the rates of both primary and secondary failure rates observed within our study lie within such ranges, it reiterates the clinical significance and need of identifying measures which can delineate whether an individual will respond or not respond to exenatide therapy . By doing so, those distinguished as unlikely to benefit from exenatide therapy can be initiated on the next most suitable therapeutic adjunct appropriate for managing their type 2 diabetes, thus reducing the cost and exposure to side effects associated with exenatide with no clinical benefit . Within our study, levels of hba1c were the only parameter to significantly differ between responders and nonresponders with our regression analysis identifying that higher levels of hba1c at baseline were associated with greater reductions in hba1c over the study period . Such findings have been reported in the majority of the literature within this field [26, 27, 30, 31]; however, song et al . Also noted significant correlations between changes in hba1c and the patient's age, levels of serum fasting glucose, and parameters that measure pancreatic- cell function such as homeostatic model assessment- (homa-), levels of insulin at baseline, and both basal and stimulated c - peptide levels . As we were unable to measure variables of insulin secretion, we cannot ascertain whether such a relationship in the changes in hba1c and these parameters would have existed in our study . However, with regard to age, no other study has identified such a relationship and despite the study by shin et al . Also including cohorts of south korean type 2 diabetics only therefore, this association may simply be exclusive to the study sample used by song et al . . Additionally, our predictive model noted that baseline hba1c was the sole indicator of response to exenatide therapy with higher baseline values of hba1c corresponding to 5% greater odds of exenatide response by 6 months . Although several studies have reported additional parameters as predictors of response, the most reproducible variable has been baseline hba1c . However, within the existing literature the predictive role of baseline hba1c determining response to exenatide report that a higher baseline hba1c reduces the odds of response [27, 29], yet studies by anderson et al . And observed a greater likelihood of response with higher levels of baseline hba1c [26, 31]. Although results from our study correspond to the latter studies, we believe the differing predictive role of baseline hba1c observed by preumont et al . And anichini et al . Is a result of the definition used to determine response to exenatide . Both preumont et al . And defined response to exenatide as participants achieving an absolute hba1c value 58 mmol / mol (7.5%) and hba1c 53 mmol / mol (7%), respectively . However, in both our study and those conducted by anderson et al . And shin et al ., response to exenatide was defined by a relative loss in hba1c [26, 31]. Subsequently, the variations in the predictive value of baseline hba1c may be in part attributed to whether an absolute or a relative value was used as the response definition . One explanation for this is that despite individuals with a higher baseline hba1c reporting a greater relative reduction in their hba1c, it is more difficult for these individuals to reach the absolute hba1c response cut - off due to the large reduction required to reach that point . In contrast, those with a baseline hba1c that marginally differs from the hba1c response value are, despite having only a relatively small reduction in their hba1c, more likely to reach this end - point . We believe this policy of defining response based upon attainment of an absolute value should not be used to define response to exenatide, but rather a relative reduction . Studies such as the uk prospective diabetes study (ukpds) have shown that an 11 mmol / mol (1%) reduction in hba1c reduces the risk of microvascular complications by 25%; thus it would be clinically unwise to omit individuals with a high baseline hba1c from receiving exenatide despite knowing these are more likely to show a significant relative reduction overall . In our study we also identified patients with a baseline hba1c <60.3 mmol / mol (7.7%) as unlikely to respond to exenatide, whereas those with an hba1c at baseline 60.3 mmol / mol (7.7%) were likely to respond . Such findings have been reported by anderson et al . Which noted that individuals with a baseline hba1c <56.3 mmol / mol (7.3%) were less likely to respond to exenatide compared to those with a baseline hba1c 56.3 mmol / mol (7.3%). The significance of these observations correlates to the requirements for use of exenatide as defined by nice . Nice state that exenatide should be utilised when both first - line metformin and second - line sulphonylureas have failed, in addition to the patients' hba1c being 58.5 mmol / mol (7.5%). Subsequently, our results coincide with such criteria for exenatide use as patients with a baseline hba1c <60.3 mmol / mol had no response to exenatide; thus clinicians should avoid utilising exenatide in such scenarios as it only exposes patients to the possible side effects and the financial implications associated with exenatide with no improvement in glycaemic control . Although identifying individuals with a baseline hba1c value <60.3 mmol / mol (7.7%) served as one means of delineating nonresponse to exenatide therapy, it only accounted for 12.2% of the cohort that eventually failed to respond following 6 months of exenatide administration . Unlike the previous studies within this area, we are the only study to utilise three time - points, that is, baseline, 3 months, and 6 months . Therefore, analyses in the percentage change in hba1c at 3 months compared to baseline identified another measure of predicting response to exenatide therapy . In our study, which we believe is the first to report such an observation, a reduction in the baseline hba1c by 1520% (or greater) at the 3-month period resulted in a greater proportion of the cohort following on to be classed as responders at 6 months . Furthermore, individuals who have had an hba1c reduction between 0 and 5% (or less) at 3 months resulted in a significantly greater proportion being deemed as nonresponders by 6 months . At present, it is only at this point that clinicians are deemed appropriate to make a judgement regarding whether an individual is suitable to continue exenatide therapy and if not, an alternative therapy will then be commenced which will also be monitored to observe for any glycaemic benefit . This attitude within healthcare to periodically observe and in some situations fail to alter or adequately titrate upwards treatment strategies in the face of an uncontrolled disease is referred to as therapeutic inertia . In our study we identified that a significant proportion of the initial cohort could be identified as responders by the 3-month period simply by quantifying the percentage difference in hba1c from baseline . Therefore, in the clinical setting clinicians would only have to wait for 3 months to determine whether an individual was likely to respond, thus allowing for the rapid intensification or alteration of treatment if shown to be not working whilst also ameliorating a further three - month period of exposure to the theoretical side effects and cost attributed to exenatide . During the course of this study we encountered limitations which may have influenced our results . Firstly, we did not envisage such a large proportion of the identified sample to be excluded on the basis of incomplete data . This in turn led to a reduced sample size available for statistical analyses and thus may have resulted in fewer variables being identified as predictors of response . Secondly, all our participants were derived from one institute and so this puts into question whether our predictive model is applicable to other institutes . Furthermore, the patients we included within our study were identified from a cohort under the care of the endocrine department at the coch, thereby exposing our study to selection bias . As such patients in our clinics reflect individuals with difficult cases of t2 dm, and not the typical cases seen in primary care, inclusion of such cases m ay have skewed our results to reflect a predictive model only applicable for such treatment resistant cases . Another limitation of our study is that we did not measure adherence to exenatide during the study period . As exenatide is associated with both side effects and the possibility of stigma arising due to use of injections, patients may have not adhered to the agreed upon treatment plan . This limitation may be responsible for the significant rise in hba1c identified between 3 months and 6 months in the nonresponder group . Additionally, in our study we cannot ascertain whether the weight loss observed is a direct consequence of exenatide suppressing appetite or as a result of concurrent dietary and lifestyle interventions undertaken by the patient . Finally, despite identifying other medications prescribe alongside exenatide, we did record the initial dosage and whether medication regimens changed over the course of the study . Therefore, this may have meant that any observed changes may have been, in part, attributed to the additional medications given alongside exenatide and not exenatide alone . Although our study provides another dimension, applicable to the real world setting that clinicians should consider when determining if an individual will respond to exenatide, we believe additional research examining the other glp-1 analogues is warranted; there is currently only one study on predictors of response to liraglutide . Furthermore, it would be of clinical relevance to determine if the predictors of response to exenatide differ depending upon inclusion of the 3% weight to our existing definition of response to exenatide . Patients with t2 dm represent a diverse heterogeneous population with varying demographic, anthropometric, and metabolic characteristics . Despite exenatide being shown to be efficacious, there remains a subset of individuals who fail to respond . Our results reinforces what has been documented previously with regard to the negative linear relationship between baseline hba1c and changes in hba1c but additionally sheds further insight into the conflicting nature of baseline hba1c as a predictor of response . Furthermore, our study provides a novel insight into the possibility of using the percentage change in hba1c observed at 3 months of treatment to predict response by 6 months . We hope that our findings reinvigorate this field of research as with supplementary research we can ultimately develop a predictive model which both outlines and quantifies a series of predictor variables and changes allowing clinicians to identify, with a significant degree of confidence, individuals likely to respond to exenatide therapy.
To report a case of recurrent idiopathic frosted branch angiitis (fba) successfully treated with adalimumab . A 14-year - old otherwise healthy boy was referred to the uveitis clinic for bilateral panuveitis with diffuse retinal vascular sheathing and severe macular edema . Two years ago, a similar episode had been treated with oral prednisolone, however it was complicated by adverse psychiatric effects . The progressive course of the condition mandated considering other therapeutic measures; adalimumab was chosen based on its purported efficacy for treatment of childhood uveitis and a favorable safety profile . The patient responded dramatically to a single subcutaneous injection of adalimumab without any side effect during and after injection . To the best of our knowledge, this is the first case of idiopathic fba treated successfully with adalimumab without adjunctive steroid therapy . Frosted branch angiitis (fba) is a rare disease characterised by visual disturbance associated with anterior chamber and vitreous inflammation and severe sheathing of retinal vessels resembling the appearance of frosted branches of a tree . It was originally described in a six - year - old child by ito in 1976.1 fba can be associated with ocular and systemic diseases . Cytomegalovirus retinitis, acquired immunodeficiency syndrome, and toxoplasma chorioretinitis are frequent ocular associations, while systemic lupus erythematosus, crohn's disease, behcet s disease, large cell lymphoma, and acute lymphoblastic leukemia have been described as systemic associations.2 the idiopathic type typically occurs in young and healthy persons and is associated with good recovery of vision . A 14-year - old boy, who was otherwise healthy, was referred to the uveitis clinic for progressive blurring of vision in both eyes since 1 month before . On examination, anterior segment examination revealed mild ciliary injection and anterior chamber reaction (2 + cells) but no keratic precipitates or posterior synechiae . Both eyes had inflammatory reaction in the vitreous (2 + cells) but there were no snow balls or snow bank formation . Fundus examination disclosed extensive retinal vascular sheathing and scattered intraretinal hemorrhages with severe macular edema in both eyes (fig . 1). Systemic work - up and investigations, including a complete blood count (cbc), erythrocyte sedimentation rate (esr), c - reactive protein (crp) titer, angiotensin converting enzyme (ace) titer, tuberculin skin test (tst), urine analysis and chest x - ray were unremarkable . Rheumatologic tests including antinuclear antibodies (ana), anti double strand dna antibody (anti ds - dna), rheumatic factor (rf), antiphospholipid antibody, anti - neutrophil cytoplasmic antibodies (ancas), and cryoglobulin assay were all negative . Blood cd4 and cd8 counts were within normal range and serology for the human immunodeficiency virus was also negative . Blood serology for possible infectious causes including herpes family viruses, i.e. Herpes simplex virus (hsv) type 1 and 2, varicella zoster virus (vzv), cytomegalovirus (cmv), and epstein - barr virus (ebv), hepatitis b and c viruses, treponema, borrelia, and toxoplasma were checked . Immunoglobulin g (igg) levels against hsv type 2 and cmv (but not igm) were elevated . We tested the aqueous specimen after an anterior chamber paracentesis to investigate the presence of deoxyribonucleic acid (dna) of hsv type 1 and cmv viruses . The result of polymerase chain reaction (pcr) on aqueous samples was negative for both viruses . Until the results of blood tests and aqueous pcr became ready, vision continued to deteriorate down to 20/1000 and 20/800 in the right and left eyes, respectively . Based on the inconclusive work - up a diagnosis of idiopathic fba the patient s parents mentioned that two years ago, for treatment of a similar condition, oral prednisolone 1 mg / kg had been commenced but soon after initiation, the patient developed severe mood changes which persisted long after discontinuation of treatment . The progressive nature of the condition mandated considering other therapeutic measures to prevent macular scarring . Due to the safety profile of anti - tumor necrosis factor (tnf) agents and their rapid onset of effect, adalimumab was considered as an alternative to conventional treatment for which the parents provided written informed consent . A single dose of 40 mg pre - filled adalimumab (humira, abbott laboratories, north chicago, il, usa) was injected subcutaneously in the patient s right thigh . No local or systemic adverse effect was noted at the time of injection or later on . Three weeks after injection, visual acuity improved to 20/200 and 20/120 in the right and left eyes, respectively . Anterior chamber and vitreous reaction decreased to 1 + cell, and vascular sheathing and macular edema resolved dramatically . Repeat fluorescein angiography and optical coherence tomography (oct) confirmed the improvement (figs . 2 and 3). 10 weeks after injection, visual acuity improved to 20/40 and 20/30 in the right and left eyes, respectively . In both eyes, clinical improvement continued up to the last visit (6 months after the injection) when visual acuity reached 20/25 in both eyes with no sign of active inflammation . Fba is basically divided into three different subgroups.3 the first comprises patients affected by lymphoma and leukemia presenting with a frosted branch - like appearance in the fundus . The second group includes patients with associated autoimmune or viral disease which can present with fba in association with the underlying disease . The characteristic feature of this latter type is its occurrence in otherwise healthy young individuals with good recovery of vision . Clinically, there is severe sheathing of retinal vessels appearing like the frosted branches of a tree, and acute visual disturbance associated with anterior chamber and vitreous inflammation; fluorescein angiography shows leakage from sheathed vessels without signs of occlusion or stasis.4,5 systemic steroids are the main and only suggested treatment for this condition . In our patient however, prior adverse psychiatric effects of steroid treatment encouraged us to employ another therapeutic option: adalimumab, a fully humanized monoclonal anti - tnf antibody, was chosen based on its suggested effect for treatment of refractory childhood uveitis including juvenile idiopathic arthritis.6 - 8 our patient responded dramatically to a single subcutaneous injection of adalimumab . The improvement was rapid and long lasting and the patient did not report any side effect during follow up . Review of the literature revealed no other therapeutic option for treatment of idiopathic fba except systemic steroids . To our best knowledge, this is the first case of idiopathic fba treated successfully with an anti - tnf agent without adjunctive steroid therapy.
From december 2007 to may 2013, 59 h. pylori - infected patients with two previous eradication failures were enrolled for this study prospectively . The study design was based on a single - centered, randomized, open - label, and controlled clinical trial . The study protocol was approved by the institutional review board at yongin severance hospital and confirmed to the ethical guidelines of the declaration of helsinki, 1964, as revised in 2004 . The requirement for informed consent was waived, and all subjects signed written informed consent . The intended sample of 55 recruited subjects (32 patients for group a and 27 patients for group b) provided a power of approximately 90%, assuming a significance level <.01 . The dropout rate was expected to be <10%, based on the documented good tolerability of drugs . All patients received first - line eradication therapies (lansoprazole 30 mg bid, clarithromycin 500 mg bid, amoxicillin 1 g bid) for 7 days and received second - line eradication therapy (lansoprazole 30 mg bid, tripotassium - dicitrato - bismuthate 600 mg bid, metronidazole 500 mg tid, tetracycline 500 mg qid) for 7 days . The eligible patients were randomly assigned to either group a who received lansoprazole 30 mg bid, amoxicillin 1.0 g tid, and rifabutin 150 mg bid for 1 week or group b who received lansoprazole 60 mg bid, amoxicillin 1.0 g tid, and rifabutin 150 mg bid for 7 days . H. pylori infection was defined a positivity according to at least one of the following two tests: a positive rapid urease test (clo test delta west, bently, australia) using a specimen from antrum or histologic evidence of h. pylori in any of two specimen taken from corpus by hematoxylin and eosin stain . The eradication of h. pylori was assessed with c - urea breath test 4 weeks after the therapy . Exclusion criteria included the patients with the coexistence of serious concomitant illness 8 (e.g., liver cirrhosis with decompensation, and uremia), pregnant women, allergic history to the medications used and patients with previous gastric surgery antibiotics, bismuth and ppi within the previous 2 weeks and nonsteroidal anti - inflammatory drugs within the previous 4 weeks . Compliance with therapy was defined as intake of 80% more of the prescribed study medication . In addition, the patients were instructed to contact the study site immediately in case of any severe adverse event . From december 2007 to may 2013, 59 h. pylori - infected patients with two previous eradication failures were enrolled for this study prospectively . The study design was based on a single - centered, randomized, open - label, and controlled clinical trial . The study protocol was approved by the institutional review board at yongin severance hospital and confirmed to the ethical guidelines of the declaration of helsinki, 1964, as revised in 2004 . The requirement for informed consent was waived, and all subjects signed written informed consent . The intended sample of 55 recruited subjects (32 patients for group a and 27 patients for group b) provided a power of approximately 90%, assuming a significance level <.01 . The dropout rate was expected to be <10%, based on the documented good tolerability of drugs . All patients received first - line eradication therapies (lansoprazole 30 mg bid, clarithromycin 500 mg bid, amoxicillin 1 g bid) for 7 days and received second - line eradication therapy (lansoprazole 30 mg bid, tripotassium - dicitrato - bismuthate 600 mg bid, metronidazole 500 mg tid, tetracycline 500 mg qid) for 7 days . The eligible patients were randomly assigned to either group a who received lansoprazole 30 mg bid, amoxicillin 1.0 g tid, and rifabutin 150 mg bid for 1 week or group b who received lansoprazole 60 mg bid, amoxicillin 1.0 g tid, and rifabutin 150 mg bid for 7 days . H. pylori infection was defined a positivity according to at least one of the following two tests: a positive rapid urease test (clo test delta west, bently, australia) using a specimen from antrum or histologic evidence of h. pylori in any of two specimen taken from corpus by hematoxylin and eosin stain . The eradication of h. pylori was assessed with c - urea breath test 4 weeks after the therapy . Exclusion criteria included the patients with the coexistence of serious concomitant illness 8 (e.g., liver cirrhosis with decompensation, and uremia), pregnant women, allergic history to the medications used and patients with previous gastric surgery antibiotics, bismuth and ppi within the previous 2 weeks and nonsteroidal anti - inflammatory drugs within the previous 4 weeks . Compliance with therapy was defined as intake of 80% more of the prescribed study medication . In addition, the patients were instructed to contact the study site immediately in case of any severe adverse event . Patients who had not taken antibiotics or antisecretary drugs were fasted for 6 hours before performing the ub t - test (heliprobetm; noster system ab, stockholm, sweden). The gelatin capsule containing c - urea was swallowed with water by the patient to avoid contamination with oral bacteria . Patients were instructed to exhale into the breathcard, which was inserted into the heliprobe (noster system ab, stockholm, sweden) instrument . The result was considered> 50 positive is based on clinical trials using the 1 curie 14c - ubt . The eradication rates and their 95% confidence intervals (ci) at both intention - to - treat (itt) and per - protocol (pp) analyses were calculated for each treatment regimen . For all other variables, fisher's exact test and t - test were used as appropriate, and p values <.05 were considered significant . This study was approved by ethics committee of our institution (yongin severance hospital, yonsei university, republic of korea). Chicago, il, u.s.a). The eradication rates and their 95% confidence intervals (ci) at both intention - to - treat (itt) and per - protocol (pp) analyses were calculated for each treatment regimen . For all other variables, fisher's exact test and t - test were used as appropriate, and p values <.05 were considered significant . The difference between the proportions eradicated was estimated . Before pooling these estimates, this study was approved by ethics committee of our institution (yongin severance hospital, yonsei university, republic of korea). From december 2007 to may 2013, a total of 59 patients were enrolled in the study (mean age, 55.3 years, range, 3474 years). They took as first - line therapy with ppi, clarithromycin, and amoxicillin and as second - line therapy with quadruple therapy consisting of a ppi, a bismuth salt, metronidazole, and tetracycline . Group a had 32 patients with lansoprazole 30 mg bid including rifabutin 150 mg bid daily and amoxicillin 1 g tid for 7 days . Group b had 27 patients with lansoprazole 60 mg bid including rifabutin 150 mg bid (daily) and amoxicillin 1 g tid for 7 days (fig.1). There were 25 males (mean age 58.8 years) and 34 females (mean age 52.9 years). Indications for eradication therapy mainly included peptic ulcer disease (32/58) and nonulcer disease (27/58). A history of smoking and alcohol use was present in 9 (15.5%) and 12 (20.7%) patients, respectively . There were no statistically significant differences between the two groups in terms of demographic characteristics, history of smoking and alcohol, and indication for eradication therapy . One patient in group a was not enrolled for pp analysis due to adverse events such as abdominal pain and red - colored urine and one patient in group b due to loss of follow - up; 56 patients were fully compliant with the treatment taking more than 80% of the prescribed tablets . In group a, h. pylori eradication was achieved in 25 (78.1%) of the 32 patients in the itt analysis and in 25 (80.6%) of the 31 patients in the pp analysis . In group b, h. pylori eradication was achieved in 26 (96.3%) of the 27 patients in the itt analysis and in 27 (100%) of the 27 patients in the pp analysis . There was statistically significant difference between the two groups in terms of the eradication rates in pp analysis (p = .047), whereas a marginally statistical significance was found in terms of the eradication rates in itt analysis (p = .051; table2). Clinical characteristics of patients eradication rates of helicobacter pylori the most common adverse events were epigastric pain in the three patients (9.3%) versus one patient (3.7%), epigastric discomfort in two patients (6.2%) versus, one patient (3.7%), and nausea in one patient (3.1%) versus one patient (3.7%), in the groups a and b. there was no statistically significant difference between the two groups in terms of adverse events (table3). Reported side effects were mild, and treatment was well tolerated . No major changes in physical examination or in standard laboratory parameters were observed after treatment . As drug compliance and tolerability were optimal in all patients, we searched for other factors possibly associated with treatment failure (table4). From december 2007 to may 2013, a total of 59 patients were enrolled in the study (mean age, 55.3 years, range, 3474 years). They took as first - line therapy with ppi, clarithromycin, and amoxicillin and as second - line therapy with quadruple therapy consisting of a ppi, a bismuth salt, metronidazole, and tetracycline . Group a had 32 patients with lansoprazole 30 mg bid including rifabutin 150 mg bid daily and amoxicillin 1 g tid for 7 days . Group b had 27 patients with lansoprazole 60 mg bid including rifabutin 150 mg bid (daily) and amoxicillin 1 g tid for 7 days (fig.1). There were 25 males (mean age 58.8 years) and 34 females (mean age 52.9 years). Indications for eradication therapy mainly included peptic ulcer disease (32/58) and nonulcer disease (27/58). A history of smoking and alcohol use was present in 9 (15.5%) and 12 (20.7%) patients, respectively . There were no statistically significant differences between the two groups in terms of demographic characteristics, history of smoking and alcohol, and indication for eradication therapy . One patient in group a was not enrolled for pp analysis due to adverse events such as abdominal pain and red - colored urine and one patient in group b due to loss of follow - up; 56 patients were fully compliant with the treatment taking more than 80% of the prescribed tablets . In group a, h. pylori eradication was achieved in 25 (78.1%) of the 32 patients in the itt analysis and in 25 (80.6%) of the 31 patients in the pp analysis . In group b, h. pylori eradication was achieved in 26 (96.3%) of the 27 patients in the itt analysis and in 27 (100%) of the 27 patients in the pp analysis . There was statistically significant difference between the two groups in terms of the eradication rates in pp analysis (p = .047), whereas a marginally statistical significance was found in terms of the eradication rates in itt analysis (p = .051; table2). Clinical characteristics of patients eradication rates of helicobacter pylori the most common adverse events were epigastric pain in the three patients (9.3%) versus one patient (3.7%), epigastric discomfort in two patients (6.2%) versus, one patient (3.7%), and nausea in one patient (3.1%) versus one patient (3.7%), in the groups a and b. there was no statistically significant difference between the two groups in terms of adverse events (table3). Reported side effects were mild, and treatment was well tolerated . No major changes in physical examination or in standard laboratory parameters were observed after treatment . As drug compliance and tolerability were optimal in all patients, we searched for other factors possibly associated with treatment failure (table4). Our study shows that as empirical third - line therapy, rifabutin - based high - dose ppi and amoxicillin combined therapy is more effective in patients with refractory h. pylori infection after two eradication failures with key antibiotics such as clarithromycin, metronidazole, and tetracycline previously prescribed . High - dose ppi and amoxicillin are recommended as one of several options for empirical rescue therapy for h. pylori infection in the absence of antimicrobial susceptibility testing . Antibiotics with the beta - lactam ring, such as amoxicillin, have little postantibiotic effects on the gram - negative rods 22 . The frequent dosing of amoxicillin is required to sustain the levels of amoxicillin higher than the mic level for a long time, which makes the bioavailability of amoxicillin enhanced in comparison with the twice - daily dosing from the point of view of pharmacology of antibiotics 23 . 24 reported in pp analysis, eradication rate of high - dose rabeprazole (40 mg trice) and amoxicillin 1.0 g trice for 14 days was 75% . Four studies from japan reported the efficacy of a second - line treatment with a ppi plus amoxicillin after the failure of the standard triple therapy 2325 . Surprisingly, three of these studies (106 patients) reported optimal results (eradication rates of 87, 91 and 100%), dosing 10 mg of rabeprazole and 500 mg of amoxicillin four times daily, for 14 days . The fourth study (63 patients), with a 58% eradication, administered 20 mg of rabeprazole plus 1 g of amoxicillin twice a day, for 14 days . The differences between these studies may be explained by the different dosage scheme, especially on the pharmacokinetics of these drugs . An oral dose of 500 mg of amoxicillin is rapidly absorbed, with a peak in plasma concentrations between 1 and 2 hours, and approximately a 75% is excreted between 6 and 8 hours after its administration . A dosage of 500 mg every 6 hours can probably maintain higher plasma dosages of amoxicillin than a dosage of 1000 mg twice daily 26 . The large multicenter surveillance studies have confirmed that resistance of h. pylori to amoxicillin is in the order of 1% 27,28 . In korea, primary amoxicillin resistance was seen in 2.2 - 5.6% (mean 2.3%, 8/350) 29,30 . Other clinical trials did not detect any pre- or post - treatment resistance to amoxicillin 31,32 . Also, it is known that the genotype of cyp2c19 is associated with the metabolism of the ppis . Not knowing whether the patients are extensive or poor metabolizers, or their cyp2c19 polymorphisms, higher ppi doses or newer ppi types might be more effective 3335 . Furthermore, acid inhibition allows h. pylori to reach its growth phase, rendering the bacteria more sensitive to antibiotics . The distribution frequencies of people with the homem genotype are 3040% in asian countries 36 . The usual doses ppis used in the standard regimen are insufficient for patients with the homem genotype, as well as that a higher dose of a ppi causes few adverse effects and is considered to be sufficiently safe 36 . The rifabutin - based high - dose ppi and amoxicillin combined therapy can resolve the problems of clarithromycin and metronidazole resistance and homozygous extensive metabolizers of cyp2c19 gene polymorphisms, the main reasons for eradication failure 37 . Rifabutin - based triple therapy was found to be highly effective and reliable as an alternative rescue therapy for the treatment of h. pylori infection after two failed eradication treatments . Showed a 12 days of half the dose of rifabutin (150 mg daily) combined with a high dose of pantoprazole (80 mg thrice daily) and amoxicillin (1 g or 1.5 g thrice daily) produced higher eradication rates of 91 and 97%, respectively, as a rescue therapy, suggesting that the treatment regimen is considered highly effective as a rescue therapy . In the presence of low - dose rifabutin (150 mg), combined frequent high dosing of ppi and amoxicillin for 12 days is effective for rescue therapy 38 . Our present open single - center study showed that high dose of ppi (lansoprazole 60 mg twice daily) and amoxicillin (1 g trice daily) combined rifabutin therapy achieved significant better eradication rate (100%) than standard dose of ppi (lansoprazole 30 mg twice daily) 80.6% in pp analysis (p = .047) after failures in two courses of previous h. pylori eradication therapy . Primary rifabutin resistance in h. pylori isolates is low, ranging from 1.3% to 2.4% . Unlike other antibiotics, rifabutin is chemically stable at a wide ph range and is not likely affected by inadequate acid suppression 39 . The combination of rifabutin with either metronidazole or amoxicillin shows additive effects 40 . For treating h. pylori infection, the length of treatment for the rifabutin regimen has controversies, as does the influence this has on the treatment outcome . A mean h. pylori eradication rate of 75% (95% ci from 68 to 83%) for the 7-day regimen was calculated, while the 10- to 14-day regimen was similar or even slightly lower (71%; 95% ci, 6379%) 21 . However, as previously reviewed, when a subanalysis was performed depending on the duration of the second - line rifabutin therapy, better results were observed with 1012 days (92%) than with 7 days (69%). Finally, therapies between 12 and 14 days have yielded results similar to the 10-day course and are likely to increase the incidence of adverse event 21 . Our data suggest that in the presence of 7 days relatively short duration, frequent dosing of amoxicillin and a high - dose ppi can improve eradication rate and reducing side effects . It has been suggested that rifabutin efficacy decreases with increasing number of failed previous therapies, perhaps due to patients who had failed at least two courses of eradication therapy and may have harbored h. pylori strains that were more difficult to eradicate 41 . Overall, mean h. pylori eradication rate (intention - to - treat analysis) with rifabutin - containing regimens (1008 patients) was 73% (6779%). Respective cure rates for second - line (223 patients), third - line (342 patients), and fourth-/fifth - line (95 patients) rifabutin therapies were 79% (6792%), 66% (5577%), and 70% (6079%), respectively 41 . Antimicrobial susceptibility testing of h. pyloriis was desirable before initiation of third - line therapy, although the culture - based antibiotic susceptibility testing for h. pylori is expensive, time - consuming, and not always available on a routine basis 42 . The sensitivity of a bacterial culture is not 100%, and therefore, the antimicrobial susceptibility cannot be obtained in all cases . Therefore, rifabutin (together with a ppi and amoxicillin) can be administered as a rescue treatment without the need for a prior antibiogram 13 . The mean rate of adverse effects to rifabutin treatment in h. pylori studies has been approximately 22% 21 . In our study, this incidence was reported to be somewhat lower (15.5%), but in most cases, symptoms were well tolerated and no side effects were considered serious . In our study, most frequent side effects were epigatric pain (7.0%), epigastric discomfort (5.2%), and nausea (3.5%). Overall, this complication is rare and is far more likely when high dose (600 mg / day) and prolonged duration therapy is used . However, myelotoxicity was not reported in most of the studies evaluating rifabutin for h. pylori infection . First, this drug is extremely expansive; second, severe leucopenia and thrombocytopenia can occur to a patient under treatment with rifabutin . Finally, there is some concern about widespread use of rifabutin, a member of class of established antimycobacterial drugs in patients with h. pylori infection 45 . Because multiresistant strains of mycobacterium tuberculosis have increased in number, indications for these drugs should be chosen very carefully to avoid further acceleration of development of resistance . Rifabutin should be used only as rescue therapy after amoxicillin, clarithromycin, metronidazole, tetracycline, and levofloxacin have failed to eradicate to patients who have experienced failure of h. pylori 42 . The main limitation of our study is that it was a single - centered study and that no antibiotic testing has been performed . In conclusion, rifabutin - based high - dose ppi - combined therapy as empirical rescue treatment is more effective than standard dose ppi - combined rifabutin - based therapy, safe and best tolerable in third - line therapy in the korean population . The key to successful rescue therapy with rifabutin amoxicillin ppi regimen may be to increase doses of ppi and amoxicillin.
Transesophageal echocardiogram (tee) has been introduced to the operating room over 30 years ago . We report a case of esophageal perforation caused by tee during an aortic valve replacement operation, treated successfully with a new endoscopic clip . To the best of our knowledge, this is the first case of post - tee esophageal perforation treated endoscopically with this new clip device . A 72-year - old female was admitted to the operating theater for aortic valve replacement . During the operation and in the first hours in the intensive care unit (icu) the patient had to undergo tee in order to assess postoperative left ventricle performance status . A deep 2 1.5 cm ulcer covered with blood thrombus was found approximately 2 cm above the gastroesophageal junction . The rest of the endoscopy was unremarkable . Within the next 48 h, thoracic and only a few blood clots were found in the area between the stomach and the left liver hilum, but no signs of mediastinitis or free intra - abdominal air . Four days later, as the patient became febrile, a second gastroscopy was performed . A 2 1.5 cm perforation was seen at the same site of ulcer with no bleeding (fig . Due to the patient's clinical condition and the size of the gap, an endoscopic intervention was decided . A new 12-mm clip (otsc; ovesco endoscopy, tbingen, germany) was engaged . The clip is made of nitinol and approximates large perforation margins like a surgical clamp . The edges of the perforation were approximated by using a specific endoscopic double grasping forceps and applying suction through the cap . Thus, the tissue was pulled into the cap and the clip was released by rotating the wheel attached to the shaft of the endoscope . A nasogastric levin tube was uneventfully left in the stomach under direct vision for long - term enteral feeding . Further, the patient was offered two intra - abdominal drainage catheters in the operating theater so that intra - abdominal air and blood clots be drained and high fever get under control . All intra - abdominal and peripheral blood cultures were sterile . The patient was kept on intravenous antibiotics, proton pump inhibitors and parenteral nutrition over the next 10 days followed by enteral feeding through the levin tube . Two weeks later she was transferred to the clinical ward, experiencing no dysphagia, and was discharged fully recovered 3 months after the operation . Tee has safely been applied in patients undergoing cardiac surgery and in icu departments for diagnostic purposes and monitoring over 30 years . Factors that may determine the way of intervention following esophageal perforation are age, clinical condition, location and size of the perforation, cost of intervention and elapsed time since the injury . The time since perforation has occurred is crucial . A diagnosis delayed for more than 36 h is associated with high mortality (50%), despite surgical intervention . Up to date, there have been no randomized controlled trials comparing surgical to endoscopic intervention regarding patient outcome . In our case, the tee performed in the operating theater and the icu led to esophageal perforation due to the patient's esophagus anatomy . The delayed diagnosis of perforation is attributed to the blood thrombus on top of the ulcer seen in the first endoscopy that behaved as a sealant . Our patient's serious clinical condition precluded major surgical intervention, and it was decided to provide her with endoscopic therapy . Additionally, due to the size of perforation and cost, a single clip that could seal the gap was very appealing . Last, by leaving this clip in place for a long time, longer than common clips, could possibly guarantee safe sealing of this perforation . According to clip manufacturers, the clip may remain in place for over 6 months and is magnetic resonance imaging - safe . Esophageal perforations closed endoscopically range from 3 to 25 mm in size . Due to the new device technology (twin grasper and the cap at the tip of endoscope), a single clip is enough and able to create a full - thickness closure of perforations up to 3 cm in diameter . This new clip provides a secure and successful esophageal perforation closure rate, up to 100% in perforations diagnosed within the first 24 h . According to this study, the clip remains in place for over 6 months and the mean repairing time of an esophageal perforation after successful multiple clipping is 18 days (interquartile range 626). Most importantly, chronicity of perforation (> 10 days) was the only independent predictive factor for successful perforation closure in univariate and multivariate analysis . However, esophageal perforation as a complication of this clip during patient intubation has been reported in up to 3% of patients, but probably this can be attributed to the learning curve . Indeed, in another study with 94 patients regarding the use of the otsc clip to narrow the pouch in patients who underwent gastric bypass surgery, no esophageal perforation occurred . In conclusion, although tee is a safe procedure, it may be complicated by esophageal perforation . We clearly showed and strongly suggest that esophageal perforation of 5 days can safely and successfully be treated endoscopically by applying this new otsc clip device.
Dentures are worn by around 20% of the population, yet if displaced they may enter the gastrointestinal or respiratory system, sometimes with grave consequences . Further understanding of the epidemiology, and possible consequences of such events could lead to more informed prescription of appropriate denture design, and hence reduce these risks . Tooth replacement is carried out to improve masticatory efficiency and aesthetics, reduce over - eruption and drifting of unopposed teeth, and to restore phonetics . Prosthodontics may be of the upper or lower jaw, may be removable or fixed, and may be made out of plastic, metal, ceramic or a combination . An appropriate prescription for tooth replacement takes into account many factors including patient s aesthetic requirements, amount of remaining alveolar bone, dental health of remaining teeth, strength of the gag reflex, and financial cost . Assessment of a patient s risk of aspirating or swallowing a particular denture is also an important part of this process . Once a denture passes posteriorly into the oropharynx, or down into the hypopharynx, it may either continue into the aerodigestive system or be expelled back to the oral cavity by the usual gag reflex . Any factor which inhibits the gag reflex could be reasonably considered to increase the risks of aspiration or swallowing of a denture . Such factors may include neuromuscular disorders such as stroke, multiple sclerosis or parkinson s disease . From the hypopharynx, the denture may pass into the larynx and then into the respiratory system, or it may enter the oesophagus . In the larynx, the vocal cords may prevent further ingress, or the denture may pass inferiorly to the carina . From there, the more direct path is into the right bronchus, and then, depending on size, into the bronchioles . In the oesophagus there are four well documented sites where foreign bodies may lodge: a) at the level of the cricoid cartilage, b) where the oesophagus is compressed by the aortic arch, c) compressed by the left main bronchus, d) at the lower oesophageal sphincter . The stomach presents few obstructions to the foreign body, though the small bowel progressively decreases in diameter up until the ileocecal valve . Different designs of dental prostheses may lodge in different areas, or may pass through the gastrointestinal tract altogether . Hooked on the mucosa of the aerodigestive tract, whilst small fixed dentures may pass out in stool . Different designs may also require varying surgical strategies for removal whilst short fixed dentures may be safely monitored as they traverse the intestines, larger dentures may require endoscopic removal, and those which have become attached to the mucosa may require open surgery . Dentures made entirely of acrylic present a particular challenge as they cannot be easily identified on plain x - rays, and therefore a ct or mri scan may be necessary to determine their position . There is clear potential for morbidity and even mortality from a swallowed or aspirated denture . Therefore it is important to minimise the risk of aspirating or swallowing dentures, and mitigate the potential damage . We review recent published literature in order to identify the epidemiology of patients and characteristics of swallowed and aspirated dentures, and propose strategies to minimise these risks . Protocol and registration a systematic review was carried out using the prisma 2009 guidelines (http://www.prisma-statement.org/prismastatement/). The following focus question was developed according to the population, intervention, comparison and outcome (pico) study design: what are the common design features, morbidities, therapeutic strategies and epidemiology of swallowed and aspirated dentures reported in case studies? Information sources and search the authors searched embase and medline, with date limits 1 january 2000 to 31 december 2015 inclusive, using the mesh terms this date range was chosen as it represented a pragmatic and contemporary sample since the widespread introduction of internet based journals . The remaining articles were screened by title and abstract by the primary author (sk). For papers where the full text could not be retrieved, types of publications the review included case studies and series published in the english language . Types of participants / population the patients in the case studies had all experienced an event of swallowing or aspirating a dental prosthesis . The case series all described at least one event of either swallowing or aspirating a dental prosthesis . Inclusion and exclusion criteria the full text of all case series of relevance was obtained for assessment against the following inclusion criteria: described at least one case of either swallowing or aspirating a dental prosthesis . The following exclusion criteria were applied to case series: case series in which data were not presented as a case by case series but were given as overall observations and averages . Data extraction and data items data were collected using a specially designed data extraction form on: 1) the circumstances of the event, 2) any patient factors identified in the case study which may have contributed to the event, 3) the presenting symptoms of the patient, 4) the design of the denture (material, clasps, maxillary or mandibular), 5) the anatomical position at which the denture got lodged, 6) whether the denture caused perforation of the viscus, 7) the procedure required to remove the impacted denture . Any available photos of the dental prostheses were also collected, and analysed for type of denture, presence of clasps, size of the denture, and material . Risk of bias assessment assessment of risk of bias was not undertaken, as the studies were descriptive case studies and were not subject to bias within themselves . Data were collected on a microsoft excel spreadsheet (microsoft, version 14.3), and analysed using statistical package (spss, ibm, version 9.2). Associations tested included whether the procedure used to retrieve the denture varied by presence of clasps, whether presence of clasps affected likelihood of spontaneously passing the denture, and thirdly whether dentures with clasps were more likely to cause perforation . 86 case reports and case series were reviewed [6 - 91]. In twelve of these the full text could not be retrieved but sufficient information was included in 11 of the abstracts to include in the final analysis . Two of the case series were large, including 47 and 15 patients, and these were not included in the final analysis because of the way the results were presented and the absence of information on denture design . Therefore 83 case reports and case series which included sufficient information on 91 separate swallowing and aspirating events were identified from 28 countries . From these all of the papers were case series and reports, and therefore the quality of evidence was rated as no risk of bias was identified in any of the studies (table 1). The mean age of patients was 55 (sd 16.9) years (range 15 - 93 years), with 74% male . There was no identifiable causative patient factor in 51 of the patients (56%), with no recording of this information in 5 (6%). Of the remaining 35 patients, eight had a stroke, seven had dementia, four had epilepsy, and the rest were split between alcohol or opiate intoxication, learning difficulties, schizophrenia, parkinson s disease and multiple sclerosis . The circumstance of swallowing or aspirating the denture was known in 53 (58%) situations . Of those that were recorded or known, 28 (31%) were whilst eating, 6 occurred during a general anaesthetic, 5 occurred during trauma (which ranged from a simple fall to a shotgun wound to the face), 4 were whilst sleeping and 4 were during seizures . The remainder occurred whilst intoxicated with alcohol or drugs, whilst fitting the denture or during a stroke . One letter (which was not included in the final analysis as it did not report on denture characteristics, demographics of the wearer, or retrieval method) reported a swallowed denture whilst diving into a swimming pool . The most common anatomical site for the denture to be lodged was the oesophagus, with 33 in the upper gastrointestinal tract (proximal oesophagus to stomach). No differentiation was made between the hypopharynx and larynx as most case reports did not differentiate . Nineteen lodged in the airways (trachea to lungs), seventeen in the middle and lower gastrointestinal tract (ligament of treitz to rectum) and seven passed through the gastrointestinal tract . In thirteen cases the patient had suffered from a perforation of the gastrointestinal tract, and in two cases a trachea - oesophageal fistula had formed . Endoscopic (n = 38 procedures, 42%) included flexible and rigid bronchoscopies, flexible and rigid oesophagoscopies, gastroscopy and colonoscopy . Open procedures (n = 33, 36%) included bowelresection and enterotomy (16), cervical oesophagectomy (8), transthoracic oesophagectomy and bronchotomy (4), tracheostomy (4), and hartmann s procedure . In three cases the denture was not retrieved due to death and due to the dentures were divided broadly into fixed (n = 18, 20%) or removable (n = 60, 67%). Thirty one (34%) were maxillary and 21 (23%) mandibular, and it was unclear in 39 (43%) cases . The fixed prostheses were mainly tooth supported bridges, between two and eleven units, with an average of four units . The most common material was acrylic (n = 24, 27%), followed by acrylic and metal (n = 17, 19%). There was no statistically significant relationship between denture design (clasped vs unclasped) and procedure used for retrieval (p = 0.13), or between denture design and perforation (p = 0.37), or between denture design and spontaneously passing a denture in stool (p = 0.55) (table 2). Swallowed and aspirated dentures are responsible for significant morbidity and occasional mortality . With an ageing population and a growing global middle class who can afford and desire tooth replacement, our systematic review examines the epidemiology of publications relating to this issue and identifies common design characteristics . Limitations of this study include the methodology- by only examining case reports we have missed all of the incidents that were not published . Straightforward situations have been missed in our systematic review, and what we have identified is in fact the tip of the iceberg. Therefore we are likely to have a selection bias for cases which fall out with the norm, be that in terms of the magnitude of intervention required, the time a denture was left undiscovered, or even the circumstances of the event . In the author s own experience, removing a denture endoscopically from the hypopharynx of a clinically stable patient who has some dysphagia and reports swallowing their denture is a relatively unremarkable presentation to the emergency department, and this is supported by bandopathy s case series, in which 43 dentures were removed endoscopically with only one requiring cervical oesophagectomy . However, by examining the reported cases over the past 15 years we are able to draw some limited conclusions on the seriousness of the problem, and design characteristics which these cases had in common . Firstly, a common theme running through many of the case reports was the diagnostic uncertainty involved in managing patients with swallowed acrylic dentures . When the patient had no recollection of the event, or was unable to express this to the clinicians, the diagnosis was often not made for days or even, in five patients [7 - 11], years . This could be easily remedied by making the acrylic used in denture production radio - opaque, or adding a metallic foil strip into the denture . This has been suggested by a number of authors, but has never been widely adopted . We conclude that by including radiopaque material in the acrylic, many of the cases of swallowed or aspirated dentures would have been resolved more quickly and with less morbidity . A second theme identified in the review was that there was no statistically significant difference in perforation rates or requirement for open surgery between clasped and unclasped dentures . It seems logical that metal clasps might act as a fish hook on the epithelium of the aerodigestive tract, and may therefore require surgical removal, and perforate the gastrointestinal tract . Whilst clasped dentures are radiopaque due to the metal components, they are difficult to remove endoscopically, and after initial attempts to do so patients often required a second visit to the operating theatre for an open procedure . However, in the literature we reviewed [6 - 91] there was no difference in rates of perforation and open surgery, suggesting that where clasps are required to provide better retention their benefits outweigh perceived risks of morbidity . A third conclusion is that a number of the dentures identified were damaged at the time of the event . Whilst some of these were broken during the incident (e. g. the acrylic fragments cause by the shotgun blast), most were damaged previously but still worn by the patients [14 - 18]. This highlights the importance of warning patients not to continue to wear dentures which have been damaged or are loose, the importance of adequate, understandable post insertion instructions and regular recall for denture maintenance . A fourth conclusion of interest was the number (56%) of people who did not have any identifiable predisposing factors to swallowing or aspirating a denture . One paper suggested that the method of eating and drinking could have an effect, with a high number of indian cases attributed to a particular method drinking (pouring liquids into the mouth from a height, to avoid lip contact with the container, seemed likely to dislodge the denture). Whilst dentists may be aware of the importance of providing a suitable dental prosthesis to patients with oropharyngeal incompetence secondary to epilepsy or stroke, the fact that so many of the cases identified involved patients with no risk factors should highlight that this is a problem which could affect anybody wearing a dental prosthesis . The diagnosis of swallowed or aspirated dentures is often difficult to make, and may be simplified by addition of radiopaque material to acrylic components . Secondly, damaged dentures should not be worn as they appear to be aspirated and swallowed more frequently . Thirdly, all patients, regardless of comorbidity, are at risk of aspirating a swallowing a denture, and fourthly that inclusion of clasps does not appear to statistically increase the morbidity caused by the denture . The development of a reporting system would assist in identification of cases, and identification of higher risk denture designs . Further research involving prospective study of large groups of patients after denture fitting, and guidelines in denture design which aim to minimise the risks and mitigate the damage caused by swallowed and aspirated dentures are required . The authors would like to thank the ear nose and throat department at aberdeen royal infirmary for suggesting the review, and miss frances parkinson for her help in organising the data.
Analysis of the mammalian transcriptome and transcriptional network in ex vivo cells requires technologies that provide a comprehensive and unbiased view of the tissue - specific promotome (the complete set of promoters) from small amounts of rna and the intron - exon structure of the transcripts associated with different transcription start sites (tsss), which are marked by a cap - site in most eukaryotic rna polymerase ii - derived rnas . Among sequencing - based techniques to measure gene expression, tag - based methods are common . They involve reading a short sequence of a transcript that is still long enough to be mapped onto the genome . We have used cap analysis gene expression (cage)13, a cap - trapping based method which allows for systematic 5 end profiling of capped rnas, for the first comprehensive single - base resolution maps of tss and promoters from human and mouse tissues4 and for deciphering transcriptional networks in the human leukemia cell line thp-15 . In contrast to classic gene models, the emerging view suggests that most genes have multiple tsss differing by multiple bases4 and driven by various core promoters and that newly capped 5 ends can also be created post - transcriptionaly6 . Transcription can be initiated by promoters that are broad in shape, often associated with cpg islands, or by sharp promoters, which are narrow in shape and are often associated with tata - boxes4 . These promoter structures have functional implications, being associated to tissue specificity, as for example sharp promoters are, different exon usages, translation initiation sites or classes of non - coding rnas (ncrnas). Within the locus of a coding gene, transcription can start within and downstream of the open reading frame such as for the non - coding rnas that can originate in genomic regions corresponding to the 3 ends of protein coding geness4 . Additionally, the capped transcriptome includes non - coding rnas that are associated with initiation and termination of transcription6,7 . However, there are outstanding problems that could not yet be addressed with the existing technologies . Cage requires a large quantity of starting material (~50g of total rna) precluding tss transcriptome analysis of small samples, such as homogeneous cells preparation after microdissection or samples derived from cellular sub - fractionation . Although cage identifies new promoters, determining their connection to either downstream known gene structures or to independent novel rnas is limited to low - throughput gene - by - gene validations . Rna shotgun sequencing approaches (rna - seq) have been unable to distinguish multiple 5 ends of a given gene, identifying only their most extreme boundaries at best . This constrains the functional annotation of promoters, from which accurate inference of transcriptional regulatory networks depends5 and limits the study of ncrnas overlapping known genes . Paired - end sequencing of full - length cdna, like the gis (gene identification signature) ditag approach8, allows for the determination of tss and termination sites in polyadenylated mrnas, but does not yield information on internal exons . Here we present nanocage and cagescan technologies, which provide a genome - wide profiling of tsss from small quantities of rna and link them to the anatomy of transcribed rnas . Nanocage was carried out with as little as 10 ng of total rna, the equivalent of the rna content of a thousand cells . Cagescan provided important insights on the complexity of the promotome - transcript structure, identifying among others, rnas that originate from a given tss but terminate in unrelated downstream genes . Our data also provide an estimate of rna types that populate the various cell compartments, suggesting a nuclear role for intron- and intergenic regions - derived rnas, as well as for retrotransposon elements and antisense rnas . The classic cage protocol consists of many biochemical processing steps2,9, whereas nanocage takes advantage of a peculiar property of reverse transcriptase, called template switching (ts) exploits the ability of the reverse - transcriptase to extend the cdna using the mrna s cap as a template: the resulting synthesized first strand cdna carries one to three c nucleotides that correspond to the cap structure11,12 . These cs hybridize to the ribo - g at the 3 end of a template switching oligonucleotide (fig . 1a). The reverse transcriptase extends cdna polymerization using the ts oligonucleotide as template, providing extra 3 sequence to the first - strand cdna (fig . Although ts has been observed for blunt dna / rna hybrids10, we show that its efficiency on capped rnas is far greater, therefore preferentially capturing capped, full - length transcripts (fig . 2). Ts does not require purification steps, and thus avoids loss of material . To target non - coding, non - polyadenylated rnas (poly - a) and rnas whose 3 end has been truncated during the isolation of specific cells from the tissue of interest, we developed conditions to allow random - priming of the reverse - transcription (rt) reaction in combination with 5 template - switching . Due to dna - dependent polymerase activity of the reverse - transcriptase, annealing of ts oligonucleotides and rt primers to each other generates small artefactual dna fragments that become the predominant pcr templates in subsequent steps, impairing libraries preparation (not shown). The prevalence of these artifacts has, so far, precluded the development of random primer - based cap - switch methods for whole transcriptome analysis with total rna . To overcome this problem method, in which the linkers at the 5 and 3 ends of the cdna carry similar (but not identical) complementary sequences (fig . 1b, supplementary fig . 1). Consequently, dna templates bearing the same ending sequences (such as non - oriented cdnas carrying two 5 or two 3 linkers) or small size templates (such as primer - derived artifacts) are less efficiently amplified during pcr . As a result, the majority of pcr products consist of long cdnas properly flanked by the adapter sequences present in the ts oligonucleotide and rt primer, as they are the most efficiently amplified templates (fig . 1b). Additionally, the removal of primer dimers by the semi - suppressive pcr enables the use of ts primer in concentrations as high as 10m . This maximizes the efficiency of template switching since the concentration of ts oligonucleotides is the reaction - limiting factor (data not shown). The ability to use random primers considerably extends the power of this technique since (i) poly - a transcripts constitute at least one third of the transcriptome13, (ii) long polyadenylated rnas are often damaged by ex vivo sample preparation, such as laser capture micro - dissection of fixed tissues and (iii) pcr of oligo - dt primed cdnas introduces strong size and representational biases regardless of potential rna degradation . Ts oligonucleotides and rt primers used in the nanocage protocol contain ecop15i restriction sites14 to systematically generate 25 bp fragments corresponding to the 5 end of the template - switched captured cdnas, thus producing nanocage tag libraries (fig . Although this enzymatic cleavage might be dispensable when reading short reads with several second - generation sequencers, the standardization of tag length overcomes biases during the second round pcr and simplifies dna molar quantification and sequencing . Additionally, ecop15i tagging allows the introduction of a dna sequence barcode at the 3 end (fig . 1c) and thus pooling of different libraries prior to their sequencing, resulting in dramatic cost savings15 . Cagescan was built upon nanocage, but modified to accommodate paired - end sequencing for tss determination at 5 ends coupled with 3 end sequencing of cdnas at random priming sites . Rather than cleaving the cdnas, in cagescan we added adapter sequences allowing for paired - end sequencing in the illumina genome analyzers16 (fig . Transcripts defined by their common 5 end, as obtained by sequencing of both the 5 end and the 3 end of the template - switched captured cdnas . Yet, unlike nanocage libraries that contain uniformly short sequences, cagescan libraries show a broader size range including fragments longer than 1 kb, which perform poorly on currently available second - generation sequencing platforms17 . This problem is minimized by exclusively using highly concentrated random primers and commercial reverse transcriptases, which show little strand displacement activity . Thus, cagescan sequencing templates are kept relatively short, regardless the length of the original mrna molecules . Due to the selectivity of the template switch for capped molecules, both protocols were used on total rna, without ribosomal rnas (rrna) depletion . Notably, the usage of 3 random primer allows the detection of non - coding, non - polyadenylated rnas13, which have been so far poorly characterized . In order to validate the reproducibility, the efficiency and the precision of nanocage, we prepared libraries from serially diluted total rna from cultured hepatocellular carcinoma cells (hep g2) and compared them to reference tss data . Two duplicate sets of nanocage libraries from 10, 50, 250 and 1,250 nanograms of total rna were synthesized and sequenced with an illumina genome analyzer . We clustered tss from all the libraries that were located less than 20 bp apart on the same genome strand4 . We then compared expression levels, measured as number of tags per promoter region in a given library, for each pair of replicates with the same quantity of rna . The pearson correlation coefficients between replicas were 0.97, 0.96, 0.97 and 0.99 for, respectively, 10, 50, 250 and 1,250 ng . Replicates sequencing depth were in some cases substantially different (supplementary table 1) and higher correlations were observed for deeper sequenced libraries (0.99 for 1250 ng replicas). We then pooled the tags into virtual libraries for each rna quantity, and compared them with each other . Pearson correlation coefficients varied between 0.987 to 0.999 (supplementary table 2), showing similar snapshots of the transcriptome with tiny quantities of starting total rna within a range of 10 to 1,250 ng . A similar template - switching approach has been used with fragmented, uncapped rna molecules19 showing that ts could also be used on uncapped 5 ends . However, this protocol required prior depletion of ribosomal - rna otherwise reverse - transcription of total rna with random hexamers yielded 9094% of ribosomal sequences20 in our hands, only 11% of hep g2 nanocage tags matched rrna sequences, showing an 8-fold reduction in non - capped rrna content . This demonstrates the strong preference of template - switching for capped over non - capped rnas . To prove efficient capture of the 5 ends of capped transcripts, we prepared nanocage libraries from 100 ng of decapped, fragmented or both decapped and fragmented total rna and analyzed the distribution of tags mapping to refseq transcript models21 . In a library prepared with untreated rna, 52% of the tags mapped to first exons or proximal promoters in refseq (defined as 500 bp to refseq tss) and 31% detected potentially new promoters in intergenic regions (fig . 2a, supplementary fig . 2 and supplementary table 3). We noted that tags mapping to internal exons and 3 utrs (15% in total) can also derive from genuinely capped transcripts co - localized within the boundaries of longer transcripts referenced by refseq4,6,9 . The proportion of tags matching the 5 end of known transcripts was halved to 23% after rna decapping . Furthermore, this number dropped to 8.5% upon rna fragmentation, suggesting that a large number of uncapped rna molecules is needed to compete with capped molecules . Upon combining decapping and fragmentation, the preferential capture of 5 ends was almost completely abolished, demonstrating that nanocage distinguishes capped ends from other 5 ends and preferentially captures the 5 end of capped transcripts . The semi - suppressive pcr did not impair the detection of relatively short transcripts, as we detected expression for 78% of refseq transcripts (23,512/29,996), including 44% (271/615) of the subset respect nanocage tags outperform the fantom3 dataset, in which only 5% (28/615) of the short refseq transcripts are detected (supplementary table 1). Furthermore, the ecop15i cleavage did not introduce any bias as we found the cagcag ecop151 restriction site in 81% of the detected transcripts for both the nanocage and the fantom3 libraries, which were made with a different restriction enzyme, mmei1 (see also supplementary fig . 3). To confirm the precision of template - switching in detecting tss we compared promoters identified by nanocage with those found by two methods that are using different protocols for cap selection and for which hep g2 libraries were available4,22: deep - race22 (rapid amplification of cdna ends), based on oligo - capping23, and cage, 4 . The deep - race data was limited to 18 different promoters in 17 loci22 . As exemplified with histone cluster 1, h3 (hist1h3c, fig . 2b), the main tss was the same between nanocage and deep - race or fantom3 cage data in four and seven cases respectively . The hist1h3c locus also exemplifies the ability of our random - primed approach to uncover tsss of non - polyadenylated transcripts (hist1h3c refseq model lacks any 5 utr information). When allowing only 4 bp discrepancy between the tss uncovered by each methodology, the results of nanocage were in agreement with deep - race and fantom3 cage for 11 out of 18 promoters (supplementary table 4) and for 17 of the 18 deep - race - validated tss respectively . Interestingly, the two alternative promoters of ppp2r4 uncovered by deep - race and cage were also detected by nanocage and their relative differential expression levels were consistent between all three approaches (data not shown). To extend this result we compared the location of all the promoters detected by the two genome - wide libraries, nanocage and cage . Although a large number of promoters are broad in size4, for 66% of the promoters the distance between tss detected by both techniques was less than 5 bp (supplementary fig . 4, supplementary table 5). Even for cells grown in culture, starting material becomes a limiting factor when cellular sub - compartments are selectively fractionated to explore specific rna content . As part of the encode project, attempts to produce cage libraries from nuclear rna subfractions of the k562 myelogenous leukemia cell line (the nucleolus, the nucleoplasm, the chromatin - bound rnas as well as from polysomal poly - a rna consisting mostly of rrna) were unsuccessful due to the paucity of mrna (not shown). Using nanocage, four libraries were synthesized and between 9.5 and 13.8 million tags were sequenced for each of them . Comparing to standard poly - a cage libraries, which were sequenced at the same depth, the complexity of detected 5 ends was consistent between the two technologies for each cellular compartment (supplementary fig . We have also found differences in tss specificity among different compartments (not shown). The functional significance of novel 5 ends is limited by the lack of information on the entire transcript . To better understand the structure of the transcripts associated with novel tss and to better characterize the differences between nuclear and cytoplasmic transcriptomes, cagescan libraries were prepared in technical duplicates from four different hep g2 cultures . In a first series of experiments, nuclear and cytoplasmic fractions were analyzed either as total rna or as a subfraction depleted of polyadenylated transcripts (poly - a) (supplementary table 1). Cagescan mate pair sequences were aligned against the human genome (ncbi build 36.1). The poly - a cytoplasmic, poly - a nuclear, total cytoplasmic and total nuclear fractions yielded together a total of 2,109,392 unique paired - end tags (supplementary table 6). Each of these associated a tss to a downstream sequence in a random location . Selecting mate pairs starting within 50 bp of refseq transcript s tss and using their intron - exon structure the resulting median length was 449 bp (1 and 3 quartile: 304 and 693 bp, supplementary fig . The median length of refseq transcript models is 2,422 bp (1 and 3 quartile: 1,509 and 3,799 bp), thus suggesting the majority of rna pol ii transcripts are competent to produce cagescan mate pairs, although cagescan is not optimal to map the 3 ends of transcript . Cagescan allows the association of tsss detected by cage to otherwise orphan intergenic, intron or 3 utr regions . Mate pairs were then annotated with respect to refseq transcript models21 complemented with a proximal promoter, defined as the region comprising the 500 bp directly upstream of their 5 end . This showed that an average of 4.24% of the transcripts were matching refseq transcripts, with 1.85% of them being strictly consistent with current gene models (that is starting within refseq promoter or 5 utr exon and ending within a refseq exon) while the rest was likely representing alternative mrna splice forms and non - coding rnas . . 3 and supplementary table 7). We observed specific differences between sub - cellular fractions and between the poly - a and total rna (dominated by poly - a molecules). Paired - end tags starting and ending within refseq promoters or 5 utrs were twice more prevalent than mate pairs ending within any refseq exons in the poly - a libraries than the total rna libraries . Those may correspond in part to promoter - associated long and short rnas (palrs and pasrs)7 . Nuclear fractions showed more paired - end tags starting and ending in intergenic and intronic regions than cytoplasmic fractions (fig . 3a). By preparing 12 additional cytoplasmic and nuclear cagescan libraries from 6 hepg2 independent biological replicas, we confirmed higher abundance of intronic (fig . 3b) and intergenic transcripts in nuclear fractions (p = 0.019 and p = 0.004 respectively, paired student s t - test). To better reconstruct transcript models, we grouped paired - end tags with overlapping 5 ends into cagescan clusters (fig . Alignment patterns of the corresponding 3 end tags on the genome recapitulate the potential structure of the transcript resulting from a common promoter . By pooling together the poly - a cytoplasmic, poly - a nuclear, total cytoplasmic and total nuclear fractions libraries, we obtained 854,849 distinct cagescan clusters, with an average of 2.47 reads per cluster . Clustered independently, the cytoplasmic libraries produced in technical duplicates yielded between 72,666 and 34,822 clusters (with 3.50.6 reads per cluster) and the corresponding nuclear libraries yielded between 309,682 and 147,963 clusters (with 1.60.3 reads per cluster). 9% of the cagescan clusters (all libraries pooled) started upstream of the translation initiation site of refseq; of them, 76.5% reached into their 3 utr or into their downsteam intergenic region, associating a promoter to a 3 utr for 11,131 refseq transcripts . Comparable ratios (9.5%2.65 and 80%11 respectively) were obtained when considering the four libraries separately (supplementary table 6). The region surrounding the ftl gene, for which the complete refseq model is tiled with paired - end sequences, illustrates such 5 mate pair driven clustering (fig . We observed subcellular compartment - specific antisense expression, with most of these antisense cagescan clusters ending in close proximity to the promoter of ftl (fig 4b and supplementary fig . A total of 37,818 clusters were antisense to 9,638 refseqs (supplementary table 6). Antisense rnas were generally more prevalent in the nuclear fractions . By aligning mate pairs to all exon - exon junction combinations of each transcript, we uncovered 8,462 splice junctions linked to 11,964 tss . Furthermore, clustering paired - end tags uncovered 1,569 cagescan clusters that initiated within the 5 utr of a given transcript, reached into the next downstream independent gene model in 1,198 pairs of distinct consecutive transcripts . Pervasive and regulated transcription of retrotransposon elements (re) have been observed in total rna extracts using cage24 . Using cagescan, we observed that expressed re are more abundant in the nuclear rna fractions than in the cytoplasmic ones . Globally, long and short interspersed nuclear elements (lines, sines) were the most highly expressed re in hepg2, followed by long terminal repeats (ltrs). All three were strongly over - represented in the nuclear poly - a fraction (fig . As expected srprna (signal recognition particle) repeats were enriched in the cytoplasmic compartment (fig . The classic cage protocol consists of many biochemical processing steps2,9, whereas nanocage takes advantage of a peculiar property of reverse transcriptase, called template switching (ts) exploits the ability of the reverse - transcriptase to extend the cdna using the mrna s cap as a template: the resulting synthesized first strand cdna carries one to three c nucleotides that correspond to the cap structure11,12 . These cs hybridize to the ribo - g at the 3 end of a template switching oligonucleotide (fig . 1a). The reverse transcriptase extends cdna polymerization using the ts oligonucleotide as template, providing extra 3 sequence to the first - strand cdna (fig . Although ts has been observed for blunt dna / rna hybrids10, we show that its efficiency on capped rnas is far greater, therefore preferentially capturing capped, full - length transcripts (fig . 2). Ts does not require purification steps, and thus avoids loss of material . To target non - coding, non - polyadenylated rnas (poly - a) and rnas whose 3 end has been truncated during the isolation of specific cells from the tissue of interest, we developed conditions to allow random - priming of the reverse - transcription (rt) reaction in combination with 5 template - switching . Due to dna - dependent polymerase activity of the reverse - transcriptase, annealing of ts oligonucleotides and rt primers to each other generates small artefactual dna fragments that become the predominant pcr templates in subsequent steps, impairing libraries preparation (not shown). The prevalence of these artifacts has, so far, precluded the development of random primer - based cap - switch methods for whole transcriptome analysis with total rna . To overcome this problem method, in which the linkers at the 5 and 3 ends of the cdna carry similar (but not identical) complementary sequences (fig . 1b, supplementary fig . 1). Consequently, dna templates bearing the same ending sequences (such as non - oriented cdnas carrying two 5 or two 3 linkers) or small size templates (such as primer - derived artifacts) are less efficiently amplified during pcr . As a result, the majority of pcr products consist of long cdnas properly flanked by the adapter sequences present in the ts oligonucleotide and rt primer, as they are the most efficiently amplified templates (fig . 1b). Additionally, the removal of primer dimers by the semi - suppressive pcr enables the use of ts primer in concentrations as high as 10m . This maximizes the efficiency of template switching since the concentration of ts oligonucleotides is the reaction - limiting factor (data not shown). The ability to use random primers considerably extends the power of this technique since (i) poly - a transcripts constitute at least one third of the transcriptome13, (ii) long polyadenylated rnas are often damaged by ex vivo sample preparation, such as laser capture micro - dissection of fixed tissues and (iii) pcr of oligo - dt primed cdnas introduces strong size and representational biases regardless of potential rna degradation . Ts oligonucleotides and rt primers used in the nanocage protocol contain ecop15i restriction sites14 to systematically generate 25 bp fragments corresponding to the 5 end of the template - switched captured cdnas, thus producing nanocage tag libraries (fig . Although this enzymatic cleavage might be dispensable when reading short reads with several second - generation sequencers, the standardization of tag length overcomes biases during the second round pcr and simplifies dna molar quantification and sequencing . Additionally, ecop15i tagging allows the introduction of a dna sequence barcode at the 3 end (fig . 1c) and thus pooling of different libraries prior to their sequencing, resulting in dramatic cost savings15 . Cagescan was built upon nanocage, but modified to accommodate paired - end sequencing for tss determination at 5 ends coupled with 3 end sequencing of cdnas at random priming sites . Rather than cleaving the cdnas, in cagescan we added adapter sequences allowing for paired - end sequencing in the illumina genome analyzers16 (fig . Transcripts defined by their common 5 end, as obtained by sequencing of both the 5 end and the 3 end of the template - switched captured cdnas . Yet, unlike nanocage libraries that contain uniformly short sequences, cagescan libraries show a broader size range including fragments longer than 1 kb, which perform poorly on currently available second - generation sequencing platforms17 . This problem is minimized by exclusively using highly concentrated random primers and commercial reverse transcriptases, which show little strand displacement activity . Thus, cagescan sequencing templates are kept relatively short, regardless the length of the original mrna molecules . Due to the selectivity of the template switch for capped molecules, both protocols were used on total rna, without ribosomal rnas (rrna) depletion . Notably, the usage of 3 random primer allows the detection of non - coding, non - polyadenylated rnas13, which have been so far poorly characterized . In order to validate the reproducibility, the efficiency and the precision of nanocage, we prepared libraries from serially diluted total rna from cultured hepatocellular carcinoma cells (hep g2) and compared them to reference tss data . Two duplicate sets of nanocage libraries from 10, 50, 250 and 1,250 nanograms of total rna were synthesized and sequenced with an illumina genome analyzer . We clustered tss from all the libraries that were located less than 20 bp apart on the same genome strand4 . We then compared expression levels, measured as number of tags per promoter region in a given library, for each pair of replicates with the same quantity of rna . The pearson correlation coefficients between replicas were 0.97, 0.96, 0.97 and 0.99 for, respectively, 10, 50, 250 and 1,250 ng . Replicates sequencing depth were in some cases substantially different (supplementary table 1) and higher correlations were observed for deeper sequenced libraries (0.99 for 1250 ng replicas). We then pooled the tags into virtual libraries for each rna quantity, and compared them with each other . Pearson correlation coefficients varied between 0.987 to 0.999 (supplementary table 2), showing similar snapshots of the transcriptome with tiny quantities of starting total rna within a range of 10 to 1,250 ng . A similar template - switching approach has been used with fragmented, uncapped rna molecules19 showing that ts could also be used on uncapped 5 ends . However, this protocol required prior depletion of ribosomal - rna otherwise reverse - transcription of total rna with random hexamers yielded 9094% of ribosomal sequences20 in our hands, only 11% of hep g2 nanocage tags matched rrna sequences, showing an 8-fold reduction in non - capped rrna content . This demonstrates the strong preference of template - switching for capped over non - capped rnas . To prove efficient capture of the 5 ends of capped transcripts, we prepared nanocage libraries from 100 ng of decapped, fragmented or both decapped and fragmented total rna and analyzed the distribution of tags mapping to refseq transcript models21 . In a library prepared with untreated rna, 52% of the tags mapped to first exons or proximal promoters in refseq (defined as 500 bp to refseq tss) and 31% detected potentially new promoters in intergenic regions (fig . 2a, supplementary fig . 2 and supplementary table 3). We noted that tags mapping to internal exons and 3 utrs (15% in total) can also derive from genuinely capped transcripts co - localized within the boundaries of longer transcripts referenced by refseq4,6,9 . The proportion of tags matching the 5 end of known transcripts was halved to 23% after rna decapping . Furthermore, this number dropped to 8.5% upon rna fragmentation, suggesting that a large number of uncapped rna molecules is needed to compete with capped molecules . Upon combining decapping and fragmentation, the preferential capture of 5 ends was almost completely abolished, demonstrating that nanocage distinguishes capped ends from other 5 ends and preferentially captures the 5 end of capped transcripts . The semi - suppressive pcr did not impair the detection of relatively short transcripts, as we detected expression for 78% of refseq transcripts (23,512/29,996), including 44% (271/615) of the subset respect nanocage tags outperform the fantom3 dataset, in which only 5% (28/615) of the short refseq transcripts are detected (supplementary table 1). Furthermore, the ecop15i cleavage did not introduce any bias as we found the cagcag ecop151 restriction site in 81% of the detected transcripts for both the nanocage and the fantom3 libraries, which were made with a different restriction enzyme, mmei1 (see also supplementary fig . 3). To confirm the precision of template - switching in detecting tss we compared promoters identified by nanocage with those found by two methods that are using different protocols for cap selection and for which hep g2 libraries were available4,22: deep - race22 (rapid amplification of cdna ends), based on oligo - capping23, and cage, 4 . The deep - race data was limited to 18 different promoters in 17 loci22 . As exemplified with histone cluster 1, h3 (hist1h3c, fig . 2b), the main tss was the same between nanocage and deep - race or fantom3 cage data in four and seven cases respectively . The hist1h3c locus also exemplifies the ability of our random - primed approach to uncover tsss of non - polyadenylated transcripts (hist1h3c refseq model lacks any 5 utr information). When allowing only 4 bp discrepancy between the tss uncovered by each methodology, the results of nanocage were in agreement with deep - race and fantom3 cage for 11 out of 18 promoters (supplementary table 4) and for 17 of the 18 deep - race - validated tss respectively . Interestingly, the two alternative promoters of ppp2r4 uncovered by deep - race and cage were also detected by nanocage and their relative differential expression levels were consistent between all three approaches (data not shown). To extend this result we compared the location of all the promoters detected by the two genome - wide libraries, nanocage and cage . Although a large number of promoters are broad in size4, for 66% of the promoters the distance between tss detected by both techniques was less than 5 bp (supplementary fig . Starting material becomes a limiting factor when cellular sub - compartments are selectively fractionated to explore specific rna content . As part of the encode project, attempts to produce cage libraries from nuclear rna subfractions of the k562 myelogenous leukemia cell line (the nucleolus, the nucleoplasm, the chromatin - bound rnas as well as from polysomal poly - a rna consisting mostly of rrna) were unsuccessful due to the paucity of mrna (not shown). Using nanocage, four libraries were synthesized and between 9.5 and 13.8 million tags were sequenced for each of them . Comparing to standard poly - a cage libraries, which were sequenced at the same depth, the complexity of detected 5 ends was consistent between the two technologies for each cellular compartment (supplementary fig . We have also found differences in tss specificity among different compartments (not shown). The functional significance of novel 5 ends is limited by the lack of information on the entire transcript . To better understand the structure of the transcripts associated with novel tss and to better characterize the differences between nuclear and cytoplasmic transcriptomes, cagescan libraries were prepared in technical duplicates from four different hep g2 cultures . In a first series of experiments, nuclear and cytoplasmic fractions were analyzed either as total rna or as a subfraction depleted of polyadenylated transcripts (poly - a) (supplementary table 1). Cagescan mate pair sequences were aligned against the human genome (ncbi build 36.1). The poly - a cytoplasmic, poly - a nuclear, total cytoplasmic and total nuclear fractions yielded together a total of 2,109,392 unique paired - end tags (supplementary table 6). Selecting mate pairs starting within 50 bp of refseq transcript s tss and using their intron - exon structure, we estimated the length of the rna from which mate pairs were derived . The resulting median length was 449 bp (1 and 3 quartile: 304 and 693 bp, supplementary fig . The median length of refseq transcript models is 2,422 bp (1 and 3 quartile: 1,509 and 3,799 bp), thus suggesting the majority of rna pol ii transcripts are competent to produce cagescan mate pairs, although cagescan is not optimal to map the 3 ends of transcript . Cagescan allows the association of tsss detected by cage to otherwise orphan intergenic, intron or 3 utr regions . Mate pairs were then annotated with respect to refseq transcript models21 complemented with a proximal promoter, defined as the region comprising the 500 bp directly upstream of their 5 end . This showed that an average of 4.24% of the transcripts were matching refseq transcripts, with 1.85% of them being strictly consistent with current gene models (that is starting within refseq promoter or 5 utr exon and ending within a refseq exon) while the rest was likely representing alternative mrna splice forms and non - coding rnas . Furthermore, the latter represented 87.5% of the total signal (fig . 3 and supplementary table 7). We observed specific differences between sub - cellular fractions and between the poly - a and total rna (dominated by poly - a molecules). Paired - end tags starting and ending within refseq promoters or 5 utrs were twice more prevalent than mate pairs ending within any refseq exons in the poly - a libraries than the total rna libraries . Those may correspond in part to promoter - associated long and short rnas (palrs and pasrs)7 . Nuclear fractions showed more paired - end tags starting and ending in intergenic and intronic regions than cytoplasmic fractions (fig . 3a). By preparing 12 additional cytoplasmic and nuclear cagescan libraries from 6 hepg2 independent biological replicas, we confirmed higher abundance of intronic (fig . 3b) and intergenic transcripts in nuclear fractions (p = 0.019 and p = 0.004 respectively, paired student s t - test). To better reconstruct transcript models, we grouped paired - end tags with overlapping 5 ends into cagescan clusters (fig . Alignment patterns of the corresponding 3 end tags on the genome recapitulate the potential structure of the transcript resulting from a common promoter . By pooling together the poly - a cytoplasmic, poly - a nuclear, total cytoplasmic and total nuclear fractions libraries, we obtained 854,849 distinct cagescan clusters, with an average of 2.47 reads per cluster . Clustered independently, the cytoplasmic libraries produced in technical duplicates yielded between 72,666 and 34,822 clusters (with 3.50.6 reads per cluster) and the corresponding nuclear libraries yielded between 309,682 and 147,963 clusters (with 1.60.3 reads per cluster). 9% of the cagescan clusters (all libraries pooled) started upstream of the translation initiation site of refseq; of them, 76.5% reached into their 3 utr or into their downsteam intergenic region, associating a promoter to a 3 utr for 11,131 refseq transcripts . Comparable ratios (9.5%2.65 and 80%11 respectively) were obtained when considering the four libraries separately (supplementary table 6). The region surrounding the ftl gene, for which the complete refseq model is tiled with paired - end sequences, illustrates such 5 mate pair driven clustering (fig . We observed subcellular compartment - specific antisense expression, with most of these antisense cagescan clusters ending in close proximity to the promoter of ftl (fig 4b and supplementary fig . A total of 37,818 clusters were antisense to 9,638 refseqs (supplementary table 6). Antisense rnas were generally more prevalent in the nuclear fractions . By aligning mate pairs to all exon - exon junction combinations of each transcript furthermore, clustering paired - end tags uncovered 1,569 cagescan clusters that initiated within the 5 utr of a given transcript, reached into the next downstream independent gene model in 1,198 pairs of distinct consecutive transcripts . Pervasive and regulated transcription of retrotransposon elements (re) have been observed in total rna extracts using cage24 . Using cagescan, we observed that expressed re are more abundant in the nuclear rna fractions than in the cytoplasmic ones . Globally, long and short interspersed nuclear elements (lines, sines) were the most highly expressed re in hepg2, followed by long terminal repeats (ltrs). All three were strongly over - represented in the nuclear poly - a fraction (fig . As expected srprna (signal recognition particle) repeats were enriched in the cytoplasmic compartment (fig . So far, an unbiased analysis of the transcriptome and promoter usage from challenging samples such as biopsies, homogeneous population of ex vivo cell types or subcellular compartments has been hampered by the low quantity of rna and by the use of fixatives that are detrimental to rna integrity . The simplicity of both nanocage and cagescan combined with decreasing sequencing cost opens the possibility for a truly high - throughput library production of pooled, multiplexed libraries followed by parallel sequencing, with applications ranging from drug screening, biopsy analysis, and whole transcriptome association studies . Therefore, we expect nanocage to become the technique of choice for micro - dissected samples in experimental biology and molecular pathology . Identification of novel 5 ends that are compartment - specific demonstrates the need and usefulness of nanocage . As an added advantage, the fixed length tags generated by nanocage can easily be turned into concatemers that will be advantageous when sequenced with long - read high throughput sequencers . By linking tsss to downstream sequences, cagescan provides insights into the architecture of transcripts and thus into their possible functions . Although the ability of fully scanning the 3 end of long transcripts is currently limited by the paired - end read length range that can be simultaneously sequenced, we believe that further development of sequencing technology will overcome this limitation . Cagescan profoundly differs from traditional inferences based on gene models such as refseq, which fails to grasp the complexity of the transcriptional landscape . Cagescan analysis is data - driven and hypothesis - free, which allowed us to find non - coding rnas, evidence of transcriptional read - through between neighboring loci, as well as novel forms of protein - coding genes . The expression level of cagescan promoters is indicated by the frequency of the 5 read of the mate pairs . Furthermore cagescan offers a unique perspective into the relations of non - coding rnas to the neighboring genomic / transcriptomic elements . Such novel transcription maps will be instrumental in identifying the functions of novel ncrnas that overlap regulatory regions and are likely to regulate transcription25 or processing and recapping of rnas26.
Regulation of protein expression is orchestrated through the interaction of trans - acting factors with cis - regulatory elements . Different factors are targeted to specific sequence regions or secondary structures in the transcript to promote different outcomes, including initiation of translation and degradation . Key regulatory steps in generating differential protein expression include the co - transcriptional events of pre - messenger rna (mrna) splicing, capping of the mrna 5 end, and polyadenylation . Post - transcriptional control can be exerted through de - capping, deadenylation, and cytoplasmic polyadenylation . Additionally, mrnas in the cytoplasm will often undergo transport, storage, and quality control, which influence the location and level of protein expression . All of these processes can be modulated by interactions with rna - binding proteins and non - coding rnas . For over half a century, research has focused on how these processes function individually and synchronously to control when and where proteins are made . Biochemical experiments using oocytes, embryos, yeast, and cultured cells first identified the factors involved and established the role of proteins in post - transcriptional regulation . Diligent exploration of individual rnas then enabled detailed models to be built for the different mechanisms of regulation . A burst in genome - wide and big - data approaches is now challenging previous assumptions and is uncovering levels of complexity in post - transcriptional control of protein expression . Based on recent breakthroughs, this article will discuss how new types of data are reshaping our understanding of post - transcriptional regulation in embryogenesis, with a focus on poly(a) tails and the 3 untranslated region (utr). I will consider outstanding questions such as the following: how do length, inclusion, and exclusion of the 3 utr influence mrna fate? Does poly(a) tail length directly influence translational outcomes? How is the balance between polyadenylation and deadenylation managed in the cytoplasm? When and at what level do micrornas (mirnas) function through mrna decay, repression, and deadenylation? The priority of resolving these and other queries is bolstered by increasing data linking human disease progression and severity to transcript regulation [24]. In the future, unraveling the nuances and intricacies of post - transcriptional regulation will once again require mechanistic analyses of individual transcripts . The scope of this article is limited, and the aim is to provide a glimpse of the emerging questions . In the early 1970s, fractionation experiments on mammalian cells isolated polysome - associated mrnas . An intriguing observation was made when these mrnas were cut with rnases specific to c and u as well as g; there was a long, resistant fraction . This fraction was proposed to consist of a poly(a) sequence not encoded by the dna template . This was confirmed upon the discovery of poly(a) polymerase (reviewed in). During the same period, researchers showed that poly(a) elongation commonly takes place in oogenesis, providing an important biological model . By the end of the 1980s, experiments in frog and mouse oocytes showed that regions of the 3 utr of mrna had the capacity to confer polyadenylation and initiate translation . This was somewhat surprising as the convention at the time was that the 5 utr regulated translation . It was quickly established, in both vertebrates and invertebrates, that specific mrnas were dependent on lengthened poly(a) tails for translation and that deadenylation, in some cases, directly resulted in translational repression (figure 1). Moreover, the binding of trans - acting factors to the regulatory element in the 3 utr can result in recruitment of different downstream proteins and enzymes . Changes in the poly(a) tail length are important for translation and mrna stability . Abbreviations: mrna, messenger rna; utr, untranslated region . In many organisms, there is a delay after fertilization before the zygotic genome is transcribed, and this is why maternal mrnas and post - transcriptional regulation are so important . This remains the case until inhibitors are removed or outcompeted by other factors for elements in mrna utr . Interaction between the 5 and 3 utr - associated proteins often circularizes the mrna, thus preventing the small ribosomal subunit from positioning on the transcript and initiating translation . The oocyte and early embryo of drosophila have been the focus of decades of research on mrna translational control . With a broad - scale approach taken by many researchers in the field, gene expression was recently measured by microarray and demonstrated that a specific poly(a) polymerase is required for the elongation of thousands of mrnas at the transition from oocyte to embryo . A post - fertilization wave of gene expression due to maternal mrna polyadenylation is also observed in frogs by high - resolution profiling of gene expression . These data are consistent with other work and suggest that cytoplasmic polyadenylation is a general regulator of translation at this developmental transition . Once maternal mrnas have been translated, they are degraded and the onset of zygotic transcription, known as the maternal - to - zygotic transition, follows . How these mrnas are cleared was tested by transcriptome - wide analysis in oocytes, one - cell embryos, and two - cell embryos of c. elegans and revealed that clearance of mrna following fertilization is mediated by poly(c) motifs in the 3 utr of the mrna . It will be important to test what, if any, other components are also required at this stage and whether this mechanism of clearance is unique to worms . Both the identification and authenticity of the c. elegans 3 utr have been areas of debate . Differing methods resulted in contrasting size, site specificity, and the number of 3 utrs reported . The evidence seems clear now that worm 3 utrs are much shorter than those of mammals (approximately one sixth) but are twice as dense in conserved mirna sites . As work in worms moves forward, it has important links to the evolution of the genome architecture and is generating new techniques for studying post - transcriptional gene regulation . The same method used to accurately map poly(a) regions in c. elegans was used in a vertebrate model, zebrafish, to show that mrnas experience cleavage and polyadenylation through development . A large wave of cytoplasmic polyadenylation is detected 2 hours post - fertilization, but why and exactly how this event takes place remain unclear . One possible model is that transcripts could be sites of competitive de - adenylation and re - adenylation, resulting in differential expression or decay . In mouse development, mrnas with short 3 utrs are preferentially targeted for degradation, demonstrating again the importance of regulating the length in the 3 utr . It is clear that translational regulation of mrnas is essential in early animal development and is a highly conserved mechanism for temporal control of gene expression . Although the presence of a non - templated poly(a) tail on mrna is presumed to lead to translation, a recent advancement in globally and accurately measuring the length of poly(a) tails has led to new models for the outcome of polyadenylation . In a survey of the individual length of millions of rnas from different plants, animals, and developmental stages therein, the results show that on large - scale poly(a) tail length and translation are not always coupled . As predicted, frog and zebrafish embryos displayed a strong correlation between poly(a) tail length and translation prior to gastrulation but, importantly, not after . This work challenges the assumption that poly(a) elongation is synonymous with translation . As the understanding and importance of post - transcriptional regulation, especially of the poly(a) tail and utr, have expanded, research has increased in depth as well as breadth . Examples of ongoing broad research in the field include the following: work on the maternal 3 utr of atlantic cod seeking to increase egg viability, an ongoing debate in plants on how factors that control poly(a) site choice are regulated, and testing of therapeutics that alter mrna half - life through manipulation of utrs, which could potentially block the progression of breast cancer, bipolar affective disorder, hereditary thrombocythemia, fragile x syndrome, and alzheimer's disease . An emerging mechanism adding a level of complexity to the transcriptome is alternative polyadenylation (apa) (figure 2). This is a general term inclusive of multiple classes of events that through cleavage and polyadenylation generate mrna isoforms with different 3 utrs . In addition to changes in the 3 utr, less common classes of apa involve alternation of introns and exons . The full implication of apa is unclear; however, more targeted mechanistic analysis in the future will be key to understanding this process and its outcomes . Experimental observations that could lead to further breakthroughs include work showing that ubiquitously transcribed genes in human cells gain tissue specificity through apa and that the rna localization of brain - derived neurotrophic factor and camkii both involve apa . It is exciting to hypothesize that apa could be involved in mrna localization in oocytes and embryos as well . Moreover, during cell proliferation, apa has been shown to play a role in generating mrnas with shorter 3 utrs and in turn less mirna target sites, likely conferring unique regulation . Alternative polyadenylation can change the length of the mrna 3 utr resulting in the inclusion or exclusion of regulatory elements . This results in the binding of different trans - acting factors, including proteins and mirnas, which confer specific post - transcriptional regulation . Abbreviations: apa, alternative polyadenylation; mirna, microrna; mrna, messenger rna; utr, untranslated region . A direct link to embryogenesis has been shown in the mouse model . As a result of apa, during the progression of development, mrnas with longer 3 utrs are detected . With longer 3 utrs and more au - rich sequences, it is suggested that mrnas later in development are likely to undergo more extensive post - transcriptional regulation . There is also evidence from zebrafish that high levels of apa occur throughout development and in drosophila where additional regulation is conferred in neural tissue by the inclusion of more repressor - binding sites in the 3 utr of mrna . Deep sequencing of the transcriptome has been paramount in identifying and annotating apa regulation in varied conditions, but the core biological mechanism remains elusive . Despite these examples, few transcripts show stability changes in association with apa . Therefore, questions remain as to what the role of apa is with respect to localization and translational efficiency . However, over half of human genes are predicted to generate variable 3 utrs this way, raising the possibility of major medical implications through manipulation of apa function . Therefore, the ability to shorten or lengthen the 3 utr by apa could lead to the inclusion or exclusion of different types of regulatory sequences in the mrna . In this way for example, a neuronal isoform with an extended 3 utr might contain mrna stability elements, whereas in a different cell type, polyadenylation at an upstream site would generate an isoform that has a short half - life . Given the many different regulatory sequences present in 3 utrs, including cytoplasmic polyadenylation elements, (a+u)-rich elements, and rna localization elements in addition to proteins binding the 3 utr, non - coding rnas also recognize and bind target sequences . Individual mirnas have the capacity to downregulate many different target mrnas and in some examples promote clearance of maternal mrnas in embryogenesis [2730] (figure 1). To test for the global impact of mirnas in protein production, quantitative mass spectrometry on cultured cells was used to assay changes in protein levels before and after the addition of mirnas . The results show that destabilization was the major cause of repression for the highly repressed mrna . This work also led to the proposal that mirnas act to make fine - tuning adjustments on protein levels . Correlation of ribosomal profiling data with measured mrna levels tested this question in mammalian cells . This work showed that lowered mrna levels account for a majority of the change in protein abundance . Ribosomal profiling was also used to decipher how mirnas regulate gene expression in zebrafish embryos . It was shown, by testing the ribosome occupancy on transcripts in different conditions, that the specific mirna tested functioned first to regulate initiation of translation and then in decay of the target mrna . These examples highlight a level of complexity to the system where one factor can influence multiple aspects of post - transcriptional regulation . Moreover, mirnas have been shown to function by different post - transcriptional mechanisms of regulation depending on the stage of development . In pre - gastrulation zebrafish embryos, mirnas function to reduce poly(a) tail length, resulting in translational inefficiency, and after this stage mirnas are thought to regulate mrna though destabilization . Although a full discussion is outside the scope of this article [3437], unraveling precisely how, and to what extent, non - coding rnas regulate mrna is crucial when considering their prevalence . Given all of the possible layers of regulation in a 3 utr and the mechanisms of generating the utr, post - transcriptional regulation is a complex and compelling area of research . The genome era introduced new approaches to longstanding questions in post - transcriptional control, many of which began in yeast . For example, microarray - based measurements correlated the length of mrna poly(a) tail lengths and the binding of a key protein with general translation in budding yeast . Also, ribosomal profiling enabled genome - wide analysis of translation and showed substantial translational control in response to environmental stress and total protein levels . Owing to conserved mechanisms, research in yeast is important for building the foundation for further understanding in mrna regulation in embryogenesis . One intriguing mechanism of regulating gene expression from yeast shows a connection between synthesis and decay of mrna through shuttling of mrna decay factors back into the nucleus . Whether similar factors are found in other systems to have a dual role is not clear, but this leads to the exciting hypothesis that feedback from the cytoplasm is regulating events in the nucleus . A second possible research area is comparative dynamic transcriptome analysis, which can monitor mrna levels without perturbing metabolism . In specific yeast strains tested, it was shown that when mrna degradation rates are altered, the cell's synthesis rate was reduced as well, resulting in buffering of the system . This work again suggests a level of interconnected surveillance for registering mrna levels in the cell to take subsequent action and could to be applied outside of yeast . The application of approaches from different systems, as well as establishing new protocols for testing changes in 3 utr composition, is important to decipher the molecular mechanism of these regulatory processes . However, establishing how different mechanisms are coordinated and how they relate to one another will be key to fully understanding post - transcriptional regulation not only in the early embryo but in all cells . Questions about post - transcriptional regulation are being asked more rapidly than answered . However, as demonstrated by a global comparison of mrna and protein abundance in different nematode species, separated by 30 million years of evolution, these mechanisms are important and widespread . Genomic techniques are uncovering exciting levels of control previously unknown and are important to the progression of the field . Alongside this work, classic biochemical and genetic approaches are essential to fully understanding mechanisms of mrna post - transcriptional regulation.
The current research on human diseases primarily focuses on the molecular, microbiological, immunological, and pathological influences . The mechanical basis of disease is now often being explored to decipher any direct contributions toward the physiological response [1, 2]. In functionally loaded tissues such as cartilage and bone, cells (chondrocytes and osteocytes) experience multiaxial forces (hydrostatic, compressive, tensile, and shear), which play a significant role in modulating the biological function through maintenance of the phenotype and production of a neotissue . Conversely, abnormal mechanical forces (either static or dynamic) can lead to altered cell behavior resulting in pathological matrix synthesis, increased catabolic activity (degradation), and ultimately osteoarthritis or osteoporosis (apoptosis). Our previous investigations have indicated that chondrocytes and likely other cell types respond to their stress - strain environments in a temporal and spatial manner . It has also just been shown that individual cellular mechanical properties may indicate the regenerative potential of mesenchymal stem cells . Investigations of the biomechanics at the cellular level have also identified the biomarkers of disease . Cytoskeletal stiffness of metastatic cancer cells was reported as more than 70% softer than the benign cells that line the body cavity in patients with suspected lung, breast, and pancreatic cancer . These approaches highlight the utility of single - cell biomechanics as a critical component of advancing microscale therapeutics . Advancements in laser technology and microfluidics now allow the use of optical tweezers or traps and fluid mechanics to manipulate isolated single cells [8, 9]. Isolated loads can be applied experimentally to single cells in culture to quantify cellular and cytoskeletal biomechanics . One can then apply forces and displacements as small as pico - newtons and nanometers, respectively [1012]. The local microenvironment can therefore be precisely manipulated to facilitate biomechanical test sequences on individual biological cells and molecular structures . In order to explore the connection between external mechanical stimulation and cellular regeneration or degeneration, we developed a three - dimensional, multiphysics computational model to fully characterize a unique micromechanical environment . The applied stress state within our custom - fabricated optical and hydrodynamic (optohydrodynamic) trap have been mathematically developed from the fundamental equations describing microfluidic creeping flows past a suspended sphere . The objective described in the following paper is to explore the full - field cellular strain response to a range of applied stresses and cellular moduli . The described computational framework will now allow us to develop more realistic cellular models, whose intracellular structures are distinctly identified . Living non - adhered, suspended osteoblasts, chondrocytes, fibroblasts, and myoblasts have recently been isolated and mechanically manipulated [12, 16]. Primary cultures of chondrogenic and osteogenic tissues were generated directly from rat long bones, while muscle cells were acquired from the mouse - derived myoblast c2c12 cell line (atcc, crl-1772, manassas, va, usa). All cells were tested at room temperature experiments (~20.5c) in a flow media consisting of a physiological buffer resulting in a media viscosity of ~1 mpa s. this single cell biomechanical manipulation was made available by combining optical trapping with microfluidics to create the optohydrodynamic trap . This work was facilitated by an instrument, which integrates two laser - based techniques for the mechanical characterization of cellular and biomolecular structures [8, 12]. The optical tweezers or the trap component of the device utilizes an infrared laser (= 1,064 nm) to suspend micron - sized objects with nanometer position control and pico - newton constraining forces . In the mie regime, where objects are larger in dimension than the wavelength of the trapping laser (here biological cells), a ray optics description indicates the transfer of refracted light and the associated momentum into trapping forces (figure 1). Micron - particle image velocimetry can be engaged by incorporating two frequency - doubled lasers (= 532 nm) aligned through the same optical path as the ot for full - field flow velocity characterization . However, the nanoparticles associated with velocity imaging have proven deleterious to cellular health, but provide useful experimental validation of flows around synthetic micron - sized particles . The hydrodynamic component of this approach is facilitated through a microfluidic chip design configured in the form of a cross - junction channel (figure 2). This geometry creates an extensional flow environment and a stagnation point at the channel's geometric center . Cells are positioned at the centroid with the optical trap and manipulated with microfluidics, thus creating the optohydrodynamic trap . The cell experiences a total drag force equal to zero, confirmed by integrating the stress tensor as defined by the normal (form drag) and shear (friction drag) stresses (figure 3). This reflects the mechanical stability or the saddle - point nature of the optohydrodynamic trap . Previous studies describe chip fabrication in detail including the control of the gravity - driven flow initially associated with microfluidic manipulation . The two- and three - dimensional stress states were previously developed as applied to the surface of a nonrotating spheroid cell of radius a, within the optohydrodynamic trap [2, 12, 14, 15]. Briefly, the full - field fluid velocity vector u was constructed from the constitutive equations describing a non - rotating sphere suspended in a general linear flow with viscosity and pressure distribution p . In the polar - spherical components (r-- magnitudes and er - e-e vectors), the generalized flow field produces the individual velocity components: (1)u = u(ra52(ar)2 + 32(ar)4)sin2cos2er+u(ra(ar)4)sincoscos2e+u(ra(ar)4)sincos2e including the pressure distribution (2)p = p5ua(ar)3sin2cos2. The velocity gradients can be converted into the applied fluid stresses by applying the constitutive equation for an incompressible, newtonian fluid: (3)t=pi+2e, where t is the stress tensor and i is the identity matrix associated with the local isotropic (hydrostatic) pressure distribution p. the strain rate tensor e can be characterized by the flow velocity gradient tensor and its transpose: (4)e=(12)[u+ut]. By incorporating the velocity and pressure fields into the gradient analysis and then in turn into the constitutive equation, the fluidic stress tensor can be fully defined as (5)trr=p+ua(2 + 15(ar)312(ar)5)sin2cos2er, tr=ua(25(ar)3 + 8(ar)5)sincoscos2e,tr=ua(25(ar)3 + 8(ar)5)sinsin2e. Defining the stress tensor at the cellular surface, r = a, produces the volumetric fluidic stress state applied to the cell: (6)tr = ter=rrer+re+re, where the full - field stress state can then be defined in terms of distinct normal, rr, and shear, r and r, stress components, respectively, in polar spherical coordinates: (7)rr=p+5uasin2cos2,r=5uasincoscos2,r=5uasinsin2. A three - dimensional presentation of the stresses was developed (matlab, mathworks, inc ., natick, ma) for demonstration of the site - specific nature of the stress distributions (figure 4). The six deviatoric stresses were combined as an effective stress value, eff, as a means to model the three - dimensional stress state: (8)eff=12[(xy)2+(yz)2+(zx)2 + 6(xy2+yz2+zx2)]1/2 . Here, the maximum polar coordinate derived stresses were converted to the cartesian coordinate stresses such that the maximum normal stresses are located along the x - y - z axes and the maximum shear stresses are defined along the 45 orientation off - axis locations . Volumetric strain, e, based on the strain invariants, ii, was defined to encompass the full - field deformation response of the cell within the multiaxial fluidic loading environment and does not apply the small - displacement theory assumption . The cartesian axes associated with the maximum normal strains were determined from a mohr's circle analysis [12, 20]: (9)e = i1+i2+i3=x+y+z+xy+yz+zx+xyz . The experimental approach described earlier is a planar - wise measurement technique; thus the optical depth strain value, z, can be determined through a transposition of the planar loading scenario, again through a mohr's circle analysis: (10)z=1(x+y). However, in this modeling presentation here, the full - field stress state and the corresponding strains are fully characterized . The optohydrodynamic deviatoric stress state was applied to an isotropic, homogenous biological cell (20 m in diameter) within a multiphysics computational environment (comsol v4.0, palo alto, ca, usa) in order to determine the individual principal strains and in turn the volumetric strains . In this work, we continue to examine the cellular biomechanics induced within a controlled micromechanical environment . Three - dimensional cellular strains were computationally modeled in silico with multiphysics software applying the analytically defined stress state (figure 5). Volumetric stress and strain relationships indicated both the nonlinear response of the spherical cellular structure as well as the logarithmic increase in strain with subsequently softer cellular moduli (figure 6). This relationship is further explored when examining the direct relationship of strain with elastic moduli (figure 7). The extreme strain response induced in diseased cells indicates their further vulnerability when passively or actively resisting applied stresses . We explored the full - field cellular strain response to a range of applied hydrodynamic stresses and cellular moduli, representing various degrees of functional loading and health / disease, respectively . The computational framework now allows us to develop more realistic cellular models with intracellular and membranous structures, distinct in spatial, elastic, and active transport characteristics . The mechanical properties of a single cell are often formulated using either macroscopic or microscopic approaches . Macroscopic approaches, as partially described in this work, homogenize every cellular component to produce an isotropic or anisotropic yet homogeneous continuum model so that the mechanical properties of cells can be formulated using temporal and spatially continuous partial differential equations . Future efforts will incorporate a more microscopic approach, which regards the cell as a biocomposite material consisting of randomly or uniformly spaced anisotropic reinforcement cytoskeletal materials within an isotropic medium . The microscopic approach generally obtains the biomechanical response of a single cell by applying the mechanical boundary conditions at the individual fiber and matrix level, scaling up to the cellular level . Microscopic approaches often provide much more detail into the subtle interaction between the cytoskeletal fibers and matrix, which potentially leads to a more accurate model of the cellular behavior, such as characterizing the irreversible deformation of the cellular skeleton . Unfortunately, refined microscopic models suffer from inhibitory computational and storage costs [26, 27]. When interpreting the potential geometric changes in cellular shape associated with mechanical loading, the cell may experience some interesting membrane transitions triggering unique biologic cues . Under suspension and hydrostatic loading, the cell's volume (v) can be defined as a sphere, v = 3/4abc, with radii a = b = c. as seen here during the hydrodynamic extensional loading state, the isotropic, homogenous cell model deformed into a scalene ellipsoid (a b c) with equal and opposite tensile and compressive strains in the horizontal plane . However, with the future inclusion of intracellular structures and organelles as well as nonlinear elastic properties assigned to the membrane and cytoskeleton, cellular models may also deform into either an oblate spheroid (formed when an ellipse is rotated about its minor axis, a = b> c) or a prolate spheroid (formed when an ellipse is rotated about its major axis, a = b <c). The resulting shape here replicated a scalene ellipsoid likely due to different maximum stresses applied in the three orthogonal axes . Ongoing experiments and modeling will continue to explore multiaxial single - cell biomechanics as well as the biologic triggers associated with geometric shape - shifting . The described multiphysics computational framework will facilitate more realistic cytoskeletal model interpretations, whose intracellular structures can be distinctly defined, including the cellular membrane substructures, nucleus, and organelles . Future results will provide mathematical outcomes supporting the ongoing investigations in tissue and cellular engineering.
Thrombotic microangiopathies result from damage to the endothelium leading to a cascade of thrombosis and resultant anemia, thrombocytopenia, and renal damage . Atypical hemolytic uremic syndrome (ahus), a rare genetic disorder of this class stems from a rapid inappropriate activation of the complement system, termed atypical due to the lack of a triggering event akin to conventional hus which starts with exposure to the shiga - like toxin . The mortality in pediatric - onset ahus is reported to be more (6.7%) while adult onset has higher chances of progression to end - stage renal disease . Newer therapy such as the complement binding antibody eculizumab is financially unviable at present in the indian setting . A 14-year - old male, presented with complaints of sudden reddish discoloration of urine 5 days back followed by yellowing of eyes and skin, nausea, and vomiting associated with feeds . On routine investigation, he was anemic (hemoglobin 4.9 g%) with thrombocytopenia (platelets 97,000). Renal functions were deranged with a serum creatinine of 4.27 and blood urea of 116.8 . A peripheral blood smear revealed dimorphic anemia with thrombocytopenia with polymorphonuclear leukocytosis with abundant schistocytes and tear drop cells suggestive of hemolytic uremic syndrome with a reticulocyte count of 30% . Anti - nuclear antibodies were absent with low complement 3 (c3) and normal c4 levels . Anti - complement factor h (cfh) antibodies were found to be significantly raised at 2043 au / ml . He was initiated on plasma exchange with 7 initial daily cycles and a total of 16 cycles titrated to clinical response . Anti - cfh antibodies were repeated and found to have decreased to 191.07 au / ml . He later developed sudden onset severe headache followed by loss of vision progressing to generalized tonic he was intubated and later successfully weaned off mechanical ventilation . A magnetic resonance imaging (mri) brain revealed posterior reversible encephalopathy with altered signal intensity in a subcortical white matter of the bilateral parietal, occipital, posterior temporal lobes and cerebellar hemispheres [figure 1]. Severe skin rashes developed almost 2 months into admission: centripetal in development with mucosal involvement and was diagnosed as steven he was started on further immunosuppression with steroids and azathioprine and discharged on day 91 of admission in complete clinical and hematological remission . (b) thermal - infrared images show symmetrical hypointense lesions in bilateral parietal lobes . (c) fluid - attenuated inversion recovery hyperintensities in occipital lobe typical of posterior reversible encephalopathy . Ahus is a thrombotic microangiopathy resulting from mutations in cfh, complement factor i, membrane cofactor protein (cd46), c3, thrombomodulin, cfh - receptor 5, or from autoantibodies to cfh . Autoantibodies to cfh reported in 4%14% of all cases, however, are much more common in cases with early onset of disease and are present in up to 25% of such cases . Autoantibodies to cfh prevent cell surface protection by cfh, chiefly by inhibiting binding to c3b . Ahus may be suspected in patients with typical history along with proven negative stool cultures for shiga - like toxin and normal adamts13 activity and can be confirmed by genetic assays, though empiric treatment is often initiated . The autoimmune variant, however, as in our case may not have a genetic focus and can be diagnosed by high levels of antibodies to cfh (2043 au / ml in our case). The overall ahus has incomplete penetrance, and an infectious trigger is often associated with the disease precipitating . Our case presented with a high - grade fever of undocumented etiology which was probably the trigger for complement activation . Globally, eculizumab, the monoclonal antibody to c5b, has found acceptance in the treatment of ahus . Guidelines issued in 2009 recommended initial daily plasma exchange (5070 ml / kg) with further titration of frequency according to clinical response . Our patient received an initial seven cycles of daily plasma exchange followed by gradual tapering with monitoring of hematological and renal parameters which showed complete normalization by the 12 cycle . Cfh - related ahus, similar to our patient, has been documented to respond better to plasma exchange . Our patient did not develop any requirement for renal replacement therapy with hemodialysis as is the norm in a majority of cases of ahus . Hypertension is a common complication of ahus but developed late in the course of treatment in our patient . Posterior reversible encephalopathy with associated loss of vision may have an identical presentation as cerebral thrombotic microangiopathy . The latter can be differentiated on the basis of asymmetry of the lesions on mri . Although neurological sequelae respond best to eculizumab, our patient was already in remission at the time of onset . Johnson syndrome was probably a coincidental reaction to phenytoin and does not relate to ahus in the available literature . Cyclophosphamide, mycophenolate mofetil, azathioprine and steroids, all have supporting literature for maintenance therapy . 36.5%63% progress to mortality or end - stage renal disease in the long - term . The authors certify that they have obtained all appropriate patient consent forms . In the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed . The authors certify that they have obtained all appropriate patient consent forms . In the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
The sample consisted of sixty - three female wistar rats (190 1 g), kept in individual cages, receiving water and food (ad libitum) (purina - brazil), in an artificially illuminated ambience, with a 12 hour day - night cycle and mean temperature of 24c . Initially, all animals were submitted to an adaptation period (5 days) of swimming for 5 minutes in water at thermo - neutral temperature in a pool (50 cm 45 cm). All the animals at the start of experimental procedure weighed between 180 g and 220 g and were in their reproductive maturity period . The determination of maximal work load was performed in a swimming pool with water at thermo - neutral temperature . The work load was increased every 3 minutes by weights attached to the animal's tail and corresponding to 1%, 2%, 3%, etc . Of the total body weight, until the maximal work load was reached by the animal's exhaustion (unable to surface for 10 s). All rats were kept in cages (3 - 4 females for each male) during 12 hours (night period). The copulation was confirmed the following morning by the presence of sperm in vaginal smears . The pregnant rats (n = 63) were then separated into three groups: sedentary rats (ps, n = 21; 188 2 g) submitted to water immersion but did not perform exercise during pregnancy, exercised rats (pe, n = 21; 193 2 g) submitted to swimming sessions at 80% of maximal work load supported for 30 minutes during 19 days of pregnancy, trained rats (pt, n = 21; 188 2 g) submitted to swimming sessions during 45 days before and during pregnancy for 30 minutes, for 19 days of pregnancy, at 80% of maximal work load supported into water, which was calculated for each rat before pregnancy . Each group was divided into three subgroups (n = 7), regarding water temperature: 28c, 35c, and 39c . All swimming sessions were carried out between 8:00 a.m. and 12:00 p.m. in order to determine weight gain during pregnancy, the rats were weighed on the first and 20th day of the gestational period (ohaus balance). The rats were decapitated on the 20th day of pregnancy in order to collect a blood sample (5ml) for determining plasma levels of triglycerides and glucose by the enzymatic method.8 just after decapitation, a caesarean section was carried out and the offspring were counted and weighed . Mean values standard error for rectal temperatures are shown in table 1 . Comparing rectal temperatures, prior and post conditions, showed that, there was a decrease in rectal temperature for all experimental groups at 35c and 28c, while an increase was found at 39c, and temperature (post - prior) was significantly higher at 28c than at 35c and 39c . Mean values standard error of rectal temperature (c) determined during 19 days of pregnancy for sedentary rats (ps), rats exercised during pregnancy (pe) and trained rats exercised before and during pregnancy (pt), prior to and post - immersion or swimming (80% of maximal work load supported) into water at temperatures of 28c; 35c or 39c . (n = 7 for each group) mean values standard error of weight gain (g) for sedentary pregnant rats submitted to immersion (ps), rats exercised during pregnancy (pe) and trained rats, exercised before and during pregnancy (pt) at 80% of maximal work load supported into water at 28c, 35c or 39c . (n = 7 for each group) at 28c, there was no difference between ps, pe and pt groups, but at 35c, pt and pe presented smaller weight gain than the ps group, and at 39c, only pt presented a smaller gain . Plasma glucose was higher and plasma triglycerides were lower at 28c and 39c than at 35c for sedentary and exercised rats . Trained rats did not present differences in these variables at any of the water temperatures and had different values than sedentary rats at 35c and 39c (table 3). The litter size was not different among the groups at any of the water temperatures (table 4). Mean values standard error of plasma glucose (mg / dl) and triglycerides (mg / dl) on the twentieth day of pregnancy, determined in blood samples collected 24 hours after the last immersion or swimming session (80% of maximal work load supported) of pregnant sedentary rats (ps), rats exercised during pregnancy (pe) and trained rats exercised before and during pregnancy (pt) in water at temperatures of 28c, 35c or 39c . (n = 7 for each group) mean values standard error of offspring weight (g) and size of litter in pregnant sedentary rats (ps) submitted to daily immersion, rats exercised during pregnancy (pe) and trained rats exercised before and during pregnancy (pt) at 80% of maximal work load supported into water at 28c, 35c or 39c . The literature demonstrates that pregnant rats have lower glucose levels than non - pregnant ones submitted to the same exercise conditions, which means they are not influenced by water temperature.8 our findings indicate that, under thermal stress (28c or 39c), sedentary and exercised pregnant rats present higher plasma glucose values than at 35c . These results suggest that they have either a smaller uptake or an increase of glyconeogenesis from lactate, alanine and piruvate.1415 an increase in homeostasis alterations (exercises in high or low temperatures) induces higher increases in catecholamine and cortisol release.1619 a raise in adrenalin induces pancreas beta cells to reduce the insulin release, which induce alpha cells to increase glucagon.20 catecholamines, cortisol and glucagon are gluconeogenese and hepatic glycogenolysis inducers, contributing to glicemic levels rise.16192122 yet, for trained animals, plasma glucose was kept steady, suggesting that daily thermal stress did not modify the regulatory mechanism of plasma glucose in these animals . The results obtained from pregnant sedentary rats and exercised rats showed alterations of plasma glucose according to the temperatures to which they were submitted daily . It suggests that the duration of immersion or swimming was more important than the temperature concerning the determination of plasma glucose levels, since in the previous study, which used more extreme temperatures with a shorter duration of exposition, pregnant rats showed steady levels of glucose.8 by determining the glucose level during exercise performed by pregnant women, greater hypoglycemia was recorded compared to that observed in resting condition, but this hypoglycemia is smaller during water exercises than land exercises.22324 glucose returned to resting level within 24 hours of the last session of exercise, indicating the efficiency of the glucose level adjustments.25 this quick regulation is made mainly by the hyperglycemiant hormones action (catecholamine, cortisol and glucagon) that have their serum concentrations increased by exercise.1622 in the present study, trained rats had lower values of plasma triglycerides at 35c than sedentary rats, indicating an increase of lipid utilization in this condition . Plasma triglyceride levels were also lower at extreme temperatures rather than at 35c for exercised and sedentary rats, but trained rats did not present differences in plasma triglycerides at any of the water temperatures . When the current results were compared to our previous study,8 in which pregnant rats were submitted to more extreme temperatures but for a smaller duration, it was observed that both glucose and triglycerides were more influenced by the duration of exposure (30 minutes) rather than by the temperature, which was warmer than in the previous research . Increased serum concentrations of triglycerides during the exercise are also induced by the hormones cited above . Catecholamines, cortisol and glucagon act on adiposity cells rising the lipolysis and lipids release to blood . Concomitantly the decrease in insulin concentration increases the hormone - sensitive lipase, augmenting the fat acids availability too.26 increased triglyceride levels were observed in pregnant rats, when compared with non - pregnant animals, especially at the end of pregnancy.225 this increase was produced by the endogenous triglycerides from the blood stream.27 therefore, a lipid represents an alternative substrate for the mother, safeguarding glucose for the fetus . On the other hand, aerobic training modifies the substrate utilization during exercise, increasing lipid mobilization and safeguarding glucose.1 in a previous study carried out in our laboratory,8 we did not observe any alteration in weight gain of rats submitted to daily swimming sessions of 10 - 15 minutes, at different water temperatures (22c, 35c and 40c). In the present study, using water at more agreeable temperatures for a longer exercise period, it was observed that exercised rats (pe) at 35c and trained rats (at 35c and 39c) had smaller weight gain than sedentary pregnant rats, indicating that substrate mobilization for exercise interferes with total weight gain . A smaller weight gain was found by some authors.252833 nonetheless, other authors did not observe any alterations.3436 in the present study, as in our previous paper,8 after immersion or swimming, pregnant rats modified their rectal temperature with relation to water temperature: decreasing at 28c and 35c and increasing at 39c . In spite of these alterations, a reduction in the litter size was not observed at extreme temperatures, although the duration of immersion or exercise was greater than that employed in our previous study . Nevertheless, if the length of exposure is much longer, where the rats exercise daily for one hour at temperatures of 34.6c and 37.6c, fetal abnormalities and reabsorption can be found, probably due to an increase of maternal core temperature.37 regarding exposition to the cold and heat during pregnancy, some studies showed that hypothermia as well as hyperthermia could produce deleterious effects in fetus.673842 there is a temperature gradient between mother and fetus (the fetus presents a temperature 0.4 to 0.6c greater than the mother), allowing heat transference from the fetus to the mother.43 during high - intensity exercise, this gradient could be reversed, modifying the heat exchange between mother and fetus.67 this response could lead to fetal hyperthermia and consequent deleterious effects on offspring.44 when the weight of offspring was investigated, it was verified that sedentary pregnant rats submitted daily to water immersion at 28c presented litters with smaller weight in relation to offspring from mothers submitted to immersion at 35c and 39c . This result suggests that maternal hypothermia, leading to reduction in utero - placentary flow, could induce an inadequate delivery of substrate to the fetus and, in consequence, decrease its weight . Besides, such an effect was not found in exercised and trained rats, suggesting that compensatory mechanisms to safeguard an adequate delivery of nutrients to the fetus were developed by training . Artal et al45 published a guideline for exercise during pregnancy of the american college of obstetricians and gynecologists and advocated the benefits of an exercise program to the pregnant and to the fetus . In a recent study barakat et al46 concluded that the regular practice of exercise during the pregnancy did not influence the gestational age, but this study included only sedentary participants . Our results demonstrated that an exercise program before and during the pregnancy produced better results than an exercise program only during pregnancy . Further studies are necessary to find the effect of pre pregnancy training on other variables (hormonal responses, cardiovascular and respiratory responses, gestational age, etc .) In animal and human models, considering the great similar physiology between rat and human pregnancy . We concluded that physical training before pregnancy induces a greater stability of plasma triglycerides and glucose, regardless of daily swimming sessions at different water temperatures . These exercise conditions before pregnancy interfere with total weight gain during pregnancy, without deleterious effects on offspring . Dalo and idcp carried out the design and coordinated the study, participated in most of the experiments and prepared the manuscript . Jsc, akr and wr carried out all the experiments and participated in manuscript preparation . Mm and rp provided assistance for all experiments and participated in manuscript preparation specially the interpretation and description of the results and discussion . All authors have read and approved the content of the manuscript.
The simple gas ethylene has been recognized as a plant growth regulator for a century (crocker and knight, 1908; knight et al ., 1910; neljubov, 1901). Ethylene influences many aspects of plant growth and developmental processes, including germination, fruit ripening, flower senescence, leaf abscission, nodulation, lateral root initiation, and the response to a variety of abiotic and biotic stresses (abeles et al ., 1992; mattoo and suttle, 1991). The russian scientist neljubov first demonstrated that ethylene is the responsible component caused the early defoliation of the tree nearby a leaking illuminating gas main in a small german town in the late1800 s (neljubov, 1901). Neljubov used pea seedlings to determine that ethylene is the active component of the illuminating gas by exposing the filtered illuminating gas to pea seedlings . The pea seedlings exhibited distinctive morphology changes which include shortening of hypocotyl and root, swelling and thickening of hypocotyl, and formation of exaggerated hook . This seedling phenotype was later defined as the triple response, which is a hallmark of the ethylene response of dark - grown seedlings (knight et al ., 1910). 30 years later, gane showed that ethylene is naturally produced by plants (gane, 1934). Through the efforts of yang and co - workers, 1) (kende, 1993; yang and hoffman, 1984; zarembinski and theologis, 1994). Ethylene is synthesized from the amino acid methionine via two intermediates, s - adenosyl methionine (sam) and 1-aminocyclopropane-1-carboxylic acid (acc) (adams and yang, 1977; lieberman and mapson, 1964). In the first step conversion of sam to acc, which is catalyzed by a family of acc synthase (acs) enzymes, is the first committed and rate - limiting step in ethylene biosynthesis (boller et al ., 1979; yang and hoffman, 1984). During this conversion, 5-methylthioadenosine (mta) is generated as a by - product which is readily recycled back to the yang cycle to conserve the methlythio group of mta into the methionine (murr and yang, 1975). This salvage step enables plants to maintain a constant level of cellular methionine, which can be utilized during rapid ethylene production (sauter et al ., 2013). Acc is then converted to ethylene by acc oxidase (aco), a member of the oxygenase / oxidase superfamily of enzymes (dong et al ., 1992). The levels of free acc are regulated by formation of acc derivatives, malonyl - acc, -glutamyl - acc, and jasmonyl - acc, which likely affect the pools of free acc for ethylene biosynthesis (van de poel and van der straeten, 2014). As gaseous ethylene is diffusible and not degraded in plant cells, strict regulation of ethylene biosynthesis is necessary to its diverse function in plant growth and development . Due to its role in the rate - limiting step of the ethylene biosynthesis pathway, acs has been considered as a major point of the regulation in the pathway . Regulation of the transcript levels of acs genes appears to be a key mechanism to control changes in ethylene production in plants (argueso et al ., 2007; harpaz - saad et al ., 2012 . However, recent studies suggest that post - translational modifications, such as phosphorylation and ubiquitination, serve as an important mechanism to regulate the stability of the acs proteins, thus controlling the levels of ethylene in plants (argueso et al ., 2007; chae and kieber, 2005; chae et al ., 2003). The family of acs proteins can be grouped into 3 types, type-1, type-2, and type-3, based on the presence of distinct consensus sequences including phosphorylation target sites near the non - catalytic c - termini (fig . 2) (chae and kieber, 2005; mcclellan and chang, 2008; yoshida et al ., 2005). Type-1 acs proteins contain a relatively long c - terminal domain that shares highly conserved sequences and the target sites for a mitogen - activated protein kinase (mapk) and calcium - dependent protein kinase (cdpk) (hernndez sebasti et al ., 2004; kim et al ., 2003; liu and zhang, 2004). However, type-2 acs proteins contain a unique regulatory motif called a target of ethylene overproducer 1 (eto1) (toe) which overlaps with the cdpk target site . Toe motif mediates interaction with eto1 e3 ligase and its two paralogs, eto1-like (eol1 and eol2) and is required for degradation of type-2 acs (chae et al ., 2003; christians et al ., 2009; wang et al ., 2004; yoshida et al ., 2005; 2006); type-3 acs contains no target sites for a cdpk and mapk, with only a short stretch of amino acids in the c - terminal extension (chae and kieber, 2005). Here, i focus on recent advances and current knowledge on the protein turnover regulation of acs in ethylene biosynthesis . New insights into the role of phosphorylation, ubiquitination and recently identified novel components governing the stability of acs proteins are discussed . Finally, i conclude with the prospects regarding the cross - talk between ethylene and other cellular biosynthetic / signaling pathways . Transcriptional regulation in ethylene biosynthesis has been well documented, therefore not discussed in this review . Treatment of arabidopsis etiolated seedlings with ethylene results in the triple response (guzman and ecker, 1990). The triple response has been extensively used to identify arabidopsis mutants with defects in ethylene perception and signaling, as well as mutants affecting ethylene biosynthesis . The ethylene biosynthesis mutants can be further categorized into two groups: (1) cytokinin - insensitive (cin) mutants which are impaired in response to cytokinin, resulting in reduced levels of ethylene production in response to cytokinin; (2) ethylene - overproducer (eto) mutants which show a constitutive ethylene response due to increased ethylene biosynthesis (chae et al ., 2003; the recessive cin5 mutant was identified as the first example of the cin mutants, and further characterization of the mutant revealed that the corresponding cin5 mutation is a loss of function allele of the acs5 gene . The cin5 mutant is severely insensitive to exogenous cytokinin, and as a result, it fails to display the triple response . However, it shows normal triple response to ethylene, suggesting acs5 is the primary target of cytokinin - mediated ethylene induction in etiolated seedlings (vogel et al ., 1998). Other phytohormones, such as auxin, brassinosteroids and aba are also known hormonal triggers that increase ethylene production (arteca and arteca, 2008; woeste et al ., 1999a; yi et al ., 1999 auxin promotes ethylene production mainly through the increase of mrna levels of specific acs genes in various plant species . In arabidopsis, the most of acs genes are transcriptionally induced in response to auxin, and auxin treatment also alters the spatial expression pattern of the acs genes (tsuchisaka and theologis, 2004). Aba has been shown to regulate ethylene production in apples, tomato and various plant tissues (lara and vendrell, 2000; tari and nagy, 1996; zhang et al ., 2009). In tomato, ethylene levels increase remarkably after aba treatment and this coincides with the increase in the expression of leacs2, whereas application of ndga, an inhibitor of a key enzyme in aba biosynthesis, suppresses the expression of leacs2 (zhang et al ., 2009). Brassinosteroid is another phytohormone that enhances ethylene production by increasing the transcript abundance of acs genes, but brassinosteroid in part promotes ethylene production by stabilizing acs protein (hansen et al ., 2009; yi et al ., 1999 cytokinin, however, stimulates ethylene production by acting on the stability of acs proteins, thereby increasing the ethylene production in plants (chae et al ., 2003; hansen et al ., 2009 analysis of eto mutants has provided further evidence that the stability of acs proteins is regulated . Three eto mutants have been identified via genetic screens based on the constitutive triple response phenotype due to ethylene overproduction: eto1, eto2, and eto3 (chae et al ., 2003). Etiolated eto mutants exhibit the constitutive triple response and this phenotypes is rescued by treatment of mutant seedlings with ethylene biosynthesis inhibitor aminoethoxyvinylglycine (avg). The dominant eto2 and eto3 mutations alter the c - terminal domain of acs5 and acs9, both type-2 acs proteins, as the result of a single base insertion and a missense mutation, respectively . The eto2 and eto3 mutants significantly produce more ethylene than wild - type seedlings, but this increase in ethylene production is not correlated to the acs5 or acs9 gene expression, thus suggesting that the mutants control the acs function at the post - translational level similar to the action of cytokinin . These results reveal that the c - terminal domain of both acs proteins is a target for post - translational modification for degradation (chae and kieber, 2005). Characterization of the eto1 revealed that ubiquitination via the 26s proteasome pathway is involved in regulating ethylene biosynthesis by modulating the protein stability of type-2 acs proteins . Recessive eto1 produces a nearly 10-fold excess of ethylene compared to wild - type etiolated arabidopsis seedlings and exhibits the constitutive triple response (woeste et al ., 1999b). Epistasis analysis demonstrates that acs5 acts downstream of eto1; the eto1cin5 double mutant produces significantly reduced amount of ethylene compared to eto1 itself (chae et al ., 2003), indicating eto1 plays a role as a negative regulator by acting through acs5 in ethylene biosynthesis . Eto1 encodes a novel plant - specific protein that contains a broad - complex, tramtrack, bric - - brac (btb) and tetratrico - peptide repeat (tpr) with a coiled - coil domain (wang et al ., 2004). Proteins containing btb motifs have been shown to participate in substrate recognition via their protein - protein interaction motifs and bridge substrates to cullin 3 (cul3)-based ubiquitin ligase complexes for degradation (albagli et al ., 1995; pintard et al ., 2004). The tpr motifs are involved in diverse protein - protein interactions, and also serve as a scaffold for the assembly of high - order protein complexes (blatch and lassle, 1999). Eto1 interacts with acs5 and cul3, and in the case of acs5, this interaction is dependent on the c - terminus of acs5, as eto1 fails to interact with acs5 (pintard et al ., 2004; wang et al ., 2004). Consequently, these studies suggest that eto1 serves as a substrate - specific adaptor to bridge acs5 to the cul3 to regulate acs5 protein degradation . Analysis of eto1 function in plants shows that eto1 suppresses cytokinin - induced acs protein stabilization via the c - terminus of acs5 . Overexpression of eto1 inhibits cytokinin - induced ethylene biosynthesis, and overexpression of acs5 results in a partial constitutive triple response which is suppressed by co - expression of eto1 (wang et al ., 2004). However, additional data indicate that cytokinin - mediated stabilization and eto1-mediated destabilization are at least partially independent effects; exogenous cytokinin treatment still increases acs5 protein stability in etiolated eto2 and eto3 seedlings, suggesting cytokinin partially acts via an alternative mechanism that is independent of eto1 and acs5 c - terminus (hansen et al ., 2009). Other factors that modify e3 ligase function involved in ethylene biosynthesis are the related to the ubiquitin (rub) and rub1 conjugating enzyme (rce1) (bostick et al ., 2004; larsen and cancel, 2004). Like ubiquitin, rub functions through a covalent attachment to target proteins . In arabidopsis, rub attaches to the cullins, thereby promoting the activity of the scf (for skp, cdc53p / cul1, and f - box protein) ubiquitin ligase complex for polyubiquitination of target proteins . Interestingly, rna interference lines of rub exhibit the partial triple response in etiolated seedlings due to the increase in ethylene biosynthesis, implying that conjugation of rub to cul3 is required for the activation of eto1 containing cul3 e3 ligase complex (bostick et al ., 2004). Analysis of the rce1 mutant also demonstrated that the modification of rub is required for regulating ethylene biosynthesis (larsen and cancel, 2004). Rce1 encodes a rub conjugating enzyme, and has been shown to conjugate rub to the scf complex to modify the activity of the complex . Interestingly, the basis of ethylene overproduction phenotype of rce1 is not due to enhanced acs activity, but due to an increase of aco activity . Aco is generally not a rate - limiting step in ethylene biosynthesis during vegetative arabidopsis tissues; however, ethylene biosynthesis in r. palustris is limited by aco activity during submergence . Elevated ethylene levels have been shown to increase aco activity, which could explain the increased aco activity in rce1 (vriezen et al ., 1999). Xbat32, a ring domain - containing ankyrin repeat subfamily of e3 ligases, also plays a role in ethylene biosynthesis by controlling the turnover of acs4 (type-2) and acs7 (type-3) (lyzenga et al ., 2012). Xbat32 was previously identified as a positive regulator of lateral root development and the xbat32 mutant produces significantly less ethylene than wild type (nodzon et al ., 2004; prasad et al ., 2010). Xbat32 interacts with the acs proteins and it catalyzes the attachment of ubiquitin to the acs proteins (prasad et al ., 2010), suggesting xbat32 negatively regulates the ethylene biosynthesis by regulating the stability of acs proteins . Phosphorylation is one of the most abundant post - translational modifications which affect many important aspects of protein function, including activity, stability, subcellular localization and protein - protein interaction (holt et al ., 2009). Mounting evidence suggests that ethylene biosynthesis is regulated by phosphorylation events which likely influence acs protein turnover . Studies from the application of kinase and phosphatase inhibitors to tomato suspension cell cultures and tissues indicated that phosphorylation regulates the activity and/or turnover of acs (kamiyoshihara et al ., 2010). A cdpk present in the extracts of wounded tomato fruits phosphorylates leacs2 (mayfield et al ., 2007). The extract containing cdpk activity phosphorylates the c - terminal domain of leacs2 in vitro, but the activity of the leacs2 does not show a significant increase, suggesting phosphorylation regulates the turnover of acs rather than affecting the activity . The c - terminus of leacs2 contains a consensus phosphorylation target site for a cdpk, and this cdpk recognition site is present in a subset of acs isoforms (fig . 2) (kamiyoshihara et al ., 2010). Unlike the target sites of mapk in the c - terminal domain of the type-1 acs proteins, phosphorylation of the cdpk target site, which lies immediately upstream from the target site for mapk has not been shown to be phosphorylated in vivo and in vitro . Among three types of acs, protein stability of the type-1 and genetic and biochemical studies of a mapk pathway have revealed that pathogen - activated arabidopsis mpk6 phosphorylates the type-1 acs2 and acs6, which leads to increased accumulation of these acs proteins and, hence, increases ethylene production (joo et al ., 2008; liu and zhang, 2004). Mpk6 phosphorylates 3 serine residues residing within the consensus mapk target site in the c - terminus in vitro, suggesting mpk6-mediated phosphorylation of acs2 and acs6 prevents their degradation, resulting in an increase in ethylene biosynthesis in response to pathogen attack . 2014) report a similar result that rice mpk3 and 6 are involved in ethylene production via the salt - intolerance 1 receptor - like kinase (sit1), but acs stability was not discussed in their work . Until recently, the effect of phosphorylation on type-2 acs protein stability was not clear; neither direct phosphorylation nor responsible kinase has been identified . However, a recent study demonstrated that a casein kinase isoform 1.8 (ck 1.8) phosphorylates the type-2 acs5 protein, which in turn promotes the interaction between eto1 and acs5, resulting in the degradation of acs5 protein (tan and xue, 2014). The ck1.8 mutant displays the constitutive triple response due to overproduction of ethylene similar to the eto mutant . Interestingly, the triple response of ck1.8 seedlings is only observed in the hypocotyl and hook, not in the roots, implying that ck1.8 affects specific aspects of ethylene - mediated seedling growth responses . Several recent studies have identified novel regulatory factors which target multiple acs isoforms belonging in different types of acs (fig . 3). This regulatory feature is distinct from that of previous known regulatory proteins with a type - specific targeting (e.g. Eto1/eol e3 ligase for type-2 and mapk3/6 for type-1 acs). 14 - 3 - 3 proteins, novel regulator of ethylene biosynthesis, target all three types of acs (fig . 14 - 3 - 3 proteins are a family of evolutionarily well - conserved dimer proteins that specifically interact with phosphoproteins and are involved in a diverse array of physiological processes (darling et al ., 2005; dougherty and morrison, 2004; freeman and morrison, 2011). Upon interaction with target proteins, 14 - 3 - 3 proteins change their localization, stability, and activity, resulting in changes in physiological responses (freeman and morrison, 2011). There are 13 functional 14 - 3 - 3 genes in arabidopsis and their encoded proteins possess a highly conserved target binding domain, which can recognize a short stretch of peptide containing phosphoserine or phosphothreonin on target proteins (aitken et al ., 1992; de boer et al ., 2013; denison et al ., 2011 this could allow different 14 - 3 - 3 isoforms to function redundantly by recognizing similar sets of target proteins . However, increasing results suggest that a defined subset of 14 - 3 - 3 isoforms display specific functions, such as the regulation of stomatal opening, flowering time, and phytochrome signaling in arabidopsis (mayfield et al ., 2007; paul et al . It is unclear whether this is a result of biochemical specificity or simply differences in expression patterns of 14 - 3 - 3 isoforms in plants . 14 - 3 - 3 interacts with all three types of acs proteins via a non - c - terminal domain of the proteins and there is no specificity in the interaction between 14 - 3 - 3 isoforms and acs proteins in bimolecular fluorescence complementation assay (yoon and kieber, 2013a). 14 - 3 - 3 stabilizes acs protein by direct interaction and by negatively regulating the stability of the e3 ligases, eto1/eols, which specifically target the type-2 acs proteins for degradation (yoon and kieber, 2013a). Studies from mammalian and yeast systems have suggested that the stability of f - box proteins which promote ubiquitination in the ubiquitinproteasome pathway, is regulated based on the availability of substrates through an autocatalytic process (ho et al . It is possible that 14 - 3 - 3 proteins preferentially interact with type-2 acs proteins, which in turn leads to the depletion of the acs proteins for the eto1/eols, thus regulating the turnover of both sets of proteins . Alternatively, the interaction with dimeric 14 - 3 - 3 proteins may cause the eto1/eols to dimerize, thereby promoting self - ubiquitination and subsequent degradation . Finally, the interaction with 14 - 3 - 3 proteins could enhance the interaction with distinct e3 ligases, such as xbat32, leading to the ubiquitination and subsequent degradation of the eto1/eols . Intriguingly, in mammalian cells, 14 - 3 - 3 interacts with and regulates the protein stability of a short - lived p53 tumor suppressor protein and its cognate e3 ligases cop1 and mdm2 (su et al ., 2011; yang et al ., 2007). 14 - 3 - 3 stabilizes p53 by down - regulation of mdm2 and cop1 protein stability . This 14 - 3 - 3-mediated inverse stability regulation on p53 and mdm2 and cop shows a similar regulatory mechanism by which arabidopsis 14 - 3 - 3 control the protein stability of acs5 and eto1/eols, suggesting that the function of a subset of 14 - 3 - 3 isoforms in protein stability regulation is evolutionarily conserved between mammalian and plants . Several findings imply that 14 - 3 - 3 also regulates acs stability independently of eto1/eols (yoon and kieber, 2013a; 2013b). First, 14 - 3 - 3 interacts with acs5, a type-2 acs with a lack of toe motif for eto1/eol interaction . Secondly, 14 - 3 - 3 directly interacts and stabilizes the sole type-3 acs7 and type-1 acs2, whose protein stability is not regulated by the eto1/eols . Thus, there is at least one other system acting to degrade type-2 acs proteins in addition to the eto1/eols, but in the manner dependent on 14 - 3 - 3 function . This is consistent with the observation that cytokinin and brassinosteroid increase type-2 acs function partly through a toe - independent mechanism (hansen et al ., 2009). While 14 - 3 - 3 positively regulates acs protein stability (yoon and kieber, 2013a; 2013b), studies from characterization of the rare cold inducible 1a (rci1a), which encodes a 14 - 3 - 3 isoform suggest that 14 - 3 - 3 negatively regulate the protein stability of acs in response to cold stress (catala et al ., 2014). Rci1a mutant displays increased levels of acs6 protein in response to cold treatment, and this change is not due to the changes of the acs6 mrna levels . However, the direct effect of 14 - 3 - 3 on acs protein stability was not demonstrated in the study . Suggest that the discrepancy of two studies might be contributed to the functional specificities of 14 - 3 - 3 isoforms; yoon and kieber (2013) used 14 - 3 - 3, while catala et al . (2014) used 14 - 3 - 3 isoform (bornke, 2005; fu et al ., 2000; a recent study also reported that single 14 - 3 - 3 isoform could have distinct functions depending on the binding sites on a given target protein (ganguly et al ., 2005). Identification and characterization of the role of ck1.8 have also brought new insights into the post - translational regulation in ethylene biosynthesis (tan and xue, 2014). Ck1.8 is a conserved serine / threonine protein kinase that plays role in various physiological processes, including blue light signaling, flowering, microtubule organization and brassinosteroid signaling in rice (ben - nissan et al ., 2008; dai and xue, 2010; liu et al ., 2003; tan et al ., 2013). As briefly discussed in the previous section, the ck1.8 mutant overproduces ethylene resulting from accumulation of acs proteins, suggesting ck1.8 is a negative regulator of acs protein stability . Ck1.8 phosphorylates acs5 at threonine residue 463 (t463) which is located within the toe motif, and phosphorylation on this site promotes the interaction with eto1, indicating the phosphorylation of t463 on acs5 plays a negative role in acs5 protein stability . It can be found in only in a subset of arabidopsis type-2 acs (acs5 and acs9), type-1 (acs6), and tomato type-1 (leacs2), suggesting that ck1.8 could target different acs types rather than committing to regulate a specific type of acs (fig . The role of ck1.8 is somewhat in contrast to what has been observed from other studies indicating that phosphorylation promotes acs protein stability . The roots curl in 1-n - naphthylphthalamic acid 1 (rcn1) gene encodes one of three regulatory / scaffolding a subunits of arabidopsis pp2a, and targets the type-1 protein acs2 and acs6 for regulating their stability (skottke et al ., 2011). Genetic studies revealed that the function of rcn1 requires acs2 and acs6 and the rcn1 mutant exhibits increased accumulation of the acs6, suggesting phosphorylation promotes the protein stability of type-1 acs . Strikingly, rcn1 shows different effects on type-2 acs5 protein stability . In etiolated rcn1 seedlings, the accumulation of myc - tagged acs5 is significantly reduced, whereas accumulation and turnover of the myc - acs5 is not affected, indicating that rcn1 plays a positive role in acs5 stability through the toe motif of acs5 . However, rcn1-directed dephosphorylation on acs5 has not been evidenced, suggesting the possibility that rcn1 may dephosphorylate the eto1 complex . Both rcn1 and ck1.8 regulate the stability of acs5 through the toe domain, but it is not clear whether the effects of rcn1 on acs5 are dependent on ck1.8 or eto1/ eols . Due to the lack of regulatory motifs in the c - terminal domain, including phosphorylation sites, it was considered that acs7, the sole type-3 acs, may not be subjected to proteasome - mediated degradation pathway, and that it may be more stable than other acs proteins (chae and kieber, 2005). However, lyzenga et al ., recently showed that the protein stability of acs7 is also governed by the ubiquitin - mediated proteasomal degradation, and that degradation requires the ring - type e3 ligase xbat32 (lyzenga et al . Interestingly, xbat32 also confers protein instability to the type-2 acs4, suggesting xbat32-mediated degradation mechanism is not specific for the type-3 acs . A cell - free degradation assay shows that changes in 4 lysine residues in the c - terminal domain of acs4 results in accelerating degradation of acs4 protein . This result is similar to the observation that k435r in the c - terminus of flag - acs7 promotes the turnover rate of the acs7, suggesting the c - terminal lysine residues are not for ubiquitination, but for stabilization of acs4 and acs7 . Shortly after, xiong et al . They showed that destabilization sequences of the acs7 are located in the n - terminus of acs7 . The n - terminal 54 residues of the acs7 confer significant instability to acs7-gus and first 14 amino acids are responsible for negative regulation of the acs7 protein stability (xiong et al ., 2014). One possible explanation for this may be due to the nature of the c - terminal fusion of the acs7-gus used in the study . Traditionally, the n - terminal fusion of acs has been routinely utilized for studying the turnover of acs to avoid masking the c - terminal regulatory domain and this may be blamed for concealing the destabilization signals located at the n - termini and making only the c - terminal signals available to degradation machinery . It is interesting to further study the role of the n - terminal domain of other types of acs whether they also contain putative degradation sequences in their n - termini . Several studies indicate that there are molecular components acing on the non - c - terminal domain of acs proteins to regulate their stability . Cytokinin and brassinosteroid stabilize acs5 and acs9 and the effects of these two hormones on the protein stability are additive, suggesting cytokinin and brassinosteroid act through distinct toe - independent mechanisms on these acs proteins (hansen et al ., 2009). Genetic studies showed that cytokinin - mediated acs stabilization requires a functional cytokinin signaling pathway (hansen et al ., 2009). Mutation in the signaling components, cytokinin receptors, ahps, and type - a and type - b transcription factors, in the cytokinin signaling pathway produce reduced amounts of ethylene in response to exogenous cytokinin . The effect of brassinosteroid in ethylene biosynthesis is somewhat distinguished from the typical triple response that has been observed with cytokinin treatment; br treatment results in a shortened and thickened hypocotyl formation; but it does not induce an exaggerated hook formation; and shortening of the root and hypocotyl is less severe than for cytokinin - treated seedlings . Interestingly, the ethylene - insensitive mutant ein2 - 5 still shows cytokinin and br induced hypocotyl phenotypes, although the extent is not as severe as in wild type, indicating this process is independent of the ethylene signaling pathway . Although it has not been demonstrated whether gibberellin regulates the turnover of acs proteins, studies from the characterization of a gai;eto2 (gibberellin insensitive;ethylene overproducing) double mutant showed that ga signaling is required for acs stabilization via the toe - independent manner, as the overproduction phenotype of eto2 is abolished in the gai;eto2 double mutant (de grauwe et al . Furthermore, 14 - 3 - 3-mediated eto1/eol - independent stabilization of acs proteins also indicates the existence of an alternative mechanism to stabilize acs proteins (yoon and kieber, 2013a). It is not clear whether the eto1/eol - independent mechanism acts in the same pathway that is utilized by the other factors, but is it of great interest to further study to identify molecular elements involving in these regulatory pathways . Together, these studies indicate that there are molecular components that act as the points of cross - talk between ethylene biosynthesis and other hormonal signaling pathways . Identification of these elements will bring new insights into understanding the mechanism by which protein turnover of acs is regulated to coordinate and merge different hormonal inputs to regulate ethylene production which effects on many diverse ranges of plant growth and development.
Skeletal muscle metastasis as the initial presentation of an unknown primary lung cancer is unusual . F-18 fluorodeoxyglucose (fdg) positron emission tomography / computed tomography (pet / ct) imaging is useful in the identification of primary in carcinoma of unknown origin . We describe a patient showing fdg - avid metastases to the skeletal muscles along with a previously unknown primary tumor in the right lung, in a patient presenting with initial muscular symptoms without any pulmonary manifestations . The patient underwent a whole body f-18 fdg pet / ct to identify the site of the primary malignancy . Increased fdg avidity (standardized uptake value [suvmax] 9.0) was detected in an irregular heterogeneously enhancing soft - tissue mass in the right paravertebral region in the upper lobe of the right lung with a focus of calcification within the mass [figure 1b and d, white arrow]. Abnormal fdg uptake was also noted in a presacral mass [figure 1c and e], the bulky left adrenal gland, several dorsal vertebrae and multiple lesions in the trapezius [figure 1b and d, red arrow], right brachioradialis [figure 2], deltoid, and right external oblique muscles [figure 3], suggestive of metastatic involvement . A diagnosis of primary squamous cell carcinoma of the lung was pathologically the patient was treated with 4 cycles of chemotherapy, after which significant decrease in fdg uptake (suvmax = 5.1) was seen in the primary as well as the right brachioradialis muscle lesion (not shown here). Fluorodeoxyglucose (fdg) positron emission tomography / computed tomography (pet / ct) maximum intensity projection (mip) image (a) showing multiple foci of abnormal tracer uptake . Transaxial thoracic ct (b) and fused pet / ct image (d) show increased fdg uptake in an irregular, heterogeneously enhancing soft - tissue mass (white arrow) in the paravertebral region in the upper lobe of the right lung with calcification within the mass . Increased fdg uptake is also seen in a peripherally enhancing ring like lesion in the trapezius (red arrow). Axial ct (c) and fused pet / ct (e) images at the level of the rectum show increased fdg uptake in a heterogeneously enhancing pre - sacral soft - tissue deposit coronal and transaxial computed tomography (ct) (a and c) and fused positron emission tomography / ct (b and d) images of the right hand showing increased fluorodeoxyglucose uptake in the right brachioradialis muscle with no increase in attenuation (probably because the images were acquired after completion of the whole body pet scan) axial computed tomography (a) and fused positron emission tomography / computed tomography (b) images showing increased tracer uptake in a ring - like hyperenhancing lesion in the right external oblique muscle suggestive of muscle metastasis found that only 4 patients (0.16%) among 2,557 patients with lung cancer developed metastasis to the skeletal muscle . Whole - body fdg pet / ct imaging is useful in detection of muscle metastases in lung cancer patients . Multiple muscle metastases from lung cancer are rare, and fdg pet / ct imaging is useful in the identification of unsuspected metastatic sites . Primary presentation of a skeletal muscle metastasis, such as in our case, remains an unusual occurrence . The present case, where the initial presentation was of metastatic muscular involvement, highlights the role of fdg pet / ct in tracing the location of primary lung malignancy and unsuspected sites of multiple muscle metastases in a patient with muscle metastases of unknown primary.
The lung is an organ that performs a multitude of vital functions every second of our lives . This fact leads to considering lung abnormalities, life - sustained diseases that have high priority in detection, diagnosis, and treatment if possible . Our focus in this paper will be on two popular abnormalities within the lung, which are pulmonary edema and lung tumor . Pulmonary edema (water in the lungs) is caused by fluid building up in the air sacs of the lungs [1, 2]. On the other hand, lung cancer / tumor is a disease where uncontrolled cell growth in tissues of the lung occurred . Computer - aided diagnosis (cad) schemes for thoracic computed tomography (ct) are widely used to characterize, quantify, and detect numerous lung abnormalities, such as pulmonary edema and lung cancer [4, 5]. An accurate lung segmentation method is always a critical first step in these cad schemes and can significantly improve the performance level of these schemes . Although manual or semiautomatic lung segmentation methods for ct images were used in some early cad schemes [610], they are impractical for current cad schemes because multidetector ct (mdct) scanners can generate hundreds of ct slices for a patient . An automated method for lung segmentation in addition, the eye identification / detection of the abnormality type (pulmonary edema or tumors) in computed tomography (ct) images is very difficult even for the experienced clinicians because of its variable shape along with low contrast and high noise associated with it . As the final stage of treating the lung cancer is surgical removal of the diseased lung, hence it is necessary to identify the cancer location, which can be useful before they plan for the surgery . The aim of our work is to develop an automated novel texture analysis based method for the segmentation of the lungs and the detection of the abnormalities, whether pulmonary edema or lung tumor . Haralick's features based on the gray level cooccurrence matrix (glcm) are applied to capture textural patterns in lung images . The objective of this work is the selection of the most discriminating and finding out the significant texture features that can differentiate between these two types of abnormalities, in comparison to normal . These measurements are utilized to describe the overall texture of the image using measures such as entropy and sum of variance . Propose an approach, based on haralick's features, to detect and classify colon cancer cells . A study to investigate the feasibility of using haralick features to discriminate between cancer and normal subimages within a patient is illustrated in . In this paper, ct images are first preprocessed for noise reduction and image enhancement, followed by segmentation techniques, as the tools to segment the lungs, and finally haralick texture features [1315] are calculated . Statistical analysis is done to detect the most significant haralick features that will characterize the type of the abnormality within the lungs . Despite the low contrast and high noise existence in the images, the proposed algorithms introduce promising results in detecting the abnormality of lungs in most of the patients in comparison with the normal . This paper presents a new automatic lung cancer detection system based on haralick texture features extracted from the slice of dicom lung ct images . The proposed system is accomplished in four stages: image preprocessing, lung image segmentation, feature extraction, and classification . Statistical analysis is used to obtain the best features for classification to differentiate between lung cancer patients, ordered edema patients, and control subjects . Patients with either a lung cancer tumor or pulmonary edema were encompassed in the study . This study included two datasets, the first dataset referred to the radiology department at new elkasr elainy teaching hospital, university of cairo . The other dataset was obtained from the cancer imaging archive (tcia) sponsored by the spie, nci / nih, aapm, and the university of chicago . Ct image, we separate the left lung from the right lung automatically, and each separated lung is labeled as normal or edema / cancer based on the dataset information . The main goal of preprocessing is to improve the quality of an image as well as make it in a form suited for further processing by human or machine . This is accomplished by enhancing the visual appearance of an image besides removing the irrelevant noise and unwanted parts in the background . The proposed enhancement process, which is based on combining filters and noise reduction techniques for pre- and postprocessing as well, is carried out applying histogram equalization (he) [1820] followed by wiener filtering [21, 22]. Figure 1 presents the enhancement in the lung image contrast attained by applying the histogram equalization . However, the obtained gray scale image contains noises such as white noise and salt and pepper noise . Thus, wiener filter is utilized to remove these noises from the enhanced lung image . Figure 1(c) shows the effect of applying weiner filter on the contrast enhanced lung image . Lung segmentation step aims to basically extract the voxels corresponding to the lung cavity in the axial ct scan slices from the surrounding lung anatomy . This technique is based on the fact that there is a large density difference between air - filled lung tissues and surrounding tissues . Furthermore, both lungs are almost looking like mirror images of themselves in a human body . The segmentation of lung regions is achieved through the following steps . In the first step, the preprocessed ct image is converted into a binary image; a threshold of 128 was selected . Values greater than the threshold are mapped to white, while others less than that are marked as black . Consequently, the two lungs are marked and the area around them is cropped out . Second, an erosion morphological operation is employed in order to eliminate any white pixels within the two lungs . Afterward, black pixels for each region in the eroded image are counted; the region with the largest black area will be deemed as a lung mask . The attained lung mask is reflected in the opposite direction . As a result these masks are multiplied with the corresponding original image regions; this will project the lung masks on the original two lungs images . Finally, update each black pixel in the obtained images by its original value; other pixels are set to 255 . Figure 2 illustrates the lung extraction process . Feature extraction is the process of obtaining higher - level information of an image such as color, shape, and texture . Statistical texture methods analyze the spatial distribution of gray values, by computing local features at each point in the image and inferring a set of statistics from the distributions of the local features . This technique has been widely used in image analysis applications, especially in the biomedical field . The glcm is computed in the first step, while the texture features based on the glcm are calculated in the second step . Glcm shows how often each gray level occurs at a pixel located at a fixed geometric position relative to each other pixel, as a function of the gray level . The horizontal direction 00 with a range of 1 (nearest neighbor) was used in this work . The 9 texture descriptions used are presented in (4) to (13), where ng is the number of gray levels, pd is the normalized symmetric glcm of dimension ng ng, and pd(i, j) is the (i, j)th element of the normalized glcm . Contrast (moment 2 or standard deviation) is a measure of intensity or gray level variations between the reference pixel and its neighbor . Large contrast reflects large intensity differences in glcm:(1)contrast=ijij2pdi, j.homogeneity measures how close the distribution of elements in the glcm is to the diagonal of glcm . As homogeneity increases, the contrast, typically, decreases:(2)homogeneity=ij11+ij2pdi the value of entropy is the largest when all elements of the cooccurrence matrix are the same and small when elements are unequal:(3)entropy=ijpdi, jlnpdi, j.energy is derived from the angular second moment (asm). The asm measures the local uniformity of the gray levels . When pixels are very similar, the asm value will be large . Consider(4)energy = asmasm=ijpd2i, j.correlation feature shows the linear dependency of gray level values in the cooccurrence matrix: (5)correlation=ijpdi, jixjyxy, where x; y and x; y are the means and standard deviations and are expressed as(6)x=ijipdi, jy=ijjpdi, jx= ijix2pdi, jy=ijjy2pdi, j.the moments are the statistical expectation of certain power functions of a random variable and are characterized as follows . Moment 1 (m1) is the mean which is the average of pixel values in an image and it is represented as(7)m1=ijijpdi, j.moment 2 (m2) is the standard deviation that can be denoted as (8)m2=ijij2pdi, j.moment 3 (m3) measures the degree of asymmetry in the distribution and it is defined as (9)m3=ijij3pdi, j.and finally moment 4 (m4) measures the relative peak or flatness of a distribution and is also known as kurtosis:(10)m4=ijij4pdi, j.furthermore, difference statistics that are a subset of the cooccurrence matrix are also used . These features are based on the distribution of probability pxy(k) which is defined as follows:(11)pxyk=cdi, j, k=0,1,,ng1,where cd(i, j) is the (i, j)th element of the glcm . The most basic difference statistic texture descriptions are the asm, mean, and entropy:(12)asm=kpxyk2.when the pxy(k) values are very similar or close, asm is small . Asm is large when certain values are high and others are low:(13)mean=kkpxyk.when pxy(k) values are concentrated near the origin, mean is small and mean is large when they are far from the origin:(14)entropy=kpxyklogpxyk.entropy is smallest when pxy(k) values are unequal and largest when pxy(k) values are equal . The calculation of the haralick texture features using the previous equations for the ct images volume sequences for every segmented lung (right and left) separately was performed . For each participant the gray level cooccurrence texture features: contrast, homogeneity, entropy, energy, correlation, and m1, m2, m3, and m4 accompanied by the difference statistical features: asm, contrast, mean, and entropy were obtained for each segmented lung (right and left). For the purpose of random lung assignment in healthy volunteers, the left lung represented the diseased lung in the same percentage of cases as the patient population . For the acute data, two single factor analyses of variance (anova) tests were conducted for each haralick texture feature measurement between affected (either left or right) and fellow lung (either left or right) for both categories cancer and edema patients . A single factor analysis of variance (anova) other between - subject single factor analyses were conducted to find out the significant haralick features that could differentiate cancer from edema ., we separate the left lung from the right lung automatically as discussed before in section 2.3, and each separated lung is labeled as normal or edema / cancer based on the dataset information . The haralick texture features measurements for each lung separately are calculated (the gray level cooccurrence texture features: contrast, homogeneity, entropy, energy, correlation, and moments along with the difference statistical features: asm, mean, and entropy). The mean and the standard deviation of the haralick texture features measurements calculated as well as the anova results are given for tumor patients affected lung versus fellow lung in table 1 and for pulmonary edema patients in table 2 . The anova summary of statistics for either pulmonary edema or tumor patients versus normal is given in table 3 . The significant haralick texture features that can differentiate between pulmonary edema and tumor are found in table 4 . From table 1, we can conclude that haralick texture features measurements (homogeneity, energy, correlation, and entropy) of the affected cancer lung were significantly different than that of the fellow lung . The homogeneity, energy, and correlation were significantly less than those of the normal fellow lung . While entropy of the cancerous lung is approaching being significantly more than that of the fellow lung, moment 3 and the difference statistical feature asm (diff_asm) texture feature measurement of the cancerous lung is approaching being significantly less than that of the normal lung . Table 2 showed that haralick texture features measurements (homogeneity, entropy, and moments calculated from the cooccurrence matrix as well as mean and asm computed from the difference statistics) of the pulmonary edema affected lung were also significantly different than those of the control subject lung; moreover contrast and entropy computed from the difference statistics were significantly more than those of the fellow lung . Considering tables 1 and 2, we can conclude from table 3 that the homogeneity, energy, entropy, m3, m4, diff_asm, diff_mean, and diff_entropy are good biomarkers to significantly differentiate between diseased and normal lungs without any disease specification . On the other hand, the results illustrated in tables 1, 2, and 4 show that entropy and the entropy calculated from the difference statistics would be a good candidate to significantly differentiate between pulmonary edema and cancer . The texture features analyses are well known approaches to quantify and express the heterogeneity that may not be appreciated by clinical naked eyes, and it was presented before as good imaging biomarkers to differentiate between diseases . In this paper an evaluation of the haralick texture features is done in order to identify the most significant features that can be used in order to detect and differentiate abnormalities within the lungs for cancer and edema versus normal . Our results indicate that entropy determined by gray level cooccurrence matrix and asm is significantly different in edema patients versus normal while it is not in cancer patients versus normal . Since the entropy is the degree of randomness or the degree of disorder in the image, and the angular second moment represents the uniformity in the image, this may be interpreted as the cancer disease causing a localized heterogeneity in the diseased specified area in the lung while the edema causes heterogeneous disorder in the whole lung image . High entropy values calculated implies that the elevated level of disorder and disorganization occurred due to the edema diseased lung versus the cancer diseased lung . The energy feature that is derived from the angular second moment measures and representing the local uniformity of the gray levels is a good biomarker to differentiate between cancer and edema diseases . From table 2, contrast is a good biomarker for the pulmonary edema disease and this agrees with the texture feature meaning which means high contrast values for heavy texture changes . Gray level cooccurrence matrix textural properties such as homogeneity, correlation, mean, and moments are good significant biomarkers for diseased lung versus normal ones in general without any specification for the disease type . Our results agree with other articles indicating that textural analysis has the potential to develop into a valuable clinical tool that improves the diagnosis, tumor staging, and therapy assessment . While our results are promising, there is still further work that can be done in the detecting of the abnormality within the lungs to detect the type of that abnormality whether it will be a lung cancer or edema . A preliminary investigation has been done using statistical analysis to identify the most useful texture features that can be fed to any classification technique later . This statistical analysis is done using anova . After selecting these features we can feed them for better localization and classification as further work.
Precise control of the adsorption of proteins on solid surfaces is a key to a wide variety of biological and technological applications . Proteins are commonly immobilized on surfaces both in microarrays and other studies of protein function and in the creation of biosensors and biocatalysts . The success of these applications depends on proteins maintaining their native state and function when adsorbed to the surface and on the prevention of nonspecific protein binding . Protein adsorption also plays an important role in the outcome of biomaterials (e.g., biomedical implants, artificial tissue scaffolds, and nanoparticles for drug delivery) in vivo, as proper protein adsorption contributes to cell adhesion and the integration of the biomaterial with the circulatory system, while the adsorption of undesired proteins can contribute to failure due to immune responses or fouling . Surface interactions are also relevant to understanding many diseases, as they are the first step in many biological processes, including blood clotting and the formation of protein aggregates, such as the amyloid plaques found in alzheimer s disease . Because of the importance of protein adsorption in these many applications, investigation of the interplay between the folding and adsorption processes and how adsorption impacts protein conformations is highly valuable . Experiments, including a variety of spectroscopic methods, have been able to show that surface adsorption can result in changes in the conformation and thermodynamic stability of a protein and that these changes are dependent on a variety of factors, such as temperature, ph, protein concentration, and the hydrophobicities of the protein and surface . However, experiments have not been able to fully address many aspects of the relationship between protein adsorption and conformational changes and are complicated by the complex heterogeneity of interactions between real protein chains and surfaces . Therefore, theoretical and computational efforts that often utilize simplified, coarse - grained protein models have been used to supplement experiments and provide a basic understanding of protein adsorption . One minimalist model used to investigate protein folding and adsorption is the hydrophobic - polar (hp) model, in which protein monomers are modeled on a lattice as either hydrophobic (h) or polar (p) beads . Within the hp model, the many thermodynamic factors underlying complex processes, such as protein folding and adsorption, are reduced to a few basic terms (i.e., enthalpic interactions between chain segments or between the chain and surface and entropically excluded volume interactions). One route through which the hp model can be used to understand protein adsorption and/or folding is the study of two transitions, the coil globule and the critical adsorption transitions, that are the result of a balance between these thermodynamic terms . The coil globule or collapse transition is one of the first steps in the protein folding process and occurs when attractive interactions between hydrophobic protein monomers become strong enough to balance the conformational entropy lost by the protein adopting compact globule conformations . A recent experiment using synthetic polymers containing hydrophobic and polar monomers, mimicking hp model chains, confirmed that attractions between hydrophobic monomers are sufficient to be the driving force of the collapse transition . The critical adsorption point (cap) on the other hand marks the transition of a protein which prefers being in solution to being adsorbed on the surface and also involves a balance between entropic and enthalpic effects . The cap is the point at which a polymer just becomes adsorbed to a surface and occurs when the conformational entropy lost by a polymer chain near a surface is offset by attractive interactions with the surface . Thus, the thermodynamics and potential conformational changes of the process through which a folded protein adsorbs on a surface, for example, can be understood in terms of these two transitions, as some hydrophobic interactions underlying the collapsed conformation of the protein can unravel to allow for additional chain surface attractive interactions . Over the last quarter century, the hp and other simple coarse - grained models have been successfully used to provide insight into the conformational changes of proteins and other macromolecules during adsorption both in terms of these transitions and in a variety of other ways . First, the adsorption of hp - like chains with various sequence types on surfaces with various patterns has been studied to understand pattern recognition . These studies have revealed that the adsorption of copolymers on heterogeneous surfaces can proceed via an initial nonspecific adsorption similar to the critical adsorption transition, followed by a reorganization in which the surface pattern is recognized by the copolymer, and that such a two - stage adsorption process depends on the chain sequence, surface pattern, and interaction parameters . Additionally, moghaddam and chan investigated the adsorption of block copolymers on patterned surfaces and showed that the sharpness of the adsorption transition was enhanced through the introduction of additional either attractive or repulsive chain surface interactions . However, these studies did not include intrachain interactions and, therefore, could not consider the balance between intrachain and chain surface interactions that underlies protein adsorption . Second, the adsorption of a homopolymer with intrachain interactions (i.e., basically, a chain consisting of only the h beads of an hp chain) has been considered, and it was shown that the presence of the surface promoted chain collapse and increased the internal structural organization (e.g., helices and antiparallel sheets) in the chain . Also, chains with strong intrachain interactions were shown to undergo two types of adsorption transitions: a docking transition, in which a collapsed chain does not deform upon adsorption, for weak chain surface attractions and a flattening transition, in which the chain adopts two - dimensional conformations after adsorption for strong chain surface attractions . Finally, several studies have examined the adsorption of hp chains directly . Rybicka and sikorski compared the adsorption of several hp sequences with that of a homopolymer containing only h - type beads on a homogeneous surface . They showed that the collapse of chains weakly adsorbed on a surface was roughly independent of the chain sequence; however, under strong adsorption, chains underwent a sequence - dependent rearrangement similar to the previously discussed flattening transition . Studies of hp chains interacting with surfaces have also confirmed the experimental observation that the presence of the surface can significantly alter the lowest energy conformation of a folded protein and have been used to determine conformational pseudophase diagrams of hp chains near a surface that shows how temperature and the strength of attractive interactions impact adsorption and folding . In this work, we seek to increase understanding of the relationship between protein adsorption and conformation change through a systematic examination of the interplay between the collapse and critical adsorption transitions . Specifically, we study the protein folding and adsorption processes by determining the temperatures of collapse, tc, and critical adsorption, tcap, transitions for hp chains end - grafted to a solid surface that equally attracts h and p monomers . In contrast with most previous simulations of the adsorption of hp chains that have used a specific short sequence with a well - defined ground state, we use a simple alternating hp sequence and vary the chain length from 10 to 400, allowing us to investigate the potential influence of chain length on collapse and adsorption . The current study also investigates the behavior of the chain at more intrachain attraction strengths and over a wider temperature range than was considered in previous studies of the relationship between chain collapse and surface adsorption . We show that, while the critical adsorption point can always be observed and is roughly independent of chain length, tcap is affected by the presence of intrachain attractions in some cases . Specifically, if tc> tcap (i.e., the chain is already collapsed when the adsorption transition is attempted), the critical adsorption transition occurs at a higher temperature than a corresponding homopolymer without intrachain attractions, indicating that collapsed chains are more easily adsorbed . In contrast, if tc <tcap (i.e., the chain is already adsorbed when the collapse transition is attempted), tc is suppressed and it is more difficult to collapse the adsorbed chain than a corresponding chain that is free in solution . Finally, we examine how the strength of the hydrophobic intrachain and chain surface attractions impact chain conformations . Our simulation system is embedded in the three - dimensional (3d) simple cubic (sc) lattice . A self - avoiding walk (saw) other sequences representing different protein types could have been used; however, since the current study focuses on the interplay between the two transitions, we focus on a generic alternating hp chain that can be easily extended to long chains . Additionally, we note that both the collapse and adsorption of an alternating hp sequence have been shown to differ from a homopolymer with intrachain attractive interactions . The protein of length n is composed of n/2 h monomers and n/2 p monomers . Bond lengths between monomers fluctuate among 1, 2, and 3 lattice constants . The bond can be taken from 26 allowed bond vectors obtained from the set {(1,0,0), (1,1,0), (1,1,1)} by symmetry operations of the sc lattice . However, bond crossing is not allowed . In this coarse - grained model, the monomers do not correspond to specific atoms in a polymer but rather to small groups of atoms, and the bonds do not represent specific covalent bonds between two atoms but, instead, the linkages between monomers . The simulation box is a cuboid with sizes lx, ly, and lz in the x, y, and z directions, respectively periodic boundary conditions are employed in the x and y directions, while the z direction is confined by an infinitely large flat surface located at z = 0 . The surface is impenetrable to the polymer, so polymer monomers are restricted to lie in the upper half space (z> 0). Polymer chain lengths studied are in the range of n = 10 to 400 . The simulation box is always large enough to ensure no finite size effects on the simulation results . To this end, the dimensions of the simulation box in all three directions are always larger than the chain length n. the first monomer, which is always an h, is considered to be adsorbed to the impenetrable surface and is grafted at the center of the z = 1 layer . Then the chain is subjected to brownian motion achieved by the dynamic monte carlo (mc) technique . Polymer monomers that are located on the z = 1 layer are considered to be adsorbed to the surface . Surface interaction is assigned for all monomers on the z = 1 layer next to the surface . A two - dimensional (2d) sketch of our 3d simulation system is presented in figure 1 . 2d sketch of our 3d simulation model for an end - grafted hp protein model chain . Monomers are numbered from 1 to n for a polymer with length n. the first monomer h is grafted to the surface . The nearest neighbor interactions are ehh, ehp, epp, ehs, and eps, as shown . The energy of a conformation is a summation of all nearest - neighbor (nn) contact interactions among the chain and all nearest - neighbor contact interactions between the chain and surface . Rij is the spatial distance between two nonbonded monomers i and j and zi is the distance of monomer i away from the surface . It is known that the adsorption of a copolymer chain is influenced by the properties of the surface . Here we set ehp = epp = 0, while the value of ehh is negative and varied . Therefore, the energy of polymer can be expressed as2where nhh, nhs, and nps represent the nn contact numbers of h h, h surface, and p surface pairs, respectively . \|ehs\| is used as the unit of energy while \|ehs\|/kb is the unit of temperature, where kb is the boltzmann constant . H interaction ehh and temperature t. variation in ehh from zero to negative numbers will allow us to investigate the adsorption of the hp chain on the surface in the absence (ehh = 0) or presence of the intrachain hydrophobic interaction . The brownian motion of the polymer chain is attributed to local moves of chain monomers . Polymer dynamics is achieved by bond fluctuation, similar to that used for one - site and eight - site polymer models on the sc lattice . For each trial move, a monomer is chosen randomly to move to one of its six nn sites . If the chosen site is already occupied by another monomer, or such a move will violate bond crossing and bond length restriction, the trial move is abandoned . Otherwise, the trial move will be accepted with a probability p = min[1, exp(e / kbt)], where e is the energy difference between new and old configurations . It has been pointed out that the metropolis method may have problems in describing the behavior of hp chains at low temperatures (kbt <0.3\|ehh\|) in comparison with the wang landau method . At these low temperatures, the sampling efficiency using the metropolis algorithm can be poor, as the polymer can become trapped in low - energy states . As we use the metropolis method in this work, we focus on polymer behavior near tcap and tc, which are higher than 0.3\|ehh\| . The results of the metropolis and wang landau methods are very similar for kbt> 0.3\|ehh\|, and ergodicity can be satisfied by a long simulation run using the metropolis algorithm . The time unit is one monte carlo step (mcs) during which n 1 trial moves are attempted since the first monomer is always adsorbed . To avoid correlation between two configurations, we measure the chain s statistical properties only after a regular time interval = n mcs . Typically, each simulation is run as long as 1000, and 5000 independent runs are simulated . We also adopted an annealing process in simulations of chain configuration at different temperatures . Simulations begin at a high temperature with the chain in a desorbed state and in a random coil configuration . The temperature decrement step is not a constant but is specially chosen in advance for clearly presenting the collapse and adsorption transitions and for saving calculation time simultaneously . To this end, we first roughly estimate the two transition temperatures using a simulation with a large temperature decrement step and then adopt a small temperature decrement step around the transition temperatures in a second simulation . At each temperature, the final configuration at the previous temperature was used as the initial configuration for the subsequent temperature . Every independent simulation run ends at a low temperature far below the cap, where the chain is in a deeply adsorbed state . We at first determine the collapse transition of the alternating hp model chain in a dilute solution . The dependence of the mean square end - to - end distance r on the temperature t is presented in figure 2 for the case where ehh = 1 . The inset shows that the scaling r n at high temperature changes to r n at low temperature, indicating a collapse transition from a random coil to a compact sphere . R/n has the steepest decrease at t = 0.75, that is, the temperature at which dr/dt is at a maximum . Therefore, we identify a coil globule transition at tc = 0.75 when ehh = 1 for the hp chain in dilute solution . Since the temperature t and ehh are interrelated through the boltzmann factor, a variation in ehh would shift tc according to tc = 0.75\|ehh\| for the hp chain in dilute solution . Here globule transition temperature of the hp chain in the dilute solution in the absence of any surface . Dependence of mean square end - to - end distance r on temperature t for the free hp chain with ehh = 1 in dilute solution . The inset presents the log log plot of r as a function of the chain length n at t = 2, 1, and 0.5 (from top to bottom). The straight lines are the best fits with slopes 1.19, 1.12, and 0.65 for t = 2, 1, and 0.5, respectively . Next, we simulate the adsorption of the end - grafted hp chain with ehh = 1 by annealing the chain with the head h monomer grafted on a flat surface and estimate the collapse transition and the critical adsorption transition temperatures . The critical adsorption transition temperature is estimated from the temperature dependence of the mean surface contact number m of the chain . M as a function of chain length at different temperatures is plotted in log log scales in figure 3 . On the basis of the eisenriegler, kremer, and binder (ekb) scaling theory, the scaling relation m n is satisfied at the critical adsorption point tcap . Meirovitch and livne have estimated that tcap = 3.44 0.01 and the crossover exponent = 0.530 0.007 for a saw chain with fixed bond length (b = 1) on the sc lattice with mc simulations . The plot of m versus chain length n has a concave upward curve at temperatures below tcap and a convex downward curve at temperatures above tcap . For the present bond - fluctuation saw hp model, we estimate tcap = 1.65 0.02 and = 0.54 0.01 . The results are close to that estimated for the adsorption of a bond - fluctuation saw homopolymer without intrachain attractive interactions, where tcap = 1.625 and = 0.52 have been estimated by using a finite - size scaling formula m c)n)], indicating that hydrophobic interactions have little effect on tcap as long as the attractive interactions with the surface are the same for both h and p monomers . Log log plot of the surface contact number m versus chain length n at temperatures t = 1.55, 1.6, 1.65, 1.7, and 1.75 for an hp polymer with ehh = 1 . The statistical error of each monte carlo datum is smaller than the symbol size . Dependence of mean square end - to - end distance r on temperature t for the end - grafted hp chain with ehh = 1 . Chain lengths are n = 50, 100, 200, and 400 from bottom to top . The vertical straight lines show the locations of tcap = 1.65 and tc = 0.5, respectively . The inset presents the heat capacity per monomer for the end - grafted hp chain with n = 200 and n = 400 . Globule transition temperature for an end - grafted chain is estimated from the temperature dependence of the mean square end - to - end distance r. figure 4 shows the dependence of r on t for the end - grafted hp polymer with ehh = 1 . At the critical adsorption temperature tcap = 1.65, we find that r tends to be a local minimum, which is in agreement with the results of adsorption of a homopolymer chain . However, unlike the adsorption of a homogeneous saw polymer on a surface where the r increases monotonically as the temperature is lowered below tcap due to the flattening of the chain on the surface, r for the end - grafted hp chain increases when t is lowered from tcap to about t = 1 and then decreases afterward . The sharp decrease of r below t = 1 is a result of the coil in contrast to the coil globule transition of free chains (figure 2), the plots of r/n versus t for different chain lengths of end - grafted chains do not cross, and we, therefore, cannot use the crossing point to define the coil we can however still find the steepest decrease of r, which takes place at about t = 0.5 and is roughly independent of the chain length . We also find a peak in the heat capacity at t = 0.5 for the hp chain as shown in the inset of figure 4 . We therefore identified this temperature as the collapse transition temperature of a surface - absorbed chain tc = 0.5 . This tc = 0.5 of the end - grafted hp chain is lower than tc = 0.75 for the free hp chain . We therefore conclude that the collapse transition of the hp polymer is suppressed by being adsorbed to the surface . When tc <tcap, the chain undergoes the adsorption transition before the collapse transition can occur . Surface adsorption makes it more difficult for the chain to adopt conformations that provide a sufficient number of h h contacts for collapse to occur, reducing tc from its value in a bulk solution . Figure 5 presents the number of h h contacts for the hp chains in dilute solution and end - grafted on the surface . Above tcap of the end - grafted chain, nhh is small and the same for both chains . At temperature tc <t <tcap, nhh of the end - grafted hp chain is slightly larger than that of the free chain, indicating that the adsorption of the chain promotes the formation of h h pairs . Below tc, however, we find that nhh of the end - grafted hp chain is significantly smaller, clearly indicating that surface adsorption prevents the collapse of chain at low temperatures . This reduces tc for the end - grafted hp chain . On the other hand, the critical adsorption of the polymer is not influenced by the collapse transition of the polymer if tc <tcap . From the heat capacity the temperature at the shoulder is consistent with the cap, determined from the location of the minimum of r, for the finite chain . Dependence of the number of h h contacts, nhh, on the temperature t for hp chains in dilute solution and end - grafted on a surface . The length of the hp chain is n = 400, and the h comparing data in figures 2 and 4 for t <tc, one can notice that r/n in figure 4 increases with n and is much bigger than that of a free hp chain in solution, as shown in figure 2 . The reason is that the adsorbed chain adopts a roughly 2d conformation at t tcap, and the subsequent coil globule transition driven by the intrachain hydrophobic attraction now occurs within this 2d conformation . As has been previously shown for a homopolymer with intrachain attractions, the collapse of a chain in 2d takes place at a lower temperature than collapse in 3d, since a 2d chain conformation will have less pairwise attraction . As will be shown, we find that the adsorbed chain is anisotropic, since its asphericity parameter a is even bigger than that of an adsorbed hp chain without h h attraction . Since tc tcap, the chain at tc is already trapped in the random coil state achieved at tcap; this would probably result in a more anisotropic conformation because the collapse would likely occur at higher density of h monomers . H attractions (ehh = 2, 3, and 4), while the monomer surface attractions are fixed as ehs = eps = 1 . Globule transition of the hp chain when free in solution to higher temperatures, following tc = 0.75\|ehh\| . Therefore, tc occurs at 1.5, 2.25, and 3 for ehh = 2, 3, and 4, respectively . These conditions allow us to examine the interplay between the transitions when the collapse transition occurs at a higher temperature than the critical adsorption transition (ehh = 3 and 4) and when the transitions occur at approximately the same temperature (ehh = 2). First, we determine the collapse transition of end - grafted chains when intrachain attractions are stronger than chain surface attractions (ehh <ehs = eps) and find that tc is not influenced by the presence of the surface . As shown in figure 6, a plot of r/n versus temperature for ehh = 2 and 4 shows the same crossover point as expected for the transition temperature of free chains based on tc = 0.75\|ehh\| . This behavior is different from the case where ehh = ehs = eps = 1 shown in figure 4, where the chains with different lengths do not cross over with each other . Although tc is not influenced by the surface, the conformational size of the chain r is influenced by the attractive surface, as can be observed through comparison of figures 2 and 6 . We next examine the impact of increasing the strength of intrachain attractions on the critical adsorption transition . Figure 7 presents the surface contact number as a function of chain length at different temperatures for ehh = 4 . A scaling relation m n is observed at tcap = 2.55 with an exponent = 0.34 . We find that both tcap and are different from those of ehh = 1 . The results of tc, tcap, and for the hp chains with different intrachain interactions ehh are listed in table 1 . The tcap for ehh = 2, 3, and 4 is obviously affected by the collapse of chain when tc is close to or larger than tcap of the hp chain with weak h h interactions . The simulation results show that the presence of intrachain interaction shifts the tcap to a higher temperature . Dependence of mean square end - to - end distance r on temperature t for the end - grafted hp chain with ehh = 2 (black) and 4 (red). The vertical straight lines show the locations of tc = 1.5 for ehh = 2 and tc = 3.0 for ehh = 4, respectively . Log plot of the surface contact number m versus chain length n at temperatures t = 2.3, 2.4, 2.5, 2.55, 2.6, and 2.7 for an hp polymer with ehh = 4 . The statistical error of each monte carlo datum is smaller than the symbol size . Surface interactions ehs = eps = 1 . At the end of this subsection figure 8 shows the coil globule transition line and the adsorption / desorption transition line for the end - grafted hp chain with polymer surface interactions ehs = eps = 1 . There is a specific interaction, named ehh, at which the two lines intersect . For intrachain interactions stronger than ehh, as temperature decreases, the hp chain changes from a desorbed 3d coil at high temperature to a desorbed collapsed structure at tc and, finally, to an adsorbed collapse structure at tcap . For interaction strengths below ehh, the hp chain changes from a desorbed 3d coil at high temperature to an adsorbed 2d coil at tcap and at last to an adsorbed collapse structure below tc . Here ehh is estimated to be about 2.6 times the polymer surface attraction, and the corresponding temperature is about 2.0 . Symbols are estimated from simulation, while lines are guides for the eyes . To further investigate the interplay between the two transition temperatures, figure 9a presents the mean surface contact number m at different temperatures t for different intrachain interactions ehh . At high temperature t> tcap, the chain is in a desorbed state with m = 0 . At low t, one of the ways to observe the tcap is the substantial increase in m as t is lowered . At t = 0, we have m = n for the case ehh = 0, indicating that all monomers are adsorbed on the surface, whereas for ehh <0, m is less than n due to the collapse of the hp chain, indicating that the conformation of the adsorbed polymer is of a multilayer structure because of the intrachain attraction . The number of h h contact pairs, nhh, always increases with the decrease of temperature as shown in figure 9b . Moreover, we find that nhh increases with \|ehh\|, whereas m decreases with \|ehh\| . Similar to behavior that has been observed for homopolymers with intrachain attractions, there are less surface contacts but more intrachain contacts as the intrachain attraction increases . This reflects the fact that the adsorbed chain adopts more compact spherical shapes for stronger intrachain interactions . Dependence of (a) mean surface contact number m and (b) the number of h h contacts nhh on temperature t for different internal interactions ehh . The length of the hp chain is n = 400 . From table 1, we find that the crossover exponent in the scaling relation m n is about 0.5 for tc <tcap while it decreases for tc> tcap at strong h h attraction . For the former case (tc <tcap), the conformation of the chain is a random coil near tcap, and m behaves similarly near tcap as shown in figure 9a, resulting in the same value of the crossover exponent for tc <tcap . For the latter case (tc> tcap), the contact number of the compact chain at tcap is reduced, since the contact monomers are located on the globule surface, as can be observed by comparing data plotted in figures 3 and 5 . For the same reason, the crossover exponent is reduced for the case tc> tcap . The reason is that the difference between tc and tcap increases with ehh as shown in table 1, and the chain becomes more compact at lower temperature below tc . In order to learn more about the conformation of the chain, we have monitored the mean square end - to - end distance r and its two components parallel to the surface rxy and normal to the surface rz at different internal interactions ehh, as shown in figure 10 . Different behaviors are exhibited for three different cases: (1) a chain with no collapse transition when ehh = 0, (2) tc <tcap with ehh = 1, and (3) tc tcap with ehh = 2 and 4 . For the first case in the absence of intrachain interaction, a slight minimum in r is found at tcap that is a result of two changes, as a sharp decrease in rz is partially offset by a sharp increase in rxy . As the temperature is further reduced, the increase in rxy outcompetes the decrease in rz, resulting in an overall increase in r. the behavior of r is similar to an earlier finding by exact enumeration of all configurations for a short homogeneous saw chain . For the second case where tc <tcap, r first increases as the temperature is lowered just as in the previous case but then r decreases because of the collapse of the chain . In this second scenario, r exhibits a maximum at a temperature close to tc, a distinct feature absent in the other cases . The maximum is also presented in the plot of rxy as a function of temperature . For the third case where tc tcap, we find that r, rxy, and rz all decrease monotonically with the decrease of t. the chain is already in a compact state at tcap; therefore, it deforms little when it adsorbs on a surface, similar to the docking transition for a compact chain adsorbed on a weak attractive surface . Dependence of (a) the mean square end - to - end distance r and (b) its components parallel and normal to the surface, rxy and rz, respectively, as a function of temperature t for hp polymers with different h h interactions . L1, l2, and l3 are three eigenvalues of the radius of gyration tensor4where si = col(xi, yi, zi) is the position of monomer i of polymer in a frame of reference with its origin at the center of mass . The asphericity parameter a ranges from zero for 3d spherically symmetric chain conformations, 0.25 for 2d circular shapes, and one for rod - shaped . It was found that a 0.391 for a linear rw chain and a 0.431 for a linear saw chain . Values a of the hp chain in dilute solution (i.e., free hp chain) and the end - grafted hp chain are calculated . The dependence of a on temperature t is presented in figure 11 for the end - grafted hp chains with different intrachain h h interactions . Plot of the asphericity parameter a vs temperature t for free hp chains with ehh = 1 and end - grafted hp chains with different h the arrows indicate the location of tc and tcap, and the value in parentheses is ehh . For the free hp chain with ehh = 1, a is about 0.44 at t tc and decreases steeply at tc = 0.75 . A is about 0.12 at low temperatures (t <tc), clearly showing that the chain is roughly a sphere at temperatures below tc . For the end - grafted hp chain, the behavior of a, like that of size r, is dependent on the intrachain attraction ehh . Moreover, the behavior of a is quite complicated due to the competition between tc and tcap in the hp chain . For ehh = 0, a increases at tcap due to the transition from a 3d random coil to a 2d random coil . For ehh = 1, a first increases at tcap = 1.65 and has a second increment at tc = 0.5 . The collapse at tc tcap happens locally and makes the chain configuration more aspherical . For the case with ehh = 2 where tc is close to tcap, we find that a begins to increase when the temperature drops below tcap and continues to increase at tc . But, as temperature continues to decrease further below tc, we find that a begins to decrease due to the strong collapse of chain as \|ehh\|> \|ehs\| . For ehh = 4, a decreases at tc because of collapse and then increases at tcap because of adsorption; finally, a plateaus as the chain becomes frozen at low temperatures . From these four behaviors, we conclude that adsorption of the chain increases a, whereas the effect of collapse is dependent on the strength of intrachain h if the intrachain h h attraction is weak where we have tc tcap, the adsorption of the chain increases a and the adsorbed configuration is a random coil . For this case, the collapse at low temperature will induce extra anisotropy and increase a. if the intrachain h h attraction is moderate where we have tc tcap, a is increased due to the adsorption as well as collapse of the chain but will decrease at low temperatures below tc . Finally, if the intrachain h h attraction is strong where we have tc> tcap, a first decreases due to the collapse and then increases due to the adsorption of the chain . Moreover, for the last two cases, a at low temperature reaches a plateau with a value close to 0.25, indicating that the adsorbed configuration is roughly a 2d circle . We have studied the interplay between the critical adsorption of a lattice hp protein with alternating h and p monomers and the coil globule transition with the dynamical monte carlo method . Simulations are carried out in the simple cubic lattice where bond length can be fluctuated among 1, 2, and 3 lattice units . We find that the critical adsorption temperature tcap is influenced by the presence of intrachain attractions responsible for the collapse transition of the polymer . If the coil globule transition tc is lower than tcap then tcap for the hp polymer is roughly the same as that of a homopolymer without monomer monomer attractions, but the coil it is therefore more difficult for a surface absorbed hp polymer chain to go through the coil . On the other hand, if the intrinsic coil globule transition temperature tc is higher than tcap, the tcap for the hp polymer occurs at a higher temperature than a homopolymer without monomer monomer attraction; that is, a collapsed chain can be more easily adsorbed . The conformational properties of the end - grafted hp chain are strongly influenced by the pairwise h first, the hp model itself is limited in that it does not consider several factors, such as desolvation effects, that have been shown to be relevant to the behavior of real proteins, such as the cooperativity observed during the folding of many proteins . Second, the sequence we have studied is the hp polymer with a fully alternating sequence in which the surface interactions of h and p monomers are treated as the same . This is a significant simplification and probably does not represent the real experimental situation very well . In most applications, a further extension of our study is to treat the surface interactions of h and p monomers differently . However, the overall conclusion about the mutual impact on the coil globule transition and the critical adsorption transition would probably still be valid.
Grey mould, caused by botrytis cinerea (sclerotiniaceae family), is an important plant disease that affects a large number of plant species and is particularly important in greenhouse production of tomatoes in the mediterranean basin . In greenhouse tomato, the fungus infects flowers, fruits, and leaves and can grow through the petiole into the stem [2, 3]. This problem is one of the major stresses especially in arid and semiarid regions and can severely limit plant growth and productivity [5, 6]. In algeria, a wide range of environmental stresses (such as high and low temperature, drought, alkalinity, salinity, and pathogen infection) soil salinity and irrigation water are two of the main serious problems hindering the development of most plant species . Thus, the effect of these factors may result from structural and physiological changes in the plant, an increased incidence, and severity of diseases caused by various species pathogen . Reference showed that relatively low levels of salinity (2550 meq) could increase the severity of phytophthora root rot of tomato with high na: ca ratios (10: 1), phytophthora [912], f. oxysporum f. sp . The ability of ca to form intermolecular linkages gives it an important role in maintaining the integrity and structure of membranes and cell walls . Ca is also used as a second messenger in many signal transduction pathways within the cell . Several studies have reported that ca treatment of plant tissue induces an increase in tissue ca content, resulting in reduced fungal diseases . The mechanisms by which calcium salts inhibit the development and severity of diseases are not known . One hypothesis is that high external ca concentrations may increase the concentration of ca in the cytosol, which can be toxic to the fungus . The ability of calcium to reduce the development of postharvest diseases of fruit has been attributed mainly to the formation of calcium cross - linkages in the cell wall, resulting in decreased effectiveness of cell wall - macerating enzymes secreted by the pathogen . Reference also demonstrated a relationship between increasing levels of calcium in the cell walls of potato tubers and a reduction in the macerating activity of erwinia carotovora . All the studies on the effect of ca on botrytis showed that it has an inhibitory effect on growth of this fungus at high concentrations [2325]. This effect is thought to be mainly due to the role of calcium in ameliorating physiological disorders and thus indirectly reducing pathogen activity [26, 27]. Reference has indicated that calcium chloride reduced germination and germ tube elongation of b. cinerea and penicillium expansum in vitro . Reference also has reported a similar effect of calcium in reducing the susceptibility of rose flowers to gray mold caused by botrytis cinerea . For the most effective control of disease, it seems necessary to examine the impact of salinity on the development of pathogen . The objective of this study was to determine the in vitro effect of sodium and calcium salts on spore production, conidia germination, and mycelial growth of b. cinerea . B. cinerea isolates were obtained from decayed tomato (lycopersicon esculentum) in northwestern algeria . The leaf fragments were placed on filter paper moistened with sterile water in a petri dish . Conidia were harvested from 14-day - old cultures by agitating small pieces of agar, bearing mycelia and conidia, in a glass tube . Conidia of b. cinerea were obtained from 2 week old pda cultures incubated at 25c in 12/12 hours light / dark . Culture plates were vortexed in a tube containing 10 ml sterile distilled water and 0.05 ml tween 80 . A sterile magnetic stir bar was placed on the agar and set stirring for 5 minutes to loosen the spores . The suspension enriched in spores finally the conidial concentration was determined using a malassez cell and adjusted to 10 spores per ml . To determine the influence of nacl and cacl2 on spore germination of b. cinerea, a drop containing 100 conidia was transferred onto water agar plates enriched with nacl and cacl2: 0, 50, 100, 150, and 300 meq . A conidium was considered as germinated if the germ tube length was at least twice the length of the conidium . The influence of nacl and cacl2 on the diameter growth was determined by growing the isolates in a pda medium at four nacl and cacl2 levels (50, 100, 150, and 300 meq); control medium was not amended with salts . Mycelium growth inhibition was evaluated by placing a plug (4 mm diameter) from an actively growing culture in the centre of a pda agar plate of 9 cm plastic petri dishes . Cultures were incubated for 714 days at 25c in the dark, and each treatment had four replications . All statistical analyses were analyzed by the software of statistics (statbox 6.0.4, grimmersoft). Statistical significance was assessed at the level of p = 0.05 or p = 0.01 . All the isolates exhibited variation in their colony characteristics such as color, shape, and texture (figure 1). B. cinerea colonies from tomato on pda at 25c were visually classified into three morphological groups, gi, gii, and giii, based on colony color and pycnidial distribution . Gi (f27) isolate produced white to light grey colonies, where colony texture was generally cottony, and was present at the center of the petri dish . Gii (fa13, s27, b27, and tr13) isolates had off - white to pale gray color . Based on the morphological parameter of length, pycnidia can be classified into two groups: length greater in isolates fa13, s27, b27, and f27 and length less in isolates tr13 and r13 . They scattered all over the medium in petri dish, covering the entire surface of the agar (b27 and tr13 and f27). In some isolates sclerotia were produced on concentric rings, formed along the edges of the petri dish (fa13, s27, and r13). This study evaluated the activity of 2 salts against b. cinerea in vitro at 4 concentrations . The effect of salts at different concentrations on mycelial growth of colonies of six isolates of b. cinerea in pda was observed after 3 days . All concentrations, except 300 ppm, of nacl significantly stimulated growth of b. cinerea ., reduced growth has been correlated with the increasing in the nacl of the medium . The calcium chloride at 50 and 100 ppm stimulated mycelial growth of b. cinerea relative to the control . However, higher concentrations of calcium chloride (150 and 300 ppm) caused f27, b27, r13, tr46, s27, and fa13 to reduce growth by 26.72, 24.26, 23.24, 34.78, 15.07, and 17.04%, respectively . There was a significant reduction in growth of isolates (p <0.001) with increasing calcium salt . At 300 ppm, calcium chloride was the most inhibitory, reducing growth on pda by 34.78% for the isolate . The interaction between salt and concentration was significant for r13, tr45, s27, and fa13 (p <0.001), but not significant for f27 and b27 isolates . All concentrations were significantly different from the control (p <0.001); 150 ppm cacl2 and 300 ppm nacl or cacl2 were similar to each other and different from other concentrations in reducing mycelial growth . In the absence of salt (table 2), isolates of b. cinerea do not present the same profile of conidial production . The optimum density for spore production of this fungus was from 1.97 10 to 2.2 10 spores / ml, of r13 and b27, respectively . Data in table 2 indicate that application of chloride salts and sodium salt caused a significant increase in the production of conidiogenesis at various concentrations tested compared with control (p <0.001). However, these observations indicate that the conidial production of the six isolates might increase, even at high salinity . There was a significant increase in these characters between the control and 50 ppm concentration of salinity . Under these salt conditions, sodium chloride is most favorable to the sporulation of all isolates of b. cinerea, especially at high concentrations of the culture medium . By adding 300 ppm of nacl to the culture medium, an increase in spore production by 1.21 10 spores calcium chloride stimulates little sporulation of the isolates, with only 9.2 10/ml to isolate r13 . The amount of spore production is in direct proportion to the concentration of salinity in the culture medium . The interaction between salt and concentration was significant for all salts isolates (p <0.05) except fa13 (table 2). The results for the percentage germination of different isolates studied in terms of germination capacity under the effect of different salt concentrations are shown in table 3 . The six isolates presented dissimilar percentages of germination in the presence of sodium and calcium chloride . The analysis shows that salinity affects the percentage of germination for each value of salt . The highest germination percentage, 78.33% and 63.67%, was obtained in the absence of salt after 24 h incubation at 25c, from isolates fa13 and tr46, respectively . Concerning the salts, the 50 and 100 ppm also increased the conidial germination except tr13 and fa13 isolates, which was significantly different from the stimulation caused by nacl . Conversely, spore germination was decreased for sodium concentrations of 150 and 300 ppm relative to the control . The application of lower concentrations of cacl2 (50 ppm) to wounds did not inhibit their percentage germination . The toxicity of calcium chloride (ec50 = 100 ppm) to spores was higher than that of sodium chloride (ec50 = 150 ppm). The interaction between salt and concentration was significant for all isolates (p <0.001) except f27 and tr46 . The purpose of this study was to compare the effect of sodium and calcium salts against b. cinerea . Our in vitro tests showed that sodium chloride stimulates the development of the fungal up to 150 ppm . In contrast, only calcium salts were effective at low concentrations as compared to sodium chloride . However, higher concentrations of calcium chloride reduced mycelial growth of b. cinerea pda medium . Our data indicate that salinity stimulated growth of all six isolates at high concentration (nacl at up to 150 ppm). On the contrary, high salinity (more than 300 ppm) of several reference showed that increasing the salinity of the medium promotes the in vitro mycelial growth of phytophthora citrophthora and p. parasitica, agents of root rot of citrus, with an optimum between 1.44 and 3.11 bars . Similarly, showed that all calcium salts tested (except formate, calcium pantothenate, and dibasic calcium phosphate) reduced the growth of monilia fructicola responsible for brown rot of peach, on amended potato dextrose agar (pda). In the comparison of the inhibitory effect of the various salts, higher concentrations with 300 ppm to nacl or cacl2 reduced mycelial growth to 16 and 23%, respectively . Regragui and lahlou showed that the stimulator effect of salinity was observed on the mycelial growth, conidia production, and conidia germination of the tested stain of v. dahliae, respectively, in concentrations 170, 120, and 256 mm of nacl . Oppositely, pelizza et al . Showed that the presence of nacl in the medium culture reduces the growth of an isolate of leptolegnia chapmanii . However, reid et al . Reported that sodium chloride was more effective than other chloride salts (calcium chloride, ammonium chloride, and manganese chloride) in controlling fusarium crown and root rot caused by f. oxysporum f. sp . Van bruggen and semenov reported that on a long - term basis there is a decrease in the genetic diversity of fungi as a result of stress . On the other hand zahran mentioned that the hydric stress has to deal with the increase in osmotic pressure and may therefore change their physiology and morphology in response to this . Two strategies used by microorganisms to adapt to osmotic stress were described by killham, both of which result in an accumulation of solutes in the cell to counteract the increase in osmotic pressure . One is the selective exclusion of the solute incorporated (e.g., na, cl), thus accumulating the ions necessary for metabolism (e.g., nh4). The results of the present study demonstrate that calcium salts also have been shown to reduce mycelial growth in vitro; the percentage of reduction varied between 15 and 34% as compared to the control . Several studies have reported that calcium applications can suppress diseases caused by several pathogens [23, 27, 28, 36]. Our result further supports the results of maouni et al ., who found that calcium chloride significantly reduced pear fruit decay caused by a. alternata and penicillium expansum when used at 4 and 6% . Tian et al . Recorded that calcium chloride at 2% inhibited the growth and spore germination of r. stolonifer, although cacl2 was tolerated by alternaria alternata and p. expansum in vitro . It was reported that 1,000 mg of calcium (calcium chloride) enhanced the growth of botryosphaeria dothidea . Calcium salts also have been shown to reduce mycelial growth in vitro and reduce incidence and severity of infection of peach fruits and shoots by monilinia fructicola and leucostoma persoonii, respectively [23, 40]. Reported that mycelia cultured with 100 to 200 mm ca exhibited a lower viability compared with mycelia grown with 10 mm ca for some isolates of botrytis spp . While little information is available on the role of ca in fungi, results of experiments with yeasts have shown that mutants that have defective intracellular ca transport systems or defective vacuolar h - atpase that produces the proton motive force necessary for the activity of the vacuolar ca / h exchanger could not grow in high ca concentrations [4245]. Maintenance of low basal concentrations of free cytosolic ca, in the submicromolar range, is essential for normal cell functions [46, 47]. In the case of the evaluation the effect of nacl and cacl2 on the spore production by the fungus, all isolates of b. cinerea are able to sporulate in salinity tested, but to varying degrees . In fact, sporangium formation of phytophthora parasitica in vitro appeared to be stimulated by salinity, as the numbers of sporangia were generally higher (120% to 225%) in the salt - amended treatments than the distilled water controls . With regard to conidiogenesis, reference showed that stimulation of sporulation under the effect of salinity is due to a specific effect of ions . According to this author, na and cl stimulate the production of sporangia of p. citrophthora and p. parasitica while the osmotic effect inhibits biological activity . In verticillium, increased sporulation under the effect of the salt appears to be not only solely due to the effect of na and cl ions, but also due to the osmotic effect . However calcium salts did not reduce spore production of b. cinerea spores in this study . Similarly, a minimum concentration of calcium is necessary for the production of zoosporangia or zoospore release by phytophthora spp ., it was reported that, at low concentration (50 ppm), the germination capacity for most isolates increased compared with the control in both types of salt . Beyond this concentration, similarly, a low reduction of conidial germination was observed for two salt types at the maximum concentration used . Reference showed that increasing the concentration of calcium chloride (25175 mm) causes a decrease in germination and germ tube growth in vitro of b. cinerea and penicillium expansum, respectively, causing the gray and blue mold in apples stored . Incubating b. cinerea spores in increasing concentrations of cacl2 (426 calcium was effective in inhibiting spore germination of c. gloeosporioides, rhizopus stolonifer, and alternaria alternata and penicillium expansum . Physiologically, the maintenance of low basal concentrations of internal ca is essential for normal cell functions of organisms, and the inability to regulate ca may affect the organisms' normal growth . Calcium ions may reduce the incidence of fungal infection by directly inhibiting fungal growth and by inhibiting cell wall degrading enzymes produced by the pathogens [45, 54, 55]. The effects of calcium in reducing spore germination were probably due to toxicity, with high concentrations likely affecting the osmotic balance in fungal cells.
Although it comprises only 10 - 15% of all hepatobiliary neoplasms, its incidence is increasing.1 frequent metastatic sites of biliary cancer are the liver, peritoneum, intra - abdominal lymph nodes, and lungs.2 here, we describe a very rare case in which metastatic cholangiocarcinoma of the stomach was mistaken for primary gastric cancer in a patient who underwent whipple's operation . However, histopathologic and immunohistochemical findings suggested that the gastric tumor was a metastatic adenocarcinoma originated from a distal cholangiocarcinoma . A 67-year - old woman presented with a 4-month history of abdominal pain after meals . Laboratory findings was as follows: aspartate aminotransferase 141 iu / l (normal; 10 - 40 iu / l) and total bilirubin 0.9 mg / dl (0.2 - 1.0 mg / dl). Computed tomography showed a malignant tumor of the common bile duct (cbd), located just above the intrapancreatic segment, with consequent dilatation of the proximal biliary tree including the gall bladder (fig . 1). The periportal, common hepatic, and portocaval lymph nodes were enlarged . Endoscopy revealed a 2-cm, flat, elevated lesion with convergence of the surrounding folds, situated at the gastric angle (fig . Based on the biopsy results, an adenocarcinoma, thought to be an early gastric cancer, was diagnosed . The patient underwent whipples's operation . The surgical specimen consisted of a whitish mass, 2.11.3 cm in diameter, removed from the distal cbd and showing invasion of the pancreas and peripancreatic fat . Pathologic examination of the resected stomach demonstrated that the tumor was very clearly demarcated from the surrounding nontumorous gastric mucosa and submucosa, and also showed invading lymphovascular spaces . In addition, the adenocarcinomatous tissue had infiltrated into the gastric mucosa and submucosa, while the gastric superficial mucosa was intact (fig . Immunohistochemical stains showed that the tumor tissue was strongly positive for cytokeratin (ck)-7 and weakly positive for ck-19, while the surrounding gastric mucosa was negative for both cytokeratins (fig . According to a number of reports in the literature, metastasis to the stomach is a rare occurrence, with a reported incidence of less than 1% . The main primary metastatic tumors are those of the breast (33%) and lung (25%), and malignant melanoma (22%).3 in our patient, it was difficult to distinguish gastric metastasis of a cholangiocarcinoma from a primary gastric cancer on the basis of clinical, endoscopic, and radiologic features . Thus, results obtained from complete histopathologic and immunohistochemical studies of gastric biopsies should be compared with the characteristic features of cancer of the cbd . The anatomic distribution of ck-7 and ck-19 is generally restricted to epithelia of primary sites and their neoplasm . Primary gastric cancer has the same staining pattern, because ck-7 and ck-19 expressions are reported to be positive in about 80% of stomach cancer cases . However, both the normal gastric mucosa surrounding the tumor and the gastric carcinoma, if it originates from the stomach, are positive for ck-7 and ck-19.4,5 metastasis to the gastrointestinal tract initiates from the serosa and submucosa and progresses to cause intraluminal lesions, as occurred in this case.6 based on these considerations, we diagnosed metastatic gastric carcinoma from a distal cholangiocarcinoma . Another important immunohistochemical marker, cdx-2 is a very useful marker to distinguish stomach cancers from bile duct cancers, because cdx-2 expression is reported to be positive in 61% of stomach cancer cases but only in 13% of bile duct cancers, however cdx-2 was not performed in our study.7 until now, unusual metastastic sites from cholangiocarcinoma were reported to be the colon, adrenal gland, skull bone, epididymis, corneal limbus, meninges, ovary, skeletal musle, and skin.2,8 - 15 metastasis of a distal cholangiocarcinoma to the stomach has not been reported previously to our knowledge but, as in the above - mentioned sites, appears to be a rare site of metastasis of this tumor . In conclusion, a careful histopathologic and immunohistochemical review is very important in diagnostic differentiation of metastatic tumors from primary sites in this case.
Vitamin d deficiency is a global health problem even in sunny regions as the middle eastern countries (1).vitamin d is a steroid hormone and plays a key role in minerals metabolism, specially calcium and phosphorus, as well as in bone strength (2). The active type of this vitamin is synthesized in the skin, liver and kidneys . In addition to its multi - functions in the body, vitamin d reinforces the immune system . Receptors for vitamin d exist in most organs including pancreas, stomach, genital system, skin, brain etc (3 - 8). 25-hydroxy vitamin d (25(oh) d) level is the best index to determine vitamin d status in the body, with a half - life of 2 - 3 weeks (9). Adequate exposure to sunlight and dietary intake of foods rich in vitamin d are necessary to satisfy the daily need of the body and prevent hypovitaminosis d (10,11). Many studies have indicated the association of serum 25(oh) concentrations with serum levels of other vital elements . From the several elements found in the body, only a few number as zinc, copper, magnesium, iron and calcium play important roles in the body's chemical and physiological functions . Zinc has several important roles in the human body; for instance, it is used in the process of synthesizing insulin and some enzymes as superoxide dismutase . Zinc is a trace element, which after iron, has the highest amount in the body . It is mainly accumulated in the muscles but can also be found in the blood cells, retina, bones, skin, kidneys, liver and the pancreas . Zinc is the second most vital element after iron and its deficiency during pregnancy may cause serious feto - maternal complications (12) including impaired cognitive development in the infant during the first six months of life, immunological complication in fetus, low birth weight, prematurity, miscarriage, fetal or infant death, postdate pregnancy, premature rupture of membranes, cleft palate and neural tube defects in the fetus (13). A study on 150 iranian pregnant women showed that 37% of them were vitamin d deficient and 23% were zinc deficient, with a statistically significant association between serum zinc and 25 (oh) d levels (14). Such experience is scarce in the pediatric age group . Given the high prevalence of hypovitaminosis d and zinc deficiency, as well as their possible interactions, this study aimed to assess the relationship of serum zinc and 25 (oh)d levels in adolescents . This study was conducted as a sub - study of the national survey of school students high risk behaviors, which was performed as the third survey of the school - based surveillance system entitled: childhood and adolescence surveillance and prevention of adult non - communicable disease study (caspian - iii). Caspian - iii is a school - based nationwide health survey that includes 5,528 students aged 10 - 18 years from 27 provinces in iran . In this case - control study, as a sub - study of the caspian iii study, 165 students with hypovitaminosis d as the case group and 165 normal students without hypovitaminosis d as the control group were randomly selected from the sample of frozen sera (70c) of the participants in which 25-hydroxy vitamin d (25(oh) d) was checked (15). Serum concentration of 25(oh) d was analyzed quantitatively by direct competitive immunoassay chemiluminescene method applying liason 25 oh vitamin d assay total (diasorin, inc . ), with a coefficient of variation of 9.8%.25 (oh) d levels of less than 10ng / ml as vitamin d deficiency and levels between 10 and 30ng / ml as vitamin d insufficiency (16). Sera of the two groups of participants with or without vitamin d deficiency were randomly selected . Zinc level of the samples was determined by atomic absorption spectrophotometer using hollow cathode lamps of zn . Sera were analyzed for glucose and lipid profile including total cholesterol, high - density lipoprotein cholesterol (hdl - c), ldl cholesterol (ldl - c) and triglycerides (tg), as well as liver function tests including serum glutamate oxaloacetate transaminase (sgot) and serum glutamate pyruvate transaminase (sgpt) using pars azmoon reagent kits (tehran, iran). The research and ethics committee of isfahan university of medical sciences, isfahan, iran, approved this study . Statistical analysis: the relationship between serum vitamin d and zn levels was measured through linear regression analysis considering zn as an independent variable and 25(oh) d as a dependent variable . Chicago, il, usa) by applying t test, one - way analysis of variance (anova), and pearson s correlation tests . To assess the relationship between serum zn concentration and vitamin d level, logistic regression was used to calculate odds ratios (or) for vitd level between various quartiles of serum zn concentration; the mean age of the participants was not significantly different among groups with and without hypovitaminosis d (14.74 2.52 vs.14.74 2.66 years, respectively, p>0.05). No significant difference was observed in the baseline characteristics of the participants of the two groups (table 1). Bmi: body mass index, sbp: systolic blood pressure, dbp: diastolic blood pressure, hdl - c: high density lipoprotein - cholesterol, ldl - c: low density lipoprotein - cholesterol, tg: triglyceride, fbg: fasting blood glucose, sgot: serum glutamate oxaloacetate transaminase, sgpt: serum glutamate pyruvate transaminase, sd: standard deviation the serum meansd level of 25(oh) d was 6.341.47ng / ml in the group with hypovitaminosis d and 39.276.42ng / ml in the control group (p<0.001). The meanzn level in hypovitaminosis d group was significantly lower than in controls (1.15 0.26 vs. 1.430.32g / ml, respectively, p<0.0001). The pearson correlation of serum zn concentration and vit d with metabolic factors and existence of metabolic disease showed that zn levels had a significant positive correlation (p<0.001) with vit d level . As demonstrated in table 2, the or of higher levels of vit d levels was higher in the second (q2) and fourth (q4) quartiles significantly as compared to the reference quartile (q1). The main aim of this study was to determine the relationship between serum vitamin d and serum zn levels in children and adolescents . We found a positive correlation between serum zn and serum vitamin d levels, and this finding was consistent with a previous study conducted on children (14). This might have been caused by the prominent effects of lifestyle factors as inadequate exposure to sunlight, air pollution and poor nutrition . Aside from hazardous effects on the respiratory system and pollution caused diseases, living in populated cities means less sunlight for children, especially during cold seasons when there is higher air pollution and weaker sunlight intensity (16,17). A nationwide study in iranian adolescents demonstrated a high prevalence of hypovitaminosis d and an association of serum 25(oh) d levels with cardiometabolic risk factors . It showed an inverse association of 25(oh)d with systolic blood pressure, diastolic blood pressure, total cholesterol and low - density lipoprotein cholesterol . In contrast, this association was significantly positive with high - density lipoprotein cholesterol, but not with fasting plasma glucose and mets (19). The first evidence of zn deficiency in humans was reported from central part of iran (21). Zn deficiency has several etiologies; one of them might be the consumption of white flour and white rice as the main dishes in iran . The zn intake from these diets is high at 15 mg a day, but it is not available because of the high phytate content of this kind of diet . Phytate, the phosphorus storage compound of plant seeds, bind zn and other bivalent ions in insoluble complexes, making them unavailable to human and other monogastric species (22). Iron, when present in large amounts in the diet, also inhibits zn absorption . Epidemiological studies in iran showed a considerably high prevalence of zn deficiency, as high as 31.1% among school students and 28.1% in children (23). The most important causes of zn deficiency are soil content of zn, dietary deficiency (phytate containing whole grains), malabsorption due to impaired transport across intestinal absorptive surface, damaged or absent intestinal absorptive surface, increased loss, increased utilization, and chronic disease (24). The vitamin d receptor (vdr) binds zinc, and the activity of vitamin d dependent genes in cells is influenced by intracellular zinc concentrations . It is also important to ensure that the calcium from foods or supplements is used in your bones . Hence, low level of both zn and vitd can affect many important body functions (25). However, studies in animals have shown positive effects of vitd on zinc increases (26) the effects of zinc and vitd on absorption - desorption and eventually the amount of each other, but their basic metabolic pathways in humans are not clear . Our results revealed that hypovitaminosis d was accompanied with low serum zn level . Comparing our results with those of other studies, it could be concluded that hypovitaminosis d is probably due to inadequate exposure to sunlight . In addition, it seems inappropriate diet and lack of absorbable zn in our foods lead to zn deficiency in our schoolchildren . Finally, conducting future studies to identify the factors affecting vitamin d and zinc deficiency in schoolchildren, their relationship and the real causes of simultaneous lack of vitd and zinc study limitations and strengths: the main limitation of this study was its cross - sectional nature . Moreover, we did not obtain the detailed dietary intake of the participants . The strengths of the study were its novelty in the pediatric age group and recruiting a nationally representative sample.
Diphenyl cyclo propenone (dpcp) is used as a topical immunomodulator in alopecia areata . It is a potent contact allergen in humans and animals; 98 - 99% of the cases of alopecia areata can be sensitized on the scalp skin . Although its use has been increasing of late, the process of procuring, dilution and storage at a particular concentration is cumbersome and limits its wide use . This short communication aims to make the dpcp preparation and application easy for the readers . Dpcp is available as 1 and 5 g powder in amber - colored glass bottles . The standard solvent, acetone, is a strong uv light absorber and inhibits this process . For dilution, first, the dpcp is weighed in a weighing scale and then diluted with the required quantity of acetone . Initial sensitization is carried out with a 2% solution, which is made by dissolving 20 mg in 1 ml of acetone . Further dilution can be prepared by making a stock solution of 2% and diluting with acetone taken in a pipette as per the concentration [tables 1 and 2]. Dilution for diphenyl cyclo propenone dilution from the stock solution 2% percentage = weight / volumedilution100 (% = w / vdilution100) to prepare a 0.001% solution, 0.1 ml of stock solution is mixed with 200 ml of acetone, and 5 ml of the resulting solution can be used for application over the scalp . The rest of the solution should be stored in air tight, screw - capped amber - colored containers for further use . Storage of the diluted solution is difficult as acetone often evaporates, leading to a change in the concentration . Fresh solution can be made every time to avoid this . For concentrations of 0.01% and above, dpcp can be weighed prior and stored in glass containers to which acetone can be added in the required amount at the time of application as this saves time and prevents wastage (e.g., 0.01% can be made by adding 10 ml acetone in 1 mg preweighed dpcp containers). The diluted solutions are placed in a wide - mouthed glass beaker and applied on the scalp with cotton swab . Application needs to be done fast to cover the entire scalp before acetone starts evaporating in the beaker . Health care professionals should take proper precautions of wearing a glove, face mask and apron during the dpcp application as the spill of solution may cause an irritant / urticarial reaction . Patients are advised to wait for 5 - 10 min in the out patient department before covering their head with a cap or cloth to protect from sunlight . After 48 h, patients should be advised to shampoo the scalp to remove the residual dpcp . During these 48 h, patients should be advised not to touch the scalp accidentally either by themselves or others . The common adverse effects after dpcp applications are local eczema with blistering, regional lymphadenopathy and contact urticaria . Rare adverse effects include an erythema multiforme - like reaction, hyperpigmentation, hypopigmentation and vitiligo.
Relativistic thermodynamics is needed, because in relativity the mass of a body depends on how hot it is and the temperature is not necessarily homogeneous in equilibrium . But unlike classical thermodynamics the relativistic theory cannot be constructed on the intuitive notions of heat and work, because our intuition does not work well with relativistic effects . Therefore we must rely upon logic, or what would seem logical: the cautious and careful extrapolation of the tenets of non - relativistic thermodynamics . A pioneer of this strategy was carl eckart [14, 15, 16] who as early as 1940 established the thermodynamics of irreversible processes, a theory now universally known by the acronym tip . Eckart s theory is an important step away from equilibria toward non - equilibrium processes . It provides the navier - stokes equations for the deviatoric stress and a generalization of fourier s law of heat conduction . The latter permits a heat flux to be generated by an acceleration, or a temperature gradient to be equilibrated by a gravitational field . But eckart s theories the relativistic and non - relativistic ones have one draw - back: they lead to parabolic equations for the temperature and velocity and thus predict infinite pulse speeds . Naturally relativists, who know that no speed can exceed c, are particularly disturbed by this result and they like to call it a paradox . Cattaneo proposed a solution of the paradox as far as it concerns heat conduction1 . He reasoned that under rapid changes of temperature the heat flux is somewhat influenced by the history of the temperature gradient and he was thus able to produce a hyperbolic equation for the temperature actually a telegraph equation . Mller [35, 37] incorporated this idea into tip and came up with a fully hyperbolic system for temperature and velocity . He calculated the pulse speeds and found them to be of the order of magnitude of the speed of sound, far removed from c. and indeed, neither cattaneo s nor mller s arguments have anything to do with relativity, although mller also formulated his theory relativistically . The theory became known as extended thermodynamics, because the canonical list of fields density, velocity, temperature is extended in this theory to include stress and heat flux, 14 fields altogether . The pulse speed problem may not be the most important question in thermodynamics but it is a question that can be answered, and has to be answered, and so there was a series of papers on the problem all using extended thermodynamics of 14 fields . Israel who reinvented extended thermodynamics in 1976 and kranys and stewart, and boillat, and seccia & strumia all calculate the pulse speed for classical as well as for relativistic gases, degenerate and non - degenerate, for bosons and fermions, and for the ultra - relativistic case . Actually in some of these gases the pulse speed reaches the order of magnitude of c but it never exceeds it . So far, so good! But now consider this: the 14 fields mentioned above are the first moments in the kinetic theory of gases and the kinetic theory knows many more moments . In fact, in the kinetic theory we may define infinitely many moments of an increasing tensorial rank . And so mller and his co - workers, particularly kremer [25, 26], weiss [49, 51, 50] and struchtrup, came to realize that the original extended thermodynamics was not extended far enough . Guided by the kinetic theory of gases they formulated many - moment theories . These theories have proved their validity and relevance for quickly changing processes and processes with steep gradients, in particular for light scattering, sound dispersion, shock wave structure and radiation thermodynamics . And each theory predicts a new pulse speed . Weiss, working with the non - relativistic kinetic theory of gases, demonstrated that the pulse speed increases with an increasing number of moments . Boillat & ruggeri proved this observation and very recently boillat & ruggeri also proved that the pulse speed tends to infinity in the non - relativistic kinetic theory as the number of moments becomes infinite . As yet unpublished is the corresponding result by boillat & ruggeri [6, 3] in the relativistic case by which the pulse speed tends to c as the number of moments increases . These results put an end to the long - standing paradox of pulse speeds - 50 years after cattaneo; they are reviewed in section 3 and 4 . The quest for macroscopic field theories with finite pulse speeds has proved heuristically useful for the discovery of the formal structure of thermodynamics, relativistic and otherwise . This structure implies basis equations are of balance type; hence there is the possibility of weak solutions and shocks, constitutive equations are local in space - time; hence follow quasilinear first - order field equations, entropy inequality with a concave entropy density; this implies symmetric hyperbolic field equations . Basis equations are of balance type; hence there is the possibility of weak solutions and shocks, constitutive equations are local in space - time; hence follow quasilinear first - order field equations, entropy inequality with a concave entropy density; this implies symmetric hyperbolic field equations . The latter property is essential for finite speeds and for the well - posedness of initial value problems which is a feature at least as desirable as finite speeds . The formal structure of the theory is described in section 2; it was constructed by ruggeri and his co - workers, particularly strumia and boillat, see [43, 41, 4]. A convenient presentation may be found in the book by mller & ruggeri of which a second edition has just appeared . Section 5 presents extended thermodynamics of viscous, heat - conducting gases due to liu, mller & ruggeri, a theory of 14 fields . That section demonstrates the restrictive character of the thermodynamic constitutive theory by showing that most constitutive coefficients can be reduced to the thermal equation of state . Also new insight is provided into the form of the transport coefficients: bulk- and shear - viscosity, and thermal conductivity, which are all explicitly related here to the relaxation times of the gas . It is true that some of the tenets of extended thermodynamics are strongly motivated by the kinetic theory of gases, for instance the choice of moments as variables . But even so, extended thermodynamics is a field theory in its own right, it is not kinetic theory . The kinetic theory, complete with boltzmann equation and sto\zahlansatz, offers another possibility of discussing finite propagation speeds or speeds smaller than c in the relativistic case . Such discussions are more directly based on the observation that the atoms cannot be faster than c. thus cercignani has directly linked the phase speed of small harmonic waves to the speed of particles and proved that the phase speeds are smaller than c. cercignani & majorana in a follow - up paper have exploited the full dispersion relation to calculate phase speeds and attenuation as functions of frequency, albeit for a simplified collision term . Earlier works on the kinetic theory which address the question of propagation speeds include sirovich & thurber and wang chang & uhlenbeck . This section explains the formal structure of modern extended thermodynamics, relativistic or otherwise . Its key ingredients are field equations of balance typelocal constitutive equationsentropy balance inequalityconcavity of entropy density field equations of balance type local constitutive equations entropy balance inequality concavity of entropy density thermodynamic processes are defined and characteristic speeds and the pulse speed are introduced . Thermodynamics, and in particular relativistic thermodynamics is a field theory with the primary objective to determine the thermodynamic fields . These are typically the 14 fields of the number density of particles, the particle flux vector and the fields of the stress - energy - momentum tensor . However, in extended thermodynamics we have generally more fields and therefore it is better at least for the initial arguments we have x = ct and x = (x, x, x)2 . For the determination of the n fields u we need field equations generally n of them and these are based on the equations of balance of mechanics and thermodynamics . The generic form of these balance equations reads (1)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${f^a}{,_a} = \pi .$$\end{document} the comma denotes partial differentiation with respect to x, and f is the n - vector of densities, while f is the n - vector of flux components . Thus f represents n four - fluxes, and is the n - vector of productions . Obviously the balance equations (1) are not field equations for the fields u, at least not in this form . These relate the four - fluxes f and the productions to the fields u in a materially dependent manner . We write (2)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${f^a} = {\hat f^a}(u)\;\;\;\;\;{\rm{and}}\;\;\;\;\;\pi = \hat \pi (u).$$\end{document} ^f and ^ denote the constitutive functions . Note that the constitutive quantities f and at one event depend only on the values of u at that same event . In particular there is no dependence on gradients and time derivatives of u. if the constitutive functions ^f and ^ are explicitly known, we may eliminate f and between the balance equations (1) and the constitutive relations (2) and obtain a set of explicit field equations for the fields u. these are quasilinear partial differential equations of first order . Every solution of the field equations is called a thermodynamic process . Since, however, the constitutive functions ^f and ^ are generally not explicitly known, the major task of thermodynamics is the determination of these functions, or at least the restriction of their generality . In simple cases it is possible to reduce the constitutive functions to a few coefficients which may be turned over to the experimentalist for measurement . The tools of the constitutive theory are certain universal physical principles which have come to be accepted by the extrapolation of common experience . Above all the entropy density h and the entropy flux h combine to form a four - vector h = (ch, h), whose divergence h, a is equal to the entropy production . The four - vector h and are both constitutive quantities and is assumed non - negative for all thermodynamic processes . Thus we may write h = ^h(u), = ^(u) and (3)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${h^a}_{,a} = \sigma \ge 0\;\;\;\;\forall \;\;\;\;{\rm{thermodynamic processes}}{\rm{. }}$$\end{document} this inequality is clearly an extrapolation of the entropy inequalities known in thermostatics and thermodynamics of irreversible processes; it was first stated in this generality by mller [36, 38].the principle of relativity . The principle of relativity requires that the field equations and the entropy inequality have the same form in all galilei frames for the non - relativistic case, or in alllorentz frames for the relativistic case . The formal statement and exploitation of this principle have to await a specific choice for the fields u and the four - fluxes f.the requirement of concavity of the entropy density . It is possible, and indeed common, to make a specific choice for the fields u and the concavity postulate is contingent upon that choice . In the non - relativistic case we choose the fields u as the densities f. the requirement of concavity demands that the entropy density h be a concave function of the variables f: (4)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{{{\partial ^2}{h^0}}}{{\partial {f^0}\partial {f^0}}} \sim {\rm{negative}}\;{\rm{definite}}.$$\end{document}in the relativistic case we choose the fields u as the densities f = fa in a generic lorentz frame that moves with the four - velocity ca with respect to the observer . We cannot be certain that in all these frames the entropy density h = ha is concave as a function of f . Therefore we assume that there is at least one a a privileged one, denoted by a such that h = ha is concave with respect to f = fa, viz . (5)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{{{\partial ^2}{h_{\bar \zeta}}}} {{\partial {f_{\bar \zeta}} \partial {f_{\bar \zeta}}}} \sim {\rm{negative}}\;{\rm{definite}}{\rm{. }}$$\end{document} the privileged co - vector a remains to be chosen, see section 4.1 . The entropy inequality . The entropy density h and the entropy flux h combine to form a four - vector h = (ch, h), whose divergence h, a is equal to the entropy production . The four - vector h and are both constitutive quantities and is assumed non - negative for all thermodynamic processes . Thus we may write h = ^h(u), = ^(u) and (3)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${h^a}_{,a} = \sigma \ge 0\;\;\;\;\forall \;\;\;\;{\rm{thermodynamic processes}}{\rm{. }}$$\end{document} this inequality is clearly an extrapolation of the entropy inequalities known in thermostatics and thermodynamics of irreversible processes; it was first stated in this generality by mller [36, 38]. The principle of relativity requires that the field equations and the entropy inequality have the same form in all galilei frames for the non - relativistic case, or in alllorentz frames for the relativistic case . The formal statement and exploitation of this principle have to await a specific choice for the fields u and the four - fluxes f. galilei frames for the non - relativistic case, or in all lorentz frames for the relativistic case . It is possible, and indeed common, to make a specific choice for the fields u and the concavity postulate is contingent upon that choice . In the non - relativistic case we choose the fields u as the densities f. the requirement of concavity demands that the entropy density h be a concave function of the variables f: (4)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{{{\partial ^2}{h^0}}}{{\partial {f^0}\partial {f^0}}} \sim {\rm{negative}}\;{\rm{definite}}.$$\end{document}in the relativistic case we choose the fields u as the densities f = fa in a generic lorentz frame that moves with the four - velocity ca with respect to the observer . We cannot be certain that in all these frames the entropy density h = ha is concave as a function of f . Therefore we assume that there is at least one a a privileged one, denoted by a such that h = ha is concave with respect to f = fa, viz . (5)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{{{\partial ^2}{h_{\bar \zeta}}}} {{\partial {f_{\bar \zeta}} \partial {f_{\bar \zeta}}}} \sim {\rm{negative}}\;{\rm{definite}}{\rm{. }}$$\end{document} the privileged co - vector a remains to be chosen, see section 4.1 . In the non - relativistic case we choose the fields u as the densities f. the requirement of concavity demands that the entropy density h be a concave function of the variables f: (4)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{{{\partial ^2}{h^0}}}{{\partial {f^0}\partial {f^0}}} \sim {\rm{negative}}\;{\rm{definite}}.$$\end{document} in the relativistic case we choose the fields u as the densities f = fa in a generic lorentz frame that moves with the four - velocity ca with respect to the observer . We cannot be certain that in all these frames the entropy density h = ha is concave as a function of f . Therefore we assume that there is at least one a a privileged one, denoted by a such that h = ha is concave with respect to f = fa, viz . (5)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{{{\partial ^2}{h_{\bar \zeta}}}} {{\partial {f_{\bar \zeta}} \partial {f_{\bar \zeta}}}} \sim {\rm{negative}}\;{\rm{definite}}{\rm{. }}$$\end{document} the privileged co - vector a remains to be chosen, see section 4.1 . In both cases the concavity postulate makes it possible that the entropy be maximal for a particular set of fields the set corresponding to equilibrium and that is its attraction for physicists . For mathematicians the attraction of the concavity postulate lies in the observation that concavity implies symmetric hyperbolicity of the field equations, see sections 3.2 and 4.2 below . The key to the exploitation of the entropy inequality lies in the fact that the inequality should hold for thermodynamic processes, i.e. Solutions of the field equations rather than for all fields . By a theorem proved by liu this constraint may be removed by the use of lagrange multipliers themselves constitutive quantities, so that = ^(u) holds . Indeed, the new inequality (6)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${h^a}_{,a} - \lambda \; \cdot \;({f^a}_{,a} - \pi) \ge 0\;\;\;\forall \;\;\;{\rm{fields}}\;u.$$\end{document} is equivalent to (3). Liu s proof proceeds from the observation that the field equations and the entropy equation are linear functions of the derivatives u, a . By the cauchy - kowalewski theorem these derivatives are local representatives of an analytical thermodynamic process and therefore the entropy principle requires that the field equations and the entropy equation must hold for all u, a . It is then a simple problem of linear algebra to prove that \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{{\partial {h^a}}}{{\partial u}}\;\;\;{\rm{must}}\;{\rm{be}}\;{\rm{a}}\;{\rm{linear}}\;{\rm{combination}}\;{\rm{of}}\;\;\;\frac{{\partial {f^a}}}{{\partial u}}.$$\end{document} liu s proof is not restricted to quasilinear systems of first order equations but here we need his result only in that particularly simple case . We may use the chain rule on h = ^h(u) and f = ^f(u) in (6) and obtain (7)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left ({\frac{{\partial {h^a}}}{{\partial u}} - \lambda \cdot \frac{{\partial {f^a}}}{{\partial u}}} \right){u_{,a}} + \lambda \cdot \pi \ge 0.$$\end{document} the left hand side is an explicit linear function of the derivatives u, a and, since the inequality must hold for all fields u, it must hold in particular for arbitrary values of the derivatives u, a . The entropy inequality could thus easily be violated by some choice of u, a unless we have (8)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$d{h^a} = \lambda \; \cdot \;d{f^a};$$\end{document} and there remains the residual inequality (9)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\lambda \; \cdot \;\pi \ge 0.$$\end{document} the differential forms (8) represent a generalization of the gibbs equation of equilibrium thermodynamics; the classical gibbs equation for the entropy density is here generalized into four equations for the entropy four - flux . Relation (9) is the residual entropy inequality which represents the irreversible entropy production . Note that the entropy production is entirely due to the production terms in the balance equations . The system of field equations (1), (2) may be written as a quasilinear system of n equations in the form (10)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{{\partial {f^a}}}{{\partial u}}{u_{,a}} = \pi .$$\end{document} such a system allows the propagation of weak waves, so - called acceleration waves . There are n such waves and their speeds are called characteristic speeds, which are not necessarily all different . Let (x) = 0 define the wave front; thus (11)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{{\partial \phi}} {{\partial {x^a}}} = \left\| {{\rm{grad}}\;\phi} \right\|{n_a}\;\;\;{\rm{and}}\;\;\;\frac{{\partial \phi}} {{\partial ct}} = - \left\| {{\rm{grad}}\;\phi} \right\|\frac{v}{c}$$\end{document} define its unit normal n and the speed v. an easy manipulation provides (12)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{{{v^2}}}{{{c^2}}} = 1 + \frac{{{g^{ab}}{\phi _ {, a}}{\phi _ {, b}}}}{{{{\left\| {{\rm{grad}}\;\phi} \right\|}^2}}}.$$\end{document} since in a weak wave the fields u have no jump across the front, the jumps in the gradients must have the direction of n and we may write (13)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$[{u_{,a}}] = \delta u{n_{a,}}\:\:\:[{u_{,0}}] = - \frac{v}{c}\delta u,\:\:\:{\rm{where}}\:\delta u = \left [{{n_a}\frac{{\partial u}}{{\partial {x^a}}}} \right].$$\end{document} u is the magnitude of the jump of the gradient of u. the square brackets denote differences between the front side and the back side of the wave . In the field equations (10) the matrix \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{{\partial {f^a}}}{{\partial u}}$$\end{document} and the productions are equal on both sides of the wave, since both only depend on u and since u is continuous . Thus, if we take the difference of the equations on the two sides and use (13) and (11), we obtain (14)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\phi _ {, a}}\frac{{\partial {f^a}}}{{\partial u}}\delta u = 0.$$\end{document} non - trivial solutions for u require that this linear homogeneous system have a vanishing determinant (15)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\rm{det}}\left ({{\phi _ {, a}}\frac{{\partial {f^a}}}{{\partial u}}} \right) = 0.$$\end{document} insertion of (11) into (15) provides an algebraic equation for v whose solutions for a prescribed direction n determine n wave speeds v, of which the largest one is the pulse speed . Equation (15) is called the characteristic equation of the system (10) of field equations . By (11) it may be written in the form (16)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\rm{det}}\left ({\frac{{\partial {f^a}}}{{\partial u}}{n_a} - \frac{v}{c}\frac{{\partial {f^0}}}{{\partial u}}} \right) = 0.$$\end{document} it is shown in this section that the concavity of the entropy density h with respect to the fields f implies global invertibility of the map f, where is the n - vector of lagrange multipliers . Also the system of field equations written in terms of is recognized as a symmetric hyperbolic system which guarantees finite characteristic speeds andwell - posedness of initial value problems . Thus we conclude that no paradox of infinite speeds can arise in extended thermodynamics, at least not for finitely many variables . A commonly treated special case occurs when the fields u are moments of the phase density of a gas . In this case the pulse speed depends on the degree of extension, i.e. On the number n of fields u. for a gas in equilibrium the pulse speeds can be calculated for any n. also it can be estimated that the pulse speed tends to infinity as n grows to infinity . We recall the argument of section 2.2 concerning concavity and choose the fields u to mean the fields of densities f. thus equation (8), for a = 0, leads to (17)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\lambda = \frac{{\partial {h^0}}}{{\partial {f^0}}},\;\;\;{\rm{hence}}\;\;\;\frac{{\partial \lambda}} {{\partial {f^0}}} = \frac{{{\partial ^2}{h^0}}}{{\partial {f^0}\partial {f^0}}}.$$\end{document} therefore the concavity of the entropy density h in the variables f the negative - definiteness of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{{{\partial ^2}{h^0}}}{{\partial {f^0}\partial {f^0}}}$$\end{document} implies global invertibility between the field vector f and the lagrange multipliers the transformation f helps us to recognize the structure of the field equations and to find generic restrictions on the constitutive functions . Indeed, obviously, with as field vector instead of u, or f, we may rephrase (8) in the form (18)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$d{h'^a} = {f^a}d\lambda, $$\end{document} where (19)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${h'^a} = \lambda \; \cdot \;{f^a} - {h^a},$$\end{document} thus we have (20)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${f^a} = \frac{{\partial {{h'}^a}}}{{\partial \lambda}}, $$\end{document} and (21)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${h^a} = \lambda \frac{{\partial {{h'}^a}}}{{\partial \lambda}} {h'^a},$$\end{document} so that the constitutive quantities f and h result from h defined by equation (19) through differentiation . It follows from equation (20) that \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{{\partial {f^a}}}{{\partial \lambda}} \;{\rm{is}} \;{\rm{symmetric,}}$$\end{document} which implies 4n(n - 1) restrictions on the constitutive functions ^f(). Using the new variables we may write the field equations in the form (22)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{{\partial {f^a}}}{{\partial \lambda}} {\lambda _ {, a}}{\rm {=}} \pi (\lambda), $$\end{document} or, by (20): (23)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{{{\partial ^2}{{h'}^a}}}{{\partial \lambda \partial \lambda}} {\lambda _ {{\rm{,}}a}}{\rm {=}} \pi {\rm{(}}\lambda {\rm{),}}$$\end{document} we observe that the coefficient matrices in (23) are hessian matrices derived from the vector potential h. therefore the matrices are symmetric . Also the matrix \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{{{\partial ^2}{{h'}^0}}}{{\partial \lambda \partial \lambda}} $$\end{document} is negative definite on account on the concavity (4) of h with respect to f. this is so, because the defining equation of h, viz . (24)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{h'}^0} = \lambda \; \cdot \;{f^0} - {h^0}$$\end{document} represents the legendre transformation from h to h connected with the map f between dual fields . Indeed, we have by (20, 21) and (8) (25)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${f^0} = \frac{{\partial {{h'}^0}}}{{\partial \lambda}} \;\;\;{\rm{and}}\;\;\;\lambda = \frac{{\partial {h^0}}}{{\partial {f^0}}}.$$\end{document} such a transformation preserves convexity or concavity so that h is a concave function of, since h is a concave function of f. a quasilinear system of the type (23) with symmetric coefficient matrices, of which the temporal one is definite, is called symmetric hyperbolic . We conclude that symmetric hyperbolicity of the equations (23) for the fields is equivalent to the concavity of the entropy density h in terms of the fields of densities f. hyperbolicity implies finite characteristic speeds, and symmetric hyperbolic systems guarantee the well - posedness of initial value problems, i.e. Existence and uniqueness of solutions at least in the neighbourhood of an event and continuous dependence on the data . Thus without having actually calculated a single characteristic speed, we have resolved cattaneo s paradox of infinite speeds . The structure of extended thermodynamics guarantees that all speeds are finite; no paradox can occur! The fact that a system of balance - type field equations is symmetric hyperbolic, if it is compatible with the entropy inequality and the concavity of the entropy density was discovered by godunov in the special case of eulerian fluids . In general this ruggeri & strumia have found that the symmetry is revealed only when the lagrange multipliers are chosen as variables; these authors were strongly motivated by liu s results of 1972 and by a paper by friedrichs & lax which appeared a year earlier . In a gas the most plausible choice for the four - fluxes f are the moments of the phase density f(x, p, t) of the atoms . Thus we have (26)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$f_\alpha ^a = \int {{p^a}{p_\alpha} fdp}, \;\;\;\;\;(\alpha = 1,\;2,\; \ldots n),\;(a = 0,\;1,\;2,\;3).$$\end{document} p is equal to mc, where m is the atomic mass, while p denotes the cartesian coordinates of the momentum of an atom . Is a multi - index and p stands for (27)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${p_\alpha} = \left\ {\begin{array}{l} 1\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\alpha = 1\\ {p^{{i_1}}}\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\alpha = 2,\;3,\;4\\ {p^{{i_1}}}{p^{{i_2}}}\;\;\;\;\;\;\;\;\;\;\;\;\;\alpha = 5,\;6, \ldots 10\\ {p^{{i_1}}}{p^{{i_2}}} \cdots {p^{{i_n}}}\;\;\;\;\;\;\;\alpha = n - \frac{1}{2}(n + 1)(n + 2), \ldots, n \end{array} \right\}$$\end{document} so that the densities f0, (= 1, 2,...n) form a hierarchy of moments of increasing tensorial degree up to degree n. because of the evident symmetry of (27) there is a relation between n and n, viz . (28)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$n = \frac{1}{6}(n + 1)(n + 2)(n + 3).$$\end{document} the kinetic theory of gases implies that the moments (26) satisfy equations of balance of the type (1) so that the foregoing analysis holds . In particular, we have (18) which may now be written in the form (29)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$d{h'^a} = f_\alpha ^ad{\lambda _ \alpha} = $$\end{document} (30)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\int {{p^a}d({\lambda _ \alpha} {p_\alpha}) fdp =} $$\end{document} (31)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\int {{p^a}df({\lambda _ \alpha} {p_\alpha}) dp =} $$\end{document} (32)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$d\int {{p^a}f({\lambda _ \alpha} {p_\alpha}) dp .} $$\end{document} we introduce = p and note that by (30) the phase density depends on the single variable only . Also (32) implies that the vector potential has the form (33)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${h'^a} = \int {{p^a}f(\chi) dp,} $$\end{document} where, by (31), \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{{df}}{{d\chi}} = f$$\end{document} holds . The field equations (23) now read ax (34)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left [{\int {{p^a}{p_\alpha} {p_\beta}} \frac{{{d^2}f}}{{d{\chi ^2}}}dp} \right]{\lambda _ {\beta, a}} = {\pi _ \alpha}.$$\end{document} obviously the coefficient matrices are symmetric in, and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\int {{p_\alpha} {p_\beta}} \frac{{{d^2}f}}{{d{\chi ^2}}}dp}$$\end{document} is negative definite, provided that (35)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\frac{{{d^2}f}}{{d{\chi ^2}}} <0,}$$\end{document} i.e. F() must be concave for the system (34) to be symmetric hyperbolic . For moments as variables the entropy four - flux h follows from (19) and (33). We obtain (36)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${h^a} = \int {{p^a}(\chi f(\chi)} - f(\chi)) \;dp.$$\end{document} on the other hand statistical mechanics defines the four - flux of entropy by (e.g. See huang) (37)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${h^a} = - k\int {{p^a}\left ({\ln \frac{f}{y} \pm \frac{y}{f}\left ({1 \pm \frac{f}{y}} \right)\ln \left ({1 \pm \frac{f}{y}} \right)} \right)fdp\;{\rm{for}}\;} {\rm{fermions}}\;{\rm{bosons}}{\rm{. }}$$\end{document} k is the boltzmann constant and 1/y is the smallest phase space element . Comparison shows that we must have \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\chi f(\chi) - f(\chi) = - k\left ({\ln \frac{f}{y} \pm \frac{y}{f}\left ({1 \pm \frac{f}{y}} \right)\ln \left ({1 \pm \frac{f}{y}} \right)} \right)f,$$\end{document} and hence, by differentiation with respect to, (38)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$f = \frac{y}{{{{\rm{e}}^{\chi /k}} \pm 1}},$$\end{document} so that (39)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$f = \mp ky\;\ln \left ({1 \pm {{\rm{e}}^ {- \chi /k}}} \right).$$\end{document} f is the phase density appropriate to a degenerate gas in non - equilibrium . Differentiation of (39) with respect to proves the inequality (35). Therefore symmetric hyperbolicity of the system (34) and hence the concavity of the entropy density with respect to the variables f0 is implied by the moment character of the fields and the form of the four - flux of entropy . For a non - degenerate gas the term 1 in the denominator of (38) we have (40)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$f = y{{\rm{e}}^ {- \chi /k}},$$\end{document} hence (41)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$f = - kf\;\;\;{\rm{and}}\;\;\;\frac{{{d^2}f}}{{d{\chi ^2}}} = - \frac{1}{k}f,$$\end{document} and therefore the field equations (23), (34) assume the form (42)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left [{- \frac{1}{k}\int {{p^a}{p_\alpha} {p_\beta} fdp}} \right]{\lambda _ {\beta, a}} = {\pi _ \alpha}.$$\end{document} note that the matrices of coefficients are composed of moments in this case of a non - degenerate gas . We know that a non - degenerate gas at rest in equilibrium exhibits the maxwellian phase density (43)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${f_e} = \frac{n}{{{{\sqrt {2\pi mkt}} ^3}}}{{\rm{e}}^ {- \frac{{{p^2}}}{{2mkt}}}}.$$\end{document} n and t denote the number density and the temperature of the gas in equilibrium . Comparison of (43) with (40) shows that only two lagrange multipliers are non - zero in equilibrium, viz . (44)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\lambda ^e} = k\ln \frac{{y{{\sqrt {2\pi mkt}} ^3}}}{n}\;\;\;{\rm{and}}\;\;\;\lambda _ {ii}^e = \frac{1}{{{\textstyle{2 \over 3}}mkt}}.$$\end{document} we recall the discussion of characteristic speeds in section 2.4 which we apply to the system (23) of field equations . The characteristic equation of this system reads (45)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\rm{det}}\left ({{\phi _ {, a}}\frac{{{\partial ^2}{{h'}^a}}}{{\partial \lambda \partial \lambda}}} \right) = 0$$\end{document} or, by (11): (46)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\rm{det}}\left ({\frac{{{\partial ^2}{{h'}^a}}}{{\partial \lambda \partial \lambda}} {n_a}\frac{v}{c}\frac{{{\partial ^2}{{h'}^0}}}{{\partial \lambda \partial \lambda}}} \right) = 0.$$\end{document} this equation determines the characteristic speeds v, whose maximal value vmax is the pulse speed . In the case of moments and for a non - degenerate gas at rest and in equilibrium this equation reads, by (42), (47)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\rm{det}}\left ({\int {({p^a}{n_a} - vm)} {p_\alpha} {p_\beta} {f_e}dp} \right) = 0.$$\end{document} fe is the maxwellian phase density, so that all integrals in (47) are gaussian integrals, easy to calculate . Recall that, range over the values 1 through n. he has made a list of vmax which is represented here in table 1 . Vmax is normalized in table 1 by \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${c_o} = \sqrt {\frac{{5kt}}{{3 m}}} $$\end{document}, the ordinary speed of sound, sometimes called the adiabatic sound speed . N: number of moments, n: highest degree of moments, vmax / c0: pulse speed . N n vmax / c0410.774596671021.341640792031.808229483542.212999465652.574958748462.9050781112073.2103524516583.4955579122093.76412372286104.01860847364114.26098014455124.26098014560134.71528716680144.92949284816155.13625617969165.33629131140175.530205691330185.718521121540195.901689621771206.080105852024216.254116732300226.424029192600236.590116272925246.752622133276256.911766153654267.067746314060277.220741984495287.370916294960297.518418075456307.663383625984317.805938046545327.946196547140338.084265497770348.220243318436358.354221299139368.486284329880378.6165114410660388.7449764411480398.8717483312341408.9968917113244419.1204672214190429.2425318415180439.36313918 pulse speed in extended thermodynamics of moments . N: number of moments, n: highest degree of moments, vmax / c0: pulse speed . Inspection of table 1 shows that the pulse speed increases monotonically with the number of moments and there is clearly a suspicion that it may tend to infinity as n goes to infinity . This suspicion will presently be confirmed . Since in (47) the integral pnappfedp is symmetric and ppfedp is symmetric and positive definite, it follows from linear algebra3 that (48)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\int {({p^a}{n_a} - {v_{{\rm{max}}}}m){p_\alpha} {p_\beta} {f_e}dp} \;\;\;{\rm{is}}\;{\rm{negative}}\;{\rm{semi - definite}}{\rm{. }}$$\end{document} boillat & ruggeri have used this knowledge to derive an estimate for vmax in terms of n, the highest tensorial degree of the moments . The estimate reads (49)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{{{v_{\max}}}} {{{c_0}}} \ge \sqrt {\frac{6}{5}\left ({n - \frac{1}{2}} \right)}.$$\end{document} therefore, indeed, as more and more moments are drawn into the scheme of extended thermodynamics, the pulse speed goes up and, if n tends to infinity, so does vmax . The proof of (49) rests on the realization that because of symmetry \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${p_\alpha} = {p^{{i_1}}}{p^{{i_2}}} \ldots {p^{{i_l}}}$$\end{document} has only \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{1}{2}(l + 1)(l + 2)$$\end{document} independent components and they are simply powers of p, p and p, so that p may be written as (p)(p)(p) with p + q + r = l. accordingly \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${p_\beta} = {p^{{j_1}}}{p^{{j_2}}} \ldots {p^{{j_k}}}$$\end{document} may be written as (p)(p)(p) with s + t + u = k. therefore (48) assumes the form (50)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\int {({p^a}{n_a} - {v_{{\rm{max}}}}m){{({p^1})}^{p + s}}{{({p^2})}^{q + t}}{{({p^3})}^{r + u}}{f_e}dp - {\rm{negative}} \;{\rm{semi - definite}}{\rm{.}}} $$\end{document} the elements of a semi - definite matrix aij satisfy the inequalities aiiajj aij2 and therefore (50) implies (51)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{l} \left ({\int {({p^a}{n_a} - {v_{{\rm{max}}}}m){{({p^1})}^{2p}}{{({p^2})}^{2q}}{{({p^3})}^{2r}}\;{f_e}dp}} \right) \times \\ \times \left ({\int {({p^a}{n_a} - {v_{{\rm{max}}}}m){{({p^1})}^{2s}}{{({p^2})}^{2t}}{{({p^3})}^{2u}}\;{f_e}dp}} \right) \ge \\ \ge {\left ({\int {({p^a}{n_a} - {v_{{\rm{max}}}}m){{({p^1})}^{2p + s}}{{({p^2})}^{2q + t}}{{({p^3})}^{2r + u}}\;{f_e}dp}} \right)^2} \end{array}$$\end{document} since fe is an even function of p we obtain (52)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{l} {({v_{{\rm{max}}}})^2}{m^2}\left ({\int {{{({p^1})}^{2p}}{{({p^2})}^{2q}}{{({p^3})}^{2r}}\;{f_e}dp}} \right) \times \\ \times \left ({\int {{{({p^1})}^{2s}}{{({p^2})}^{2t}}{{({p^3})}^{2u}}\;{f_e}dp}} \right) \ge \\ \ge {\left ({\int {({p^a}{n_a} - {v_{{\rm{max}}}}m){{({p^1})}^{p + s}}{{({p^2})}^{q + t}}{{({p^3})}^{r + u}}\;{f_e}dp}} \right)^2} \end{array}$$\end{document} this estimate depends on the choice of the exponents p through u and we choose, rather arbitrarily p = n, s = n - 1 and all others zero . (52) implies (53)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$v_{{\rm{max}}}^2 \ge \frac{{{{\int {(p}} ^1}{)^{2n}}\;{f_e}d{p^1}}}{{{{({p^1})}^{2(n - 1)}}\;{f_e}d{p^1}}} = \frac{6}{5} \cdot \frac{5}{3}\frac{k}{m}t\left ({n - \frac{1}{2}} \right)$$\end{document} which proves (49). An easy check will show that for each n the value \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\sqrt {{\textstyle{6 \over 5}}(n - {\textstyle{1 \over 2}})} $$\end{document} lies below the corresponding values of table 1, as they must in the relativistic theory the entropy density is not a scalar, it depends on the frame . This fact creates problems: granted that an entropy density tends to be concave, which one would that be? To my knowledge we assume that there exists a privileged frame in which the entropy density is concave . And we choose the privileged frame such that symmetric hyperbolicity of the system of field equations is guaranteed . Symmetric hyperbolicity means finite characteristic speeds, not necessarily speeds smaller than the speed of light . However, for moments as four - fluxes it can be shown that all speeds are smaller or equal to c and that for infinitely many moments the pulse speed tends to c. moreover, for moments the privileged frame is the rest frame of the gas, at least, if the gas is non - degenerate . We recall the arguments of section 2.2 concerning concavity in the relativistic case and choose the fields u to mean the privileged densities the privileged entropy density is assumed by (5) to be concave with respect to the privileged fields f. the privileged co - vector a will be chosen so that the concavity of h implies symmetric hyperbolicity of the field equations . From (8) we obtain after multiplication by a (54)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\lambda = \frac{{\partial {h_{\bar \zeta}}}} {{\partial {f_{\bar \zeta}}}} + {h'^a}\frac{{\partial {{\bar \zeta} _ a}}}{{\partial {f_{\bar \zeta}}}}, $$\end{document} hence (55)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{{\partial \lambda}} {{\partial {f_{\bar \zeta}}}} = \frac{{{\partial ^2}{h_{\bar \zeta}}}} {{\partial {f_{\bar \zeta}} \partial {f_{\bar \zeta}}}} + \frac{\partial} {{\partial {f_{\bar \zeta}}}} \left ({{{h'}^a}\frac{{\partial {{\bar \zeta} _ a}}}{{\partial {f_{\bar \zeta}}}}} \right).$$\end{document} h is still defined as f - h, as in (19). From (55) it follows that the concavity of h(f) the negative definiteness of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{{{\partial ^2}{h_{\bar \zeta}}}} {{\partial {f_{\bar \zeta}} \partial {f_{\bar \zeta}}}} $$\end{document} implies global invertibility between the field vector f and the lagrange multipliers, provided that the privileged co - vector is chosen as co - linear to the vector potential h. we set (56)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\bar \zeta} ^a} = - \frac{{{{h'}^a}}}{{\sqrt {{{h'}^a}{{h'}_a}}}}.$$\end{document} indeed, in that case we have (57)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{h'}^a}\frac{{\partial {{\bar \zeta} _ a}}}{{\partial {f_{\bar \zeta}}}} = 0,$$\end{document} hence (58)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\bar \zeta} ^a}\frac{{{\partial ^2}{{\bar \zeta} ^a}}}{{\partial {f_{\bar \zeta}} \partial {f_{\bar \zeta}}}} = - \frac{{\partial {{\bar \zeta} ^a}\partial {{\bar \zeta} ^a}}}{{\partial {f_{\bar \zeta}} \partial {f_{\bar \zeta}}}} \; \sim \;{\rm{positive}}\;{\rm{semi - definite}}$$\end{document} so that, by (57), the second term on the right hand side of (55) vanishes and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{{\partial \lambda}} {{\partial {f_{\bar \zeta}}}} $$\end{document} is definite . Equation (58) will be used later . With as a field vector, instead of f, we may rephrase (8) in the form (59)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$d{{h'}^a} = {f^a} \cdot d\lambda, $$\end{document} or (60)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${f^a} = \frac{{\partial {{h'}^a}}}{{\partial \lambda}}, $$\end{document} hence (61)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${f_{\bar \zeta}} = \frac{{\partial {h_{\bar \zeta}}}} {{\partial \lambda}}, $$\end{document} where h = h = f - h. thus h is the legendre transform of h with respect to the map f . It follows that h is concave in, since h is concave in f; thus we have (62)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{{{\partial ^2}{{h'}_{\bar \zeta}}}} {{\partial \lambda \partial \lambda}} \sim \;{\rm{negative}}\;{\rm{definite}}{\rm{. }}$$\end{document} the transformation f helps us to recognize the structure of the field equations . Obviously with as the field vector, instead of f may rephrase the field equations (10) as (63)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{{\partial {f^a}}}{{\partial \lambda}} {\lambda _ {, a}} = \pi (\lambda), $$\end{document} or, by (60): (64)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{{{\partial ^2}{{h'}^a}}}{{\partial \lambda \partial \lambda}} {\lambda _ {, a}} = \pi (\lambda).$$\end{document} we observe that the coefficient matrices are hessian matrices and therefore symmetric . By the definition of symmetric hyperbolicity due to friedrichs the system is symmetric hyperbolic, if there exists at least one co - vector a for which (65)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{{{\partial ^2}{{h'}^a}}}{{\partial \lambda \partial \lambda}} {\zeta _ a} \sim \;{\rm{negative}}\;{\rm{definite}}\;\;\;\;\;({g^{ab}}{\zeta _ a}{\zeta _ b} = 1,\;\;\;\;\;{\zeta _ 0}> 0).$$\end{document} in our case with the concavity (5) of the entropy density h for \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\bar \zeta ^a} = - \frac{{{{h'}^a}}}{{\sqrt {{{h'}^a}{{h'}_a}}}} $$\end{document} it is clear that such a co - vector exists . Indeed we have (66)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{{{\partial ^2}{{h'}^a}}}{{\partial \lambda \partial \lambda}} {\bar \zeta _ a} = \frac{{{\partial ^2}{{h'}_{\bar \zeta}}}} {{\partial \lambda \partial \lambda}} + \frac{{\partial {{\bar \zeta} _ a}}}{{\partial \lambda}} \frac{{\partial {{h'}^a}}}{{\partial \lambda}} \sim \;{\rm{negative}}\;{\rm{definite}}$$\end{document} by (62) and (58). Thus symmetric hyperbolicity is implied by the concavity of the entropy density both in the relativistic and the non - relativistic case . It is true that in the relativistic case we have to rely on the privileged co - vector \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\bar \zeta ^a} = - \frac{{{{h'}^a}}}{{\sqrt {{{h'}^a}{{h'}_a}}}} $$\end{document} in this context and therefore on a privileged lorentz frame whose entropy density h is concave in f. the significance of this choice is not really understood . Indeed, we might have preferred the privileged frame to be the local rest frame of the body . In that respect it is reassuring that h is often co - linear to the four - velocity u as we shall see in section 4.3 below; but not always note that in the non - relativistic case the only time - like co - vector is a = (1, 0, 0, 0), a constant vector . In that case all the above - mentioned complications are absent: concavity of the one and only entropy density h is equivalent to symmetric hyperbolicity, see section 3 above . Also note that the requirement (65) of symmetric hyperbolicity ensures finite characteristic speeds, not necessarily speeds smaller than c as we might have wished . [in this respect we may be tempted to replace friedrich s definition of symmetric hyperbolicity by one of our own making, which might require (65) to be true for all time - like co - vectors a instead of at least one . If we did that, we should anticipate the whole problem of speeds greater than c. indeed, we recall the characteristic equation (15) which for our system (64) reads \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\rm{det}}\left ({{\phi _ {, a}}\frac{{{\partial ^2}{{h'}^a}}}{{\partial \lambda \partial \lambda}}} \right) = 0.$$\end{document} if (65) were to hold for all time - like co - vectors a, we could now conclude that,a is space - like, or light - like, so that g,a,b 0 holds . Thus (12) would imply v c. this is a clear case of assuming the desired result in a disguise and we do not follow this path .] Just like in the non - relativistic case the most plausible and popular choice of the four - fluxes f in relativistic thermodynamics is moments of the phase density f(x, p, t) of the atoms, viz . (67)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$f_\alpha ^a = \int {{p^a}{p_\alpha} fdp,\;\;\;\;(\alpha = 1,\;2, \ldots n),\;\;\;\;(a = 0,\;1,\;2,\;3)} $$\end{document} this is formally identical to the non - relativistic case that was treated in section 34 . There are essential differences, however p is now the lorentz vector of atomic four - momentum with p> 0 and ppa = mc . Thus instead of (27) we have \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${p_\alpha} = \left\ {\begin{array}{l} 1\\ {p^{{b_1}}}\\ {p^{{b_1}}}{p^{{b_2}}}\\ \vdots \\ {p^{{b_1}}}{p^{{b_2}}} \ldots {p^{{b_n}}}. \end{array} \right.$$\end{document} the element dp of phase space is now equal to dp / p0 instead of dp . P is now the lorentz vector of atomic four - momentum with p> 0 and ppa = mc . Thus instead of (27) we have \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${p_\alpha} = \left\ {\begin{array}{l} 1\\ {p^{{b_1}}}\\ {p^{{b_1}}}{p^{{b_2}}}\\ \vdots \\ {p^{{b_1}}}{p^{{b_2}}} \ldots {p^{{b_n}}}. \end{array} \right.$$\end{document} the element dp of phase space is now equal to dp / p0 instead of dp . Thus for instance in the relativistic case we still have (68)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${h'^a} = \int {{p^a}f(\chi) dp} $$\end{document} with (69)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$f(\chi) = \mp ky\ln \left ({1 \pm {{\rm{e}}^ {- \frac{\chi} {k}}}} \right),$$\end{document} just like (33) and (39). We conclude that the vector potential h is not generally in the class of moments . However, in the non - degenerate limit, where e 1 holds, we obtain from (69) (see also (41)) (70)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$f(\chi) = - ky{{\rm{e}}^ {- {\textstyle{\chi \over k}}}}\;\;\;{\rm{or}}\;\;\;f = - kf.$$\end{document} therefore h for a non - degenerate gas reads (71)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{h'}^a} = - k\int {{p^a}fdp} $$\end{document} and that is in the class of moments . In fact h is equal to the four - velocity u of the gas to within a factor . We have (72)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{h'}^a} = - \frac{{nk}}{c}{u^a},$$\end{document} where n is the number density of atoms in the rest frame of the gas . We recall the discussion in section 4.2 of the important role played by h in ensuring symmetric hyperbolicity of the field equations: symmetric hyperbolicity was due to the concavity of h(f) in the privileged frame moving with the four - velocity \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c{\bar \zeta ^a} = - c\frac{{{{h'}^a}}}{{\sqrt {{{h'}_a}{{h'}^a}}}} $$\end{document}. Now we see from (72) that for the non - degenerate gas we have c = u so that the privileged frame is the local rest frame of the gas . [there remains the question of why the rest frame is not the privileged one for a degenerate gas . We recall the form of the field equations (34) (73)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left [{\int {{p^a}{p_\alpha} {p_\beta} \frac{{{d^2}f}}{{d{\chi ^2}}}dp}} \right]{\lambda _ {\beta, a}} = {\pi _ \alpha} $$\end{document} which is still valid in the relativistic case, albeit with p as the lorentz vector of the atomic fourth - momentum rather than p = (mc, p) as in section 3 . We already know that \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{{{d^2}f}}{{d{\chi ^2}}} <0$$\end{document} holds . Also p is a time - like vector so that we have (74)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\zeta ^a}\int {{p^a}{p_\alpha} {p_\beta}} \frac{{{d^2}f}}{{d{\chi ^2}}}dp \sim \;{\rm{negative}}\;{\rm{definite}}$$\end{document} for all time - like co - vectors a . Therefore the characteristic equation of the system (73) of field equations, viz . (75)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\rm{det}}\left ({{\phi _ {, a}}\int {{p^a}{p_\alpha} {p_\beta}} \frac{{{d^2}f}}{{d{\chi ^2}}}dp} \right) = 0$$\end{document} implies that,a is space - like, or light - like and therefore by (12) all characteristic speeds are smaller than c. we conclude that the speed of light is an upper bound for the pulse speed vmax . [recall that the requirement (65) of symmetric hyperbolicity did not require speeds c. i have discussed that point at the end of section 4.2 . Now, however, in extended thermodynamics of moments, because of the specific form of the vector potential, the condition (65) is satisfied for all co - vectors . Therefore all speeds are c.] more explicitly, by (11), the characteristic equation (75) reads (76)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\rm{det}}\left ({\int {\left ({{p^a}{n_a} - \frac{v}{c}{p^0}} \right){p_\alpha} {p_\beta}} \frac{{{d^2}f}}{{d{\chi ^2}}}dp} \right) = 0$$\end{document} and this holds in particular for vmax . Obviously \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\int {{p^a}p} _ \alpha} {p_\beta} \frac{{{d^2}f}}{{d{\chi ^2}}}dp$$\end{document} is symmetric and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$- {\int {{p^0}p} _ \alpha} {p_\beta} \frac{{{d^2}f}}{{d{\chi ^2}}}dp$$\end{document} is positive definite and symmetric . Therefore it follows from linear algebra (see footnote (3)) that (77)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\int {\left ({{p^a}{n_a} - \frac{{{v_{{\rm{max}}}}}}{c}{p^0}} \right){p_\alpha} {p_\beta}} \frac{{{d^2}f}}{{d{\chi ^2}}}dp \sim \;{\rm{negative}}\;{\rm{semi - definite}}{\rm{. }}}$$\end{document} in very recent papers, boillat & ruggeri [6, 3] have used this knowledge to prove lower bounds for vmax . The lower bounds depend on n, the number of fields, and for the number of fields tending to infinity the lower bound of vmax tends to c from below . The strategy of proof is similar to the one employed in section 3.6 for the non - relativistic case . Therefore the pulse speeds of all moment theories are smaller than c, but they tend to c as the number of moments tends to infinity . This result compares well with the corresponding result in section 3.6 concerning the non - relativistic theory . In that case there was no upper bound so that the pulse speeds tended to infinity for extended thermodynamics of very many moments . While the synthetic treatment of the foregoing sections is concise and seems quite elegant, it is also little suggestive of the laws for heat flux and stress that we associate with non - equilibrium thermodynamics . Moreover, the elegance of this treatment disguises the fact that much work is needed in order to obtain specific results . The following section highlights this situation by considering a viscous heat - conducting gas, a material which is fully characterized by 14 fields, viz . The density and flux of mass, energy and momentum, and stress and heat flux . With this choice of fields we shall be able to exploit the principle of relativity and the entropy inequality in explicit form and to calculate some specific pulse speeds . It is true that much of the rigorous formal structure of the preceding section is lost when it comes to specific calculations . Linearization around equilibrium cannot be avoided, if we wish to obtain specific results, and that destroys global invertibility and general symmetric hyperbolicity . The objective of thermodynamics of viscous, heat - conducting gases is the determination of the 14 fields (78)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{l} {a^a}\;:\;{\rm{particle}}\;{\rm{flux}}\;{\rm{vector}}\\ {a^{ab}}:\;{\rm{energy - momentum}}\;{\rm{tensor}} \end{array}$$\end{document} in all events x. both a and a are lorentz tensors . The energy - momentum tensor is assumed symmetric so that it has 10 independent components . For the determination of these fields we need field equations and these are formed by the conservation laws of particle number and energy - momentum, viz . (79)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${a^a}_{,a} = 0$$\end{document} (80)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${a^{ab}}_{,b} = 0$$\end{document} and by the equations of balance of fluxes (81)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${a^{abc}}_{,c} = {i^{ab}}.$$\end{document} a is the flux tensor it is completely symmetric, and i is its production density . We assume (82)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${i^a}_a = 0\;\;\;{\rm{and}}\;\;\;{a^{ab}}_b = {c^2}{a^a}$$\end{document} so that among the 15 equations (79, 80, 81) there are 14 independent ones, which is the appropriate number for 14 fields . The components of a and a have the following interpretations (83)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{{20}{l}} {{a^0}\;\,:\,\;c\:\cdot\:{\rm{rest}}\,{\rm{mass}}\,{\rm{density}},}\\ {{a^a}\,\;:\,\;{\rm{flux}}\,{\rm{of}}\,{\rm{rest}}\,{\rm{mass}},}\\ {{a^{00}}\;\,:\,\;{\rm{energy}}\,{\rm{density}},}\\ {{a^{0a}}\,\;:\,\;{\rm{1/}}c\:\cdot\:{\rm{energy}}\:{\rm{flux}},}\\ {{a^{a0}}\,\;:\,\;c\:\cdot\:{\rm{momentum}}\,{\rm{density}},}\\ {{a^{ab}}\;\,:\,\;{\rm{momentum}}\,{\rm{flux}}.} \end{array}$$\end{document} the motivation for the choice of equations (79, 80, 81), and in particular (81), stems from the kinetic theory of gases . Indeed a and a are the first two moments in the kinetic theory and a, a = 0 and a, b = 0 are the first two equations of transfer . Therefore it seems reasonable to take further equations from the equation of transfer for the third moment a and these have the form (81). In the kinetic theory the set of equations (79, 80, 81) must be supplemented by constitutive equations for the flux tensor a and the flux production i. the generic form of these relations in a viscous, heat - conducting gas reads (84)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{l} {a^{abc}} = {{\hat a}^{abc}}({a^m},{a^{mn}})\\ {i^{ab}} = {{\hat i}^{ab}}({a^m},{a^{mn}}). \end{array}$$\end{document} if the constitutive functions and are known, we may eliminate a and i between (79, 80, 81) and (84) and obtain a set of field equations for a, a. each solution is called a thermodynamic process . It is clear upon reflection that this theory, based on (79, 80,81) and (84), provides a special case of the generic structure explained in section 2 . We recall the restrictive principles of the constitutive theory from section 2 and adjust them to the present case entropy inequality h, a 0 with h = (a, a),principle of relativity . The former principle was discussed and exploited in the general scheme of section 2, but the principle of relativity was not . This principle assumes that the constitutive functions,, generically are invariant under lorentz transformations \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${x^{a}} = {x^{a}}({x^b}).$$\end{document} thus the principle of relativity may be stated in the form (85)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c = \hat c({a^m},\:{a^{mn}})\:\:\:{\rm{and}}\:\:\:\mathop {{\rm {}} c}\limits^ = \hat c({a^{m}},{a^{mm}})$$\end{document} note that is the same function in both equations . It is complicated and cumbersome to exploit the constitutive theory but the results are remarkably specific, at least for near - equilibrium processes: will be reduced to the thermal equation of state. Will be reduced to the relaxation times of the gas, which may be considered to be of the order of magnitude of the mean time of free flight of its molecules . Will be reduced to the relaxation times of the gas, which may be considered to be of the order of magnitude of the mean time of free flight of its molecules . For details of the calculation the reader is referred to the literature, in particular to the book by mller & ruggeri [39, 40] or the paper by liu, mller & ruggeri . Here no matter how much a person may be conditioned to think relativistically, he will appreciate the decomposition of the four - tensors a, a and h into their suggestive time - like and space - like components . We have (86)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{l} {a^a} = nm{u^a}\\ {a^{ab}} = {t^{\left\langle {ab} \right\rangle}} + (p(n,\;e) + \pi) {h^{ab}} + {\textstyle{1 \over {{c^2}}}}({u^a}{q^b} + {u^b}{q^a}) + {\textstyle{e \over {{c^2}}}}{u^a}{u^b}\\ {h^a} = h{u^a} + {\phi ^a}, \end{array}$$\end{document} and the components have suggestive meaning as follows (87)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{l} n\;:\;{\rm{number}}\;{\rm{density}}\\ {u^a}\;:\;{\rm{velocity}}\\ {t^{\left\langle {ab} \right\rangle \;}}\;:\;{\rm{stress}}\;{\rm{deviator}}\\ p + \pi \;:\;{\rm{pressure}}\\ {q^a}\;:\;{\rm{heat}}\;{\rm{flux}}\\ e\;:\;{\rm{heat}}\;{\rm{flux}}\\ h\;:\;{\rm{entropy}}\;{\rm{density}}\\ {\phi ^a}\;:\;{\rm{(non - convective)}}\;{\rm{entropy}}\;{\rm{flux}}{\rm{.}} \end{array}$$\end{document} at least this is how n through are to be interpreted in the rest frame of the gas . We have defined \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${h^{ab}} = \frac{1}{{{c^2}}}{u^a}{u^b} - {g^{ab}}$$\end{document} and m is the molecular rest mass . The decomposition (86) is not only popular because of its intuitive quality but also, since it is now possible to characterize equilibrium as a process in which the stress deviator t the heat flux q and the dynamic pressure the non - equilibrium part of the pressure vanish . The equilibrium pressure p is a function of n and e, the thermal equation of state . In thermodynamics it is often useful to replace the variables (n, e) by \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\rm{fugacity}}\;\alpha \;\;\;{\rm{and}}\;\;\;{\rm{absolute}}\;{\rm{temperature}}\;t,$$\end{document} because these two variables can be measured at least in principle . Also and t are the natural variables of statistical thermodynamics which provides the thermal equation of state in the form p = p(, t). The transition between the new variables (, t) and the old ones (n, e) can be effected by the relations (88)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$nm = - \frac{1}{t}\dot p\;\;\;{\rm{and}}\;\;\;e = p' - p$$\end{document} where and here and below denote differentiation with respect to and ln t respectively . If we restrict attention to a linear theory in t, q, and, we can satisfy the principle of relativity with linear isotropic functions for a, i viz . (89)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{l} {a^{abc}} = (c_1 ^ 0 + c_1^\pi \pi) {u^a}{u^b}{u^c} + {\textstyle{{{c^2}} \over 6}}(nm - c_1 ^ 0 + c_1^\pi \pi).\\ \;\;\;\;\;\;\;\;\; \cdot ({g^{ab}}{u^c} + {g^{bc}}{u^a} + {g^{ca}}{u^b}) + {c_3}({g^{ab}}{q^c} + {g^{bc}}{q^a} + {g^{ca}}{q^b}) - \\ \;\;\;\;\;\;\;\;\; - {\textstyle{6 \over {{c^2}}}}{c_3}({u^a}{u^b}{q^c} + {u^b}{u^c}{q^a} + {u^c}{u^a}{q^b}) + \\ \;\;\;\;\;\;\;\;\; + {c_5}({t^{\left\langle {ab} \right\rangle}} {u^c} + {t^{\left\langle {bc} \right\rangle}} {u^a} + {t^{\left\langle {ca} \right\rangle}} {u^b}), \end{array}$$\end{document} (90)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${i^{bc}} = b_1^\pi \pi {g^{ab}} - \frac{4}{{{c^2}}}b_1^\pi \pi {u^a}{u^b} + {b_3}{t^{\left\langle {ab} \right\rangle}} + \frac{1}{{{c^2}}}{\hat b^4}({q^a}{u^b} + {q^b}{u^a}).$$\end{document} note that i vanishes in equilibrium so that no entropy production occurs in that state . The coefficients c and b in (89, 90) are functions of e and n, or and t. in fact, the entropy principle determines the c s fully in terms of the thermal equation of state p = p(, t) as follows (91)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{{20}{c}} {c_1 ^ 0 = \frac{{{{\gamma'} _ 1}}}{{2{c^2}t}}\;\;\;{\text{with}}\;\;\;{\gamma _ 1} = - 2{c^2}{t^6}\int {\frac{{\dot p}}{{{t^7}}}dt}} \\ {c_1^\pi = - \frac{2}{{{c^2}t}}\frac{{\left [{\begin{array}{{20}{c}} {- \ddot p}&{\dot p - \dot p'}&{{{\dot \gamma} _ 1}} \\ {\dot p - \dot p'}&{p' - p"}&{{{\gamma'} _ 1} - {\gamma _ 1}} \\ {{{\dot \gamma} _ 1}}&{{{\gamma'} _ 1} - {\gamma _ 1}}&{\frac{5}{3}{\gamma _ 2}} \end{array}} \right]}}{{\left [{\begin{array}{{20}{c}} {- \ddot p}&{\dot p - \dot p'}&{{{\dot \gamma} _ 1}} \\ {\dot p - \dot p'}&{p' - p"}&{{{\gamma'} _ 1} - {\gamma _ 1}} \\ {- \dot p} & {- p'}&{\frac{5}{3}{\gamma _ 1}} \end{array}} \right]}}} \\ {{c_3} = - \frac{1}{{2t}}\frac{{\left [{\begin{array}{{20}{c}} {\dot p} & {- {{\dot \gamma} _ 1}} \\ {{\gamma _ 1}}&{{\gamma _ 2}} \end{array}} \right]}}{{\left [{\begin{array}{{20}{c}} {\dot p} & {- {{\dot \gamma} _ 1}} \\ {p'}&{{\gamma _ 1} - {{\gamma'} _ 1}} \end{array}} \right]}}} \\ {{c_5} = - \frac{1}{{2t}}\frac{{{\gamma _ 2}}}{{{\gamma _ 1}}}\;\;\;{\text{with}}\;{\gamma _ 2} = 2{c^2}{t^8}\int {\frac{1}{{{t^3}}}{{\dot \gamma} _ 1}dt .}} \end{array}$$\end{document} the b s in (90) are restricted by inequalities, viz . (92)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$b_1^\pi \ge 0,\;\;\;{\hat b_4} \ge 0,\;\;\;{b_3} \le 0.$$\end{document} all b s have the dimension 1/sec and we may consider them to be of the order of magnitude of the collision frequency of the gas molecules . In conclusion we may write the field equations in the form (93)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${(nm{u^a})_{,a}} = 0$$\end{document} (94)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${({t^{\left\langle {ba} \right\rangle}} + (p + \pi) {h^{ba}} + \frac{1}{{{c^2}}}({q^b}{u^a} + {q^a}{u^b}) + \frac{e}{{{c^2}}}{u^b}{u^a})_{,a}} = 0$$\end{document} (95)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${a^{bca}}_{,a} = b_1^\pi \pi {g^{bc}} - \frac{4}{{{c^2}}}b_1^\pi \pi {u^b}{u^c} + {b_3}{t^{\left\langle {bc} \right\rangle}} + \frac{1}{{{c^2}}}{\hat b_4}({q^b}{u^c} + {q^c}{u^b}),$$\end{document} where a must be inserted from (89) and (91). We conclude that extended thermodynamics of viscous, heat - conducting gases is quite explicit provided we are given the thermal equation of state p = p(,t) except for the coefficients b. these coefficients must be measured and we proceed to show how . It is instructive to identify the classical constitutive relations of navier - stokes and fourier of tip within the scheme of extended thermodynamics . They are obtained from (93, 94, 95) by the first step of the so - called maxwell iteration which proceeds as follows: the n iterate \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{{20}{c}} {(n)} \\ i \end{array}ab\left ({{\text{or}}\:\begin{array}{{20}{c}} {(n)} \\ \pi \end{array},\:\begin{array}{{20}{c}} {(n)} \\ t \end{array}ab,\:\begin{array}{{20}{c}} {(n)} \\ q \end{array}a} \right)$$\end{document} results from (96)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{{20}{c}} {{a^a}_{,a} = 0}&{{\text{with}}\;{\text{the}}}&{\begin{array}{{20}{c}} {(e)} \\ a \end{array}{a_{,a}} = 0} \\ {\begin{array}{{20}{c}} {(n - 1)} \\ a \end{array}a{b_{,b}} = 0}&{{\text{initiation}}}&{\begin{array}{{20}{c}} {(e)} \\ a \end{array}a{b_{,b}} = 0} \\ {\begin{array}{{20}{c}} {(n - 1)} \\ a \end{array}ab{c_{,c}} = \begin{array}{{20}{c}} {(n)} \\ i \end{array}ab}&{{\text{agreement}}}&{\begin{array}{{20}{c}} {(e)} \\ a \end{array}ab{c_{,c}} = \begin{array}{{20}{c}} {(1)} \\ a little calculation provides the first iterates for dynamic pressure, stress deviator and heat flux in the form (97)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mathop \pi \limits^{(1)} = - \lambda [{u^a}_{,a}]$$\end{document} (98)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mathop {{t_{\left\langle {ab} \right\rangle}}} \limits^{(1)} = \mu \;[h_a^mh_b^n{u_{\left\langle {m, n} \right\rangle}}] $$\end{document} (99)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${q_a} = - \kappa \left [{h_a^m{{(\ln t)}_{,m}} - \frac{1}{{{c^2}}}\frac{{d{u_m}}}{{d\tau}}} \right]$$\end{document} with \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{l} \lambda = \frac{1}{{2tb_1^\pi}} \frac{{\left [{\begin{array}{{20}{c}} {- \ddot p}&{\dot p - \dot p'}&{{{\dot \gamma} _ 1}}\\ {\dot p - \dot p'}&{p' - p"}&{{{\gamma'} _ 1} - {\gamma _ 1}}\\ {{{\dot \gamma} _ 1}}&{{{\gamma'} _ 1} - {\gamma _ 1}}&{{\textstyle{5 \over 3}}{\gamma _ 2}} \end{array}} \right]}}{{\left [{\begin{array}{{20}{c}} {- \ddot p}&{\dot p - \dot p'}\\ {\dot p - \dot p'}&{p' - p"} \end{array}} \right]}}\\ \mu = \frac{1}{{2t{b_3}}}{\gamma _ 1}\\ \kappa = \frac{1}{{2{t^2}{{\hat b}_4}}}\frac{{\left [{\begin{array}{{20}{c}} {\dot p} & {- {{\dot \gamma} _ 1}}\\ {p'}&{{\gamma _ 1} - {{\gamma'} _ 1}} \end{array}} \right]}}{{\dot p}} \end{array}$$\end{document} these are the relativistic analogues of the classical phenomenological equations of navier - stokes and fourier ., and are the bulk viscosity, the shear viscosity and the thermal conductivity respectively; all three of these transport coefficients are non - negative by the entropy inequality . The only essential difference between the equations (97, 98, 99) and the non - relativistic phenomenological equations is the acceleration term in (99). This contribution to the fourier law was first derived by eckart, the founder of thermodynamics of irreversible processes . Thus for instance equilibrium of a gas in a gravitational field implies a temperature gradient, a result that antedates even eckart . We have emphasized that the field equations of extended thermodynamics should provide finite speeds . Below in section 5.6 we shall give the values of the speeds for non - degenerate gases . Indeed, if the phenomenological equations (97, 98, 99) are introduced into the conservation laws (93, 94) of particle number, energy and momentum, we obtain a closed system of parabolic equations for n, ua and e. this unwelcome feature results from the maxwell iteration; it persists to arbitrarily high iterates . For a relativistic gas jttner [22, 23] has derived the phase density for bosons and fermions, namely (100)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${f_e} = \frac{y}{{{\rm{exp}}\left [{\frac{m}{k}\alpha + \frac{{{u_a}}}{{kt}}{p^a}} \right] \mp 1}}\;\;\;{\rm{or}}\;\;\;{f_e} = \frac{y}{{{\rm{exp}}\left [{\frac{m}{k}\alpha + \frac{{m{c^2}}}{{kt}}\sqrt {1 + \frac{{{p^2}}}{{{m^2}{c^2}}}}} \right] \mp 1}}.$$\end{document} the latter equation is valid in the rest frame of the gas . For the non - degenerate gas he found that bessel functions of the second kind, viz . (101)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${k_n}\left ({\frac{{m{c^2}}}{{kt}}} \right) = \int\limits_0^\infty {\cos} \,h(n\rho) \,\exp \,\left ({- \frac{{m{c^2}}}{{kt}}\cos h\rho} \right)\,d\rho $$\end{document} are the relevant special functions . The thermal equation of state p = p(, t) reads (102)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$p = nkt\;\;\;{\rm{with}}\;\;\;n = \exp (- \frac{m}{k}\alpha) \;\cdot\;4\pi y{m^3}{c^3}\frac{{{k_2}\left ({\frac{{m{c^2}}}{{kt}}} \right)}}{{\frac{{m{c^2}}}{{kt}}}},$$\end{document} where 1/y is the smallest phase space element . From (102) we obtain with \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$g = \frac{{{k_3}}}{{{k_2}}}$$\end{document} and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\gamma = \frac{{m{c^2}}}{{kt}}$$\end{document} (103)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$e = nm{c^2}\;(g - \frac{1}{\gamma}) \,,\:\:\:\:\frac{{{\gamma _ 1}}}{t} = nm{c^2}\frac{2}{\gamma} g$$\end{document} and hence (104)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{l} c_1 ^ 0 = nm\left ({1 + \frac{6}{\gamma} g} \right)\\ c_1^\pi = - \frac{6}{{{c^2}}}\frac{{\left ({2 - \frac{5}{{{\gamma ^2}}}} \right) + \left ({\frac{{19}}{\gamma} - \frac{{30}}{{{\gamma ^3}}}} \right)g - \left ({2 - \frac{{45}}{{{\gamma ^2}}}} \right){g^2} - \frac{9}{\gamma} {g^3}}}{{\frac{3}{\gamma} - \left ({2 - \frac{{20}}{{{\gamma ^2}}}} \right)g - \frac{{13}}{\gamma} {g^2} + 2{g^3}}}\\ {c_3} = - \frac{1}{\gamma} \frac{{1 + \frac{6}{\gamma} g - {g^2}}}{{1 + \frac{5}{\gamma} g - {g^2}}}\\ {c_5} = \left ({\frac{6}{\gamma} + \frac{1}{g}} \right)\: . \end{array}$$\end{document} the transport coefficients read (105)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{l} \lambda = \frac{1}{3}\frac{{nm{c^2}}}{\gamma} \frac{1}{{b_1^\pi}} \frac {{- \frac{3}{\gamma} + \left ({2 - \frac{{20}}{{{\gamma ^2}}}} \right)g + \frac{{13}}{\gamma} {g^2} + 2{g^3}}}{{1 - \frac{1}{{{\gamma ^2}}} + \frac{5}{\gamma} g - {g^2}}}\\ \mu = - nkt\frac{1}{{{b_3}}}g\\ \kappa = - nkt\frac{{{c^2}}}{{{{\hat b}_4}}}(\gamma + 5 g - \gamma {g^2})\: . \end{array}$$\end{document} it is instructive to calculate the leading terms of the transport coefficients in the non - relativistic case mc kt . We obtain (106)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\lambda = - \frac{5}{{6b_1^\pi}} nkt\frac{1}{{{\gamma ^2}}}$$\end{document} (107)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mu = - \frac{1}{{{b_3}}}nkt$$\end{document} (108)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\kappa = - \frac{5}{{2{{\hat b}_4}}}\frac{{n{k^2}t}}{m}.$$\end{document} it follows that the bulk viscosity does not appear in a non - relativistic gas . Recall that the coefficients 1/b are relaxation times of the order of magnitude of the mean - time of free flight; so they are not in any way relativistically small . Note that, and are measurable, at least in principle, so that the b s may be calculated from (105). All constitutive coefficients are now explicit: the c s can be calculated from the thermal equation of state p = p(, t) and the b s may be measured . It might seem from (106) and (97) that the dynamic pressure is of order \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$o\left ({\frac{1}{{{\gamma ^2}}}} \right)$$\end{document} but this is not so as was recently discovered by kremer & mller . Indeed, the second step in the maxwell iteration for it provides a term that is of order \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$o\left ({\frac{1}{\gamma}} \right)$$\end{document}, see also . That term is proportional to the second gradient of the temperature t so that it may be said to be due to heating or cooling . Specific results of the type (104, 105) can also be calculated for degenerate gases with the thermal equation of state p(, t) for such gases . The results for 14 fields may be found in mller & ruggeri [39, 40]. We recall from section 2.4, in particular (14), that the jumps u across acceleration waves and their speeds of propagation are to be calculated from the homogeneous system (109)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\phi _ {, a}}\frac{{\partial {f^a}}}{{\partial u}}\delta u = 0.$$\end{document} in the present context, where the field equations are given by (79, 80) this homogeneous algebraic system spreads out into three equations, viz . (110)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\phi {, _ a}\delta {a^a} = 0,\:\;\;\;\;\;{\phi _ {, a}}\delta {a^{ab}} = 0,\:\;\;\;\;\;\phi {, _ a}\delta {a^{abc}} = 0.$$\end{document} by (89) and (91) this is a fully explicit system, if the thermal equation of state p = p(, t) is known . Seccia & strumia have calculated these speeds one transversal and two longitudinal ones for non - degenerate gases and obtained the following results in the non - relativistic and ultra - relativistic cases (111)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{*{20}{l}} {\frac{{m{c^2}}}{{kt}} \gg 1:{v_{{\rm{trans}}}} = \sqrt {\frac{{7k}}{{5m}}t}, \;\;\;v_{1{\rm{ong}}}^1 = \sqrt {\frac{{4k}}{{3m}}t}, \;\;\;v_{1{\rm{ong}}}^2 = \sqrt {5.18\frac{k}{m}t},} \\ {\frac{{m{c^2}}}{{kt}} \ll 1:{v_{{\rm{trans}}}} = \sqrt {\frac{1}{5}} c,\;\;\;\;\;\;\;v_{1{\rm{ong}}}^1 = \sqrt {\frac{1}{3}} c,\:\:\:\:\:\;\;\;\;v_{1{\rm{ong}}}^2 = \sqrt {\frac{3}{5}} c.} \end{array}$$\end{document} all speeds are finite and smaller than c. inspection shows that in the non - relativistic limit the order of magnitude of these speeds is that of the ordinary speed of sound while in the ultra - relativistic case the speeds come close to c. so as to anticipate a possible misunderstanding i remark that the equations (93, 94, 95) with a from (89) and (91) are neither symmetric nor fully hyperbolic . Indeed the underlying symmetry of the system (79, 80, 81), and (84) reveals itself only when the lagrange multipliers are used as variables . But (93, 94, 95, 89, 91) are equations for the physical variables a, a or in fact n, t, u, t, and q. also the hyperbolicity in the whole state space is lost, because the equations (89, 90) are restricted to linear terms . Therefore the system is hyperbolic only in the neighbourhood of equilibrium . For a more detailed discussion of these aspects,
Kienbck s disease, defined as avascular necrosis of the lunate, is a relatively rare condition with a poorly understood etiology . The disease is a relatively rare condition in which the etiology and natural history of this condition are poorly understood and even more poorly documented in the literature . A wide array of treatment recommendations is available, and reported results vary, which may hinder consistent treatment recommendations . Conservative and invasive treatments for kienbck s disease include the following: wrist immobilization with a splint during early disease stages, surgical joint - leveling procedures to attempt to reduce loading on the joint and to promote revascularization and increased blood flow, vascularized bone grafting, proximal row carpectomy, and total wrist arthrodesis . Proximal row carpectomy and total wrist arthrodesis attempt to alleviate arthritis pain due to carpal collapse; however, these 2 interventions greatly limit range of motion . None of these treatments effectively address the chronic pain, loss of range of motion, and decreased grip strength associated with kienbck s disease . The staging distinguishes only the state of collapse in an ordinal classification scheme and does not allow localization or indicate partial involvement of the lunate, which the image contrast from mri may provide . Osteochondral transfer for treatment of kienbck s disease may be possible for individuals with segmental or incomplete avascular necrosis of the lunate . Previous investigators have acknowledged the importance of donor site cartilage thickness to aid in minimizing postoperative abnormal stresses and resulting poor function . Selection of donor cartilage with an appropriate thickness may be difficult due to source availability and donor site location relative to the load - bearing region of a joint, which may lead to donor site morbidity . An alternative to matching cartilage thickness is the matching of curvature of the articular surface . Matching the curvature of donor and recipient sites has been shown to maintain preoperative contact stresses for a given loading regimen . In this short communication, we report the treatment of a patient s kienbck s disease by combining mri with mathematical modeling to optimize the congruency between the curvature of donor and recipient sites of an autologous osteoarticular plug transfer . The purpose of this study was to describe the feasibility of a novel surgical technique . The results indicate that donor site selection for autologous osteoarticular transfer using a quantitative evaluation of articular surface curvature may be beneficial for optimizing the likelihood for restoring the radius of curvature and thus joint articulation following cartilage repair . A 48-year - old, right hand dominant woman had left wrist pain associated with decreased wrist flexion since september 2009 . In november 2009, the patient experienced severe wrist pain after falling on her hand . After acquiring standard radiographs, the woman was diagnosed with kienbck s disease, and a proximal row carpectomy was recommended by an initial orthopedic surgeon . The patient sought a second opinion after understanding that the carpectomy would not only reduce pain during activities of daily living but would also limit range of motion . Upon presentation to our institution, physical examination of the left wrist revealed no focal swelling, dystrophic changes, or evidence of muscular atrophy . The left wrist range of motion was 20 of flexion and 30 of extension; the patient had full, painless range of motion in her right wrist . Grip strength in her left hand was 111.2 n as compared with 333.6 n in her right hand . Mri of the wrist was performed utilizing a clinical superconducting 3-t imaging unit (sigma hdx, general electric healthcare, waukesha, wi) and a dedicated wrist coil . Pulse sequencing included a 3-dimensional gradient - recalled sequence with tr / te of 39/14 milliseconds, field of view of 9 cm, slice thickness of 1 mm with no gap, flip angle of 10, and band width of 41.7 khz . Coronal, sagittal, and axial cartilage sensitive fast spin echo sequences were obtained with a long tr (4,000 - 5,500 milliseconds), moderate te (31 - 38 milliseconds), field of view of 8 to 9 cm, slice thickness of 2 to 3 mm, acquisition matrix of 512 512 (320 - 352), 2 excitations, a band width of 14.7 to 41.7 khz, and echo train length of 10 to 12 . Coronal fast inversion recovery sequence was performed with a tr of 5200 milliseconds, te of 23 milliseconds, inversion time of 170 milliseconds, field of view of 9 cm, slice thickness of 2.5 mm, matrix of 288 192, 2 excitations, band width of 31.2 khz, and echo train length of 12 . The examination demonstrated collapse of the proximal pole of the lunate, with subchondral sclerosis and a mild bone marrow edema pattern (fig . 1). Foci of completely devitalized marrow were noted in the proximal pole with an associated intense synovitis . Moderate partial wear of cartilage was seen over the collapsed lunate with preservation over the radial side of the lunate fossa . A best - fit circle was manually placed on the lunate with its articulation with the distal radius to determine the joint curvature . Preoperative coronal (a) and fat - suppressed (b) fast spin echo images of the wrist demonstrate sclerosis and partial collapse of the lunate (arrows) with hyperintensity in the adjacent cartilage . One - year postoperative sagittal (d) and fat - suppressed (e) fast spin echo images and computerized tomographic evaluation (f) demonstrate partial bony incorporation expected for the time interval . Computerized tomographic examination was performed utilizing axial acquisition with a slice resolution of 0.8 mm, subsequently reformatted into sagittal and coronal reformations . Concurrent computerized tomography was performed to quantify the extent of subchondral collapse, which measured up to 1 mm . Mri of the left knee was subsequently acquired to provide a digital template for the planned osteochondral transplantation . A 3-dimensional t1-weighted spoiled gradient echo data set with frequency - selective fat suppression was acquired for cartilage segmentation: tr of 13.6 milliseconds, te of 3.0 milliseconds, field of view of 16 cm, flip angle of 20, slice thickness of 1.5 mm, matrix of 512 512, receiver bandwidth of 41.7 khz, and 1.2 excitations . The cartilage was segmented from the image data using custom - written semiautomated software (general eelectric healthcare), with minimal manual editing performed as needed . A 3-dimensional mesh representation of the segmented lateral condyle image data was constructed and smoothed for curvature calculations . Curvature calculations were performed on the 3-dimensional surface mesh using a custom - written matlab (mathworks, natick, ma) program . 2a), and a best - fit paraboloid (z(x, y) = ax + bxy + cy) was fit to each vertex and the surrounding 2-ring neighborhood (fig . 2b). The analytical solutions for maximum curvature (max) and minimum curvature (min) were determined from the best - fit paraboloid (fig . This process is repeated for each vertex on the 3-dimensional surface mesh to generate max and min maps . Regions with large values of min display concavities on a surface, and regions with large values of max display convexities of a surface . The model of a paraboloid was chosen due to the computational efficiency as well as the verified accuracy of the curvature calculation as compared to other numerical methods . (a) each mesh vertex (red point) had a 2-ring neighborhood (blue points) calculated . (b) the coefficients of a best - fit paraboloid through the vertex and 2-ring neighborhood were used to calculate the maximum and minimum curvatures . The directions of maximum curvature (red arrow) and minimum curvature (green arrow) were also calculated . (c) the direction and magnitude of the maximum curvature and minimum curvature at the vertex are shown by the orientation and radius of the pink and green circles, respectively . The radius of each circle () is the inverse of the calculated curvature (= 1/). At the indicated vertex (red dot), the anterolateral femoral condyle had the maximum curvature in the mediolateral direction and minimum curvature in the anteroposterior direction . The patient underwent osteochondral transfer from her left femoral condyle to her left lunate . Using a tourniquet to control bleeding, the necrotic area of the lunate, which measured approximately 8 mm 1 mm 1 cm, was debrided . After thorough debridement of the avascular necrotic bone, intraoperative fluoroscopy was used to identify and better assess the defect in the lunate . The donor site was filled with a synthetic acellular biphasic copolymer plug (trufit, smith and nephew, andover, ma). Using the digital template acquired from the algorithm, an osteochondral plug from the left femoral condyle that closely matched the radius of curvature of the lunate, 12.7 mm from the manual measurement, was harvested during knee arthroscopy (fig . The location of the plug was assessed from the previously constructed 3-dimensional model of the articular surface, relative to the local anatomy . A 10 blade and rongeur were used to fit the dimensions of the osteochondral plug with those of the lunate . 2 fiberwire (arthrex, naples, fl) stitch was placed through the scapholunate ligament . Internal fixation was performed with a 1-mm drill, drilled antegrade through the lunate to fix the osteochondral plug; a 1.5-mm 8-mm screw was then compressed across the plug with fixation in the distal aspect of the lunate . There was no block in flexion, extension, or radial deviation, and the osteochondral fragment remained stable through a full range of motion . The patient was placed in a sling and discharged with instructions to return for regular follow - up visits . Maximum (left) and minimum (right) principal curvature maps of the anterior portion of the lateral femoral condyle . Postoperative radiographs demonstrated that the wrist was in good alignment and the hardware was intact . Follow - up radiographs and physical examinations were performed at 2, 4, 6, 8, and 10 weeks; 3, 4, 7, and 10 months; and 1 year postoperatively . We treated kienbck s disease with the aid of a 3-dimensional model of femoral articular surface and calculated the principal curvatures of the surface . This was performed to determine the optimal congruency of the chondral donor and recipient sites . Surgical challenges for autologous osteoarticular transfer include restoration of the articular surface as well as the tidemark . Koh et al . Have shown in a preclinical model that plugs left elevated or angled result in increased contact pressures, thus presenting potential risk to the cartilage on the opposite side of the joint . The mesh model created in the knee allowed for objective assessment of the articular surface to ensure more optimal plug alignment, provide reconstitution of joint architecture, and promote biologic incorporation of the plug into the recipient site . We did not seek to evaluate the functional outcome of the surgical procedure in this short report but to introduce the feasibility of this novel surgical planning technique for osteochondral repair . This case report presents the unique application of mri and mathematical modeling when applying the autologous osteoarticular transfer procedure to the lunate . These techniques require only an additional limited 3-dimensional model of the knee with minimal scan time beyond the routine examination of the wrist for assessment of marrow viability and the integrity of the articular surfaces . Additional applications would include osteochondral lesions of the distal femur, capitellum, talus, and potentially, over the femoral head . Future studies will refine the image acquisition and 3-dimensional modeling processes for optimal selection of the osteochondral plug based on local tissue morphology.
Urinary tract infections (utis) are one of the inflammatory diseases produced by high multiplication of many pathogens in the urinary apparatus, resulting in alterations in the perfect function of the urinary tract and kidneys . Uti is particularly a major problem for females; nearly 5080% of the female population endures from uti at least once in lifetime and 20 - 50% of them will have recrudescent events (1, 2). Escherichia coli is the most frequent pathogen responsible for up to 80% of utis (3). This bacteria is responsible for 85% and 50% of community and hospital acquired utis, respectively (4). Uropathogenic e. coli (upec) strains have special virulence factors, including pili or fimbriae, which mediate attachment to uroepithelial and vaginal cells, resistance to human serum bactericidal activity, haemolysin production, and increased amounts of k capsular antigen (5). Furthermore, virulence factors of upec strains have a significant role in development of utis . The most virulence factors dependent upon the upec include adhesions (type 1 fimbriae, p fimbriae, curli mbriae, ambrial adhesion and flagellum), aerobactins, hemolysins, and cytotoxic necrotizing factor 1 . The mentioned virulence factors are important in colonization of upec, extra - intestinal survival, and creation of cytopathic effects . In addition, the expression of special virulence factors of upec can contribute to uropathogenicity, as well as worsening of utis (1, 6, 7). An essential step for beginning and development of uti is bacterial attachment to uroepithelial cells . E. coli attachment is mediated by ligands of bacteria (generally small proteins placed at the tips of bacterial fimbriae) which bind to host cell wall carbohydrate residues, working as receptors (5). Therefore, the adherence of e. coli to host receptors is a function, usually mediated by adhesions of bacteria to host cell receptors (8). The bacterial attachment permits bacteria to resist mechanical elimination by the flow of urine and bladder emptying and increasing persistence of e. coli . Upec strains produce different types of adhesins, including type 1 fimbriae, which are essential for recognition and attachment to urinary tract receptors (9). Among adhesions of upec, the adhesive subunit of type 1 fimbriae, fimh, is a major determinant, which has high tropism for urinary tract receptors; thus, fimh adhesion is important in colonizing different niches of e. coli (10). In addition, single - nucleotide polymorphism (snp) analysis of fimh is a screening tool for epidemiological typing of upec (11, 12). Therefore, the research on bacterial virulence factors can result in expansion and development of new methods for diagnosis and prevention of utis . For more subsequent investigations, the fimh gene was detected in upec strains isolated from hospitalized and out - patients with uti, referred to educational hospitals of shahrekord . The present study was conducted for detection of the fimh virulence gene from upec isolated from both hospitalized patients and outpatients with uti, referred to educational hospitals of shahrekord, iran . In this study, 140 isolated e. coli strains from patients with uti were evaluated . The isolates were collected from both hospitalized and non - hospitalized patients with utis from april to july 2012 from kashani and hajar hospitals, shahrekord, iran . Hospitalized infections were characterized as patients who were confined to bed in hospital and non - hospitalized infections were characterized as infections in patients who had no prior contacts with hospitals or long - term care facilities two weeks prior to admission . The samples were cultured on macconkey agar, blood agar and eosin methylene blue (emb) agar (hi media, india). The plates were incubated at 35c for 24 hours and pure isolates were characterized and identified according to gram staining and biochemical tests such as catalase, oxidase, indole production, citrate utilization, triple iron sugar, ortho - nitrophenyl--galactoside (onpg) test, and methyl red - voges proskauer, as described in standard bacteriological methods . All the above chemicals and media were purchased from sigma - aldrich (germany). Genomic dna templates for pcr amplification were gained from overnight growth of bacterial isolates on luria - bertani agar (hi media, india), pelleted by centrifugation, resuspended in 500 l of sterile deionized water, and boiled for 10 minutes . After centrifugation of the boiled samples at 19000 g for five minutes, the supernatant was applied as the dna template for pcr . The bacterial isolates were subjected to screening for the presence of the fimh gene by pcr . The nucleotide sequence of the primers and the annealing temperature for amplification of the fimh gene by pcr are shown in table 1 . Pcr was carried out in 25 l containing 2.5 l of 10 pcr reaction buffer with mgcl2 (1.6 mm), 0.5 l (200 m) of deoxynucleoside triphosphates mixture (dntps, 10 mm), 0.5 l of each primer (10 pm/l), 2 l of the dna template (50 ng) with 0.5 l (3 u/l) taq dna polymerase . The amplification condition included an initial denaturation (96c for three minutes), 37 cycles (96c for 30 seconds, 64c for five minutes, 72c for 60 seconds) and a final extension (72c for fiv eminutes). The pcr amplifications were performed on a thermocycler tc-512 (bio - techne, cambridge uk) and the pcr products were analyzed by polyacrylamide gel (8%) electrophoresis . A molecular marker (fermentas sm0321, 100 bp) was used to assess the pcr products sizes . In this study, 140 isolated e. coli strains from patients with uti were evaluated . The isolates were collected from both hospitalized and non - hospitalized patients with utis from april to july 2012 from kashani and hajar hospitals, shahrekord, iran . Hospitalized infections were characterized as patients who were confined to bed in hospital and non - hospitalized infections were characterized as infections in patients who had no prior contacts with hospitals or long - term care facilities two weeks prior to admission . The samples were cultured on macconkey agar, blood agar and eosin methylene blue (emb) agar (hi media, india). The plates were incubated at 35c for 24 hours and pure isolates were characterized and identified according to gram staining and biochemical tests such as catalase, oxidase, indole production, citrate utilization, triple iron sugar, ortho - nitrophenyl--galactoside (onpg) test, and methyl red - voges proskauer, as described in standard bacteriological methods . All the above chemicals and media were purchased from sigma - aldrich (germany). Genomic dna templates for pcr amplification were gained from overnight growth of bacterial isolates on luria - bertani agar (hi media, india), pelleted by centrifugation, resuspended in 500 l of sterile deionized water, and boiled for 10 minutes . After centrifugation of the boiled samples at 19000 g for five minutes, the supernatant was applied as the dna template for pcr . The bacterial isolates were subjected to screening for the presence of the fimh gene by pcr . The nucleotide sequence of the primers and the annealing temperature for amplification of the fimh gene by pcr are shown in table 1 . Pcr was carried out in 25 l containing 2.5 l of 10 pcr reaction buffer with mgcl2 (1.6 mm), 0.5 l (200 m) of deoxynucleoside triphosphates mixture (dntps, 10 mm), 0.5 l of each primer (10 pm/l), 2 l of the dna template (50 ng) with 0.5 l (3 u/l) taq dna polymerase . The amplification condition included an initial denaturation (96c for three minutes), 37 cycles (96c for 30 seconds, 64c for five minutes, 72c for 60 seconds) and a final extension (72c for fiv eminutes). The pcr amplifications were performed on a thermocycler tc-512 (bio - techne, cambridge uk) and the pcr products were analyzed by polyacrylamide gel (8%) electrophoresis . A molecular marker (fermentas sm0321, 100 bp) was used to assess the pcr products sizes . The fimh gene was amplified using specific primers and appeared as a band of about 164 bp on polyacrylamide gel (figure 1). The fimh gene was found in 130 isolates (92.8%) of upec . Of the 130 isolates positive for the fimh gene, 62 (47.7%) and 68 (52.3%) belonged to hospitalized patients and outpatients, respectively . Coli clinical isolates containing the fimh gene (164 bp); lane 13,negative control (without dna template); lanes 14 - 17: e. coli clinical isolates containing the fimh gene (164 bp). The onset of uti results from the ability of upec attachment to urinary tract epithelial cells by specific adhesions including type 1 fimbriae (13). Upec strains have many virulence factors which enhance their capacity to colonize in the urogenital tract . Attachment to the urothelial cell surface is mediated by fimh adhesion, placed at the tip of the type 1 fimbriae, which prevents bacterial washout by urine flow and starts bacterial invasion (4, 14). Since the fimh virulence factor associated with uti cases was not widely determined from the upec isolated from both hospitalized patients and outpatients with utis referred to educational hospitals of shahrekord, iran, the prevalence of the fimh gene was examined . The presence of the fimh gene was confirmed by pcr and the results indicated that the fimh gene was present in 130 upec isolates (92.8%); 62 isolates (47.7%) of hospitalized patients and 68 (52.3%) of outpatients . This showed that most of the upec strains had the fimh gene and our results were almost in accordance with the results of previous literature . (15) reported that among the studied virulence genes of upec strains, the fimh gene was the most prevalent virulence gene and was found in 68% (61/90) of the uti isolates . (16) studied 18 upec isolates collected from females and found that the fimh gene was the most prevalent virulence factor and 100% of the isolates had that gene . In another study, 17) demonstrated that the fimh gene was the most frequent virulence gene and was detected in 98% of e. coli strains isolated from patient with utis . (7) evaluated the distribution of virulence genes among the studied upec and showed that the prevalence of different virulence genes varied from 10% for the cnf gene to 80% for the fimh gene . In addition, arabi et al . (18) investigated the frequency of fimh and other adhesions genes in upec and determined the fimh gene frequency as 87.7% . Apart from investigation on evaluation of the fimh gene in upec strains, this gene has been detected in other strains of e. coli . For example, kaczmarek et al . (13) evaluated and detected the genes encoding virulence factors among e. coli strains with k1 antigen as well as the non - k1 e. coli strains . They found that the fimh gene existed in the whole tested e. coli k1strains as well as in 97.0% of non - k1 strains . (19) also reported that the fimh gene was present in 54.55% of avian pathogenic e. coli (apec) strains which are pathogenic for birds . In another study, 20) showed that in enterohemorrhagic e. coli (ehec) strains, the fimh gene was conveyed by the majority of non - o157:h7 e. coli strains (97%) and by all the o157:h7 e. coli strains . (21) detected the fimh gene in all the e. coli isolates from bovine mastitis . Moreover, fimh snp analysis is a typing tool for epidemiological studying of community- and hospital - associated e. coli isolates and could be used as an easy, cheap screening test for genotypic analyses of upec (11, 12). We can conclude that type 1 fimbriae is present among upec as well as other strains of e. coli, to the extent that the fimh gene was detected in more than 90% of the e. coli strains . The high binding ability of fimh could result in increased bacterial binding to target cells and increased pathogenicity of e. coli; thus, fimh could be used to design vaccine for prevention of e. coli infections by blocking the bacterial attachment and colonization . In addition, fimh could be used as a tool for the extension of rapid detection - based assays.
Tryp is the least abundant amino acid in mammalian organisms, and accounts for only 11.5% of the total protein amino acid content . However, it was not until the late 1970s and 1980s that kp generated considerable interest among neuroscientists, since it was discovered that quin and kynurenine acid (kyna), (two metabolites of the kp) exhibited significant and opposing actions on neuronal cells . Quin is a selective agonist at the n - methyl d - aspartate (nmda) site of the excitatory nmda (glutamate subtype) receptor, while kyna is an antagonist at both the nmda and glycine site of this ionotropic receptor (guillemin, 2012). Activation of the nmda receptor has been shown to permeate cells to ca, na and k ions . Increased intracellular ca influx has been shown to activate several secondary messenger signalling pathways leading to synaptic alterations . Moreover, increased intracellular ca influx due to excessive nmda receptor activation can induce excitotoxicity and neuronal cell death in several neurodegenerative diseases (braidy et al ., 2010). In humans, cns quin levels are increased in several neurological disorders including ad, depression, epilepsy, autism, schizophrenia, and patients are more susceptible to suicide risk (brundin et al ., 2016). This has led to the hypothesis that the increase in cns levels of quin is pivotal to the pathogenesis of these disorders through its mode of action at the nmda receptor . Quin may also induce toxicity to brain cells via exogenous free radical production . On the contrary, reduced levels of kyna have been reported in neurological disorders such as huntington's disease (hd), and ad . It has been suggested that the development of inflammatory mediated neuropathology is correlated to changes in the ratio of kyna to quin rather than quin levels alone . Given the extent that the kp influences neuronal function, identifying the site of tryp metabolite production during cns inflammation is beneficial to gain a sound understanding of inflammatory mediated neuropathology . These metabolites may have originated either from upregulated tryp catabolism within the cns during inflammation, or may cross the blood - brain barrier after being systemically produced . It has been reported that cns levels of kp metabolites increase independently of their systemic concentration during neuroinflammation, thus suggesting that kp metabolites can be present at upregulated levels locally within the cns . Quin is converted to nicotinic acid mononucleotide (namn) by the enzyme quinolinic acid phosphoribosyl transferase (qprt) in the presence of mg2 + . Further transformation leading to the synthesis of the parent molecule of the pyridine nucleotide, nad, appears to be nuclear, mitochondrial, and golgi specific, by nicotinamide mononucleotide adenyl transferase (nmnat1, 2, and 3) in the presence of atp to produce desamido - nad . In the presence of glutamine desamido - nad is amidated to the parent pyridine nucleotide, nad, as the final product of the kp in addition to its de novo synthesis for tryp, nad can also be synthesised from either one of three routes: (1) nicotinic acid (na), which is then converted to nad via the three - step preiss - handler pathway; (2) the enzyme nicotinamide phosphoribosyl transferase (nampt) converts nm to nicotinamide mononucleotide (nmn) and then to nad by the action of nmnat1, 2, and 3 in the presence of atp, or (3) phosphorylation of nicotinamide riboside (nr) to nmn by nr kinases (nrks) (ratajczak et al ., 2016) (figure 1a and b). In spite of the potential for nad production from vitamins, the de novo synthesis of nad from tryp appears to be more important than nad production from vitamins under normal physiological conditions . (a) the de novo synthesis of nad from tryp appears to be more important than nad production leading to the production of several neuroreactive compounds such as the neurotoxin and nmda receptor agonist, quinolinic acid, and the nmda receptor antagonist, kynurenine acid . (b) nad can also be produced by the salvage of nicotinic acid, nicotinamide and nicotinamide riboside . (c) parp activity following dna damage utilized nad as the essential substrate to repair damaged dna . Hyperactivation of parp due to accumulation of free radicals can lead to cell death by nad depletion, and energy restriction . Nmda: n - methyl d - aspartate; parp: poly(adp - ribose) polymerase; ros: reactive oxygen species; tryp: tryptophan . The kp is not fully expressed in all brain cells . To date, the only cells in the cns demonstrated to possess the enzyme 3-hydroxyanthranilic acid oxygenase (3-hao) which generates quin are astroglial and microglia / macrophages / dendritic cells (braidy et al ., 2016). Therefore, the increase in tryp catabolism observed during neuroinflammation must necessarily involve these two cell types . Ifn- is the primary activating factor of macrophage / microglial / dendritic cells in the cns and elsewhere, increasing their antimicrobial activity through the modulation and upregulation of a variety of activities including, enhanced production of reactive oxygen species (ros), increased nitric oxide synthase activity, upregulation of mhc antigens, secretion of cytokines such as interleukin (il)-1, il-6, tumour necrosis factor- (tnf-), platelet activating factor (paf), macrophage chemotactic protein (mcp-1), and secretion of other biologically active proteins such as complement pathway components . Indoleamine 2,3 dioxygenase (ido) is the primary enzyme of the kp, and is potently induced by ifn- in both astrocytes and inflammatory cells leading to a marked increase in kp metabolites in these cells . Ifn- activated microglial / macrophages / dendritic cells will readily catabolize tryp through induction of ido, producing significant amounts of metabolic neuroreactive intermediates such as kyn, anthranilic acid (aa), 3-hydroxykynurenine (3-hk), 3-hydroxyanthranilic acid (3-haa), and quin . The kp also leads to the production of the metal chelating agent, picolinic acid (pic) (schwarcz and stone, 2016). Astrocytes also readily degrade tryp in response to ifn- treatment through the induction of ido . Kyn appears to be the main metabolite found in the supernatants of ifn- treated astroglial cells . However, significant but trace amounts of aa from an astrocytoma cell line, and quin, from human foetal brain cultures have also been reported (guillemin et al ., 2000). It has been suggested that tryp catabolism increased in some cells to decrease tryp concentrations in the microenvironment thereby reducing the availability of this essential amino acid for microbial metabolism . One of the products of tryp catabolism, 3-haa, can function as an effective antioxidant, and potential nitric oxide synthase inhibitor . Production of this metabolite may therefore serve to reduce non - specific oxidative damage at the site of neuroinflammation induced by activated mononuclear phagocytes, and may explain the increased tryp catabolism under these conditions (krause et al ., 2011). However, increased secretion of kyn and quin under these circumstances has not been confirmed . Moreover, ifn- induced ido tryp catabolism has been shown to increase cellular nad concentrations in an astroglioma cell line (grant et al ., 1999). Despite the consolidated role of the classic kp metabolites (e.g., kyn, kyna, 3-haa, and quin) during immune responses, it has recently emerged that cinnabarinic acid (ca) is crucial for immune system functions (hiramatsu et al . Ca can also be produced via non - enzymatic reactions under oxidative conditions . In inflammatory cells such as neutrophils, co - expression of ido and other enzymes involved in the formation of free radicals may divert the kp away from the production of quin, and instead towards ca over pic or quin (lowe et al ., 2014). Poly(adp - ribose) polymerase and nad depletion: double strand dna breaks caused by excessive oxidative damage activate the parp enzyme . Neuronal cells exposed to pathological concentrations of the excitotoxic neurotransmitter, glutamate show both an increase in intracellular oxidative stress and parp activity . Parp is a protein modifying nucleotide polymerising enzyme found abundant in the nucleus, with approximately one molecule of enzyme per 1,000 base pairs . Parp, along with dna dependent protein kinases appears to play an important role in maintaining genomic integrity (massudi et al ., 2012). However, the precise physiological roles of parp are not completely understood . As a dna nick sensor, parp rapidly binds to dna strand breaks and is activated . Activated parp uses up nad (and nadp+), exclusively as substrate to poly(adp - ribosylate) itself and a number of nuclear proteins that are involved in the repair of dna, releasing nicotinamide as a by - product . Recent evidence suggests that polyadp - ribosylation of histones or transcription factors may also be involved in nuclear receptor signalling (morales et al ., 2014) a significant decrease in intracellular nad levels has been reported in the brain and a variety of cell types as a result of dna strand breaks and parp activation following exposure to free radical generators, excitotoxins, infections, ad, and during inflammation or ageing (abeti and duchen, 2012; braidy et al ., 2014). Increased parp activity resulting in decreased nad has been shown to decrease atp and neurotransmitter levels in the brain, as well as cell lysis and death . Inhibition of parp activity following oxidant injury has been shown to preserve nad and atp levels, prevent cell lysis, although damage to the dna was not prevented . Additionally, at low levels of oxidant, parp cells survived better than parp cells, suggesting that loss of nad may be a cause of cell death . Elevated levels of free radicals, pro oxidants, and excitotoxins have been reported in inflammatory brain disorders, and in most cases, dna damage has been observed . This suggests that nad depletion through parp activation may play a crucial role in cns dysfunction and pathology under these conditions (massudi et al ., 2012). Immune activation of macrophage / microglial / dendritic cells results in a marked increase in their generation of ros . Elevated extracellular fluid levels of these free radicals have been implicated in the cause of tissue damage during inflammation in several disorders . Damage to surrounding tissue, including astrocytes and neurons in the cns, may occur in response to inflammation in the cns . As well, the non - discriminating nature of these free radicals may induce damage to the activated macrophage itself . At least one study has reported an increase in parp activity in ifn- activated macrophages, suggesting that dna damage has occurred with a corresponding increase in the rate of nad catabolism . The cellular immune response potentially increases nad catabolism in cells at the site of inflammation . A mechanism by which this important nucleotide could be regenerated appears to be essential to the continued viability of cells within this environment . Therefore therapeutic strategies targeted at inhibiting kp metabolism alone may not be sufficient for the resolution of symptomatic disease . However, inhibition of quin production may need to be coupled to the administration of a suitable nad precursor.
Incidence of ep ranges between 0.25% to 2% of all pregnancies . In 95% of eps, fertilized ovum implants in the tubes, but rarely in other organs like abdomen, ovaries, cervix, spleen, omentum, cessarian section scar, and intramural . The death rate is about 1 per 2000 eps and 15% of all maternal deaths . Incidence of ep is nearly double in black women who also have a four times greater risk of ep - related mortality than white women . The risk factors for ep include pelvic inflammatory disease, previous tubal surgery, previous tubal pregnancy, progestin contraceptive, assisted reproduction, ovulation induction, induced abortion, salpingitis isthmica nodosa, smoking, and diethyl stilbestrol exposure . Most of the tubal pregnancies become symptomatic within 12 weeks, but a small number of them progress beyond this gestation and are diagnosed late . Proper management of ep needs early diagnosis, resuscitation, timely treatment, and follow - up . Early diagnosis of ep is a difficult task but can be possible with the help of quantitative -human chorionic gonadotropin (-hcg) level, transvaginal ultrasonography, and laparoscopy . Knowledge of the risk factors for ep helps in rapid diagnosis and could reduce the need for surgery and suggest actions to improve prognosis . However, several studies assessing risk factors for ep have been published from developed countries . Saudi women have different characteristics, like cultural, religious, sociodemographic, sexual behavior, beliefs, and contraception practices, than those of developed countries and may have different risk factors for ep . The purpose of this study was to highlight the frequency and predisposing factors of ep, and to elucidate the outcome after different treatment modalities in a general hospital . This retrospective descriptive study was conducted at hera general hospital, makkah, saudi arabia, over a duration of 18 months from july 1, 2009 to december 31, 2010 . The study subjects included all females in their reproductive age who were admitted at the obstetrics and gynaecology department with suspicion of ep . Data were retrieved from the medical record office by exploring the patients files in detail . Patients files were looked for age; gravidity; gestational age; clinical presenting features; and ep risk factors, like history of previous abortion, infertility treatment, current use of intrauterine contraceptive device (iucd), history of previous tubal surgery and pelvic surgery, previous ectopic and/or pelvic inflammatory disease . Peak level of -hcg, transvaginal ultrasonographic findings, mode of treatment, and type of surgical procedure were also retrieved . A woman experiencing several eps during the study period generated multiple case entries, one for each ep episode . In this study, seven women experienced two, and two women experienced three eps . The duration from symptoms onset to admission and admission to definite treatment was also recorded . Numerical data were subjected to descriptive analysis, with mean standard deviation (sd). The institutional review board of hera general hospital, makkah, saudi arabia, granted us permission to conduct this study . The institutional review board of hera general hospital, makkah, saudi arabia, granted us permission to conduct this study . Incidence was 0.58% out of 7654 pregnant women admitted at the department of obstetrics and gynaecology (5.8/1000 pregnancies). The mean age of the women was 27.9 7 years and only 2 (4.5%) were more than 40 years old . Twenty - five (56.8%) were multigravida whereas 21 (47.7%) presented at 7 - 8 weeks of gestation . The range of gestational age was 3 - 9 weeks at presentation . Only 24 (54.5%) patients presented within 24 h, 6 patients within 1 - 2 days, and 14 patients presented after 2 days of onset of symptoms of ep . The most common presenting feature was amenorrhea (41, 93.2%), followed by abdominal pain (39, 88.6%) and abdominal tenderness (38, 86%). Fluid in pouch of douglas was found in 21 (55.3%) cases and ectopic mass in 13 (34.2%) cases [tables 1 and 2]. Demography and obstetrical history clinical presentation of ectopic pregnancy sixteen (36.3%) females presented with hemoglobin levels of 8 - 10 gm%, followed by 13 (29.5%) with 10 - 12 gm%, 9 (20.5%) with 12 - 14 gm%, and 6 (13.6%) with 8 gm% . Twenty - three (52.3%) patients did not receive any blood transfusion during their hospital stay, but 11 patients received one, 5 received two, 3 received three, and 2 patients received 4 units of blood transfusion . Out of 44 patients, 25 underwent emergency laparotomy whereas the remaining 19 patients were admitted for observation and further diagnosis . When diagnosis was proved, 12 underwent laparoscopy whereas seven received medical treatment by methotrexate (mtx) and it was successful in all cases . On the other hand, salpingectomy was performed in 33 (89.1%) cases followed by salpingostomy in 2 (5.4%), whereas one case underwent salpingotomy and resection of ectopic mass was performed for the remaining one patient . Pre - operative findings for 37 cases revealed that 35 (94.6%) females had ep in the fallopian tube, whereas one case had within the ovary and one had heterotopic pregnancy, i.e., combined intra- and extra - uterine . Average hospital stay after surgery was 4 days in laparotomy and 2 days in laparoscopy . Eighty percent (n = 35) had some risk factors, whereas previous pelvic surgery was most frequently seen (13, 29.5%) followed by history of pelvic inflammatory disease (10, 22.7%) [table 3]. The rate of ep was 1.9% as reported by lozeau and potter in usa, and 1.04% and 1% by bangash and ahmad, and waseem, respectively, in pakistan . Our study was conducted in a secondary healthcare center, which is why we were not receiving referral cases from the whole of the makkah region, and incidence represented the incidence in hera general hospital rather than the general population of the region . Mtx treatment of ep was safe and effective for selected patients with unruptured tubal ep, with no major side effects as found by dhar et al . A paper presented in a conference proceeding highlighted management of advanced ep, i.e., in bahrain, managements of ep were as follows: laparotomy and salpingectomy (84.2% and 5.3%), mtx alone (5.3%) and mtx followed by laparotomy and salpingectomy (10.5%), whereas in qatar it was laparoscopy and salpingectomy (77.6%), mtx alone (19.4%), and mtx followed by laparoscopy and salpingectomy (3%). Laparoscopy in qatar and laparotomy in bahrain were the treatment of choice to treat advance ectopic cases . Lozeau and potter reported a success rate of 88% in a single - dose versus 93% in a multiple - dose regimen of mtx . Salpingostomy was concluded as a better option than salpingectomy regarding the future fertility outcome in one study . We tried in our study to increase the clinical knowledge and the degree of suspicion of the physicians to diagnose the ep as early as possible . This will avoid delay in presentation and diagnosis, which is vital to avoid morbidity and mortality in patients with ep . After a review of the literature and our study results, we are in a position to recommend the following steps at three levels i.e., public, primary healthcare, and specialist center . Aim should be early diagnosis and prompt treatment of ep without unnecessary delay in presentation, diagnosis, and treatment . We should launch a public awareness program about the risk factors to educate all females through electronic media like tv, radio, or newspapers . All these patients should register themselves at a specialist hospital for care of their pregnancy where specialist gynecologists and facilities for diagnosis and treatment of ep are available . General practitioners working in primary healthcare centers should be educated to have a high index of suspicion for ep . At specialist - level hospitals, all females (at their child - bearing age) presenting with hemodynamic instability or pain in the lower abdomen should be admitted and immediate investigations like pregnancy test, -hcg, and ultrasound (both trans - abdominal and trans - vaginal) should be ensured even if there is no history of amenorrhea . This study found that previous pelvic surgery, infertility treatment, and pelvic inflammatory disease were the major predisposing factors for ep . The most common site was the fallopian tube and the success rate of mtx was found to be 100% . To avoid emergency laparotomy that has its own post - operative complications, timely diagnosis of ep should be encouraged.
Drugs were not new to asylums in the 1950s . While opium and morphine have long been used in psychiatry, the second half of the nineteenth century witnessed the emergence of a large number of other substances considered more effective and less dangerous, such as chloral and bromides . After the success of these sedatives in the second half of the nineteenth century, the early 1890s saw the rise of barbiturates . These were considered more active and were seen as having fewer side effects than their predecessors, although concerns were regularly voiced about their addictive properties . The use of therapeutic drugs was widespread within the institut ever since its foundation in the early 1930s . In 1931, at least 55 per cent of patients received some drugs during their stay, the most prominent being luminal, a barbiturate distributed by bayer . In 1935, this percentage dropped to around 40 per cent; in 1940, it rose to around 80 per cent; in 1945, approximately 50 per cent of patients were medicated; and by 1950, two thirds of them were on some form of medication . Despite the significant variations due to its rather small size, this sample shows that, before the introduction of neuroleptics, more than half of the patients at the institut consumed therapeutic drugs . Well before the 1950s, drugs thus rhythmically structured the daily lives of patients at this institution; and, indeed, the users were well aware of the material nature of these medications . Their distribution whether in the form of pills or injections took place two or three times a day and represented a moment of negotiation between patients and caregivers . The doctor prescribed the framework within which the drugs were administered (frequency and maximum amount), but every day it was the nurses who decided on these parameters and negotiated the actual taking of the drugs with the residents . By the time psychotropics entered psychiatric wards, the first neuroleptic was introduced into french psychiatry in 1952 under its generic name chlorpromazine; it was sold under the name largactil . But only in 1954 did the psychiatric field begin to consider the drug as a breakthrough, and its use in most western - european countries began to transcend the experimental framework . That same year, largactil was administered to the first patients at the institut . At this point, it had been marketed for about a year in belgian medical journals by specia, the pharmaceutical branch of rhne - poulenc and the belgian distributor of largactil . The first accounts of its use appeared in belgian scientific journals published in 1954 . At first, largactil was not sold exclusively as a psychiatric drug, but also as a treatment for surgery and obstetrics . Pathogenic specificity, that is, on the drug s effect on symptoms rather than actual healing . Over the years, largactil was more specifically defined as a psychiatric drug, although the indications for use still remained very broad: psychomotor agitation, confusion, anxiety, depression, obsessions, cenestopathy, intractable insomnia, sleep therapy, neuralgia, various pains, dysautonomia. In the second half of 1954, largactil was classified as category a by the belgian national health and disability insurance fund (fonds national dassurance maladie - invalidit). This classification offered patients in belgium a refund of 70 per cent of the drug s cost, providing an important argument for its use given the high cost . It should be mentioned that the introduction of neuroleptics happened in a context of collective enthusiasm for medical drugs . Since the triumphant march of penicillin in the mid-1940s, the medical community seemed to find a new miracle cure every year: for example, the first anti - tb medicine (late 1940) and extended spectrum antibiotics . As a symbol of postmodern medicine, the doctor no longer merely affixed a diagnosis, but also healed organic diseases . In parallel with these discussions at national and international levels and across different arenas medicine, pharmaceutics and welfare state neuroleptics also made their way into asylums at a local level . At the institut, largactil was used very broadly to treat schizophrenia as well as anxiety and delirium tremens . Within ten years, treatment patterns went through some major changes, as indicated in the table below . Table 1:psychotropic drugs and electroconvulsive therapy at the institut de psychiatrie de brugmann (percentage of patients receiving treatment). 195054 () 195559 () 196064 () 196569 () ect20%13% 6% 3%psychotropic drugs24%68%82%86% psychotropic drugs and electroconvulsive therapy at the institut de psychiatrie de brugmann (percentage of patients receiving treatment). While electroconvulsive therapy (ect) was still the cure used most frequently in the late 1940s, it saw a significant and sustained decrease from the second half of the next decade . At the same time, the growing and widening diffusion of psychotropic medication allows for two conclusions . First, never in the history of psychiatry had any treatment experienced such a widespread use; in the late 1960s, nearly nine out of ten patients received psychotropic drugs . Second, their use led to the virtual disappearance of all other biological therapies such as cardiazol and insulin shock therapy . These developments had an impact on patient experience as well as on the dynamics among members of the institut . The detailed nursing notes, taken three times a day, as well as the letters kept in patient records, can be used to gain insight into how patients perceived the change in medication practice during the 1950s ., a nurse or psychiatrist has noted down the patient s reaction to the medication . Patient response is extremely heterogeneous and a far cry from clichd images (depictions of inmates being force - fed drugs by doctors on the pay list of pharmaceutical companies or, in stark opposition, the introduction of neuroleptics as a universally hailed innovation). To be sure, many patients were opposed to taking drugs during their stay . The administration of medication usually at 8 am, 2 pm and 8 pm was a daily ritual . Since the patients were left to themselves most of the time, this was also one of the few moments when they interacted with the staff . Indeed, administering drugs not only structured the day as an obligatory meeting with the caregivers, but it also represented a moment when an opposition to hospitalisation could be expressed especially at a time when this still happened mostly without the patient s consent . A 65-year - old retired officer of the belgian army, he was not given a very precise diagnosis and the reasons for his admittance cannot be found in his medical records . At no point did the staff take any detailed notes on his consuming any drugs . The word medicine appears in one single instance within a more general comment: the patient refused all food and medicine . [?] Active bites his fingers refuses to urinate in toilets . [?] On the hands after the injection at noon, rests on the bed, in a theatrical position, feigns total inertia . The patient refused all food and medicine . [?] Active bites his fingers refuses to urinate in toilets . [?] On the hands after the injection at noon, rests on the bed, in a theatrical position, feigns total inertia . In this note, not taking one s medication was a way to show a general opposition to one s stay in the hospital, in the same way as a refusal to eat or urinate properly . For many patients, disease, medication and healing formed a closely interlaced triangle . It would be interesting to study whether administering drugs helped psychiatrists convince patients of their illness . At the same time, receiving medication offered patients some hope for their approaching remission . Refusing drugs was thus a way to tell the physician that one did not (any longer) consider oneself sick . Joseph r., for example, was a german worker in his forties . In february 1958, twenty years after leaving germany, he was interned at the institut for the first time . Paranoid, he received his first dose of largactil after two weeks . Over the following two weeks his daily dose was quadrupled from three administrations of 50 mg to three administrations of 200 mg . Although joseph r. had no access to his file, he realised that the dose was increasing . It thus became progressively more difficult to convince him to take his pills: wants to leave but wonders how on earth he can, considering his medication was increased. A few days later: unhappy wants to go home if his medication is not reduced. For this patient who was convinced that he was not ill, the administration of drugs reminded him daily that he would not soon be leaving the institut . The dosage increase was interpreted as a sign of his deteriorating condition, and implied that his stay would be prolonged . Another frequent complaint for joseph r. and others concerned the unwanted physical reactions of the drugs . Indeed, the first neuroleptics produced quite serious side effects, affecting both patients (who developed tremors similar to parkinson s disease) and nurses (who developed skin rashes). The medical records regularly mention secondary symptoms commonly attributed to psychotropic drugs from the 1950s and 1960s . These include stomach aches, headaches and sleep problems . Since physicians were not in regular contact with the drugs, at first they paid little attention to these side effects . Before the thalidomide scandal, which broke out in west germany and austria in the early 1960s and then spread throughout europe, this issue was hardly debated in the public space, and its ethical implications were not discussed among medical doctors . At the institut a first sign of concern about side effects appears in a patient record of 1962 . After complaining repeatedly about tremors caused by haloperidol, a neuroleptic that had been produced in belgium since 1958, dirk m. caused the head physician to reduce the prescribed dose used by the attending psychiatrist this earned the physician letters of thanks: dear madam, thank you for your lovely visit and for your fortunate intervention as well as for the cogentin, crucial in stopping internal and external tremors the side effects of r1625 [which produced in me a purely artificial anxiety]. Dear madam, thank you for your lovely visit and for your fortunate intervention as well as for the cogentin, crucial in stopping internal and external tremors the side effects of r1625 [which produced in me a purely artificial anxiety]. Dirk m. is not the only patient who linked his medication to physical problems . In february 1955, depression. She had repeatedly received psychotropics before: there is just one thing that is unpleasant about my health; i sleep very well, but every morning i wake up with a crazy headache . And there is no way to get rid of it even though i dine at 6 pm and i go to bed at 10 or 11 pm the latest . Lately dr dewale prescribed me quatane but with these tablets i only managed to wake up completely at 10 oclock . While i was working, i always became tired with these drugs and the headaches did not go away . There is just one thing that is unpleasant about my health; i sleep very well, but every morning i wake up with a crazy headache . And there is no way to get rid of it even though i dine at 6 pm and i go to bed at 10 or 11 pm the latest . Lately dr dewale prescribed me quatane but with these tablets i only managed to wake up completely at 10 oclock . While i was working, i always became tired with these drugs and the headaches did not go away . It is perhaps not surprising, therefore, that patients should make up various strategies to escape what some of them saw as poison: [patient] is to be monitored for taking pills, tries to throw them or hide them between his fingers, under his tongue; basically tries every possible trick, even spitting them into a cup so as to throw them out at the slightest inattention . Thirty - one - year - old christian d. was admitted to the institut in june 1958 . The scion of a bourgeois family from brussels, he had attended university and worked as an artist . Very quickly he challenged the authority of the psychiatrist and opposed his forced hospitalisation by writing letters to family members and friends who held influential positions in brussels society . In a call to a relative (who also happened to be a physician), he wrote: i apologise [for this unstructured letter] - they gave me, i should say fed me, largasil [largactil], a revolting pill that makes it difficult to follow up on two ideas . Since you re a doctor, i hope you can intervene and demand explanations from the doctors of the hospital . I apologise [for this unstructured letter] - they gave me, i should say fed me, largasil [largactil], a revolting pill that makes it difficult to follow up on two ideas . Since you re a doctor, i hope you can intervene and demand explanations from the doctors of the hospital . Nursing notes are filled with the questions he raised about his drugs: the reasons for their use, their dosage, their frequency, the point of the treatment, and so on . Often enough requests explanations about medications treatment, etc . ; would prefer not having to take medication and leave from here. Christian d. remained one of the few patients who voiced specific questions about the use of drugs and the reasons for their use . Mental illness; rather, they are evidence of a social milieu that saw its relationship with the medical world not only paradigmatically as patients, but also as customers . The nurses he had to face throughout the day were not part of his social class in the outside world, and christian d. seems to have had particular difficulty recognising their authority . As seen above, patient opposition to the medication also gave rise to a whole range of ruses to escape treatment . But these tactics of resistance, in turn, encouraged the nurses to develop tactics of their own: a form of knowledge that was obviously not included in their textbooks . To be sure, nursing notes such as the one below (about an s., who was admitted to the psychiatric ward after an acute psychotic state) are common: [patient] has just complained to the nurse because must take agarol [largactil] at night, said it makes her ill, afterwards came complaining that she passes no stool and that it has been lasting all day . Just bored everyone with her supposed constipation . [patient] has just complained to the nurse because must take agarol [largactil] at night, said it makes her ill, afterwards came complaining that she passes no stool and that it has been lasting all day . Just bored everyone with her it is therefore not surprising that, for some patients, taking drugs became a symbol of the physicians negative power. Yet refusing medication did not mean escaping medication . Even within the voluntary service, interestingly, however, a first refusal was not automatically penalised by forced administration (a measure that is nowhere mentioned in this sample). Convinced of suffering from joubert syndrome, he was admitted to the institut for the first time in november 1954 at the age of forty - three . After a week, he began to be opposed to taking drugs: very suspicious, constantly asking why he has to take medication, at first does not want to take them, took them finally, does not sleep yet, calm. These notes are found in several reports . Sometimes nurses were even willing to accept that patients reject their drugs if they remained calm, as shown in the example of nordin f., diagnosed as senile demented: evening refuses his medication [25 mg of largactil] falls asleep nonetheless. But these instances remain exceptional, even for nordin f. indeed, the following day he refused his neuroleptic once again: did not want to take his larg [largactil] phen [phenergan]. Larg[actil] at 21 oclock. The nurses notes remain silent on the means of persuasion used at the institut . The records say close to nothing on the strategies and persuasive techniques of nursing and medical staff . They do, however, suggest that, sometimes, the administration of neuroleptics in 1950s psychiatric clinics was due to administrative or practical reasons . In a later file, dating from 1960, a letter from a patient to the league of human rights makes explicit the possible strategies for constraint used by medical officers . These included the (more painful) threat of administering the drug intravenously in case a patient refused to swallow the pill: they wanted me to take a drug called haloperidol (1625), which they had wanted me to take before and to which i am opposed if you do not take your drug or drugs they make you take them as an injection however, in recent days, they make me take largactif [largactil] in large doses . Upon my refusal to take the drug again comes the threat of injections . They wanted me to take a drug called haloperidol (1625), which they had wanted me to take before and to which i am opposed if you do not take your drug or drugs they make you take them as an injection however, in recent days, they make me take largactif [largactil] in large doses . Upon my refusal to take the drug again comes the threat of injections . Despite these tensions, records show that not all patients perceived the introduction of these drugs in solely negative terms . Karine o. (discussed above) was admitted to the institut for the fourth time in september 1954 . Until then, the only treatment she had received was electroconvulsive therapy; but this time, the psychiatrist decided to have her try a new drug that had just been released: chlorpromazine . A letter to a friend shows her delight: since saturday, as i ve said, i have a new treatment with lacartine [largactil] 6 per day and from tomorrow wednesday i get an additional injection in the morning to calm me because i can only sleep from 9 pm until 2 or 3 am; thus i stay awake in my bed, it s long () i ll tell you about the treatment that they give me . The morning some beladomme at about 9 am 1 cup protecum 2 promenal 2 lacartine [largactil]3am: 2 promenal, 2 lacartine [largactil], 1 cup protenum6am: 2 promenal, 2 lacartine [largactil]8pm: 1 fnergant [phenergan]9am 1 injection 1 in the morningso you see that i am cared for and that i am treated very well . That way i ll leave the hospital completely healed . Since saturday, as i ve said, i have a new treatment with lacartine [largactil] 6 per day and from tomorrow wednesday i get an additional injection in the morning to calm me because i can only sleep from 9 pm until 2 or 3 am; thus i stay awake in my bed, it s long () i ll tell you about the treatment that they give me . The morning some beladomme at about 9 am 1 cup protecum 2 promenal 2 lacartine [largactil] 3 am: 2 promenal, 2 lacartine [largactil], 1 cup protenum 6 am: 2 promenal, 2 lacartine [largactil] 8 pm: 1 fnergant [phenergan] 9 am 1 injection 1 in the morning so you see that i am cared for and that i am treated very well . That way i ll leave the hospital completely healed . In this case, receiving drugs was taken as a sign that the disease was being dealt with . For karine o. it acted as a synonym for good treatment and it was closely linked to the hope of healing . The introduction of new medication therapies and their widespread use in psychiatric hospitals, therefore, not only led to new hopes within the medical field, but also among patients . We still lack studies on their consumption in private during the 1950s and 1960s, but several patients of the institut did regularly take medication at home . Thus anne - marie c. expressed from the first day her desire to receive drugs: sick, came in at noon, clean . Insists on taking something to sleep, received one pill of [vitamin] b complex at 8 pm . Insists on taking something to sleep, received one pill of [vitamin] b complex at 8 pm . In this example, the patient no longer appears as a passive subject who merely waits for the psychiatrist s instructions . Instead, she turns into what we might now consider a psychiatric user. One can even wonder if these attitudes have not become more widespread since the 1980s, when patients came to be represented not simply as objects without agency, but, indeed, as active customers . Anne - marie c. was not the only person who had very specific ideas about the therapies that suited her best . In the admission report of gilles s., suffering from oligophrenia [joubert syndrome], we find similar comments: pat[ient] well oriented in time and space, extremely agitated and anxious, constantly gets up from his bed, despite all the injunctions, asks plaintively for sedatives, or inquires about his release date you re surely not restraining me with the belt, i was wrapped like rotten fish give me a tranquilliser and let me out tell doctors several times to consultations at the hospital of etterbeek where one would have given him 1 bottle and tablets . Pat[ient] well oriented in time and space, extremely agitated and anxious, constantly gets up from his bed, despite all the injunctions, asks plaintively for sedatives, or inquires about his release date the examination is constantly interrupted by anxious questions, always the same: you re surely not restraining me with the belt, i was wrapped like rotten fish give me a tranquilliser and let me out tell doctors several times to consultations at the hospital of etterbeek where one would have given him 1 bottle and tablets . Gilles s. had already been interned in psychiatric hospitals by the time he arrived in the institut . During short stays in other asylums, as soon as he was admitted, he required a sedative; and to give more weight to his demand, he mentioned his experience at another hospital . For him, taking medication was a way to escape internment: give me a tranquilliser and let me leave. He was aware of being agitated, but knew that his agitation could be stopped with medication . The administration of the drug was thus perceived, instead, as a possibility for escaping the hold of the psychiatric hospital . Compared to other psychiatric treatments of the time, such as ect, taking drugs was an act that paradoxically served to weaken the medical grip; this was a therapy that could be administered outside the hospital walls . For medical staff, there also remained the issue of finding out how much access patients had to drugs prior to their hospitalisation at the institut . A few days after his first appointment in december 1953, he was admitted to the institut in january 1954 with a diagnosis of depression . Since he was particularly agitated, francis was administered hyoscine, a sedative, almost every night . In fact, for two consecutive nights he destroyed the sheet in which he was wrapped . The attending psychiatrist then prescribed bromide and chloral, two other sedatives, but francis c. remained very disturbed until his release on 8 february 1954 . The two drugs were administered to calm him down and he received five electroshocks . During that first stay, his record remains silent as to how he perceived the medication . During a conversation with his wife, however, it emerged that he had taken largactil even before coming to brugmann . The case of francis c. therefore not only poses the question of how many patients had been on medication before entering the institut, but also whether (and how widely) largactil was distributed outside asylums before being used in those institutions . In fact, the records lack enough detail to provide a precise answer, but they do reveal practices that have hardly been addressed by historians until now: namely, the use of medications outside hospital . Six months later, francis c. went back to the institut, this time without a precise diagnosis . His admission was justified on the basis of some very general symptoms: anxious states of melancholia . Mental disorders, dangerous to himself. The man now seemed used to taking medication . Upon admission, he asked for an injection to help him sleep, a request that was denied by the nurse on duty . The following night, he received two tablespoons of largactil . In late august, after thirteen days, he left the hospital . The physician made the following diagnosis: old agitation, tendency to depression and suicide . Insomnia. This was the first time he received neuroleptics on a regular basis and after ten days he started to complain . The nurses noted that he spends much time resting on his bed. The next day the psychiatrist wrote: remembers having had ect in 54 and it was better than the pills. The same day, the nurse recorded in the morning: seems a little depressed today, never agrees with what he is given as a medicine thinks it s too much, also complains of headache. And at night: around 6 pm received 1 cibalgine did not want to take (suspicious), said he will be dead tomorrow . Very depressed cries said the doctor had promised him another treatment . During his third stay and a few days after he had asked several questions about drugs, francis c. received his first electroshock . From that moment remains a little confused in the afternoon . Constantly asks for his injection in evening . Remains a little confused in the afternoon . Constantly asks for his injection in evening . The above indicates that, at a time when therapies were not explained to patients, they could actually appropriate a part of medical knowledge . It is particularly striking, in this respect, that patients often referred to drugs by their names sometimes even their industrial names, for example, r1625 for haloperidol . These regularly appear in the files, generally with a more or less correct pronunciation and spelling . (it is to be noted, however, that patients did not describe drugs through their effects: the terms antipsychotics, neuroleptics and some even declared themselves psychiatric specialists inside their family circles by virtue of their stay at the institut . Depressed who stayed thrice at brugmann while several of her family members also suffered from mental illnesses, once wrote to her grandmother: what i was especially happy about is that c[.] And you granny both take a seresta; which seems to do you well because i found you all 4 very well. More generally, these records highlight the varied and complex tactics employed by the various protagonists in this story . The nurses were interested in keeping their wards calm or had other practical interests . As for patients, they had some power of compliance or non - compliance, and integrated these drugs into their daily lives and into different understandings of healing . Naturally, elements such as social class often played a role on the degree to which their demands were met by medical staff . In any event, the vast majority of patients who asked for some medication received it . In 200 cases studied in detail, this article has aimed to follow the proposition made in 2007 by flurin condrau in his rather pessimistic assessment of the historiography of patients . If historians want to have a voice, he noted, they must engage with debates that have raged elsewhere and to claim intellectual, empirical and theoretical importance in the field. The few pages of this contribution raise questions linked to four broader debates: the bias due to observing the sufferer solely through the institutional archive, the multiplicity of players in the hospital setting, the critical and often anti - psychiatric genealogy of scholars interested in psychiatric history and the question of whether a historical investigation can only reveal what is heard, not what is said. I would like to return to roy porter s seminal article, the patient s view . Doing medical history from below. In this piece the author did not really lay his cards on the table . Indeed, his was far from a plea to consider the patient only within the hospital . Porter aimed further, by also addressing aspects beyond the patient that is, the sufferer outside the hospital . Herein lie also the limits of this article . The wealth of information in the institut s archives allows one to appreciate patient narratives, not only because of the medical reports, nursing notes, and so on that were recorded, but also because of the institut s tendency to archive documents produced directly by patients . These files contain the direct voices of these key players through therapy - related material (for example, drawings and results of writing workshops), but there are also traces that were illegally preserved such as letters that the hospital management decided never to send . Yet our knowledge remains extremely limited on the use of antipsychotics and antidepressants in non - hospital settings, since these practices do not necessarily give rise to archival material . As such, the patient records provide a few small glimpses into what appears to be a high consumption of these new drugs outside the institution . On a purely empirical level, the integration of the patient s voice into a history of psychiatric medication expands the characters that ought to appear in this narrative . That these now include patients seems obvious, as evidenced by the theme of this special issue dedicated to porter s seminal article . But the same applies to caregivers, whose central role in patients accounts should now be clear . In the past three decades, historians of psychiatry have sought to integrate the patient into the classic narrative but still often present it as a dance for two . It is time to recognise the multiplicity of roles within psychiatric institutions and to include, in particular, the heterogeneous figure of the nurse (for example, guardians, servants, caregivers and nurses). After world war ii, when these new drugs spread through the asylum, there was a proliferation of new professions in psychiatry (for example, social workers, psychologists and occupational therapists) who changed the patients experiences of their stay in psychiatric institutions . If, for the nineteenth century, roy porter could still claim that it takes two for a medical encounter, this is definitely not the case for the twentieth century especially since this period witnessed the move of medical encounters and healthcare from patients private houses to hospitals . The change in scale that often goes along with an interest in patients attempts to make sense also takes this story to the ground level. This change of perspective moves away from a medical history that is embedded (as is so often the case) in a sterile and moralising opposition between, on the one hand, a narrative that focuses on the remarkable progress of medicine, and, on the other hand, one that only sees a history of rulers and ruled. This particular historiographical understanding of the past is particularly strong in psychiatry . The humanities remain strongly marked by this anti - psychiatric genealogy, even more than sixty years after the publication of the two great classical critical studies: erving goffman s asylums and michel foucault s madness and civilisation . Integrating the patient as a key player and not just as an object of the medical gaze renders narratives less linear and more complex . The psychiatric ward of the 1950s was certainly marked by an asymmetry of structuring power . By choosing to focus on daily life, however, we perceive the margins of interpretation and manoeuvring for all those involved: patients, nurses, doctors and families . This article also questions a particularly strong current among sociologists who emphasise two phenomena: first, that the patient supposedly exists only through medicine, or, in the words of michel foucault, through the medical gaze. From this perspective, the patient s experience is only transmitted by a technique demanded by medicine. Second, they argue that, over the last hundred years, the voice of the patient has largely disappeared from the medical field, whose representatives no longer rely exclusively on the patient to establish a diagnosis but use other techniques to psychiatrists still depend largely on the patient s word to determine the efficacy of psychiatric therapies, particularly with regard to psychoactive drugs, because their effect cannot be fully measured by bodily reactions . Despite the development of tools supposed to decipher mental illness first electroencephalograms (eeg), then computerised axial tomography (cat) and magnetic resonance imaging (mri) scans this voice from below remains essential for gaining insight into various forms of disease experience and its management and representation.
Children under five years of age are an important population group in regard to their physiological and psychosocial development . As a consequence, they are more susceptible to the development of various morbidities, such as diarrhea [2, 3], respiratory infections [2, 4], anemia, and malnutrition [4, 6]. Most cases of acute diarrhea and mortality from this disease occur in children under five years of age . This increased prevalence is due to a combination of poor hygiene habits, poor sanitation [2, 7], and unfavorable socioeconomic conditions [2, 7]. Respiratory infections are another frequent morbidity in this group . Children suffer the greatest impact from air pollutants, increasing the risk of respiratory illness . Additionally, contact with other children in daycare facilities promotes the acquisition of respiratory diseases due to overcrowding and exposure to possible infectious agents . In relation to anemia, children under five years are the most vulnerable because of their increased need for iron, higher nutritional requirements during this period of life, their transition in the types of food they intake, and a higher susceptibility of the acquisition of intestinal parasites . The main causes of malnutrition are inadequate intake of nutrients and infectious diseases, specifically respiratory and gastrointestinal diseases . Since this age group is very susceptible to morbidity, attention must be paid to ensure that these children have access to health services . Provision of prenatal and childbirth care is effective measures in reducing infant mortality, as well as access to health care in both routine situations and emergencies . Peru, located in south america and bordered to the east by brazil, was ranked 82nd by the human development index (hdi) in 2013 . This is an index that considers wealth, education, and health as proxies for human development . In 2011, 36.19% of visits to peru's health services were from children under 11 years of age . According to the 2010 national demographic and health census of peru, 17.1% of peruvian children under five years of age showed symptoms of acute respiratory infection and 14.9% had acute diarrheal disease . Anemia is also one of the most prevalent nutritional problems in peru . In 2010, 50.3% of children under three years of age were anemic, with 56.6% being from rural areas . Malnutrition is also a large health problem in peru . According to the national institute of statistics and informatics (inei), the prevalence of chronic malnutrition in peruvian children under five years was 23.2% in 2010 . This study aims to evaluate the prevalence of reported morbidities in children under five years of age in the municipality of iapari (peru) and their access to health services in the municipality . This study was conducted in the urban area of iapari, a municipality located in the peruvian amazon, in the triborder region between brazil (assis, brazil), bolivia (bolpebra), and peru . Iapari is a district of the amazon province of tahuamanu and is located in the madre de dios department (figure 1). The study selected as a criterion for inclusion children under 5 years of age, because they are at greater risk of health inequalities and a greater susceptibility to the development of diseases because of that . Children eligible for the study were identified using a census of households carried out by the health workers of iapari, which identified children under the age of five years living in the urban area of the city in 2011 . Data collection for the study took place between january and february 2011 using structured interviews that evaluated the socioeconomic and demographic conditions of the family (family income, maternal / guardian education level, presence of government scholarship, and maternal / guardian paid work in the last 30 or 90 days), household environmental conditions (type of housing construction, predominant material on the floor of the house, presence of electricity, presence of paved street, and type of toilet in the household), peridomestic environmental conditions (garbage collection, flooding of the peridomestic area during rain, and presence of open sewers near the house), water supply and treatment (water supply for domestic use, presence of running water inside the house, and type of water treatment), the child's demographic information (age, sex, and ethnicity), reported infant morbidity (morbidities in the last 15 days, morbidities in the last 12 months, morbidities ever in life, and hospitalization cases), and access to health services (health care, medication). Interviews were conducted by medical students who spoke spanish, supervised by a peruvian medical doctor and a peruvian biologist with a m.s . Interviews were performed with the biological mother, and when she was not present, the biological father or the guardian of the child was interviewed . A few variables contained missing values (one or two unknown responses). The variable is the child living with the biological father? Was a sensitive question and therefore resulted in a larger number of absences of response . Interviews were conducted by medical students who spoke spanish, supervised by a peruvian medical doctor and a peruvian biologist with a m.s . Chicago, il). The anova test was used to compare means, and pearson's chi - square test or fisher's exact test was used to compare frequencies or proportions at the level of 5% . The study was approved by the ethics committee for human research of acre federal university (brazil) and of the instituto nacional de salud (lima, peru) (processes 23107.008153/2010 - 92 ufac and 2010-ci-59-ins). Informed consent was obtained from the participants in the study or from the parents or legal guardians of the minors prior to the interviews . The study was conducted with all children living in the urban area of the city . There were 108 children under five years of age living in the urban area of iapari . Of these, 46.3% were females and 53.7% were males, with a mean age of 2.11 years (standard deviation of 1.36 years and median of 2.24 years). Regarding ethnicity, 7.4% declared themselves white, 2.8% indigenous, 38.9% brown (pardos), and 50.9% mestizo . Almost all children (93.5%) were living with their biological mother, and 95.6% lived with their biological father . Maternal education was as follows: 68.9% of the mothers had more than eight years of schooling and 1.9% had no schooling (table 1). Houses were made predominantly of wood (82.4%), with latrines (67.6%) and unpaved streets or undefined streets (76.9%). Almost half (47.7%) of households reported being in flood - prone areas and 22.2% had open sewage in the peridomestic environment (table 1). In the study group, the most frequently reported morbidities were anemia (23.1%) and intestinal parasites (worms, 18.5%), and less frequent were wheezing (3.7%) and asthma (2.8%). As for the occurrence of these morbidities in the last 12 months, anemia (20.6%) and intestinal parasites (13%) were still the most frequent morbidities, and less frequent were wheezing (3.7%) and asthma (1.9%) (table 2). The prevalence of children who had ever been consulted by a doctor or nurse was 89.8%, and the prevalence of consultation in the last 12 months was 81.5% . Only 16.7% of children were reported to have been hospitalized at least once in their life . In relation to morbidities occurring in the last 30 days, 56.5% of the children referred to having had some morbidity . The most common were fever (28.7%) and diarrhea (22.2%), and the least common were nausea (3.7%) and shortness of breath (3.7%) (table 3). Regarding the previous 15 days prior to the interview, 53.7% of children reported some type of morbidity . Respiratory symptoms were the most frequent (38.9%) and included wheezing (4.6%), runny nose (22.2%), dry cough (14.8%), productive cough (16.7%), and sore throat (18.5%). Other morbidities were diarrhea (23.4%), fever (23.1%), and vomiting (15.7%) (table 4). About 63.1% of children who became ill used the health service, and 97.2% of these had access to health care . A drug prescription was filled in 91.1% of cases, and in these cases the drug was purchased from the public health care system (37.5%), purchased by the patient at the pharmacy (53.1%), or in a few cases purchased at the pharmacy by the health service (9.3%) (table 5). For 36.8% of children who did not seek health care for their morbidities, the main reasons for such behavior were that parents did not find it necessary (68.2%), there was lack of time to seek the health service or self - medication was practiced (31.8%), or parents thought there would be no available health care (4.5%). In iapari, 18.5% of the children reported the presence of worms in their lifetime and 13% reported this condition in the last 12 months . There was an association between infection and age (p = 0.006, chi - square test), showing that, in children older than two years, the prevalence of worms was higher . There was also an association between previous intestinal parasites and no piped water into the household (p = 0.038, fisher's test) and failure to consult with and/or little follow - up by the health service, confirming the hypothesis of little access to antiparasitic drugs . Respiratory symptoms in the last 15 days prior to the interview were associated with the presence of diarrhea in the same period of time (p = 0.001, fisher's test), maternal smoking (p = 0.037, chi - square test), the type of street the house was located on (brick or asphalt) (p = 0.022, fisher's test), and the type of sidewalk (brick or cement) (p = 0.022, fisher's test). Diarrhea in the last 15 days prior to interview was also associated with the presence of fever (p = 0.018, fisher's test), vomiting in the last 15 days (p = 0.002, chi - square test), presence of stomachache in the last 30 days (p = 0.002, chi - square test), and lack of maternal paid work over the past 90 days (p = 0.045, fisher's test). When analyzing the presence of diarrhea in a longer period of time (in the 30 days preceding the interview), it was possible to detect an association with other symptoms: fever (p = 0.003, fisher's test), vomiting (p = 0.047, chi - square test), and weakness in the last 30 days (p = 0.014, chi - square test). Some socioeconomic variables were also associated, such as absence of maternal paid work over the past 90 days (p = 0.05, fisher's test), lack of water piped into the household (p = 0.026, fisher's test), not boiling water before consumption (p = 0.016, fisher's test), and having a latrine or no toilet at home (p = 0.049, fisher's test) (table 7). Variables associated with the occurrence of diarrhea in the last 30 days are shown in table 7 . Having been hospitalized was significantly more frequent in females, children older than two years, low maternal education, and poor prior access to health care . Children whose mothers did not have morbidities during pregnancy were admitted for hospitalization more frequently than those who did . The main recently referred morbidities in this study were diarrhea, fever, vomiting, and respiratory complaints . In relation to morbidity that occurred sometime in their lives, the most frequent were anemia and intestinal parasites . According to the world health organization, brazil and peru are among the latin american countries with the highest prevalence of anemia in preschool children . According to the national institute of statistics and informatics, the prevalence of anemia in children under five years of age in peru was 37.7% in 2010 . The prevalence of anemia was higher in rural areas (45.7%) than in urban areas of the country (33.0%). In the department of madre de dios, where iapari is located, the prevalence of anemia in children was 44.7%, while it was much lower (28.3%) in the department of lima . Grantham - mcgregor et al . Performed a review of 33 studies and found that school and preschool children were more susceptible to the development of anemia and that anemic children had lower income level in relation to nonanemic children . Found a prevalence rate of anemia of 35.9% in brazilian children from minas gerais, which was associated with iron deficiency, parasitic infections, being at risk of or being a low length / height - for - age, and lower retinol intake . A review of studies from recent, large population health surveys, carried out in 11 french - speaking african countries (benin, burkina faso, cameroon, congo brazzaville, ivory coast, gabon, guinea, mali, niger, democratic republic of congo, and senegal) identified factors associated with anemia . Anemia (hb <11 g / dl) was found in 72.4% of the children (60.287.8%), with no gender difference but a slightly lower incidence in older children (62% at age of 4 - 5 years versus 85% at age of 9 months). In these countries, anemia was associated with location (rural areas), income (lower quintile), and low maternal education . In acre, brazil, the prevalence of anemia in children under five years of age living in small communities was 57.3% in 2005, and it was primarily associated with nutritional deficiency in this age group . In other municipalities of acre, including the neighboring city of assis, brazil, the prevalence of anemia in children younger than five years of age was 29.2% in 2003, being higher in children under two years of age and also associated with diarrhea . The prevalence of anemia during the course of life in children under five years of age was 23.1%, and the prevalence in the last 12 months was 20.6% . This is a lower frequency than appointed by national institute of statistics and informatics for madre de dios (44.7%). This difference may be explained because our data is based on referred morbidities, and there might be undiagnosed cases among our study population since anemia is a silent disease in some cases, especially in those who do not seek health services on a regular basis . Anemia was not associated with age in this study, as it was in other studies in acre [17, 21]. Children are most vulnerable to developing parasitic intestinal infections [22, 23], especially when in contact with other people at home or school, due to a lack of hygiene . Intestinal parasites are also associated with poor socioeconomic [17, 23, 24] and sanitation conditions [23, 24]. According to the national institute of statistics and information (peru), in 2013 the proportion of people with basic sanitation in the region of madre de dios was only 43.8%, while those with access to potable water were only 93.1% . These conditions are worse than those found in the department of lima, the capital of peru, where basic sanitation was available to 90.3% of children from this age group and access to potable water was available to 99.3% [22, 23], thus demonstrating a higher exposure of the population of madre de dios to poor sanitation and related infectious diseases . In iapari, 67.6% of the children had a latrine at home, and 22.2% were living in houses near open sewage . Such associations are reported in children from other developing countries as well [2, 26]. Conducted a trial in rural areas of ghana, to estimate the effects of water treatment in the incidence of diarrhea, adjusting the analysis in the presence of sanitation . The adjusted prevalence ratio of diarrhea in the intervention compared with the control communities was 0.82 (95% ci 0.710.96) for krachi west, 0.95 (0.861.04) for krachi east, and 0.89 (0.820.97) for both districts . Data from the national institute of statistics and informatics show that in peruvian children under five years of age, the prevalence of diarrhea in the last 15 days prior to the interview was 14.9% in 2010, with a higher prevalence in children living in the region of the peruvian amazon . In madre de dios, the prevalence of diarrhea in the last 15 days in children was 22.4% in 2010, a smaller prevalence than that found in the department of lima or on the peruvian coast (13.9%). Therefore, the prevalence found in the present study in iapari (23.4%) in 2011 is similar to that reported for the whole peruvian amazon and higher than reported on the peruvian coast . One possible cause of this discrepancy may be related to the growing urbanization of the amazon without adequate infrastructure and sanitation . Diarrhea was mainly associated with unfavorable socioeconomic conditions and poor hygiene habits, such as drinking untreated water . Children who consumed no boiled water had more diarrhea (32.7%) than those that consumed boiled water (13.6%). Ochoa et al . Also found that in peruvian children diarrhea was associated with age, breastfeeding, and natural immunity obtained by previous infections . In tanzania, diarrhea was associated with unfavorable socioeconomic conditions, younger age, and lack of toilet at home . In ghana, data from 3466 children showed that diarrhea was associated with household wealth quintiles, child sex and age, being the poorer, male children, and younger children more likely to have diarrhea . A study with 348,706 children from 40 nations found that the prevalence of acute diarrhea was 14% . The main factors associated with child diarrhea were poor household wealth and mother's lack of education . Other factors associated with diarrhea were female sex of the child, age of the child, immunization status, maternal age, and working status of the mother . A large case - control hospital - based study conducted in brazil showed that lack of adequate excreta disposal (paf = 12%), untreated drinking water (paf = 11%), and a history of previous hospitalization due to acute diarrhea (paf = 21%) were the main factors attributed to hospitalization due to diarrhea . Low socioeconomic conditions, no public water supply, crowding, and low weight - for - age were of less but still significant importance . A study with 5,828 indigenous brazilian children found an overall prevalence of recent diarrhea of 23.5%, being the highest rate in the north of the country (38.1%). Higher risk of diarrhea was observed among younger children and those who had less maternal schooling, lower household socioeconomic status, undernutrition (weight - for - age deficit), presence of another child with diarrhea in the household, and occurrence of upper respiratory infection . The diarrheal process may be associated with other clinical manifestations such as fever, vomiting, and abdominal pain, as shown by other studies [3335]. These clinical manifestations can suggest possible infectious agents involved in gastroenteritis, such as rotavirus, e. coli, shigella, and salmonella species . Intervention studies focusing on education about how to prevent infant diarrhea were conducted with 600 mothers in egypt . Results showed that knowledge of mothers about diarrhea (etiological factors and preventive measures) had improved significantly after the intervention and that health and nutrition education sessions were successful in improving mothers' knowledge regarding preventive measures and management of diarrhea . A meta - analysis of 22 randomized clinical trials conducted in low- and middle - income countries found that hand washing promotion probably reduces diarrhea episodes in both child daycare centers in high - income countries and among communities living in lmics by about 30% . Such measurements could be implemented by health care workers in other countries, such as peru, in order to reduce the incidence of diarrhea in young children . In a study conducted in brazilian nurseries, pedraza et al . Showed that acute respiratory disease is a group of important diseases in children, as they are more vulnerable due to the immaturity of the immune system and their rapid growth . Comment that, in the united states, hospital admission still continues to be a significant health care problem for children . In iapari, the prevalence of respiratory symptoms in the last 15 days was 38.9% . In the country as a whole, the prevalence of respiratory disease in the last 15 days in children under five years of age in 2010 was 17.1%, in the madre de dios region it was 15.4%, and it was 18.3% in the region of lima, suggesting a relationship between respiratory symptoms and the degree of urbanization of the area . This difference in prevalence may also be the result of differences in the concept of respiratory disease among the studies . In the approach adopted by inei in peru, the concept of respiratory disease was reported as illness associated with the presence of fast and/or troubled breathing, while, in the study in iapari, respiratory symptoms were defined as the occurrence of one or more symptoms such as wheezing, runny nose, cough (dry or productive), or sore throat . Found association of respiratory symptoms of children to environmental pollution in the netherlands . In this study, the prevalence of respiratory symptoms was different from one region to another suggesting that regions with greater development and urbanization had higher incidence of children with respiratory symptoms, corroborating the findings in peruvian children described above . The association found in iapari between child respiratory symptoms and having tobacco users at home has been documented in previous studies [34, 36]. Gonalves - silva et al . Showed that not only can parental smoking be associated with child respiratory symptoms, but also smoking by any other resident of the household . In iapari, the frequency of respiratory symptoms in children whose mothers were smokers was 77.8%, while, in the study by gonalves - silva et al . A review of studies from 11 french - speaking african countries also found that respiratory disease and diarrhea are common in children under 5 years of age . In the study countries, acute respiratory diseases and diarrhea affected 6.2 and 15.6% of children aged between 6 and 59 months, respectively . In india, a community - based cross - sectional study found an overall prevalence of acute respiratory infections of 59.1%, which was associated with overcrowding (adjusted odds ratio (aor) = 1.492), urban residence (aor = 2.329), and second birth order (aor = 0.371). Data from the peruvian ministry of health showed a hospitalization rate of 3.03 per 100 inhabitants in the general population in 2010 . Influenza (flu) and pneumonia accounted for the largest amount of hospitalization cases in that year . In madre de dios, in the peruvian amazon, intestinal infections prevailed in relation to influenza and pneumonia as a cause of hospitalization in the general population in the same year . In iapari, hospitalization of children under five years of age was mainly due to diarrhea and other intestinal diseases, confirming the data obtained by the ministry of health in the region . Estimated that 84,000 to 144,000 hospitalization cases due to lower respiratory tract disease occur annually in the usa in children under 5 years of age . In the study of quigley et al ., conducted in the uk, approximately 12% of children in the first eight months of age were hospitalized at least once . The most common causes of hospitalization were infection of the lower respiratory tract (3.2%) and diarrhea (1.1%). The study demonstrated a related protection factor to breastfeeding for hospitalization of protection related to injury . Factors associated with child hospitalization in iapari were related to child age and sex, socioeconomic characteristics, and access to health care . Children whose mothers had no formal education or low education were more likely to have been hospitalized . Macedo et al . Also found that child hospitalization increased when maternal education was low . In iapari, male children had a higher frequency of hospitalization (26.3%), a finding that was reported in other studies such as macedo et al ., with 53.3%, and caetano et al ., with 56.9% . Also, the frequency of hospitalization was higher in children older than 2 years (26.3%) than in those younger than 2 years (5.9%). This could be explained by the fact that as time goes by, there are more chances to get sick . . Found that hospitalization was higher in children younger than six months (60.3% in public hospitals and 47.3% in private hospitals), while caetano et al . Found that hospitalization was higher in children younger than 1 year (47.7%). A study in rural tanzania showed that children aged two years and older were more likely than children under one year of age to receive care at home, rather than to receive care at the health facility . Also, children aged two years and older were less likely to experience morbidity than children under one year . Describe a frequency of 56.9% of child hospitalization in children younger than 5 years, being the main causes of respiratory and parasitic diseases . In other latin american countries, diarrhea and respiratory diseases are also a public health problem in childhood . In haiti, respiratory diseases accounted for 9183 (29%) hospitalization cases and 301 (17%) deaths between 2011 and 2013 . Children aged 623 months had the highest percentage of hospitalization cases attributable to respiratory diseases (38%), whereas children aged 3647 months had the highest proportion of deaths attributable to respiratory diseases (37%). Diarrheal diseases accounted for 8063 (26%) hospitalization cases and 224 (13%) deaths . Children aged 611 months had the highest percentage of diarrhea - associated hospitalization cases (39%) and deaths (29%). About 27.3% of children with no history of maternal morbidity during pregnancy were hospitalized at some point during life, while only 9.8% of those without maternal history of gestational morbidity had a hospitalization . This could be explained by a modified maternal behavior in child care after a gestational morbidity, leading to improved maternal care or more access to health services during pregnancy and after birth . Children with poor health access after birth were also more likely to have a hospitalization . About 26.7% of those without a medical appointment in the previous year had hospitalization, against only 9.5% of those who attended a medical appointment in the previous year . Hospitalization cases were more frequent in children who seek health care services only when sick or who never sought health services . Access to health care was high in iapari when the child was sick but lower for routine consultations . It is known that health service usage usually varies according to the degree of economic development of an area or region, and it can also be different in urban and in rural areas . According to pssoa, consultations and physical exams are more complete in iapari than in the neighboring brazilian city of assis, but the working conditions for health care personnel and the diagnostic infrastructure are worse, with low salaries and lack of diagnostic exams . Another important difference is that in peru access to consults and medication within the health service require payment, while in brazil these are free . It has the merit of being a population - based census on child health performed in the peruvian amazon, which is usually an understudied type of population because of the difficult physical access . Interviews were performed in spanish by peruvian researchers, and one of them was living in the peruvian amazon, so it was performed by personnel that were used to the habits and local culture, facilitating the interviews . The limitations of the study are that since it was a small community, the number of children was small and some associations may have not been identified . Also, because it was based on referred morbidities, there may be some degree of recall bias which could not be avoided . This study showed that the main morbidities in children referred to the iapari municipality were related to diarrheal morbidity, respiratory symptoms, anemia, and vomiting . There was an association of these morbidities referred to as low socioeconomic conditions, precarious sanitation, and the presence of other comorbidities . Measurements to reduce morbidity and promote child health in such small peruvian amazonian communities would be improvements in sanitary infrastructure, water treatment, educational programs on child health for targeted families, and a more comprehensive health care with lower costs . Another important point is that free care is not always provided in peru, which may hinder access to health care for some children . Health policies that target free universal care for infants at risk may help promote child health in this part of the amazon.
Cardiovascular disease (cvd) is the leading cause of mortality in people with type 2 diabetes mellitus (t2 dm) (1). In fact,> 50% of patients with t2 dm die of coronary artery disease (2,3). Early vascular disease can be detected using pulse wave velocity (pwv), augmentation index (aix), and brachial distensibility (brachd), measures of arterial stiffness (4,5). Adolescents with t2 dm and obese youth have been shown to have greater arterial stiffness than their lean (age-, sex-, and race - matched) counterparts, suggesting the risk for a premature onset of cvd (6). 25-hydroxyvitamin d (25[oh]d) has an established role in calcium and phosphorus metabolism and bone health . Recent discovery of 25[oh]d receptors and the enzyme that converts 25[oh]d to its active form in vascular smooth muscle and endothelial cells points to a role in vascular health as well (7). 25[oh]d has been shown to have antiproliferative effects in both vascular endothelial and smooth muscle cells . Furthermore, 25[oh]d limits monocyte and macrophage differentiation and the release of inflammatory cytokines, blunting the inflammatory response, a known mediator of vascular disease (8). Finally, to support the causality of this association, vitamin d receptor mice exhibit site - specific accelerated atherogenesis (9). As a result, studies in healthy, obese, and t2 dm adults have found that low serum 25[oh]d levels are associated with increased arterial stiffness (1013). The role of 25[oh]d in arterial stiffness has not been examined in high - risk youth, namely those with obesity and those with t2 dm who are at risk for low levels of 25[oh]d secondary to decreased sun exposure and excessive storage of 25[oh]d in the adipose tissue (14). The purpose of this study was to examine the association between serum 25[oh]d levels and arterial stiffness in youth and to determine if the relationship between 25[oh]d levels and arterial stiffness differs by group . We hypothesized that low serum 25[oh]d levels would be associated with increased arterial stiffness independent of traditional cardiovascular risk factors . The population for this analysis was drawn from individuals who participated in the national institutes of health funded study cardiovascular disease in adolescents with type 2 diabetes (t2cvd) at cincinnati children s hospital medical center from 2004 to 2010 (15,16). In brief, the t2cvd study consisted of three groups: 221 individuals with t2 dm, 250 obese individuals without t2 dm, and 261 lean individuals (nonobese individuals without t2 dm). Individuals with diabetes were islet cell antibody negative (glutamic acid decarboxylase, ica512, and insulin autoantibodies), had no evidence of another specific type of diabetes, and did not require insulin in the basal state to prevent diabetic ketoacidosis . Each participant with t2 dm was matched to at least one lean (bmi <85th percentile) and one obese control (bmi 95th percentile) by age, race, and sex . All obese subjects underwent a 2-h oral glucose tolerance test to rule out subclinical t2 dm according to american diabetes association guidelines (17). Pregnant females were excluded from the study . Prior to enrollment in t2cvd, written informed consent was obtained from subjects 18 years old or the parent or guardian for subjects <18 years old . Written assent was also obtained for subjects <18 years old according to the guidelines established by the institutional review board at cincinnati children's hospital . We determined that a total sample size of 570 (190 subjects from each of the three groups) would be able to detect group differences of at least 6 20 ng / ml in 25[oh]d with 90% power at a significance level of 0.05 . After a 10-h overnight fast, participants in the t2cvd study were seen at an in - person study visit (6,15). Anthropometric measurements, venipuncture, blood pressure (bp), and arterial stiffness data were collected . Trained personnel obtained two measures of height (stadiometer; veeder - rood, elizabethtown, nc) and weight (electronic scale; health o meter), with the average of each used in analyses . Waist circumference was measured to the nearest 0.5 cm using a flexible metal research tape twice and averaged according to a standardized research protocol from the national heart, lung, and blood institute (nhlbi) growth and health study . Systolic and diastolic bp were measured according to the standards of the fourth report on blood pressure in children (18). . Standards correlating x - ray beam attenuation to amount of lean and fat mass have been developed and validated against the hydrodensitometry method, which has previously been established as the most valid measurement of lean body mass and fat mass (19). Percent body fat was calculated as total body fat mass / total body mass 100 . Plasma glucose was measured using a hitachi model 704 glucose analyzer (roche hitachi, indianapolis, in) with intra - assay and interassay coefficients of variation of 1.2 and 1.6%, respectively . Plasma insulin was measured by radioimmunoassay using an anti - insulin serum raised in guinea pigs, i - labeled insulin (linco, st . Louis, mo), and a double antibody method to separate bound from free tracer . Assays of fasting plasma lipid profiles were performed in a laboratory that is nhlbi / centers for disease control and prevention standardized, with the ldl cholesterol concentration calculated using the friedewald equation . C - reactive protein (crp) was measured using a high - sensitivity elisa . Hba1c was measured in red blood cells using high - performance liquid chromatography methods . For the current study, the following laboratory measures were performed on serum samples that had been stored at 80c . 1) 25[oh]d was measured on a diasorin liaison automated instrument by direct competitive chemiluminescence immunoassay for quantitative determination of total 25[oh]d in serum . 25[oh]d is a stable compound, and the assay is known to be reliable in frozen samples (20). 2) basic metabolic profile was performed to ensure normal calcium - phosphorus metabolism, renal function, albumin, and creatinine . Glomerular filtration rate (gfr) was then calculated using the schwarz formula [gfr (ml / min/1.73 m) = (0.41 height in cm)/serum creatinine in mg / dl]. None of the subjects had gfr <60 ml / min/1.73 m, and hence none were excluded from the study based on their renal function . Aix and pwv were obtained using the sphygmocor device (sphygmocor scor - pvx system; atcor medical, sydney, australia). Brachd was measured using the dynapulse pathway instrument (pulse metric, inc . ). A sphygmocor tonometer was used to measure aix, a measure of arterial stiffness and pulse wave reflections (6). The tonometer was placed over the right radial artery and three measures of aix were collected . A single value, calculated as an average of three aix measurements, was used in the analysis . The pressure waves were calibrated using mean arterial bp and diastolic bp obtained in the same arm . Since aix is affected by heart rate (hr), all aix values were adjusted to a standard hr of 75 bpm . The sphygmocor tonometer was also used to measure pwv, an additional measure of arterial stiffness (21). The distance from a proximal artery (carotid) to the distal artery (femoral) was measured to the nearest 0.1 cm twice, averaged, and entered into the software . A tonometer was used to collect proximal and distal arterial waveforms gated by the r - wave on a simultaneously recorded electrocardiogram . Pwv then was calculated as the distance from the carotid to distal path length divided by the time delay measured between the carotid and femoral waveforms reported in meters per second . The dynapulse pathway instrument (pulse metric, inc .) Was used to measure brachd (22). This device derives brachial artery pressure curves from distensibility arterial pressure signals obtained from a standard cuff sphygmomanometer . All analyses were performed with statistical analyses software (sas, version 9.3). Mean values for demographic, anthropometric, and laboratory data were obtained by group . Variance - stabilizing measures to transform nonnormal values were performed on hba1c, glucose, hdl cholesterol, triglycerides (tgs), crp, creatinine, and 25[oh]d . One - way anova and ancova were performed to test for mean differences between the groups . General linear models (glms) were constructed using significant covariates from correlation analyses to elucidate independent determinants of arterial stiffness . Separate models were created for each group (lean, obese, and t2 dm) because 25[oh]d has been shown to have different effects based on degree of adipose tissue . Each model potentially included age, sex, race, height, waist - to - height ratio, percent body fat (from dxa), hr (not for aix), systolic bp z score, diastolic bp z score, tg - to - hdl ratio (as a measure of small dense ldl particles), crp, glucose, and insulin . Waist to height was chosen over bmi because it has been shown to be a superior measure of adiposity compared with bmi, especially in people with diabetes (23,24). We also adjusted for potential seasonal variation in blood draw (april through september and october through march), since 25[oh]d levels have been shown to be lower in the winter months . In the final model, nonsignificant (p> 0.05) variables were removed . The population for this analysis was drawn from individuals who participated in the national institutes of health funded study cardiovascular disease in adolescents with type 2 diabetes (t2cvd) at cincinnati children s hospital medical center from 2004 to 2010 (15,16). In brief, the t2cvd study consisted of three groups: 221 individuals with t2 dm, 250 obese individuals without t2 dm, and 261 lean individuals (nonobese individuals without t2 dm). Individuals with diabetes were islet cell antibody negative (glutamic acid decarboxylase, ica512, and insulin autoantibodies), had no evidence of another specific type of diabetes, and did not require insulin in the basal state to prevent diabetic ketoacidosis . Each participant with t2 dm was matched to at least one lean (bmi <85th percentile) and one obese control (bmi 95th percentile) by age, race, and sex . All obese subjects underwent a 2-h oral glucose tolerance test to rule out subclinical t2 dm according to american diabetes association guidelines (17). Pregnant females were excluded from the study . Prior to enrollment in t2cvd, written informed consent was obtained from subjects 18 years old or the parent or guardian for subjects <18 years old . Written assent was also obtained for subjects <18 years old according to the guidelines established by the institutional review board at cincinnati children's hospital . We determined that a total sample size of 570 (190 subjects from each of the three groups) would be able to detect group differences of at least 6 20 ng / ml in 25[oh]d with 90% power at a significance level of 0.05 . After a 10-h overnight fast, participants in the t2cvd study were seen at an in - person study visit (6,15). Anthropometric measurements, venipuncture, blood pressure (bp), and arterial stiffness data were collected . Trained personnel obtained two measures of height (stadiometer; veeder - rood, elizabethtown, nc) and weight (electronic scale; health o meter), with the average of each used in analyses . Waist circumference was measured to the nearest 0.5 cm using a flexible metal research tape twice and averaged according to a standardized research protocol from the national heart, lung, and blood institute (nhlbi) growth and health study . Systolic and diastolic bp were measured according to the standards of the fourth report on blood pressure in children (18). Standards correlating x - ray beam attenuation to amount of lean and fat mass have been developed and validated against the hydrodensitometry method, which has previously been established as the most valid measurement of lean body mass and fat mass (19). Percent body fat was calculated as total body fat mass / total body mass 100 . Plasma glucose was measured using a hitachi model 704 glucose analyzer (roche hitachi, indianapolis, in) with intra - assay and interassay coefficients of variation of 1.2 and 1.6%, respectively . Plasma insulin was measured by radioimmunoassay using an anti - insulin serum raised in guinea pigs, i - labeled insulin (linco, st . Louis, mo), and a double antibody method to separate bound from free tracer . Assays of fasting plasma lipid profiles were performed in a laboratory that is nhlbi / centers for disease control and prevention standardized, with the ldl cholesterol concentration calculated using the friedewald equation . C - reactive protein (crp) was measured using a high - sensitivity elisa . For the current study, the following laboratory measures were performed on serum samples that had been stored at 80c . 1) 25[oh]d was measured on a diasorin liaison automated instrument by direct competitive chemiluminescence immunoassay for quantitative determination of total 25[oh]d in serum . 25[oh]d is a stable compound, and the assay is known to be reliable in frozen samples (20). 2) basic metabolic profile was performed to ensure normal calcium - phosphorus metabolism, renal function, albumin, and creatinine . Glomerular filtration rate (gfr) was then calculated using the schwarz formula [gfr (ml / min/1.73 m) = (0.41 height in cm)/serum creatinine in mg / dl]. None of the subjects had gfr <60 ml / min/1.73 m, and hence none were excluded from the study based on their renal function . Aix and pwv were obtained using the sphygmocor device (sphygmocor scor - pvx system; atcor medical, sydney, australia). Brachd was measured using the dynapulse pathway instrument (pulse metric, inc . ). A sphygmocor tonometer was used to measure aix, a measure of arterial stiffness and pulse wave reflections (6). The tonometer was placed over the right radial artery and three measures of aix were collected . A single value, calculated as an average of three aix measurements, was used in the analysis . The pressure waves were calibrated using mean arterial bp and diastolic bp obtained in the same arm . Since aix is affected by heart rate (hr), all aix values were adjusted to a standard hr of 75 bpm . The sphygmocor tonometer was also used to measure pwv, an additional measure of arterial stiffness (21). The distance from a proximal artery (carotid) to the distal artery (femoral) was measured to the nearest 0.1 cm twice, averaged, and entered into the software . A tonometer was used to collect proximal and distal arterial waveforms gated by the r - wave on a simultaneously recorded electrocardiogram . Pwv then was calculated as the distance from the carotid to distal path length divided by the time delay measured between the carotid and femoral waveforms reported in meters per second . The dynapulse pathway instrument (pulse metric, inc .) Was used to measure brachd (22). This device derives brachial artery pressure curves from distensibility arterial pressure signals obtained from a standard cuff sphygmomanometer . All analyses were performed with statistical analyses software (sas, version 9.3). Mean values for demographic, anthropometric, and laboratory data were obtained by group . Variance - stabilizing measures to transform nonnormal values were performed on hba1c, glucose, hdl cholesterol, triglycerides (tgs), crp, creatinine, and 25[oh]d . One - way anova and ancova were performed to test for mean differences between the groups . General linear models (glms) were constructed using significant covariates from correlation analyses to elucidate independent determinants of arterial stiffness . Separate models were created for each group (lean, obese, and t2 dm) because 25[oh]d has been shown to have different effects based on degree of adipose tissue . Each model potentially included age, sex, race, height, waist - to - height ratio, percent body fat (from dxa), hr (not for aix), systolic bp z score, diastolic bp z score, tg - to - hdl ratio (as a measure of small dense ldl particles), crp, glucose, and insulin . Waist to height was chosen over bmi because it has been shown to be a superior measure of adiposity compared with bmi, especially in people with diabetes (23,24). We also adjusted for potential seasonal variation in blood draw (april through september and october through march), since 25[oh]d levels have been shown to be lower in the winter months . In the final model, nonsignificant (p> 0.05) variables were removed . Table 1 lists the demographic, anthropometric, and laboratory data for all participants stratified by study group . There were no significant differences among the three groups in age, sex, and race . Serum 25[oh]d levels were higher in lean youth compared with their obese and t2 dm counterparts (p <0.01) with no difference in serum 25[oh]d levels between obese youth and youth with t2 dm . The 25[oh]d concentration was <20 ng / ml in 50% of lean youth and 80% of both obese youth and youth with t2 dm . There was no difference in 25[oh]d levels by sex . African american youth had a lower 25[oh]d level compared with their caucasian counterparts (p <0.01). Characteristics of the study population data are mean sd, n (%), or median (25th, 75th percentile). P values in the table represent the overall comparison between the three groups using anova; n / s, not significant . All three measures of arterial stiffness worsened from lean to obese to t2 dm (p <0.01). Negative aix in the lean group indicates that wave reflection happened late in the cardiac cycle, consistent with more pliable (less stiff) arteries . Data are mean and sd, where the white bar is the lean group, the hatched bar is the obese group, and the black bar is the group with t2 dm . The asterisks indicate a p value of <0.01 between the three groups by anova . The univariate associations between 25[oh]d levels and arterial stiffness were examined by the study group . There was a negative correlation between serum 25[oh]d and aix in lean and t2 dm only (both r = 0.24, p <0.01) and between 25[oh]d and pwv in lean (r = 0.26), obese (r = 0.27), and t2 dm (r = 0.36), all p <0.01 . Brachd was not correlated with 25[oh]d by group (data not shown), and therefore glms were not pursued . The gray line and solid black line are the regression lines for the lean group (r = 0.24, p <0.01) and the group with t2 dm (r = 0.24, p <0.01), respectively . B: the relationship between pwv (m / s) and 25[oh]d concentrations by group . The gray, dashed black, and solid black lines are the regression lines for the lean group (r = 0.24, p <0.01), obese group (r = 0.27, p <0.01), and group with t2 dm (r = 0.36, p <0.01), respectively . Glm analysis (table 2) showed that serum 25[oh]d level was an independent risk factor for aix in the t2 dm group only such that an increase in serum 25[oh]d by 3 ng / ml was associated with a 1% decrease in aix . Other independent determinants of aix in the group with t2 dm were age, height, and diastolic bp z score . In combination, the above risk factors explained 20% of the variance for aix in the group with t2 dm . The glm analysis for pwv showed that serum 25[oh]d level was an independent risk factor for pwv in the lean group only (table 2), although this was a small effect . Other independent determinants of pwv in lean individuals were age, sex, systolic bp z score, tg - to - hdl ratio, and hr . This model explained 37% of variance for pwv in the lean group . In all models, our study found that low serum 25[oh]d levels in youth with t2 dm were negatively associated with arterial stiffness, measured by aix, even after adjustment for demographic and traditional cvd risk factors . Furthermore, we show that in youth with t2 dm, a higher 25[oh]d level of 3 ng / ml is associated with a decrease in aix of 1% . Considering that aix in the group with t2 dm was 5.7 12.0% (mean sd), an absolute change of 1% may have a large impact on future cardiovascular outcomes given that an increase in arterial stiffness is known to predict future myocardial infarction and stroke (5,25). Recent epidemiologic studies in adults have shown an inverse association between serum levels of 25[oh]d and arterial stiffness in healthy adults (10), obese adults (13), and adults with t2 dm (12). Basic science research supports the potential role for 25[oh]d in the development of atherosclerosis (2628) by demonstrating that both vascular smooth muscle cells and vascular endothelial cells have 25[oh]d receptors and the enzyme to convert 25[oh]d to its active form 1,25-oh vitamin d (7). 25[oh]d has been shown to have antiproliferative effects on vascular endothelial and smooth muscle cells and the ability to limit monocyte and macrophage differentiation and the release of inflammatory cytokines, a known mediator of vascular disease (8). Finally, it has been shown that vitamin d receptor mice exhibit site - specific accelerated atherogenesis (9). Thus, there is reasonable evidence to support that 25[oh]d has a role in the development of atherosclerosis . We found serum levels of 25[oh]d to be lower in obese youth and youth with t2 dm compared with their lean counterparts . In fact, nearly 80% of both youth with obesity and youth with diabetes had a serum 25[oh]d level of <20 ng / ml (institute of medicine defines that a serum concentration of> 20 ng / ml 25[oh]d is needed for good bone health), in contrast to only 50% of lean youth . We also noted a seasonal variation in the 25[oh]d level in all three groups, with 25[oh]d levels being lower in fall and winter (october through march) compared with spring and summer (april through september). These results are consistent with prior studies (30,31) that show lower 25[oh]d in obese individuals due to decreased exposure to sunlight and excessive storage of 25[oh]d in the adipose tissue (14). Our linear regression models showed that after adjustment for risk factors, serum 25[oh]d level had a significant negative association with pwv in lean individuals and with aix in participants with t2 dm . In the obese group, although we found no association between 25[oh]d levels and arterial stiffness, it should be noted that aix was associated with season (higher aix in spring / summer vs. fall / winter), which may reflect changes in 25[oh]d levels with season . The reasons for the differential associations between 25[oh]d and arterial stiffness are unclear, but we hypothesize some potential reasons . First, each measure of arterial stiffness, although all reliable, assesses a different aspect of the vasculature . Pwv, a measure of central arterial stiffness, is considered the gold standard measure of subclinical arterial stiffness in both adults and children and has been shown to predict future cardiovascular events and mortality (32,33). Aix is a mixed measure of arterial stiffness that is influenced by central stiffness (pwv) and peripheral wave reflections (21) and has also been shown to predict all - cause mortality in adults with end - stage renal disease (34) and hypertension (5). Brachd is a nonultrasound measure of stiffness (arterial compliance) in a medium muscular artery (4) and is highly correlated with cardiovascular risk factors (4). Differential associations between risk factors and arterial stiffness have been documented previously (6,35), because although the arterial stiffness measures are correlated, each assesses a different property of the arterial tree . Second, since 25[oh]d is known to limit the release of inflammatory cytokines (8) and suppress the renin aldosterone axis (28), it is possible that the lower 25[oh]d concentrations observed in t2 dm have a larger effect on the vasculature . Confirmatory studies are needed in a larger cohort of adolescents with t2 dm . A recent study in t1 dm youth found an association between serum 25[oh]d levels and pwv after adjusting for age, sex, race, and season . However, the relationship was no longer significant after adjusting for bmi z score, lipids, and bp (36). In contrast, we found that 25[oh]d was independently associated with arterial stiffness in lean individuals and individuals with t2 dm after adjusting for adiposity (waist to height), lipids, and bp . Our study and the t1 dm study have some important differences to explain the discrepant results . First, 25[oh]d levels were much lower in this group with t2 dm compared with the previously published group with t1 dm . Second, in contrast to the t1 dm study, where pwv alone was reported as a marker of arterial stiffness, we studied the relationship between 25[oh]d levels and three measurements of arterial stiffness . Finally, there are likely inherent differences in the relationship between 25[oh]d levels and arterial stiffness in youth with t1 dm and t2 dm because of the degree of obesity in t2 dm . First, it is cross - sectional and thus represents serum 25[oh]d and arterial stiffness at one point in time . Second, it should be noted that the 25[oh]d measurements were performed on frozen samples . However, previous work has shown no difference in 25[oh]d levels with frozen storage (20). Prior studies have shown that youth with obesity and t2 dm have increased arterial stiffness, suggesting that they are at higher risk for early cvd (6). However, conventional cardiovascular risk factors do not fully explain the observed differences in arterial stiffness among lean and obese individuals and individuals with t2 dm (6). The data presented here suggest that 25[oh]d may be one of the nontraditional cardiovascular risk factors that contribute to arterial stiffness . Further randomized controlled trials in youth are needed to establish the causation and mechanism by which 25[oh]d affects arterial stiffness and to establish whether supplementation in this group may reduce, or slow, the process of arterial stiffness to some extent.
Radiofrequency ablation (rfa) has evolved as the treatment of choice for drug - resistant atrial fibrillation (af). The electrical isolation of the pulmonary veins (pvs) is regarded as one endpoint of ablation [1, 2], but more extensive approaches, such as linear- or complex - fractionated atrial electrogram (cfae) ablation, may also be employed . A remote magnetic navigation (mn) system is available and has been shown to be feasible in treating af [1, 3]. A soft rfa catheter with a magnet can be guided and positioned by directional magnetic fields . An irrigated rfa catheter has been shown to be capable of enlarging the size and improving the transmurality of ablation lesions using manual catheters, but no equivalent data are available for the mn system . A good tissue contact and positional stability are believed to be some of the advantages of the mn system, but it is not known whether af ablation using mn causes similar levels of myocardial injury as manual ablation . Rfa induces local thermal damage in the heart and previous studies have shown that traditional myocardial markers such as troponin t (tnt) and creatine kinase's cardiac isoenzyme mb (ckmb) are sensitive measures of myocardial injury [611]. Most studies have been performed when only limited ablation has been applied . In this study, we sought to determine the relative impacts of the different catheters by measuring levels of myocardial markers tnt and ckmb before and after af ablation using standard irrigated catheters and both irrigated and non - irrigated mn catheters . One hundred and fourteen highly symptomatic patients with drug - resistant, paroxysmal or persistent af referred for rfa treatment were enrolled . Demographic and clinical data were collected prior to the procedure (table 1). All patients were given oral anticoagulants for at least 1 month before the procedure . Patients underwent rfa either using a conventional manual - irrigated catheter (cir group, n = 65) or a remote magnetic navigated ablation procedure that employed irrigated (rmi group, n = 23) or non - irrigated (rmn group, n = 26) catheters, based on the operator's decision . We excluded patients with renal failure, recent myocardial ischemia or clinical signs of infection . The study was performed in accordance with the declaration of helsinki, and was approved by the local ethics committee . Table 1demographics and procedural datacir group (n = 65)rmi group (n = 23)rmn group (n = 26)male (%) 51 (79%)22 (96%)20 (77%)age (years) (sd)57 959 755 8paroxysmal af (%) 40 (61%)15 (65%)14 (54%)lone af (%) 35 (54%)12 (52%)13 (50%)prior af ablation (%) 18 (28%)7 (30%)6 (23%)pvi without additional ablation22 (34%)10 (43%)6 (46%)procedure time (min) (sd)215 61340 107324 74total ablation time (sec) (sd)3081 15156565 2206*4737 1111fluoroscopy time (min) (sd)46.1 1753.7 2254.5 21cir group conventional manual irrigated catheter; rmi group remote magnetic navigation, irrigated catheter; rmn group remote magnetic navigation, non - irrigated catheter; pvi pulmonary vein isolation; af atrial fibrillation; sd standard deviationonly those with pvi confirmed by a circular catheter were included (n = 13)significant vs cir and rmn, p <0.05; * * significant vs rmi and rmn, p <0.001; * * * significant vs rmn, p <0.001 demographics and procedural data cir group conventional manual irrigated catheter; rmi group remote magnetic navigation, irrigated catheter; rmn group remote magnetic navigation, non - irrigated catheter; pvi pulmonary vein isolation; af atrial fibrillation; sd standard deviation only those with pvi confirmed by a circular catheter were included (n = 13) * significant vs cir and rmn, p <0.05; * * significant vs rmi and rmn, p <0.001; * * * significant vs rmn, p <0.001 patients underwent electrophysiological study and rfa in a fasting, sedated state . Vascular access was obtained under local anesthesia through the right and left femoral veins . In all patients, a 7f 20-pole steerable mapping catheter (livewire, st . Paul, mn, usa) was positioned in the coronary sinus looped around the tricuspid annulus . After transseptal puncture, a 10-pole circular mapping catheter (lasso, biosense webster, diamond bar, ca, usa or optima, st . Jude medical inc) was introduced into the left atrium (la) through an 8f transseptal introducer . In the cir group, an irrigated rfa catheter (thermocool, biosense webster or navistar thermocool, biosense webster) was introduced into the la through the same puncture site without a second introducer . The ablation consisted of continuous circumferential lesions around each pv, with additional ablation between the two pvs if needed . The remainder were given additional ablation of the cavotricuspid isthmus line (n = 9), two lines connecting the two contra - lateral superior and inferior veins (n = 7), ablation on cfaes (n = 8) or a combination (n = 19). The application time of rfa was 4060 s at each site and energy was delivered with a cut - off temperature of 50c . The maximum output and irrigation rates were 3035 w, 1520 ml / min (for pv isolation), 25 w, 15 ml / min in the coronary sinus and 3040 w, 20 ml / min for linear or cfae ablation (maximum 35 w in la, 40 w in right atrium (ra)), respectively . In the patients who underwent mn ablation, the rfa catheter was introduced into the la through a second transseptal guiding introducer, and la mapping was performed by carto rmt (biosense webster). The three - dimensional mapping system was used in conjunction with the niobe ii system (stereotaxis, st . The ablation consisted of a continuous circumferential line around the two ipsilateral pvs, with additional ablation between the two pvs . Energy was applied only when good electrode - tissue contact was indicated by the system monitor . In the rmi group, a navistar rmt thermocool (biosense webster) thirteen patients underwent cfae ablation and three of these were given additional cavotricuspid isthmus ablation . Application time, output and temperature cut - offs, and irrigation rate were the same as in the cir group . In the rmn group a navistar celsius rmt (biosense webster) the circular mapping catheter was not employed and the pv isolation was confirmed by rfa catheter only . Energy was applied in temperature - controlled mode, with a cut - off temperature of 55c and a maximum output of 40 w. application time was 20 s at each site [1, 14]. Charring of the catheter tip raises its impedance and, if this was suspected, the catheter was extracted and the tip checked out during the procedure . In all three groups, a stockert - cordis (biosense webster) rfa generator was utilized . Surface ecg and intracardiac electrograms were recorded utilizing the multichannel labsystem pro (bard electrophysiology, lowell, ma, usa). Venous blood samples for baseline data were drawn from an antecubital vein before the procedure in all patients . Post - procedure samples were collected the next morning, 24 h after the start of the procedure . Tnt and ckmb were determined using an electrochemiluminescence immunoassay on a modular e system (roche diagnostics, mannheim, germany). The analytical detection limits were 0.01 g / l (tnt) and 0.1 g / l (ckmb), respectively . The tnt cut - off value for diagnosis of myocardial infarction according to acc / aha guidelines is 0.03 g / l . Follow - up was performed at the out - patient clinic or by the patients' local hospitals and referring cardiologists . All patients had clinical examination and at least one holter registration at 3 and 6 months after the procedure and further ecg - recordings if indicated by symptoms . Recurrence was defined as at least one episode of af lasting more than 60 s after a blanking period of 3 months . Patients were followed for 1 year after the relevant ablation procedure and clinical results were reported based on this procedure only . Patients who underwent a repeat procedure during the follow - up were therefore regarded as failure, independent of the outcome of the last procedure . One hundred and fourteen highly symptomatic patients with drug - resistant, paroxysmal or persistent af referred for rfa treatment were enrolled . Demographic and clinical data were collected prior to the procedure (table 1). All patients were given oral anticoagulants for at least 1 month before the procedure . Patients underwent rfa either using a conventional manual - irrigated catheter (cir group, n = 65) or a remote magnetic navigated ablation procedure that employed irrigated (rmi group, n = 23) or non - irrigated (rmn group, n = 26) catheters, based on the operator's decision . We excluded patients with renal failure, recent myocardial ischemia or clinical signs of infection . The study was performed in accordance with the declaration of helsinki, and was approved by the local ethics committee . Table 1demographics and procedural datacir group (n = 65)rmi group (n = 23)rmn group (n = 26)male (%) 51 (79%)22 (96%)20 (77%)age (years) (sd)57 959 755 8paroxysmal af (%) 40 (61%)15 (65%)14 (54%)lone af (%) 35 (54%)12 (52%)13 (50%)prior af ablation (%) 18 (28%)7 (30%)6 (23%)pvi without additional ablation22 (34%)10 (43%)6 (46%)procedure time (min) (sd)215 61340 107324 74total ablation time (sec) (sd)3081 15156565 2206*4737 1111fluoroscopy time (min) (sd)46.1 1753.7 2254.5 21cir group conventional manual irrigated catheter; rmi group remote magnetic navigation, irrigated catheter; rmn group remote magnetic navigation, non - irrigated catheter; pvi pulmonary vein isolation; af atrial fibrillation; sd standard deviationonly those with pvi confirmed by a circular catheter were included (n = 13)significant vs cir and rmn, p <0.05; * * significant vs rmi and rmn, p <0.001; * * * significant vs rmn, p <0.001 demographics and procedural data cir group conventional manual irrigated catheter; rmi group remote magnetic navigation, irrigated catheter; rmn group remote magnetic navigation, non - irrigated catheter; pvi pulmonary vein isolation; af atrial fibrillation; sd standard deviation only those with pvi confirmed by a circular catheter were included (n = 13) * significant vs cir and rmn, p <0.05; * * significant vs rmi and rmn, p <0.001; * * * significant vs rmn, p <0.001 vascular access was obtained under local anesthesia through the right and left femoral veins . In all patients, a 7f 20-pole steerable mapping catheter (livewire, st . Paul, mn, usa) was positioned in the coronary sinus looped around the tricuspid annulus . After transseptal puncture, a 10-pole circular mapping catheter (lasso, biosense webster, diamond bar, ca, usa or optima, st . Jude medical inc) was introduced into the left atrium (la) through an 8f transseptal introducer . In the cir group, an irrigated rfa catheter (thermocool, biosense webster or navistar thermocool, biosense webster) was introduced into the la through the same puncture site without a second introducer . The ablation consisted of continuous circumferential lesions around each pv, with additional ablation between the two pvs if needed . The remainder were given additional ablation of the cavotricuspid isthmus line (n = 9), two lines connecting the two contra - lateral superior and inferior veins (n = 7), ablation on cfaes (n = 8) or a combination (n = 19). The application time of rfa was 4060 s at each site and energy was delivered with a cut - off temperature of 50c . The maximum output and irrigation rates were 3035 w, 1520 ml / min (for pv isolation), 25 w, 15 ml / min in the coronary sinus and 3040 w, 20 ml / min for linear or cfae ablation (maximum 35 w in la, 40 w in right atrium (ra)), respectively . In the patients who underwent mn ablation, the rfa catheter was introduced into the la through a second transseptal guiding introducer, and la mapping was performed by carto rmt (biosense webster). The three - dimensional mapping system was used in conjunction with the niobe ii system (stereotaxis, st . The ablation consisted of a continuous circumferential line around the two ipsilateral pvs, with additional ablation between the two pvs . Energy was applied only when good electrode - tissue contact was indicated by the system monitor . In the rmi group, a navistar rmt thermocool (biosense webster) thirteen patients underwent cfae ablation and three of these were given additional cavotricuspid isthmus ablation . Application time, output and temperature cut - offs, and irrigation rate were the same as in the cir group . In the rmn group a navistar celsius rmt (biosense webster) the circular mapping catheter was not employed and the pv isolation was confirmed by rfa catheter only . Energy was applied in temperature - controlled mode, with a cut - off temperature of 55c and a maximum output of 40 w. application time was 20 s at each site [1, 14]. Charring of the catheter tip raises its impedance and, if this was suspected, the catheter was extracted and the tip checked out during the procedure . In all three groups, a stockert - cordis (biosense webster) rfa generator was utilized . Surface ecg and intracardiac electrograms were recorded utilizing the multichannel labsystem pro (bard electrophysiology, lowell, ma, usa). In the cir group, an irrigated rfa catheter (thermocool, biosense webster or navistar thermocool, biosense webster) was introduced into the la through the same puncture site without a second introducer . The ablation consisted of continuous circumferential lesions around each pv, with additional ablation between the two pvs if needed . The remainder were given additional ablation of the cavotricuspid isthmus line (n = 9), two lines connecting the two contra - lateral superior and inferior veins (n = 7), ablation on cfaes (n = 8) or a combination (n = 19). The application time of rfa was 4060 s at each site and energy was delivered with a cut - off temperature of 50c . The maximum output and irrigation rates were 3035 w, 1520 ml / min (for pv isolation), 25 w, 15 ml / min in the coronary sinus and 3040 w, 20 ml / min for linear or cfae ablation (maximum 35 w in la, 40 w in right atrium (ra)), respectively . In the patients who underwent mn ablation, the rfa catheter was introduced into the la through a second transseptal guiding introducer, and la mapping was performed by carto rmt (biosense webster). The three - dimensional mapping system was used in conjunction with the niobe ii system (stereotaxis, st . The ablation consisted of a continuous circumferential line around the two ipsilateral pvs, with additional ablation between the two pvs . Energy was applied only when good electrode - tissue contact was indicated by the system monitor . In the rmi group, a navistar rmt thermocool (biosense webster) thirteen patients underwent cfae ablation and three of these were given additional cavotricuspid isthmus ablation . Application time, output and temperature cut - offs, and irrigation rate were the same as in the cir group . In the rmn group a navistar celsius rmt (biosense webster) ablation catheter was used . The circular mapping catheter was not employed and the pv isolation was confirmed by rfa catheter only . Energy was applied in temperature - controlled mode, with a cut - off temperature of 55c and a maximum output of 40 w. application time was 20 s at each site [1, 14]. Charring of the catheter tip raises its impedance and, if this was suspected, the catheter was extracted and the tip checked out during the procedure . In all three groups, surface ecg and intracardiac electrograms were recorded utilizing the multichannel labsystem pro (bard electrophysiology, lowell, ma, usa). Venous blood samples for baseline data were drawn from an antecubital vein before the procedure in all patients . Post - procedure samples were collected the next morning, 24 h after the start of the procedure . Tnt and ckmb were determined using an electrochemiluminescence immunoassay on a modular e system (roche diagnostics, mannheim, germany). The analytical detection limits were 0.01 g / l (tnt) and 0.1 g / l (ckmb), respectively . The tnt cut - off value for diagnosis of myocardial infarction according to acc / aha guidelines is 0.03 g / l . Follow - up was performed at the out - patient clinic or by the patients' local hospitals and referring cardiologists . All patients had clinical examination and at least one holter registration at 3 and 6 months after the procedure and further ecg - recordings if indicated by symptoms . Recurrence was defined as at least one episode of af lasting more than 60 s after a blanking period of 3 months . Patients were followed for 1 year after the relevant ablation procedure and clinical results were reported based on this procedure only . Patients who underwent a repeat procedure during the follow - up were therefore regarded as failure, independent of the outcome of the last procedure . For all statistical analyses, the spss software package version 17.0 (spps inc ., il, usa) was used . Discrete variables are reported as counts (percentages) and continuous variables as mean sd . Statistical comparisons were performed using the for discrete variables and student's t test, mann whitney u test or one - way anova for continuous variables . Correlations between procedural variables and marker levels were calculated using linear regression analysis and spearman's rank correlation tests . There were no differences in the distribution of co - morbidity between the three groups . More male patients were included in the rmi group compared to the rmn and cir groups (96% vs. 77% and 79%, p <0.01). Thirty - one (27%) patients had undergone prior af ablation with an even distribution among the groups . The procedure time (skin - to - skin) and total ablation time in the rmn group were 276 76 min and 3,619 1,464 s, respectively . In the rmn group, procedure time (232 46 min vs. 324 74 min, p <0.001) and total ablation time (3,194 853 s vs. 4,737 1111 s, p <0.001) were significantly shorter in patients for whom the circular mapping catheter was not employed for confirmation of pv isolation . Procedure and total ablation times were longer in the rmi and rmn group than the cir group (p <0.001, table 1). No procedure - related complications, such as cardiac tamponade, atrioesophageal fistula, pv stenosis, transient ischemic attack or major bleeding were observed in any of the patients . Baseline myocardial marker levels were within the normal range, and displayed a statistically significant increase in both tnt and ckmb after ablation in all groups (table 2). After ablation there was a significantly lower mean tnt level in the rmn group than in the other groups (p <0.001). If the troponin level was corrected for ablation time (tntc), the mean tntc (g / l per 1000 seconds ablation) was considerably higher in the cir group than in either the rmn or rmi groups (0.61 vs. 0.30 and 0.23 g / l, p <0.001). The difference in tntc between the rmn and rmi groups was also statistically significant (p <0.05). Figure 1 shows a greater variance in tntc level in the cir group than in either the rmn or rmi groups (p <0.01). There were no differences in any group in the release of tntc between patients who underwent pv isolation alone and those who underwent additional la or ra ablation (cir group 0.63 and 0.61 g / rmi group 0.22 and 0.24 g / l, p = n.s . ; rmn group 0.35 and 0.25 g / l, p = 0.062, respectively). In all three groups there were lower levels of tntc in patients who had undergone prior af ablation than in those for the first - time procedure (cir 0.49 vs. 0.67 g / l, p <0.001; rmi 0.16 vs. 0.26 g / l, p <0.01; rmn 0.28 vs. 0.36 g / l, p <0.01). There were no significant differences between the proportions of patients undergoing repeat procedures or ablation additional to pv isolation in the three groups (table 1). Within the rmn group, there was a tendency towards higher levels of release of tnt in patients when a circular mapping catheter was used (1.35 vs. 1.03 g / l, p = 0.09). Table 2level of myocardial markers at baseline and after ablationmyocardial markercir grouprmi grouprmn groupbaselineafter rfabaselineafter rfabaselineafter rfatnt (sd) g / l<0.031.68 0.9<0.031.63 0.9<0.031.18 0.5ckmb (sd) g / l3.4 1.910.1 4.04.1 2.310.5 6.33.4 1.510.2 3.5cir group conventional manual irrigated catheter; rmi group remote magnetic navigation, irrigated catheter; rmn group remote magnetic navigation, non - irrigated catheter; rfa radiofrequency ablation; tnt troponin t; ckbm creatine kinase's myocardial isoform; sd standard deviationp <0.001, compared to baseline; * significant vs. rmi and cir, p <0.001fig . 1scattergrams of ablation time - corrected serum troponin t level . Note: there was a larger variance in time - corrected troponin t in the cir group than in either the rmi or the rmn groups (p <0.01). Cir conventional manual irrigated catheter, rmi remote magnetic irrigated catheter, rmn remote magnetic non - irrigated catheter level of myocardial markers at baseline and after ablation cir group conventional manual irrigated catheter; rmi group remote magnetic navigation, irrigated catheter; rmn group remote magnetic navigation, non - irrigated catheter; rfa radiofrequency ablation; tnt troponin t; ckbm creatine kinase's myocardial isoform; sd standard deviation * p <0.001, compared to baseline; * * significant vs. rmi and cir, p <0.001 scattergrams of ablation time - corrected serum troponin t level . Note: there was a larger variance in time - corrected troponin t in the cir group than in either the rmi or the rmn groups (p <0.01). Cir conventional manual irrigated catheter, rmi remote magnetic irrigated catheter, rmn remote magnetic non - irrigated catheter further analysis revealed a significant correlation between the total ablation time and post - ablation serum concentration of tnt (cir r = 0.61, p <0.01; rmi r = 0.74, p <0.001; and rmn r = 0.51, p <0.01, respectively; fig . 2). Post - ablation levels of ckmb also increased with total ablation time in all groups (cir r = 0.53, p <0.001; rmi r = 0.73, p <0.001; and rmn: r = 0.59, p = 0.002; fig . 2). 2correlation of troponin t (tnt) and cardiac creatin kinase (ckmb) with total ablation time . Plots show significant correlations between the myocardial marker levels and total ablation time in all groups . Y - axis serum levels of troponin t (micrograms per liter) or ckmb (micrograms per liter). Cir, rmi, rmn; see fig . 1 correlation of troponin t (tnt) and cardiac creatin kinase (ckmb) with total ablation time . Plots show significant correlations between the myocardial marker levels and total ablation time in all groups . Y - axis serum levels of troponin t (micrograms per liter) or ckmb (micrograms per liter). Cir, rmi, rmn; see fig . 1 after a mean follow - up period of 12.2 5.4 months, 68 patients (59.6%) were free from af recurrence, three patients are still on anti arrhythmic drugs (two patients in cir group and one in rmi group). Longstanding persistent af patients had significantly lower success rate than other patients (35.0% vs. 66.0%, p <0.05). There were no differences in success rate in the three different groups (cir 60.0%, rmi 61.0%, rmn 53.8%, p = n.s . ). In the rmn group, there was a significantly higher tntc in patients with successful ablation compared to patients with recurrence of af (0.43 vs. 0.25 g / l, p <0.01), but there were no such differences in the other groups (cir 0.61 vs. 0.65 g / l, p = n.s . ; rmi 0.22 vs. 0.24 g / l, p = n.s . ). No differences in total tnt was revealed between patients with or without ablation success in any group . There were no differences in the distribution of co - morbidity between the three groups . More male patients were included in the rmi group compared to the rmn and cir groups (96% vs. 77% and 79%, p <0.01). Thirty - one (27%) patients had undergone prior af ablation with an even distribution among the groups . The procedure time (skin - to - skin) and total ablation time in the rmn group were 276 76 min and 3,619 1,464 s, respectively . In the rmn group, procedure time (232 46 min vs. 324 74 min, p <0.001) and total ablation time (3,194 853 s vs. 4,737 1111 s, p <0.001) were significantly shorter in patients for whom the circular mapping catheter was not employed for confirmation of pv isolation . Procedure and total ablation times were longer in the rmi and rmn group than the cir group (p <0.001, table 1). No procedure - related complications, such as cardiac tamponade, atrioesophageal fistula, pv stenosis, transient ischemic attack or major bleeding were observed in any of the patients . Baseline myocardial marker levels were within the normal range, and displayed a statistically significant increase in both tnt and ckmb after ablation in all groups (table 2). After ablation there was a significantly lower mean tnt level in the rmn group than in the other groups (p <0.001). If the troponin level was corrected for ablation time (tntc), the mean tntc (g / l per 1000 seconds ablation) was considerably higher in the cir group than in either the rmn or rmi groups (0.61 vs. 0.30 and 0.23 g / l, p <0.001). The difference in tntc between the rmn and rmi groups was also statistically significant (p <0.05). Figure 1 shows a greater variance in tntc level in the cir group than in either the rmn or rmi groups (p <0.01). There were no differences in any group in the release of tntc between patients who underwent pv isolation alone and those who underwent additional la or ra ablation (cir group 0.63 and 0.61 g / l, p = n.s . ; rmi group 0.22 and 0.24 g / l, p = n.s . ; rmn group 0.35 and 0.25 g / l, p = 0.062, respectively). In all three groups there were lower levels of tntc in patients who had undergone prior af ablation than in those for the first - time procedure (cir 0.49 vs. 0.67 g / l, p <0.001; rmi 0.16 vs. 0.26 g / l, p <0.01; rmn 0.28 vs. 0.36 g / l, p <0.01). There were no significant differences between the proportions of patients undergoing repeat procedures or ablation additional to pv isolation in the three groups (table 1). Within the rmn group, there was a tendency towards higher levels of release of tnt in patients when a circular mapping catheter was used (1.35 vs. 1.03 g / l, p = 0.09). Table 2level of myocardial markers at baseline and after ablationmyocardial markercir grouprmi grouprmn groupbaselineafter rfabaselineafter rfabaselineafter rfatnt (sd) g / l<0.031.68 0.9<0.031.63 0.9<0.031.18 0.5ckmb (sd) g / l3.4 1.910.1 4.04.1 2.310.5 6.33.4 1.510.2 3.5cir group conventional manual irrigated catheter; rmi group remote magnetic navigation, irrigated catheter; rmn group remote magnetic navigation, non - irrigated catheter; rfa radiofrequency ablation; tnt troponin t; ckbm creatine kinase's myocardial isoform; sd standard deviationp <0.001, compared to baseline; * significant vs. rmi and cir, p <0.001fig . 1scattergrams of ablation time - corrected serum troponin t level . Note: there was a larger variance in time - corrected troponin t in the cir group than in either the rmi or the rmn groups (p <0.01). Cir conventional manual irrigated catheter, rmi remote magnetic irrigated catheter, rmn remote magnetic non - irrigated catheter level of myocardial markers at baseline and after ablation cir group conventional manual irrigated catheter; rmi group remote magnetic navigation, irrigated catheter; rmn group remote magnetic navigation, non - irrigated catheter; rfa radiofrequency ablation; tnt troponin t; ckbm creatine kinase's myocardial isoform; sd standard deviation * p <0.001, compared to baseline; * * significant vs. rmi and cir, p <0.001 scattergrams of ablation time - corrected serum troponin t level . Note: there was a larger variance in time - corrected troponin t in the cir group than in either the rmi or the rmn groups (p <0.01). Cir conventional manual irrigated catheter, rmi remote magnetic irrigated catheter, rmn remote magnetic non - irrigated catheter further analysis revealed a significant correlation between the total ablation time and post - ablation serum concentration of tnt (cir r = 0.61, p <0.01; rmi r = 0.74, p <0.001; and rmn r = 0.51, p <0.01, respectively; fig . Post - ablation levels of ckmb also increased with total ablation time in all groups (cir r = 0.53, p <0.001; rmi r = 0.73, p <0.001; and rmn: r = 0.59, p = 0.002; fig . 2correlation of troponin t (tnt) and cardiac creatin kinase (ckmb) with total ablation time . Plots show significant correlations between the myocardial marker levels and total ablation time in all groups . Y - axis serum levels of troponin t (micrograms per liter) or ckmb (micrograms per liter). Cir, rmi, rmn; see fig . 1 correlation of troponin t (tnt) and cardiac creatin kinase (ckmb) with total ablation time . Plots show significant correlations between the myocardial marker levels and total ablation time in all groups . Y - axis serum levels of troponin t (micrograms per liter) or ckmb (micrograms per liter). After a mean follow - up period of 12.2 5.4 months, 68 patients (59.6%) were free from af recurrence, three patients are still on anti arrhythmic drugs (two patients in cir group and one in rmi group). Longstanding persistent af patients had significantly lower success rate than other patients (35.0% vs. 66.0%, p <0.05). There were no differences in success rate in the three different groups (cir 60.0%, rmi 61.0%, rmn 53.8%, p = n.s . ). In the rmn group, there was a significantly higher tntc in patients with successful ablation compared to patients with recurrence of af (0.43 vs. 0.25 g / l, p <0.01), but there were no such differences in the other groups (cir 0.61 vs. 0.65 g / l, p = n.s . ; rmi 0.22 vs. 0.24 g / l, p = n.s . ). No differences in total tnt was revealed between patients with or without ablation success in any group . We measured the levels of myocardial markers tnt and ckmb in patients who underwent manual or remote mn rfa for af . Previous studies on troponin i or t have produced diverging results regarding the relationship between enzyme release and the number of rfa lesions and site of rfa [610]. Our study demonstrates a significant positive correlation between myocardial marker levels and total ablation time with both manual and mn catheters . A considerably lower time - corrected tnt level (tntc) was associated with the mn system, whether irrigated or non - irrigated catheters were employed . This suggests that myocardial injury, as indicated by enzyme release, was lower with the mn system than with the manual ablation technique . Lesions created by mn may appear to be more sharply defined because of the positional stability of the system in comparison with manual catheters, and may therefore cause less myocardial damage and still be effective . It has been suggested that similar mechanisms are involved in cryoablation, when the ablation catheter freezes to the endocardium [1619]. The manual catheters might be less stable and slide over the endocardium resulting in larger, but shallower and thus less effective, ablation lesions . Af patients are reported to have a thinner posterior atrial wall and a stable catheter position during ablation may produce transmurality more rapidly than the preset ablation time . Our results revealed an unexpectedly higher time - corrected release of tnt using a non - irrigated mn catheter than an irrigated catheter . One possible explanation is that the irrigation flow (1520 ml / min) might reduce the electrode - tissue contact for the soft catheter . In these two groups there were also different application time, temperature and output cut - off settings, with the highest output and shortest application time in the rmn group . Efficiency is lower during the first few seconds of rfa delivery, until the output reaches a plateau and the lesions might be enlarged up to 180 s of energy delivery [22, 23]. A longer time to the plateau phase and longer ablation time at each site in the rmi group may have led to a lower tntc . On the other hand, the irrigation rate and energy settings were identical in the cir and rmi groups, and the longer total ablation times in the rmi group did not result in higher levels of myocardial marker release . With the mn system, catheter - tissue contact status can be monitored but no guidance of catheter - tissue contact is yet available with manual catheter ablation . A substantially stronger contact force might create deeper ablation lesions [24, 25]. Our analysis revealed a stronger correlation between tnt and ablation time with the remote irrigated than the manual irrigated catheter . A possible explanation is that the stable positioning and the low and stable pressure obtained with a remote catheter create more discrete ablation lesions and by that a more predictable ablation effect (fig . 1). New methods of measuring contact force are being developed, and further studies need to be performed on this subject . There were no differences in tntc levels using the mn system between patients who underwent pv isolation alone or who were given additional ablation in the atria . This suggests that catheter movement and tissue contact using remote mn are similar at the pv ostia and the other atrial locations . A lower level of myocardial marker release was observed in patients who underwent a repeat procedure . Ablation on scars or partly fibrous tissue is not expected to produce a similar release of myocardial markers as ablation on healthy tissue, thus energy was not delivered to areas without electrical signals . Longer procedure and ablation times were needed if a circular mapping catheter was employed to confirm pv isolation (rmn group), supported by the tendency towards higher myocardial marker release in these patients . Since better clinical outcomes have been demonstrated with proven pv isolation [26, 27], it may be necessary to employ a circular mapping catheter also during magnetic navigated af ablation procedures . This study was non - randomized, but there were similar baseline characteristics and portions of patients with prior af ablation or ablation extensive to pv isolation in the three groups . There were no statistically significant differences in outcome using the different ablation catheters, although there was a tendency to higher af recurrence rate in the rmn group . Neither were there any differences in marker levels in patients with or without ablation success . In all groups, the proportion of complete pvi and additional tailored ablation on cfae or complete cavotricuspid isthmus block was similar . This indicates that the choice of procedural strategy is more important than the type of catheter used to reach the endpoints of ablation . This study was non - randomized which may limit the significance of the outcome on comparisons of the different catheters . The study groups were not identical in size and the numbers of patients in both groups on which the mn system was utilized were relatively small . Although similar proportions of patients underwent individually tailored ablation additional to pv isolation, this approach may limit the interpretation of the clinical results . The temperatures reached and the energy delivered to produce individual lesions was not recorded for comparison . The operators' limited experience with the mn system may have had an impact on procedure time, but all operators were experienced electrophysiologists . This study was non - randomized which may limit the significance of the outcome on comparisons of the different catheters . The study groups were not identical in size and the numbers of patients in both groups on which the mn system was utilized were relatively small . Although similar proportions of patients underwent individually tailored ablation additional to pv isolation, this approach may limit the interpretation of the clinical results . The temperatures reached and the energy delivered to produce individual lesions was not recorded for comparison . The operators' limited experience with the mn system may have had an impact on procedure time, but all operators were experienced electrophysiologists . Remote magnetic catheters may create more discrete ablation lesions with a more predictable effect as measured by myocardial enzymes . Employing this system may require longer ablation time to reach the procedural endpoints than manual procedures . Remote magnetic non - irrigated catheters do not appear to be inferior to magnetic irrigated catheters in terms of myocardial marker release and clinical outcome.
A number of studies have documented the potential benefits of probiotic bacteria which prevent and ameliorate human t - helper-2-cell- (th2-) related diseases, such as atopic dermatitis and asthma . At the helper t - cell level, a number of probiotic lactobacilli promote the production of t - helper-1-cel - l (th1-) skewing cytokines, such as interleukin (il-) 12 and interferon (ifn-), and suppress the production of ige and th2 cytokines . Other studies have demonstrated that bifidobacteria suppress th2 cytokine and ige production, without significant induction of th1 cytokines . The th2-suppressive effect of probiotic bacteria is mediated by regulatory t (treg) cells linked to the induction of regulatory cytokines, including il-10 and transforming growth factor (tgf-) [4, 5]. It has been reported that macrophages stimulated with lactococcus lactis w58 produce il-10 and that tgf- is induced by lactobacillus paracasei ncc246 in mixed lymphocyte reactions . In addition, recent clinical studies have proven the prevention and curative features of probiotics in some intestinal disorders, such as inflammatory bowel diseases (ibd) including crohn's disease and ulcerative colitis . It was demonstrated that administration of bifidobacterium longum bb536 was effective in inducing remission of patients with ulcerative colitis . Multiple mechanisms of action have been suggested to explain the protective effects of probiotics in intestinal inflammation . These can be broadly classified as follows: (1) suppression of growth or epithelial binding / invasion by pathogenic bacteria, (2) improved epithelial barrier function, and (3) immunoregulatory activities . As for immunoregulation, several probiotics can induce protective cytokines, including il-10 and tgf-, and suppress pro - inflammatory cytokines, such as tnf-, in the mucosa of patients with paucities and crohn's disease . Besides th1, th2, and treg, t helper 17 cells (th17) have received considerable recent attention as they exhibit effector functions distinct from th1 and th2 cells [9, 10]. Differentiation factors (tgf- plus il-6 or il-21), growth and stabilization factor (il-23), and transcription factors (signal transducer and activator transcription 3 (stat3), retinoic acid receptor - related orphan receptor (ror-) t and ror-) involved in the development of th17 cells have been identified . Notably, th17 cells produce the pleiotropic cytokine il-17, which potently induces tissue inflammation and is associated with the pathogenesis of many human diseases, such as ibd and rheumatoid arthritis . Thus, modulation of th17 cells is currently viewed as a potentially positive pharmacological outcome . Although some studies have revealed that lactic acid bacteria and bifidobacteria may have potential use as anti - inflammatory agents in certain chronic inflammatory diseases, such as ibd, it is unclear whether these bacteria are able to suppress excess th17 activity . We recently reported that bifidobacterium longum infantis jcm 1222 (type strain) suppresses il-17 production in murine splenocytes in vitro and in inflamed intestinal cultures ex vivo . To identify other bacterial strains with potential th17 modulating activity, we previously performed an in vitro assay with 200 strains of streptococci and found that streptococcus thermophilus st28 exhibited potent inhibitory activity towards il-17 production . The main objective of this study was to evaluate the effects of oral administration of st28 on il-17 production by murine lamina propria lymphocytes (lpls) and on the expression of th17-related surface markers in inflamed intestines . We first evaluated and compared in vitro cytokine production patterns in th17-skewed conditions following the exposure of splenocytes to st28 and atcc 19258 (type strain). The in vivo effects of st28 were also evaluated using flow cytometry and real - time pcr in dextran - sodium - sulphate- (dss-) induced colitis mice, a human ibd model in which il-17 plays a pivotal role in pathogenesis . Two strains of s. thermophilus, atcc 19258 (type strain) and st28, were obtained from the american type culture collection and glico dairy products (tokyo, japan), respectively . Each strain was cultured in m17 broth (merck, whitehouse station, nj, usa) at 37c for 1765 hours . The cultured cells were washed with sterile distilled water and incubated at 100c for 50 min . Following the incubation, the heat - killed bacteria were lyophilized and used for subsequent experiments . Six - week - old female balb / c mice were obtained from charles river japan (kanagawa, japan), and all experimental protocols involving animals were approved by the animal care committee, graduate school of biosphere science, hiroshima university . To harvest splenocytes, mice were sacrificed by cervical dislocation, spleens were removed, and suspensions of splenocytes (1.2 10 cells) were incubated with 2 ng / ml tgf- (r&d systems, minneapolis, minn, usa) plus 20 ng / ml il-6 (r&d systems) at 37c for 72 hours in 120 l rpmi 1640 medium (life technologies, foster city, california, usa) supplemented with 10% fetal bovine serum (fbs, icn biomedicals, osaka, japan), 10 m 2-mercaptoethanol, 10 mm hepes, 5 units / ml penicillin (life technologies), and 5 g / ml streptomycin (life technologies) in a 96-well cell culture plate (thermo fisher scientific, waltham, ma, usa). Heat - killed bacteria (1.2 10 cells) were then added to the cell cultures . A culture to which no tgf- plus il-6 or heat - killed bacterial cells were added was included as a control . Following the incubation, cells were applied to flow cytometry (figure 1), and culture supernatants were assayed for cytokine concentrations (table 1) as described below . The splenocytes (1 10 cells / ml) were incubated at 37c for 4 hours in rpmi 1640 medium containing 0.5 g / ml phorbol-12-myristate-13-acetate (mp biomedicals, aurora, ohio, usa), 1 g / ml ionomycin (wako pure chemical industries, osaka, japan), and 3 g / ml brefeldin - a (wako pure chemical industries). Then, anti - mouse cd32/16 antibody was added and incubated 4c for 30 min . For analysis of surface markers, fluorescein isothiocyanate (fitc) anti - mouse cd11c antibody (biolegend, san diego, calif, usa), allophycocyanin (apc) anti - mouse cd11b antibody (ebioscience, san diego, calif, usa), or apc anti - mouse cd4 antibody (ebioscience) was added to the cell suspension, which was then incubated in the dark at 4c for 30 min . For isotype controls, fitc - armenian hamster igg, apc rat igg2a (biolegend), or apc rat igg2b (ebioscience), the cells were permeabilized by intraprep (beckman coulter, marseille codex, france) for intracellular cytokine staining . Phycoerythrin (pe) anti - mouse il-17 antibody (ebioscience) or pe rat igg2a (ebioscience) was added to the cell suspension and incubated in the dark at 4c for 30 min . Following the incubation, the cells were washed twice with 5% fbs - hanks' balanced salt solution (hbss) and suspended in 300 l of 5% fbs - hbss . Fluorescence intensity was measured on a guava easycyte flow cytometry system (millipore, billerica, mass, usa). Cytokine (ifn-, il-2, il-4, il-5, il-10, il-12(p70), tumor - necrosis - factor- (tnf-), and granulocyte macrophage colony - stimulating factor (gm - csf)) concentrations of the culture supernatants were determined by the microbead method using the bio - plex suspension array system (biorad laboratories, hercules, calif, usa), according to the manufacturer's instructions . Briefly, culture supernatants were incubated with beads conjugated with anti - ifn-, -il-2, -il-4, -il-5, -il-10, -il-12(p70), -tnf-, and -gm - csf antibodies (biorad laboratories) followed by sandwich immunoassay using biotinylated secondary antibodies . The beads were washed three times after each incubation, and pe - streptavidin was used as a reporter . The relative fluorescence units were determined by counting 100 beads using the bio - plex system . Data were evaluated with the bio - plex manager software 6.0 (biorad laboratories). Separately, the il-17 concentration of culture supernatants was measured using a duoset sandwich elisa kit (r&d systems) following the manufacturer's instructions . Six - week - old female balb / c mice were housed in an air - conditioned room under a 12 hours light - dark cycle and were allowed ad libitum access to tap water and a standard diet (mf; oriental yeast, tokyo, japan). Acute colitis was induced in mice by adding 3.5% (w / v) dss (molecular weight, 36,00050,000; mp biomedicals) to their drinking water for 5 days . The experimental protocols (figure 1(a)) were approved by the animal care committee, graduate school of biosphere science, hiroshima university . Group 1 (control group) mice were treated orally with phosphate - buffered saline (pbs) from days 1 to 3, while group 2 (control group) received drinking water containing 3.5% (w / v) dss and were orally administered pbs . Groups 3 and 4 also received drinking water containing 3.5% (w / v) dss, and, 24 hours after the start of dss administration, the mice were orally administered 10 cfu / day of heat - killed st28 (group 3) or atcc 19258 (group 4) in pbs for 3 consecutive days (figure 2(a)). The severity of intestinal inflammation was assessed on day 5 by assigning a score for each of the 2 stool scores, consistency, and bleeding (figure 2(b)). The scale used for consistency was 0 (normal stool), 1 (loose stool), 2 (diarrheal stool), and 3 (watery stool), and that used for bleeding was 0 (normally colored stool), 1 (blood visibly present in the stool), 2 (adherent of blood on anus), and 3 (gross bleeding on anus). Then, the total score was expressed as the sum of the scores of the 2 items (total score, 6). After the experimental period, the entire colon was removed from mice (figure 2(c)), and lamina propria from the excised colons was removed . Pooled (n = 4) colon tissue was suspended in hbss containing 10 mm dithiothreitol (wako pure chemical industries) and incubated at 20c for 10 min with shaking . The tissue was further treated with 2.1 mg / ml collagenase d (wako pure chemical industries) at 37c for 20 min in a shaking water bath [19, 20]. Only live cells were collected from single - cell suspensions from the lpls by dead cell removal kit (miltenyi biotec, auburn, calif, usa), and used for subsequent experiments . Portion of lpls (1 10 cells) were suspended in the same rpmi 1640 medium as described above and incubated at 37c for 48 hours in the presence of 1 g / ml anti - cd3 (ebioscience) and 1 g / ml anti - cd28 (ebioscience). Following the incubation, cells were applied to flow cytometry, and culture supernatants were assayed for il-17 and ifn- concentrations (figure 3). For analysis of intracellular il-17 (figure 4(a)), lpls (1 10 cells / ml) were incubated at 37c for 4 hours in rpmi 1640 medium containing 0.5 g / ml phorbol-12-myristate-13-acetate, 1 g / ml ionomycin, and 3 g / ml brefeldin a. then, apc anti - mouse cd4 antibody or apc rat igg2a was added to the cell suspension . After the incubation, the cells were permeabilized by intraprep, and pe anti - mouse il-17 antibody or pe rat igg2a was added to the cell suspension . Following the incubation, the cells were applied to flow cytometry . For analysis of surface markers (figure 4(b)), freshly prepared lpls were incubated with fitc anti - mouse cd11c antibody and apc anti - mouse cd86 antibody (ebioscience), or with fitc armenian hamster igg and apc rat igg2a, were added to the cell suspension and applied to flow cytometry . Pooled (n = 4) colon tissue from other dss colitis mice was suspended in hbss containing 5 mm edta and incubated at 37c for 20 min in a shaking water bath . The tissue was further treated with 1 mg / ml collagenase d (roche applied science, nonnenwald, germany) and 3 mg / ml dispase ii (life technologies) at 37c for 30 min in a shaking water bath . Briefly, tissue was resuspended in 40% percoll (ge healthcare biosciences ab, uppsala, sweden) and overlaid on 80% percoll, after which density gradient was performed by centrifugation at 1,000 g for 20 min at 25c . Lpls were subsequently collected from the interface fraction between the 40% and 80% percoll's layers [19, 20]. Rna was extracted from the lpls using trizol (life technologies) following the manufacturer's instructions . Reverse transcription reactions were performed with high - capacity cdna reverse transcription kit (life technologies) at 25c for 10 min and 37c for 120 min . The reaction was terminated by heating at 85c for 5 sec, followed by cooling at 4c . Real - time pcr was performed using kapa sybr fast abi prism qpcr kit (kapa biosystems, woburn, mass, usa) and the primer sequences listed (/see/ in supplementary material available on line at doi: 10.1155/2011/378417). Reactions were performed at 95c for 2 min, followed by 40 cycles of 95c for 5 sec and 60c for 30 sec . The dissociation stage was analyzed at 95c for 15 sec, followed by 1 cycle of 60c for 15 sec and 95c for 15 sec . The fluorescence of the sybr green dye was determined as a function of the pcr cycle number, giving the threshold cycle number at which amplification reached a significant threshold . Data were analysed by the ct method and presented as fold changes in gene expression after normalization to the internal control -actin gene expression level (supplementary figure 1). Briefly, bone marrow cells were collected from femurs of six - week - old female balb / c mice . Bone marrow mononuclear cells (2 10 cells / ml) were cultured at 37c for 7 days in rpmi1640 medium containing mouse 200 units / ml granulocyte macrophage colony - stimulating factor (miltenyi biotec), 10% fbs, 10 m 2-mercaptoethanol, 10 mm hepes, 5 units / ml penicillin, and 5 g / ml streptomycin in a tissue culture flask . On day 7, bmdc (1 10 cells / ml) were seeded in 24-well cell culture plate and matured by 1 g / ml lipopolysaccharide (lps, sigma - aldrich, st . Louis, mo, usa) for 24 hours . On day 8, heat - killed bacteria (1 10 cells / well) were added to the cell cultures . After 24-hour incubation, il-6 and il-10 concentrations of culture supernatants were measured using a duoset sandwich elisa kit (r&d systems) following the manufacturer's instructions . Statistical analysis was performed using one - way anova followed by tukey's post hoc test . When live st28 was added to murine splenocytes and coincubated at 37c for 72 hours, st28 grew in rpmi 1640 medium, and it became impossible to properly examine the effect of st28 on splenocytes . Thus, in subsequent experiments, st28 and atcc 19258 were used after heat killing . Prior to the experiment, we first purified t cells from splenocytes by magnetic bead - based cell separation (macs), and stimulated the macs - purified t cells with tgf- plus il-6 . However, il-17 production was not detectable (data not shown), which suggested that il-17 production from th17 cells in splenocytes required the coexistence of non - t cells . Therefore, we alternatively stimulated whole splenocytes with tgf- plus il-6 for further analyses, after which the stimulated splenocytes were applied to flow cytometry in order to analyze cd4, cd11b, and cd11c cells separately . As a result, most of the il-17 cells were cd4 t cells, which indicated that the major source of il-17 was th17 cells . More importantly, it was confirmed that st28 decreased the percentage of cd4 il-17 cells (figure 1). As shown in table 1, st28 significantly (p <0.01, () versus st28) repressed il-17 production (78% reduction). On the other hand, atcc 19258 failed to repress its production . Based on these results, st28 was judged to be a suitable candidate strain for the treatment of th17-mediated diseases, and its suppressive effects were further examined in vivo using dss - induced colitis mice . In regard to other cytokines than il-17, the productions of all cytokines tested were changed by the stimulation with tgf- plus il-6 . Among them, ifn- and tnf- productions in the st28 group were significantly higher than those in the atcc 19258 group, and il-2 and il-4 productions were significantly lower . On the other hand, there were not significantly differences in il-10 (anti - inflammatory cytokine) and il-12 (proinflammatory cytokines) productions between the two groups . Acute colitis was induced in mice by exposure to 3.5% dss for 5 days, which resulted in an increase in colon inflammation score compared to the control group (score, 2.5 1.5 versus 0.5 0.3) (figure 2). However, oral treatment with st28 ameliorated the inflammation (score, 1.0 0.4). Atcc 19258 exerted the same ameliorative effect on the intestinal inflammation, but the effect was weaker (figures 2(b) and 2(c)). Next, lpls from the mice was applied to ex vivo culture experiment (figure 3). Although differences in ifn- concentrations in lpls culture were not prominent among four groups, lpls from dss colitis mice produced substantial level of il-17 . However, st28 markedly suppressed il-17 production from lpls culture (86% suppression), while atcc 19258 failed to suppress il-17 production . From the analyses of mrna expression in lpls, it was revealed that the expression of both il-17 and ror-t, the master regulator of th17, in lpls from the dss - administered group was drastically upregulated, compared to control mice (supplementary figure 1). Notably, st28 markedly inhibited both expressions, which indicated that st28 suppressed the differentiation of nave t cells towards th17 in lpls . Cd11c and cd86 were also found to be downregulated by the oral administration of st28 . It was also confirmed by flow cytometry that cd4il17 cells (th17 cells) disappeared in lpls by the oral administration of both st28 and atcc 19258 (figure 4(a)). On the other hand, the effects of dss and those of the administration of st28 and atcc 19258 on cd11c cd86 cells, inflammatory dendritic cells (dc), were unclear, although atcc 19258 seemed to decrease the percentage of cd11c cd86 cells . To clarify this point the effect of st28 on lps - matured bmdc was evaluated (figure 5). Il-10 production was not significantly decreased by the addition of st28 and atcc 19258 . However, both st28 and atcc 19258 significantly suppressed il-6 production . Accordingly, the percentage of cd86cells, which is a hallmark of maturation, was also decreased by st28 and atcc 19258 . Therefore, it is highly probable that st28 suppressed th17 cells via a dc - dependent mechanism . In medical therapy for ibd, the modulation of immune responses and the regulation of intestinal inflammation is essential . Although the pathogenesis of ibd is complex, involving environmental, microbial, genetic, and immunological factors, several recent studies have shown that th17 cells play a significant role in ibd pathology [23, 24]. Here, we demonstrated that s. thermophilus st28 repressed il-17 production in mouse splenocytes under th17-skewed conditions in vitro (table 1). Moreover, oral treatment of dss - induced colitis mice with st28 suppressed inflammatory th17 cells in lpls . Although s. thermophilus is not commonly considered a probiotic bacterium, the findings of the present study imply that this species would be useful in the treatment of ibd by suppressing exaggerated th17 activity in inflamed intestines . Th1/th2 balance can influence the direction and outcomes of immune responses, due to the mutually antagonistic nature of th1 and th2 responses . In this regard, the modulating activity of probiotic and commensal bacteria on th1/th2 balance is commonly evaluated [3, 4]. Here, as ifn-/il-4 production ratio in st28 group was high even in th17-skewed conditions (table 1), it is expected that st28 would suppress not only th17, but also th2 cells . This response would also affect ige production in allergic conditions, because th2 cells drive ige class switching by allergen - stimulated b cells . Suppressing both th2 and th17 cells is desirable for the repression of ige - mediated reaction, because these cell types are closely related with each other in allergic patients . To confirm that st28 also suppressed th17 cells in vivo, acute colitis was induced by dss treatment of mice . Dss induces acute inflammation and recruitment of immune cells, whose subsequent activation directly causes epithelial cell . In dss - induced colitis mice, which serve as a useful model of human ibd, st28 administration improved inflammation score (figure 1), repressed il-17 production (figure 3), and decreased th17 cells (figure 4) in lpls isolated from inflamed intestines . Moreover, st28 decreased the mrna expressions of both rort and il-17 in lpls (supplementary figure 1). Considering all of the data, although several lines of evidence indicate probiotic bacteria have beneficial effects on dss colitis [7, 14, 26], the mechanisms by which bacteria mitigate intestinal inflammation have not been fully clarified . We propose that st28 ameliorates dss - induced colitis in mice by suppression of inflammatory th17 cells . It should be noted that even heat - killed bacteria could suppress il-17 production in this study . Therefore, some heat - resistant components of st28 might exert th17 supprressive activity . It is generally accepted that bacterial components are recognized by members of the pattern recognition receptor family, such as toll - like receptors (tlrs). In regard to the epithelial barrier, some studies have shown that the tlr2 ligand pcsk ameliorated tnf--induced intestinal barrier impairment in the human epithelial caco-2 cells [28, 29] and dss colitis mice . It is likely that the recognition of heat - resistant bacterial components by these kinds of receptors is also involved in the suppression of th17 cells . It has been recently suggested that cd86 plays a critical role in the initiation of t - cell responses including th17 cells . For example, gene silencing of cd40, cd80, and cd86 protected collagen - induced arthritis, one of the th17-related inflammation . In the effector cd4 t - cell responses inducing antigen - induced arthritis, blockade of cd86 significantly suppressed il-17 production in splenocytes, and cd86 enhanced disease severity by upregulating il-17 production . Also, high cell surface expression of cd86 was observed in patients with ibd and in mice with cd4 t - cell - induced colitis . These findings lead us to examine the effect of st28 on the expression of cd86 . The mrna expressions of cd86 and cd11c were drastically upregulated in lpls from dss colitis mice, but they were downregulated by the oral administration of st28 (supplementary figure 1). St28 also significantly decreased the percentage of cd86cells in matured bmdc (figure 5). Collectively, it is highly probable that st28 ameliorated dss induced colitis by suppressing cd86 dc, which needs to be clarified in detail . It has been described that il-17 is involved in the pathogenesis of dss - induced colitis in mice, and il-17 receptor signaling plays a critical role in the development of trinitrobenzenesulfonic acid - induced acute colitis in mice, yet another report has suggested that il-17 might offer an inhibitory role in the development of dss - induced colitis . O'connor et al . Also demonstrated the data on a protective function for il-17 in t - cell - mediated intestinal inflammation . For example, buonocore et al . Have recently suggested that a novel innate lymphoid cell population accumulates in the inflamed colon and induces il-17 and ifn-. Further investigations on th17/th1 in the intestinal inflammation are required to clarify the beneficial functions of st28 on th17-mediated diseases . In conclusion, we have demonstrated that st28 ameliorates intestinal inflammation in dss - induced colitis mice at least partially through suppression of inflammatory th17 cells . The use of probiotic bacteria in ibd therapy has been investigated in a number of clinical studies . Although the efficacy of this treatment approach was demonstrated for ulcerative colitis, the results for crohn's disease are not yet clear owing to conflicting results and a paucity of trials . In any case, the majority of the demonstrated curative and palliative effects of probiotic bacteria appear to be mediated by modulation of the intestinal immune system . Clinical approaches using probiotics are appealing due to a lack of toxicity and patient desire for the use of natural physiological approaches to treating disease . Since s. thermophilus has a long history of being safely consumed in yogurt, its further application to the treatment and/or prevention of th17-mediated diseases such as ibd
The accord trial design and patient population have been described elsewhere (10). In brief, 10,251 subjects with type 2 diabetes at high - risk for cardiovascular disease (cvd) events were enrolled in 77 clinical centers across the u.s . And canada and randomly assigned to receive either comprehensive intensive glycemia therapy (int) targeting a a1c level <6% or to receive standard glycemia therapy (std) targeting an a1c level of 77.9% . The mean duration of the trial was expected to be 5 years . The int arm of the study was discontinued after 3.5 years because of excess mortality in the int group, and all participants were transitioned to the std protocol (3). The accord study and the consent forms were approved by institutional review boards at all participating institutions . Baseline clinical and laboratory investigations were obtained in the morning after an overnight fast as described (10). We used baseline standard 12-lead digitized electrocardiograms (ecgs), recorded over 10 consecutive seconds with the patient resting supine after an overnight fast (ge mac 1200 electrocardiograph system). The ecg recordings were transferred by analog phone line to the reading center and were analyzed and reviewed to determine their technical quality . Recordings that were missing (1,034) or demonstrated poor quality (362) and recordings from those with pacemakers (65), atrial fibrillation (108), premature beats / other arrhythmias (542), and atrioventricular conduction abnormalities (5) were excluded from these analyses, leaving a cohort of 8,135 participants assessed for hrv . The following time domain markers of cardiac autonomic tone were computed: heart rate and the sd of normally conducted r - r intervals (sdnn). From simultaneous lead recordings, qt intervals were measured, and the qt index (qti) was calculated as observed / predicted qt duration where predicted value was based on bazett's correction (qtc = qt / r r). Resting heart rate reflects both overall autonomic function and cardiorespiratory fitness (11), sdnn represents joint sympathetic / parasympathetic modulation of heart rate in the time domain (11,12), qt duration represents the time between the onset of ventricular activation and the end of repolarization, a process controlled in part by sympathetic input (13,14). Impaired hrv is an easily measured sensitive marker of can that may occur early in the course of diabetes (15). Lower hrv and higher resting heart rate and qti indicate poorer autonomic function (11). These measures are a reliable estimate of can and are recommended for use in large population studies (12). Dpn was documented by any pedal amputation or a score> 2 on the clinical examination portion of the michigan neuropathy screening instrument, a validated tool for assessing dpn that evaluates abnormalities in foot appearance, ankle reflexes, and vibration at the great toe of both feet (16). The following composite measures were computed to document the presence of can: 1) can1 defined as the lowest quartile of sdnn (<7.815 ms) and the highest quartile of qti (> 104.32%); 2) can2 as the lowest quartile of sdnn and the highest quartiles of qti and resting heart rate; and 3) can3 as the lowest quartile of sdnn and the highest quartiles of qti and heart rate, in the presence of dpn . Our rationale for using these composite prespecified definitions of can was that combined abnormalities in hrv and qt interval have demonstrated stronger predictive value for mortality than either abnormality alone in patients with diabetes (13,14). The presence of dpn was included in one of the composite measures because of prior evidence linking excess mortality to the presence of dpn (6,7). The outcomes were all - cause and cvd mortality (adjudicated by a blinded panel using predefined adjudication processes). Death from cvd included deaths from myocardial infarction, heart failure, arrhythmia, invasive cardiovascular interventions, cardiovascular causes after noncardiovascular surgery, stroke, unexpected death presumed to be from ischemic cvd occurring within 24 h after the onset of symptoms, and death from other vascular diseases . We hypothesized that the increased all - cause and cvd mortality with int observed in the accord trial was due to a higher risk of mortality with int in the subset of individuals with baseline can . We also assessed whether can was related to mortality risk independent of glycemia treatment and compared the strength of these relationships across the prespecified definitions of can . These analyses are based on data collected on participants at the time of randomization and all - cause and cvd mortality data submitted to the coordinating center through 10 december 2007, the cutoff date used by the data and safety monitoring board to make its recommendation to stop intensive glycemia treatment . Baseline characteristics were compared between excluded and included participants and between can - positive and can - negative groups using and two sample t tests . Analysis of all - cause and cvd mortality was performed with time - to - event methods according to the intention - to - treat principle . Risk of these outcomes was evaluated through the use of hazard ratios (hr) and 95% cis . Two - sided p values were obtained from wald tests derived from cox proportional hazards regression analysis . For both outcomes, we examined minimally adjusted models stratified for treatment allocation and history of cvd . We also fit fully adjusted models containing treatment allocation, history of cvd, and the following prespecified baseline covariates: age, sex, ethnicity, diabetes duration, a1c, bmi, systolic blood pressure (sbp) and diastolic blood pressure (dbp), ldl cholesterol, triglycerides, urinary microalbumin - to - creatinine ratio, and use of thiazolidinediones (tzds), insulin, -blockers, ace inhibitors / angiotensin receptor blockers, statins, alcohol, and cigarettes . Participants with missing covariates (n = 235) were excluded from fully adjusted analyses . Because results were similar between models, only the fully adjusted models are presented here . We assessed the consistency of the effect of glycemia treatment allocation on all - cause and cvd mortality among prespecified subgroups using statistical tests of interaction between treatment allocation and each subgroup within the cox model . Event rates are expressed as the percentage of events per follow - up year, taking into account censoring of follow - up data, with 95% poisson cis calculated using large sample methods . We have also examined can effects after adding events of severe hypoglycemia requiring medical assistance to the cox regression models as a time - dependent covariate . We used baseline standard 12-lead digitized electrocardiograms (ecgs), recorded over 10 consecutive seconds with the patient resting supine after an overnight fast (ge mac 1200 electrocardiograph system). The ecg recordings were transferred by analog phone line to the reading center and were analyzed and reviewed to determine their technical quality . Recordings that were missing (1,034) or demonstrated poor quality (362) and recordings from those with pacemakers (65), atrial fibrillation (108), premature beats / other arrhythmias (542), and atrioventricular conduction abnormalities (5) were excluded from these analyses, leaving a cohort of 8,135 participants assessed for hrv . The following time domain markers of cardiac autonomic tone were computed: heart rate and the sd of normally conducted r - r intervals (sdnn). From simultaneous lead recordings, qt intervals were measured, and the qt index (qti) was calculated as observed / predicted qt duration where predicted value was based on bazett's correction (qtc = qt / r r). Resting heart rate reflects both overall autonomic function and cardiorespiratory fitness (11), sdnn represents joint sympathetic / parasympathetic modulation of heart rate in the time domain (11,12), qt duration represents the time between the onset of ventricular activation and the end of repolarization, a process controlled in part by sympathetic input (13,14). Impaired hrv is an easily measured sensitive marker of can that may occur early in the course of diabetes (15). Lower hrv and higher resting heart rate and qti indicate poorer autonomic function (11). These measures are a reliable estimate of can and are recommended for use in large population studies (12). Dpn was documented by any pedal amputation or a score> 2 on the clinical examination portion of the michigan neuropathy screening instrument, a validated tool for assessing dpn that evaluates abnormalities in foot appearance, ankle reflexes, and vibration at the great toe of both feet (16). The following composite measures were computed to document the presence of can: 1) can1 defined as the lowest quartile of sdnn (<7.815 ms) and the highest quartile of qti (> 104.32%); 2) can2 as the lowest quartile of sdnn and the highest quartiles of qti and resting heart rate; and 3) can3 as the lowest quartile of sdnn and the highest quartiles of qti and heart rate, in the presence of dpn . Our rationale for using these composite prespecified definitions of can was that combined abnormalities in hrv and qt interval have demonstrated stronger predictive value for mortality than either abnormality alone in patients with diabetes (13,14). The presence of dpn was included in one of the composite measures because of prior evidence linking excess mortality to the presence of dpn (6,7). The outcomes were all - cause and cvd mortality (adjudicated by a blinded panel using predefined adjudication processes). Death from cvd included deaths from myocardial infarction, heart failure, arrhythmia, invasive cardiovascular interventions, cardiovascular causes after noncardiovascular surgery, stroke, unexpected death presumed to be from ischemic cvd occurring within 24 h after the onset of symptoms, and death from other vascular diseases . We hypothesized that the increased all - cause and cvd mortality with int observed in the accord trial was due to a higher risk of mortality with int in the subset of individuals with baseline can . We also assessed whether can was related to mortality risk independent of glycemia treatment and compared the strength of these relationships across the prespecified definitions of can . These analyses are based on data collected on participants at the time of randomization and all - cause and cvd mortality data submitted to the coordinating center through 10 december 2007, the cutoff date used by the data and safety monitoring board to make its recommendation to stop intensive glycemia treatment . Baseline characteristics were compared between excluded and included participants and between can - positive and can - negative groups using and two sample t tests . Analysis of all - cause and cvd mortality was performed with time - to - event methods according to the intention - to - treat principle . Risk of these outcomes was evaluated through the use of hazard ratios (hr) and 95% cis . Two - sided p values were obtained from wald tests derived from cox proportional hazards regression analysis . For both outcomes, we examined minimally adjusted models stratified for treatment allocation and history of cvd . We also fit fully adjusted models containing treatment allocation, history of cvd, and the following prespecified baseline covariates: age, sex, ethnicity, diabetes duration, a1c, bmi, systolic blood pressure (sbp) and diastolic blood pressure (dbp), ldl cholesterol, triglycerides, urinary microalbumin - to - creatinine ratio, and use of thiazolidinediones (tzds), insulin, -blockers, ace inhibitors / angiotensin receptor blockers, statins, alcohol, and cigarettes . Participants with missing covariates (n = 235) were excluded from fully adjusted analyses . Because results were similar between models, only the fully adjusted models are presented here . We assessed the consistency of the effect of glycemia treatment allocation on all - cause and cvd mortality among prespecified subgroups using statistical tests of interaction between treatment allocation and each subgroup within the cox model . Event rates are expressed as the percentage of events per follow - up year, taking into account censoring of follow - up data, with 95% poisson cis calculated using large sample methods . We have also examined can effects after adding events of severe hypoglycemia requiring medical assistance to the cox regression models as a time - dependent covariate . For the current analyses, we included 8,135 accord trial participants with complete data, including 4,050 (79%) randomly assigned to the int arm and 4,085 (80%) to the std arm (supplementary fig . The main reason for missing data was failure of the site to capture and transmit an electronic ecg record (n = 1,034). Other reasons for exclusion, including arrhythmias (n = 542), were described in the research design and methods . The baseline characteristics of these participants, comparing those with (included) and without (excluded) available can measures are shown in supplementary table a1 (available in an online appendix). This was a cohort with long diabetes duration, elevated a1c levels, and multiple associated cvd risk factors as prespecified by the accord trial design . Participants excluded from this analysis due to inadequate ecg data were older (p <0.0001), were more likely to have had a previous cardiovascular event (p = 0.0002), and had a longer diabetes duration (p = 0.045). Sex, triglyceride levels, and -blocker use also showed significant differences (supplementary table a1). Participants with can at baseline (table 1) consistently had higher a1c, bmi, dbp, and triglycerides (p 0.01 in all cases) and were more likely to use insulin and to be female (p <0.01 for all three definitions of can) than those without can . Minority status, prior cvd, diabetes duration, sbp, urinary albumin - to - creatinine ratio, smoking history, and tzd, statin, and -blocker use showed inconsistent differences across the three definitions of can . Baseline characteristics in the analyzed cohort expressed as a function of can values shown are percentages for dichotomous variables and means (standard deviations) for continuous measures . Hba1c: hemoglobin a1c, bmi: body mass index, sbp: systolic blood pressure, dpb: diastolic blood pressure, dpn: diabetic peripheral neuropathy diagnosed by any pedal amputation or score> 2 on the michigan neuropathy screening instrument, ldlc: low - density lipoprotein cholesterol, aceis: angiotensin- converting enzyme inhibitors, arbs: angiotensin receptor blockers, tzd: thiazolidinediones . Can1 was defined as the lowest quartile of sdnn and the highest quartile of qti; can2 as the lowest quartile of sdnn, the highest quartile of qti and the highest quartile of heart rate; can3 as the lowest quartile of sdnn and the highest quartiles of qti and heart rate in the presence of dpn . During a mean follow - up of 3.5 years, there were 329 deaths from all causes in this sample of 8,351 participants . In unadjusted analyses, there was a significant increase in all - cause mortality (hr 1.61 [1.142.27], p = 0.007 for can1, 2.22 [1.453.39], p = 0.0002 for can2, and 2.72 [1.564.74], p = 0.0004 for can3) (fig . A2, available in an online appendix) and in cvd mortality (1.93 [1.223.07], p = 0.005 for can1, 2.55 [1.414.60, p = 0.002 for can2, and 3.39 [1.597.26], p = 0.0002 for can3) compared with those without can . Table 2 shows fully adjusted hrs (95% ci) for all - cause and cvd mortality for participants with can1, can2, and can3 compared with participants without can . All - cause mortality remained significantly higher in participants with can after adjustment for treatment arm allocation, cvd history, and all other covariates listed in research design and methods (1.55 [1.092.21], p = 0.016 for can1, 2.14 [1.373.37], p = 0.0009 for can2, and 2.07 [1.143.76], p = 0.02 for can3). Similar results were observed for cvd mortality (1.94 [1.203.12], p = 0.007 for can1, 2.62 [1.44.91], p = 0.003 for can2, and 2.95 [1.336.53], p = 0.008 for can3) (table 2). Hr (95% ci) for all - cause and cvd mortality in participants with can compared with participants without can * adjusted for treatment allocation, cvd history, and other prespecified covariates including baseline age, sex, ethnicity, diabetes duration, a1c, bmi, sbp and dbp, ldl cholesterol, triglycerides, microalbumin - to - creatinine ratio, and use of tzds, insulin, -blockers, ace inhibitors / angiotensin - receptor blockers, statins, alcohol, and cigarettes . After adjustment for covariates, the overall hr for all - cause mortality for int versus std arm in the 8,135 participants analyzed herein was 1.17(95% ci 0.941.46, p = 0.17). The hr for mortality in the int versus std arm for participants with missing ecg data (1.25 [0.881.78]) was not significantly different from that of participants included in this analysis (pinteraction = 0.77), although participants with missing ecg data were more likely to die (1.72 vs. 1.15% per year for included participants) regardless of glycemia treatment group . Figure 1a shows the event rate for all - cause mortality by treatment group, int versus std, as a function of can in the analyzed cohort, using the three prespecified can definitions . After adjustment for all covariates, there was no significant difference in all - cause mortality among any of the subgroups defined by the presence of can1, can2, and can3 (pheterogeneity> 0.7 for all). A: effects of can and glycemia intervention on all - cause mortality: hrs adjusted for treatment allocation and baseline age, sex, ethnicity, diabetes duration, a1c, bmi, sbp and dbp, ldl cholesterol, triglycerides, microalbumin - to - creatinine ratio, cvd history, and use of tzds, insulin, -blockers, ace inhibitors / angiotensin receptor blockers, statins, alcohol, and cigarettes . B: effects of can and glycemia intervention on cvd mortality: hrs adjusted for treatment allocation and baseline age, sex, ethnicity, diabetes duration, a1c, bmi, sbp and dbp, ldl cholesterol, triglycerides, microalbumin - to - creatinine ratio, cvd history, and use of tzds, insulin, ace inhibitors / angiotensin - receptor blockers, -blockers, statins, alcohol, and cigarettes . Can1 was defined as the lowest quartile of sdnn, and the highest quartile of qti, can2 as the lowest quartile of sdnn, the highest quartile of qti, and the highest quartile of heart rate, and can3 as the lowest quartile of sdnn and the highest quartiles of qti and heart rate in the presence of dpn . The overall hr for cvd mortality (int versus std) in the subgroup of individuals analyzed herein was 1.30 (95% ci 0.931.82). As with all - cause mortality, in fully adjusted analyses, the hr for the treatment difference in cvd mortality was not affected significantly by can status for any of the definitions examined (fig . We have also adjusted for the presence of severe hypoglycemia during the trial in the individuals with baseline can . The lack of a significant effect of can on all - cause mortality in the int arm compared with the std arm persisted after controlling for the presence of severe hypoglycemia in the model (pinteraction> 0.25 for all three definitions of can). During a mean follow - up of 3.5 years, there were 329 deaths from all causes in this sample of 8,351 participants . In unadjusted analyses, there was a significant increase in all - cause mortality (hr 1.61 [1.142.27], p = 0.007 for can1, 2.22 [1.453.39], p = 0.0002 for can2, and 2.72 [1.564.74], p = 0.0004 for can3) (fig . A2, available in an online appendix) and in cvd mortality (1.93 [1.223.07], p = 0.005 for can1, 2.55 [1.414.60, p = 0.002 for can2, and 3.39 [1.597.26], p = 0.0002 for can3) compared with those without can . Table 2 shows fully adjusted hrs (95% ci) for all - cause and cvd mortality for participants with can1, can2, and can3 compared with participants without can . All - cause mortality remained significantly higher in participants with can after adjustment for treatment arm allocation, cvd history, and all other covariates listed in research design and methods (1.55 [1.092.21], p = 0.016 for can1, 2.14 [1.373.37], p = 0.0009 for can2, and 2.07 [1.143.76], p = 0.02 for can3). Similar results were observed for cvd mortality (1.94 [1.203.12], p = 0.007 for can1, 2.62 [1.44.91], p = 0.003 for can2, and 2.95 [1.336.53], p = 0.008 for can3) (table 2). Hr (95% ci) for all - cause and cvd mortality in participants with can compared with participants without can * adjusted for treatment allocation, cvd history, and other prespecified covariates including baseline age, sex, ethnicity, diabetes duration, a1c, bmi, sbp and dbp, ldl cholesterol, triglycerides, microalbumin - to - creatinine ratio, and use of tzds, insulin, -blockers, ace inhibitors / angiotensin - receptor blockers, statins, alcohol, and cigarettes . After adjustment for covariates, the overall hr for all - cause mortality for int versus std arm in the 8,135 participants analyzed herein was 1.17(95% ci 0.941.46, p = 0.17). The hr for mortality in the int versus std arm for participants with missing ecg data (1.25 [0.881.78]) was not significantly different from that of participants included in this analysis (pinteraction = 0.77), although participants with missing ecg data were more likely to die (1.72 vs. 1.15% per year for included participants) regardless of glycemia treatment group . Figure 1a shows the event rate for all - cause mortality by treatment group, int versus std, as a function of can in the analyzed cohort, using the three prespecified can definitions . After adjustment for all covariates, there was no significant difference in all - cause mortality among any of the subgroups defined by the presence of can1, can2, and can3 (pheterogeneity> 0.7 for all). A: effects of can and glycemia intervention on all - cause mortality: hrs adjusted for treatment allocation and baseline age, sex, ethnicity, diabetes duration, a1c, bmi, sbp and dbp, ldl cholesterol, triglycerides, microalbumin - to - creatinine ratio, cvd history, and use of tzds, insulin, -blockers, ace inhibitors / angiotensin receptor blockers, statins, alcohol, and cigarettes . B: effects of can and glycemia intervention on cvd mortality: hrs adjusted for treatment allocation and baseline age, sex, ethnicity, diabetes duration, a1c, bmi, sbp and dbp, ldl cholesterol, triglycerides, microalbumin - to - creatinine ratio, cvd history, and use of tzds, insulin, ace inhibitors / angiotensin - receptor blockers, -blockers, statins, alcohol, and cigarettes . Can1 was defined as the lowest quartile of sdnn, and the highest quartile of qti, can2 as the lowest quartile of sdnn, the highest quartile of qti, and the highest quartile of heart rate, and can3 as the lowest quartile of sdnn and the highest quartiles of qti and heart rate in the presence of dpn . The overall hr for cvd mortality (int versus std) in the subgroup of individuals analyzed herein was 1.30 (95% ci 0.931.82). As with all - cause mortality, in fully adjusted analyses, the hr for the treatment difference in cvd mortality was not affected significantly by can status for any of the definitions examined (fig . We have also adjusted for the presence of severe hypoglycemia during the trial in the individuals with baseline can . The lack of a significant effect of can on all - cause mortality in the int arm compared with the std arm persisted after controlling for the presence of severe hypoglycemia in the model (pinteraction> 0.25 for all three definitions of can). These data confirm in one of the largest and most carefully characterized cohorts of patients with type 2 diabetes that the presence of can strongly predicts all - cause and cvd mortality independently of baseline cvd, diabetes duration, and multiple other important cvd risk factors . However, the increased risk of either all - cause or cvd mortality in individuals with can compared with those without can at baseline was similar in the int versus the std glycemia treatment group . Associations between measures of can and mortality, including sudden death, have been described previously (5,17,18). In a recent large meta - analysis, maser et al . (5) reported that the presence of can was associated with a greater than threefold increase in mortality and sudden death . Silent ischemic heart disease or cardiac arrhythmias have both been invoked as contributors to sudden death . In the detection of ischemia in asymptomatic diabetics (diad) study of 1,123 patients with type 2 diabetes, can was a strong predictor of silent ischemia and subsequent cardiovascular events (4). Because can is associated with multiple factors including duration of diabetes, severity of hyperglycemia, and the presence of coronary artery disease, the exact contribution of can to the increased mortality risk has been difficult to quantify in prior studies . The present analysis in the accord cohort provided a unique opportunity to evaluate and confirm the independent effect of can on all - cause and cvd mortality . Study strengths were the large sample size of high - risk participants analyzed, the balanced sex randomization, diverse ethnic participation, high rate of follow - up, and rigorous cvd end point adjudication procedures . These data document that the presence of can strongly predicts all - cause and cvd mortality independently of multiple important cvd risk factors in this high - risk cohort with type 2 diabetes . These observations suggest that documenting can in individuals with type 2 diabetes identifies a subset at higher risk for cvd events and that can may explain at least in part the increased risk of cvd events observed in type 2 diabetes . Furthermore, these data indicate that can should be considered in models of cvd and mortality risk in type 2 diabetes cohorts . Although clinical symptoms of autonomic dysfunction usually occur late in the course of diabetes, subclinical can, manifested as impaired hrv, may be detected within 1 year of diagnosis in type 2 diabetes and within 2 years of diagnosis in type 1 diabetes (15). Traditionally, the diagnosis of can involves a number of tests, including the r - r response to deep breathing, postural changes and during valsalva maneuver, or 24-h ecg recordings, which may be cumbersome to perform uniformly, especially in large multicenter trials such as accord . This study demonstrates that using a combination of hrv and qti measurements derived from a standard 10-s ecg can identify subsets of patients at increased mortality risk independently of traditional cvd risk factors . Hrv and qt abnormalities have different origins, as reflected by a weak correlation between the two parameters demonstrated by this study (data not shown) and by others (13). The qt interval abnormalities have a different pathophysiological background, representing the consequences of sympathetic tone on cardiac depolarization and repolarization (13). The accord findings in type 2 diabetes are in line with another recent report in patients with type 1 diabetes showing an increased mortality risk with combined abnormalities in hrv and qt interval (13). This finding has important implications because these measures obtained from a standard ecg can be used as a noninvasive and objective method for assessing can in other large trials as well as in clinical practice . Despite the significant increase in the mortality risk in all subgroups of participants with can, we did not find that the presence of can at baseline contributed significantly to the increased mortality observed with the intensive versus standard treatment of glycemia in this cohort . Control of blood glucose is a cornerstone of diabetes management because more intensive glycemic control decreases the incidence and progression of diabetic microvascular (1,2) and, in some studies, macrovascular complications (19). The reported excess mortality in the intensive arm of the accord trial (3) has led to controversy about implementation of intensive glucose control in patients with type 2 diabetes although two other major trials, action in diabetes and vascular disease: preterax and diamicron modified release controlled evaluation (advance) and veterans affairs diabetes trial (vadt), reported no increase in mortality with intensive treatment compared with standard treatment in type 2 diabetes (20,21). Furthermore, the long - term follow - up of the ukpds cohort reported risk reductions for myocardial infarction and death from any cause associated with intensive glucose control (22). One of the most feared consequences of rigorous glycemic control is an increased incidence of hypoglycemia (1,2). Prior and more recent reports have shown strong associations between hypoglycemia and increased mortality (23,24). Hypoglycemia can impair hormonal and autonomic responses to subsequent hypoglycemia (8), and hypoglycemia may promote a reduced threshold for malignant arrhythmias and subsequent sudden cardiac death . Likewise, a recent study reported that exposure to hypoglycemia leads to impaired cardiovascular autonomic function in healthy volunteers (9). In the accord trial, even though participants with severe hypoglycemia were at higher risk of death, this risk was not explained by can; after adjusting for the effects of postrandomization hypoglycemia, we could not document that can was an independent determinant of the higher mortality associated with intensive glycemia treatment . Therefore, our data imply that type 2 diabetic patients with can are not necessarily at increased risk with intensive versus standard glucose management . Hrv measures were limited by the short duration of the standard ecg recordings and by the absence of control for respiration . The prevalence of can may be higher in the excluded subset because of their older age, longer diabetes duration, and higher prevalence of cvd . Although the large sample of participants analyzed and their uniform characterization probably provides a reasonable estimate of can prevalence, it is possible that we underestimated the true effect of can on mortality in this cohort . There was limited statistical power to detect an interaction between subgroups defined by the presence or absence of can because the study was not designed for this purpose . There remains a theoretical possibility that can was worsened by intensive treatment during the trial and did account for the increased mortality with intensive treatment . We believe that this possibility is unlikely, considering that recent evidence showed a beneficial effect of intensive glucose treatment on can in type 1 diabetes (25). Last, because the accord trial was not designed to ascertain interactions between can and a rapid lowering of a1c or / and hypoglycemia and considering the post hoc nature of our analysis, we cannot draw more specific conclusions regarding the precise mechanisms involved by which can increases mortality . Incomplete understanding of the role of can in the pathogenesis of cvd and the precise mechanisms underlying its associations with mortality are areas deserving further research . In summary, although can was associated with increased all - cause and cvd mortality in the accord trial, these analyses indicate that among the subset with can, assignment to intensive compared with standard glycemia treatment was associated with similar mortality outcomes . Considering the vast array of variables that could have contributed to the increased mortality in the int arm, it will be difficult and perhaps impossible to sort out the true determinants of this outcome . However, the presence of can, defined by simple, easily derived resting ecg measures, identified a subset of type 2 diabetic patients at higher all - cause and cvd mortality risk independent of multiple traditional cvd risk factors.
After obtaining approval from institutional ethics committee, a cross - sectional questionnaire - based study was undertaken from august 25, 2014, to september 25, 2014, including 200 pharmacies in various residential (r) and commercial (c) areas of bengaluru . Two hundred and twenty - five pharmacies were screened for the study by convenient sampling . A total of 25 pharmacies (r-12, c-13) were not included as they were either not willing to participate or were closed at the time of visit . A total of 200 pharmacies, 100 each in various residential and commercial areas of bengaluru willing to participate were selected for the study . It was pretested on twenty pharmacies, and cronbach's alpha of 0.71 was obtained . The questionnaire had two sections, practice, and knowledge with 14 and 4 questions, respectively . Informed consent was taken in an attached consent form describing the objectives of the study and participants were assured of confidentiality to elicit an unbiased response . The prevalidated semi - structured questionnaire was administered to the pharmacist or to the person - in - charge in case of nonavailability or absence of the chief pharmacist by the investigators . The data collected in the form of completed questionnaires was categorized, coded, and analyzed . Data were expressed as percentages / proportions, tests of proportions were done with chi - square wherever deemed necessary, and p <0.05 was considered statistically significant . The data collected in the form of completed questionnaires was categorized, coded, and analyzed . Data were expressed as percentages / proportions, tests of proportions were done with chi - square wherever deemed necessary, and p <0.05 was considered statistically significant . Dispensing without prescription at pharmacies was 45% of the total dispensing encounters, with analgesics (90%) being the most commonly dispensed drugs without prescription followed by antipyretics (68%), antihistaminics (49%), and antacids (46%) [figure 1]. Classes of drugs dispensed without prescription dispensing without prescription was higher among pharmacies located in residential areas (46.64%) as compared to commercial areas (43.64%), which was statistically significant, = 15.2, p <0.001, and df = 1 [table 1]. Dispensing encounters without prescription in residential and commercial areas of bengaluru (n=200) only about 19% of the pharmacists checked for all the particulars (patient particulars, date of prescription, drug name, drug dose and frequency, signature of the doctor, and register number of the doctor) in the prescription before dispensing the drugs . Almost all of them (98%) maintained an inventory, computerized (41%), and manual (57%) while 2% did not [table 2]. Dispensing practices at pharmacies in bengaluru (n=200) although 97% (194) of the pharmacies had a refrigerator, 31% (66) of these did not have power back - up . Sixty - two percent (124) checked the pharmacy for drugs nearing expiry on a monthly basis . The pharmacists advising patients about the various aspects of medication use voluntarily, on patient request or no advice given is shown in figure 2 . Medication counseling in pharmacies in bengaluru (n = 200) only 50.5% were aware of schedule h. about 60.5% did not know that prescription in duplicate and stringent dispensing records have to be maintained while dispensing schedule x and h1 drugs [table 3]. Among who knew, 22% thought that they have to be maintained for schedule x drugs only, 4.5% for schedule h1 drugs only, and very few (13%) knew that these have to be maintained for both . Knowledge of the pharmacists in bengaluru (n=200) the majority of them did not know about look alike sound alike (lasa) drugs and generic substitution (88% and 73.5%, respectively, [table 3]). Dispensing without prescription at pharmacies was 45% of the total dispensing encounters, with analgesics (90%) being the most commonly dispensed drugs without prescription followed by antipyretics (68%), antihistaminics (49%), and antacids (46%) [figure 1]. Classes of drugs dispensed without prescription dispensing without prescription was higher among pharmacies located in residential areas (46.64%) as compared to commercial areas (43.64%), which was statistically significant, = 15.2, p <0.001, and df = 1 [table 1]. Dispensing encounters without prescription in residential and commercial areas of bengaluru (n=200) only about 19% of the pharmacists checked for all the particulars (patient particulars, date of prescription, drug name, drug dose and frequency, signature of the doctor, and register number of the doctor) in the prescription before dispensing the drugs . Almost all of them (98%) maintained an inventory, computerized (41%), and manual (57%) while 2% did not [table 2]. Although 97% (194) of the pharmacies had a refrigerator, 31% (66) of these did not have power back - up . Sixty - two percent (124) checked the pharmacy for drugs nearing expiry on a monthly basis . The pharmacists advising patients about the various aspects of medication use voluntarily, on patient request or no advice given is shown in figure 2 . Only 50.5% were aware of schedule h. about 60.5% did not know that prescription in duplicate and stringent dispensing records have to be maintained while dispensing schedule x and h1 drugs [table 3]. Among who knew, 22% thought that they have to be maintained for schedule x drugs only, 4.5% for schedule h1 drugs only, and very few (13%) knew that these have to be maintained for both . Knowledge of the pharmacists in bengaluru (n=200) the majority of them did not know about look alike sound alike (lasa) drugs and generic substitution (88% and 73.5%, respectively, [table 3]). India, with an abysmal doctor - population ratio of 1:1218 (minimum recommended by the who being 1:1000) is struggling to cope with the health - care needs of a growing population . People rely on alternative health - care professionals such as chemists, traditional medicine practitioners, and faith healers directly to meet their health needs and to reduce personal costs . Hence, pharmacists are one of the crucial focal points for health care in the community . They have a large outreach to the public as the number of people visiting pharmacy outlets is possibly greater than other health - care units . This although provides pharmacists with an opportunity for greater involvement in the health - care system, their fragmentary knowledge and improper dispensing practices may have undesired effects . Indiscriminate dispensing of antibiotics, sub - therapeutic quantities of antibacterials, inappropriate combination of drugs, and injectables under suboptimal conditions have also been reported in the previous studies . Hence, this study was undertaken to evaluate the drug dispensing practices at pharmacies and the knowledge of pharmacists in bengaluru . The most important finding in this study was a high proportion (45%) of dispensing encounters without prescription . This dispensing pattern is similar to another study conducted in tamil nadu, india, where 58% of the pharmacists dispensed drugs without prescription but better than practices in other countries such as egypt (72%), tanzania (77%), and vietnam (99%) where higher rates of dispensing without prescriptions were documented . While lack of universal access to health care could be a major factor, convenience, reduced time to procure medicines, and cost consumption furthermore, a higher proportion of dispensing encounters without prescription in the residential areas was observed as compared to the commercial areas . Apart from the reasons cited above, this could be due to a farther location of health - care facilities . Analgesics, antipyretics, antacids, cold and cough remedies, vitamins, nutrition supplements, and antibiotics constituted commonly dispensed drugs without prescription, in that order . Similar patterns were also seen in the egypt and tanzania studies while in the vietnam study and another study conducted in india, antibiotics, vitamins, and nutrition supplements were most commonly dispensed . In another study conducted in saudi arabia, almost all the pharmacists dispensed antibiotics without prescription . Analgesics and antipyretics are the most commonly used drugs for relief of fever, common aches, and pains . Although aspirin and paracetamol can be dispensed without prescription, they can lead to adverse outcomes under certain conditions and hence, there is a need for them to be dispensed at right doses with proper counseling of end users . Other analgesics such as ibuprofen and diclofenac are prescription drugs . While antacids are among over - the - counter (otc) drugs, most of the cold and cough remedies and all the antibiotics are schedule h drugs . Some authors have also questioned the appropriateness of widespread use of cold and cough remedies, some vitamin preparations and their irrational combinations . This scenario prevails even in the face of existing regulations as most pharmacies do not adhere to them . With bias being a known caveat of questionnaire surveys the actual situation could still be worse . In the uk, a prescription is valid for 6 months from the date of prescription, unless the medicine prescribed contains a controlled drug, in which case it is valid antibiotics and other drugs as otc medications . Under the federal law, prescriptions for schedule iii substances expire after 6 months and 5 refills are allowed within the 6-month period, whereas prescriptions for schedule ii controlled substances cannot be refilled . In this study, it was found that 77% of the pharmacists dispensed drugs for older prescriptions also . This could be due to no specific regulations in place regarding duration of validity of a prescription in our country . Although antibiotics are an essential tool for modern medicine in combating most of the infections, antibiotic resistance remains to be a worldwide problem . While misuse of antibiotics is a key factor contributing to antibiotic resistance, failure to take an antibiotic as prescribed adds no less . Only one - third of the pharmacists insisted on dispensing full course of the antibiotics prescribed and this improper dispensing can contribute to the growing health hazard of antibiotic resistance, which adds to health - care costs and the gpp guidelines by the indian pharmaceutical association require the pharmacist, on receiving the prescription, to confirm the identity of the client, review the prescription for completeness, and check for correctness of the prescribed medications . This is to ensure right dispensing and thus avoid contributing to medication errors at large, the stakes of which are high . However, in this study, 81% of the pharmacists said they would not check for all the particulars ., all of them did check for expiry of the medications before dispensing . Maintaining an inventory, especially a computerized one, in this study, we found that 41% of the pharmacies had a computerized inventory, 57% had manual inventory, and 2% did not maintain one . According to the gpp guidelines pharmacies should preferably be equipped with computers for inventory management . Herbal medicines have emerged as a common choice of therapy for self - care as they are perceived to be free of side effects . This practice is also enforced by irrational claims and aggressive promotions by manufacturers and media, peer views, and lack of well - documented unbiased studies on herbal drug use in disease conditions . A study conducted in lagos, nigeria, showed that community pharmacies frequently supplied herbal medicines . Fifty - seven percent of the pharmacies included in this study also dispensed herbal medicines . In the uk, human medicines regulations 2012 allows only long - established and quality - controlled herbal medicines to be sold . It has also implemented the traditional herbal medicines registration scheme for authorizing the marketing of herbal products . Medication counseling is the duty of the modern community pharmacists through which they can significantly contribute to medication safety and patient compliance . The gpp guidelines require the pharmacist to provide professional counseling with regard to the use of medicines, their side effects, and precautions, if any . Recognizing the importance of medication counseling, the north carolina board of pharmacy since 1993 made a rule to offer patient counseling on all new prescriptions . However, a low rate of voluntary medication counseling by the pharmacists was noted in this study . This could possibly be improved by modifying the attitude of pharmacists through educational interventions, incentives, and regulations . Drug storage practices, essential for maintaining efficacy of some drugs, were not in accordance with the gpp guidelines which states that pharmacies should be equipped with refrigerated storage facilities validated from time to time for products requiring storage at cold temperature . Also having a refrigerator in the pharmacy is a licensing requirement . About 3% of the pharmacies did not have a refrigerator and about 31% of the pharmacies which had a refrigerator did not have power back - up for the same . This scenario is better than another study conducted in pakistan where the majority of the pharmacies did not have an alternative power supply for the refrigerator . Generic substitution is the mutual substitution of medicinal products having the same active ingredient, in the same dose and pharmaceutical form . This concept especially becomes important in case of biologicals and drugs with narrow therapeutic index . Disasters such as breakthrough seizures in epileptics due to disregard to the concept of generic substitution are well known . Nevertheless, unaware of its implications, 56% said they would dispense an alternative brand of the same drug in case of nonavailability of the brand prescribed, irrespective of which drug it was . Although guidelines for the same are in place in few countries, lack of such robust system in our country and unawareness can compound the problems faced in therapeutics . Indian pharmaceutical industry is now the third - largest in the world with more than 20,000 registered units . The potential for error due to confusing names among health - care personnel has also significantly increased . Pharmacists should, therefore, be cautious while dispensing such drugs and take steps to avoid dispensing errors such as sticking warning labels and avoid keeping lasa drugs in close proximity . However, in this study, the majority (88%) did not know about lasa drugs . In all the pharmacies drugs rampant use of antibiotics and other drugs as otc medications can lead to serious adverse drug reactions and antibiotic resistance . Hence, schedule h and schedule x of the drugs and cosmetic rules, 1945, came into being . Schedule h includes substances that could be sold by retail on the prescription of a registered medical practitioner only . Schedule x drugs are those for which a retailer has to preserve the prescription for a period of 2 years . While schedule x has worked, schedule h implementation has been lax, necessitating the schedule h1, with 46 drugs in its purview . Schedule h1 came into effect from march 1, 2014, and contains prescription only drugs for which a copy of the prescription has to be preserved . Only about half of the pharmacists were aware of schedule h and 17.5% were updated with current regulations on schedule h1 . Sixty - five percent of the pharmacists are not aware that prescription in duplicate and stringent dispensing records have to be maintained while dispensing schedule x drugs . Knowledge and updated drug information is a must for people involved in drug dispensing to assure the quality of dispensing . However, most of them obtain their drug information in informal ways such as industry representatives and peers . Thus, given the gap in knowledge of pharmacists and their unawareness of current regulations, their dispensing practices may undo the beneficial effects of the health - care system . However, a drug dispensing audit at pharmacies could have avoided the bias associated with a questionnaire - based study . Furthermore, simple random sampling instead of convenient sampling could have been more representative of all pharmacies in bengaluru city . This study revealed improper dispensing practices at pharmacies in both residential and commercial areas of bengaluru city with a high proportion of dispensing encounters without prescription, a higher rate of older prescription refills and inadequate medication counseling, and storage practices . It also highlights lacunae in the knowledge of pharmacists about schedule h and current regulations pertaining to scheduled drugs . A similar scenario seems to prevail elsewhere in india and in other developing countries of the world . Hence, structured educational campaigns, generation, and implementation of otc list, and regulatory enforcement can better equip pharmacists for their main role of promoting rational drug use.
Epidermolysis bullosa (eb) is a rare genetic disorder characterized by abnormal fragility of skin and mucosal surface . The separation of skin layers occurs after application of friction or shearing forces and results in intradermal fluid accumulation and bullae formation . In addition to considerations associated with positioning, monitoring, infection, and prevention of skin and mucosal trauma, anesthetic management of eb is uniquely challenging because of the effects on the airway . This case report describes the successful anesthetic management of a patient with eb presenting for syndactyly release . A 6-year - old male child, weighing 14 kg; a known case of eb since birth presented with congenital left hand syndactyly [figure 1]. Significant preoperative findings were generalized scars, bed sores, pustules, and joint contractures . Airway assessment revealed mallampatti class iii, microstomia, loose teeth, and poor oral hygiene with thyromental distance 3 cm . Left hand shows syndactyly and right shows iv canula secured with nonadhesive technique airway assessment shows anticipated difficult airway oral antibiotics were administered preoperatively . Intravenous (iv) line 22-g secured with use of emla and fixed with vaseline gauze . Premedication with iv fentanyl 20 g, midazolam 0.4 mg, and dexamethasone 2 mg was given . Were given with iv ranitidine 1 mg / kg and cefotaxime 50 mg / kg, respectively . Intraoperative heart rate, rhythm, and oxygen saturation were monitored with lubricated clip on pulse oximeter . Noninvasive blood pressure (nibp) and electrocardiogram monitoring were avoided [figure 3]. Patient was positioned supine and pressure points were padded with cotton . Due to anticipated difficult airway, a difficult airway cart was prepared with various size masks, laryngoscope, endotracheal tubes (ets), ventilating bougie, and stylet . Though supraglotic airway devices were inappropriate in this patient they were kept ready for an emergency airway situation . Minimal monitoring was used intraoperatively preoxygenation was followed by induction with iv propofol 20 mg along with oxygen and sevoflurane under gentle mask holding with vaseline gauze . After confirming ventilation, with use of lubricated macintosh laryngoscope trachea was intubated with uncuffed et no . 5 and tube secured with nonadhesive lubricated bandage [figure 4]. Et secure with nonadhesive technique anesthesia was maintained with oxygen, nitrous oxide, and sevoflurane with manual ventilation using jackson rees circuit . As rectal suppositories, subcutaneous and intradermal routes are generally not recommended; postoperative analgesia was provided with iv diclofenac sodium 25 mg 8 hourly . Regional block was avoided due to joint contracture and scars, which made location of anatomical landmarks difficult . Neuromuscular blockade was antagonized at the end of the procedure, and trachea extubated when adequate signs of spontaneous recovery were evident . Postoperatively, oral examination revealed small intra - oral blister formation not requiring active management . Eb encompasses an array of autosomal dominant and recessive conditions that may have either localized or generalized dermatological manifestations . The loss or absence of normal intracellular bridges is due to a collagen abnormality, which makes patient susceptible for blister formation by friction / shearing forces and subsequent scarring . Equipment and techniques routinely used in the induction and maintenance of general anesthesia can be the source of serious postoperative complications . Anesthesia is frequently required for multiple surgeries like daily dressings, dental procedure, esophageal dilatation, gastrostomy, contracture release, and syndactyly release . Malnutrition, anemia, and decreased immunity are present in patients due to decrease oral intake secondary to oropharyngeal and esophageal lesions . Malnutrition can leads to hypoprotienemia, anemia and electrolyte imbalance which may affect pharmacokinetic effects of anesthetic agents . Infection is common as patients often have both poor immunity and long - term corticosteroid treatment . Antacid prophylaxis is required due to history of reflux, regurgitation, or esophageal stricture . Patients with eb are prone for ophthalmic complications like corneal erosion, conjunctival symblepharon, and ectropion . Dental problems like cleaning difficulty, poor eating patterns, enamel dysplasia, carious, and loose teeth are frequently associated . Iv or intra - arterial access should be secured with bandage, cotton wrap, or suture . All persons involved in handling these children must be aware of the extreme vulnerability of skin . During transport or mobilization of the patient, the most important task is to maintain the integrity of the skin and avoid friction and trauma . Direct pressure to the skin is not as damaging as frictional or shearing forces, so nibp and tourniquet can be used with pressure and duration limitations . As our case was of a short duration with no expected major blood loss and hemodynamic shifts, use of nibp and tourniquet therefore, clinical use of nibp and tourniquet in such patients depends on its merits and demerits . Microstomia, neck contracture, oropharyngeal lesion, ankyloglossia (decrease mobility of the tongue), thickened epiglottis, and possible tracheal stenosis make maintenance of a patent airway challenging . Fiberoptic intubation is less traumatic to the mucosa than direct laryngoscopy and should be the first choice in eb patients with a difficult airway . Laryngeal, tracheal involvement in eb is rare because that tissue is pseudostratified, columnar, ciliated epithelium and whereas oropharyngeal, esophageal mucosa is stratified squamous . Supraglottic airway increases bullae formation but can be used in difficult airway scenario with appropriate care . After application of moisturizing ophthalmic gel, eyes should be covered with moistened gauze to protect from mechanical trauma . Regional anesthesia should be considered whenever possible but contractures, scars, and infections are the issues associated with regional anesthesia . Perioperative blister should be treated with liquid paraffin, silver sulphadiazine, or steroids . In ambulatory careful monitoring, transport and positioning was instituted in the present case to avoid undue skin trauma . Atraumatic ventilation and intubation was possible with use of vaseline gauge and gentle mask holding . Minimal mandatory monitoring and no touch principle remains the key to successful anesthesia management of eb cases . Patients with eb present a unique challenge for all anesthesia - care providers . With maximal skin and mucous membrane protection, anesthesia in children with eb can be conducted with few sequelae . Hence, meticulous execution of preoperative planning is indispensable for ensuring a favorable intraoperative and postoperative course.
The subjects were enrolled in a health survey, which started in 19701973 (17). All 50-year - old men living in the municipality of uppsala at the time were invited to participate in a health survey on risk factors for coronary heart disease . About 20 years later, a follow - up investigation of the 70-year - old a total of 855 men have been investigated and a subgroup (n = 75) was randomly selected and asked to take part in the present study . The men visited the metabolic clinic at the university hospital as part of the ongoing health survey . Those who agreed to report their dietary intake during two separate weeks were included in the present study . The subjects were recruited throughout november and may and were randomly assigned to start either with the orfr or with the wr . Each subject was weighed initially and at the end of each dietary recording period including seven consecutive days, with a break of one week between the two periods . Each subject received detailed oral and written instructions from a dietician or nurse on how to record food intake using the wr or the orfr, as well as instructions on how to use the scales . Information on how to fill in the records correctly was also given in a video film that was shown to each participant . Subjects were instructed to continue their normal eating habits, and to start filling in the dietary record the following day . If further questions arose during the week of reporting, subjects were instructed to contact the dietician or nurse by phone for assistance . Each subject recorded his intake in the orfr by marking a horizontal pencil stroke in a circel. Each page of the orfr contains pre - printed food items or dishes eaten at main meals, and the subject has to mark the amount of food eaten, expressed either in household measurements or as portion sizes . The portion sizes were illustrated in a series of four photographs, as a guide to estimating the portions of the meal components (meat / fish; potatoes / pasta / rice; vegetables / salad, etc . ). Use of spreads on sandwiches was also estimated with the help of photographs showing four alternatives for the amounts used . For the other pre - printed foods, the subject indicated the amount consumed in household measures, e.g. Glasses, cups, slices, etc . Others were described by the subjects in free text and were coded into the computer by hand in accordance with the national food administration's food composition database (18). Each page was read by an optical reader (kaiser omr 32) using the omr technique, where the position of the marking on the page constitutes the necessary information . The optical reader is equipped with an extra - sensitive head for reading, which sends out visible light at a reading speed of 3,000 pages / h . The reader is linked to a pc . The kaiser lepron program transfers the data to the pc, where it is accessed and analysed . The subjects were instructed to weigh all the foods eaten during the seven - day period using a scale (soehnle, model 8020). As the subjects were retired men, the majority of their meals were eaten at home, according to a regular eating pattern . If for any reason they were to eat away from home, they were asked to bring the scale with them . In some situations where they were unable to weigh their food, they would estimate their food intake using the same set of photographs as for the orfr . The subjects were instructed to record their intake of food and beverages in a small notebook specially prepared for the present study . Further, they were instructed to record any intake as soon as possible after consumption of any food or drink . All subjects were asked to collect a 24-h urine sample during the weighed food record period . Protein intake was validated by comparing the estimated intake from the food records with the protein intake calculated from the 24-h un excretion (9). The subjects were given a plastic litre measure for collecting the urine, and two - litre plastic bottles for storing the urine collections . The first morning urine voided on the collection day was discarded and the time noted . All urine passed in the next 24 h was collected until the same time the next morning . To check the completeness of urine collection, para - amino - benzoic acid (paba) tables were used (10, 11). The subjects were instructed to take the paba tablets (380 mg) during the 24-h urine collection . One tablet was taken with each of the three main meals, i.e. Breakfast, lunch and dinner . To include all days of the week, un excretion was converted to grams of protein ingested using the formula gram n/0.81 6.25, as suggested by bingham and cummings (10), as it was found that, on average, 81% of the nitrogen is excreted with the urine . The daily intakes of energy and selected nutrients were calculated using a computerised dietary assessment program (19) equipped with a food composition database from the national food administration (20). The database includes about 1,500 food items, drinks and standard recipes, and reports data on energy and 47 nutrients . The adequacy of the recorded ei values was evaluated using the cut - off method, according to goldberg et al . A cut - off value below 1.27 for ei / estimated bmr was used to identify subjects with apparent long - term under - reporting . This value is considered as the lower limit for long - term survival (21). In addition, a cut - off value of 1.1 was used to characterise subjects with an implausibly low ei for a seven - day period based on statistical considerations described by goldberg et al . (23). The statistical analysis system (24) for personal computers for comparison of means of normally distributed variables, the paired student's t - test was used . For variables correlation analysis was performed with spearman's rank correlation to test for the trend in the different quintiles of the main variables (25). The subjects were enrolled in a health survey, which started in 19701973 (17). All 50-year - old men living in the municipality of uppsala at the time were invited to participate in a health survey on risk factors for coronary heart disease . About 20 years later, a follow - up investigation of the 70-year - old a total of 855 men have been investigated and a subgroup (n = 75) was randomly selected and asked to take part in the present study . The men visited the metabolic clinic at the university hospital as part of the ongoing health survey . Those who agreed to report their dietary intake during two separate weeks were included in the present study . The subjects were recruited throughout november and may and were randomly assigned to start either with the orfr or with the wr . Each subject was weighed initially and at the end of each dietary recording period including seven consecutive days, with a break of one week between the two periods . Each subject received detailed oral and written instructions from a dietician or nurse on how to record food intake using the wr or the orfr, as well as instructions on how to use the scales . Information on how to fill in the records correctly was also given in a video film that was shown to each participant . Subjects were instructed to continue their normal eating habits, and to start filling in the dietary record the following day . If further questions arose during the week of reporting, subjects were instructed to contact the dietician or nurse by phone for assistance . Each subject recorded his intake in the orfr by marking a horizontal pencil stroke in a circel. Each page of the orfr contains pre - printed food items or dishes eaten at main meals, and the subject has to mark the amount of food eaten, expressed either in household measurements or as portion sizes . The portion sizes were illustrated in a series of four photographs, as a guide to estimating the portions of the meal components (meat / fish; potatoes / pasta / rice; vegetables / salad, etc . ). Use of spreads on sandwiches was also estimated with the help of photographs showing four alternatives for the amounts used . For the other pre - printed foods, the subject indicated the amount consumed in household measures, e.g. Glasses, cups, slices, etc . Others were described by the subjects in free text and were coded into the computer by hand in accordance with the national food administration's food composition database (18). Each page was read by an optical reader (kaiser omr 32) using the omr technique, where the position of the marking on the page constitutes the necessary information . The optical reader is equipped with an extra - sensitive head for reading, which sends out visible light at a reading speed of 3,000 pages / h . The reader is linked to a pc . The kaiser lepron program transfers the data to the pc, where it is accessed and analysed . The subjects were instructed to weigh all the foods eaten during the seven - day period using a scale (soehnle, model 8020). As the subjects were retired men, the majority of their meals were eaten at home, according to a regular eating pattern . If for any reason they were to eat away from home, they were asked to bring the scale with them . In some situations where they were unable to weigh their food, they would estimate their food intake using the same set of photographs as for the orfr . The subjects were instructed to record their intake of food and beverages in a small notebook specially prepared for the present study . Further, they were instructed to record any intake as soon as possible after consumption of any food or drink . All subjects were asked to collect a 24-h urine sample during the weighed food record period . Protein intake was validated by comparing the estimated intake from the food records with the protein intake calculated from the 24-h un excretion (9). The subjects were given a plastic litre measure for collecting the urine, and two - litre plastic bottles for storing the urine collections . The first morning urine voided on the collection day was discarded and the time noted . All urine passed in the next 24 h was collected until the same time the next morning . To check the completeness of urine collection, para - amino - benzoic acid (paba) tables were used (10, 11). The subjects were instructed to take the paba tablets (380 mg) during the 24-h urine collection . One tablet was taken with each of the three main meals, i.e. Breakfast, lunch and dinner . To include all days of the week, un excretion was converted to grams of protein ingested using the formula gram n/0.81 6.25, as suggested by bingham and cummings (10), as it was found that, on average, 81% of the nitrogen is excreted with the urine . The daily intakes of energy and selected nutrients were calculated using a computerised dietary assessment program (19) equipped with a food composition database from the national food administration (20). The database includes about 1,500 food items, drinks and standard recipes, and reports data on energy and 47 nutrients . The adequacy of the recorded ei values was evaluated using the cut - off method, according to goldberg et al . A cut - off value below 1.27 for ei / estimated bmr was used to identify subjects with apparent long - term under - reporting . This value is considered as the lower limit for long - term survival (21). In addition, a cut - off value of 1.1 was used to characterise subjects with an implausibly low ei for a seven - day period based on statistical considerations described by goldberg et al . (23). The statistical analysis system (24) for personal computers release 6.04 was used for the statistical analyses . The results are expressed as means standard deviation . For comparison of means of normally distributed variables, correlation analysis was performed with spearman's rank correlation to test for the trend in the different quintiles of the main variables (25). Seventy - three of the 75 men completed both recordings: one week using the optical readable food record and one week using the weighed food registration method . There were no significant differences in body weight from baseline, orfr 82.7 11.4 kg and wr 82.5 11.4 kg, to the end of either of the two dietary recording periods, orfr 82.7 11.5 kg and wr 82.7 11.2 kg, or between the two methods used . The mean daily intakes of energy and selected nutrients as measured with the two methods are shown in table 1 . Higher average intakes were obtained by the wr for energy and protein, total fat, carbohydrates, - carotene, vitamin d, -tocopherol, thiamin, riboflavin, preformed niacin, vitamin b6, folate, vitamin c, magnesium, iron, zinc, selenium and dietary fibre . However, the proportion of energy (e%) from energy - yielding nutrients was similar, except for a higher e% saturated fatty acids and alcohol, and a lower e% sucrose, with the orfr method . Average daily intakes of energy, nutrients and alcohol as measured by the orfr and the weighed record (wr); mean and (sd) (n = 73) values significantly different from weighed record: * p <0.05; * * p <0.01; * * * p <0.001 . When expressed as nutrient density (i.e. Amount per mj), the orfr yielded lower nutrient density for iron, calcium, - carotene, selenium, - tocopherol and dietary fibre, while a higher nutrient density was found for potassium (table 2). Lower intakes per mj were also obtained with the orfr for the fatty acids 16:1, 18:1 and 18:3 n-3 and the long - chain n-3 fatty acids 20:5 and 22:6 (data not shown). Nutrient density (per mj) according to orfr and wr (n=73) significant difference between orfr and wr: * p <0.05; * * p <0.01; * * * p <0.001 . The correlation coefficients (spearman) between the two dietary assessment methods for intakes of energy and nutrients ranged from 0.08 to 0.68 and were all significant (p <0.01). Moderate to high correlation coefficients,> 0.400.60, were obtained for energy and most energy - yielding nutrients (table 3), while coefficients were between 0.30 and 0.50 for most micro - nutrients (table 4). Lower figures (<0.30) were obtained for vitamin a, vitamin d, - carotene, thiamin, riboflavin, preformed niacin and sodium . A comparison of the ranking of the individual intakes of energy and selected nutrients showed that the majority were classified into the same quintile or nearest quintile, i.e. The one below or the one above (table 4). Cross - classification (quintiles) and correlation of intakes of energy, energy - yielding nutrients and dietary fibre (in absolute and energy - adjusted values), according to orfr and wr percent subjects in first quintile wr classified into first and second quintiles in orfr . Percent subjects in fifth quintile wr classified into fourth and fifth quintiles in orfr . Significance of the spearman correlation: * p <0.01; * * p <0.001 . Cross - classification (quintiles) and spearman correlation for daily intakes of selected nutrients measured by the orfr and the weighed record (wr) (n = 73) percent subjects in first quintile wr classified into first and second quintiles in orfr . Significance of the spearman correlation: * p <0.01; * * p <0.001 . All subjects (n = 73) collected a 24-h urine sample during the weighed food record period . As we considered urine samples with a recovery of less than 85% incomplete, the daily protein intake estimated using orfr was 31% lower than the calculated values from the 24-h un (p <0.0667, r = 0.59), while that estimated using the wr was 22% lower (p <0.0001, r = 0.27) (table 5). Intake of protein (g / d) measured by orfr, wr and 24-h urinary nitrogen excretion (un). The figures are presented as mean and (sd) and their correlation (n = 59) significance difference from urinary protein: * p <0.0001 . Significance of spearman correlation: * * p <0.05 . The average daily consumption of foods (aggregated into major food groups) obtained by the two methods is shown in table 6 . The consumption of cheese, milk and other milk products as well as coffee, tea and alcohol was significantly higher when estimated using the orfr method than when using the wr method, while the opposite was seen for vegetables, meat and meat products, fish, bread, cereals and pasta, cakes and biscuits, jam, sweet drinks and desserts, chocolate and sugar (table 6). No differences were seen for spreads, potatoes, fruit, juice and eggs . Average consumed amounts, mean and (sd) of food among healthy, elderly men according to the optically readable food record (orfr) and the weighed record (wr) (n = 73) a large proportion of the men under - reported their ei . Use of the cut - off point <1.27 (21) showed a majority of the men reported an ei below this value when using the orfr and wr, respectively . Further, it was found that a higher percentage had an implausibly low ei according to goldberg, i.e. Cut - off <1.1 (23), when using the orfr (26%) than when using the wr (12%). Seventy - three of the 75 men completed both recordings: one week using the optical readable food record and one week using the weighed food registration method . There were no significant differences in body weight from baseline, orfr 82.7 11.4 kg and wr 82.5 11.4 kg, to the end of either of the two dietary recording periods, orfr 82.7 11.5 kg and wr 82.7 11.2 kg, or between the two methods used . The mean daily intakes of energy and selected nutrients as measured with the two methods are shown in table 1 . Higher average intakes were obtained by the wr for energy and protein, total fat, carbohydrates, - carotene, vitamin d, -tocopherol, thiamin, riboflavin, preformed niacin, vitamin b6, folate, vitamin c, magnesium, iron, zinc, selenium and dietary fibre . However, the proportion of energy (e%) from energy - yielding nutrients was similar, except for a higher e% saturated fatty acids and alcohol, and a lower e% sucrose, with the orfr method . Average daily intakes of energy, nutrients and alcohol as measured by the orfr and the weighed record (wr); mean and (sd) (n = 73) values significantly different from weighed record: * p <0.05; * * p <0.01; * * * p <0.001 . When expressed as nutrient density (i.e. Amount per mj), the orfr yielded lower nutrient density for iron, calcium, - carotene, selenium, - tocopherol and dietary fibre, while a higher nutrient density was found for potassium (table 2). Lower intakes per mj were also obtained with the orfr for the fatty acids 16:1, 18:1 and 18:3 n-3 and the long - chain n-3 fatty acids 20:5 and 22:6 (data not shown). Nutrient density (per mj) according to orfr and wr (n=73) significant difference between orfr and wr: * p <0.05; * * p <0.01; * * * p <0.001 . The correlation coefficients (spearman) between the two dietary assessment methods for intakes of energy and nutrients ranged from 0.08 to 0.68 and were all significant (p <0.01). Moderate to high correlation coefficients,> 0.400.60, were obtained for energy and most energy - yielding nutrients (table 3), while coefficients were between 0.30 and 0.50 for most micro - nutrients (table 4). Lower figures (<0.30) were obtained for vitamin a, vitamin d, - carotene, thiamin, riboflavin, preformed niacin and sodium . A comparison of the ranking of the individual intakes of energy and selected nutrients showed that the majority were classified into the same quintile or nearest quintile, i.e. The one below or the one above (table 4). Cross - classification (quintiles) and correlation of intakes of energy, energy - yielding nutrients and dietary fibre (in absolute and energy - adjusted values), according to orfr and wr percent subjects in first quintile wr classified into first and second quintiles in orfr . Percent subjects in fifth quintile wr classified into fourth and fifth quintiles in orfr . Significance of the spearman correlation: * p <0.01; * * p <0.001 . Cross - classification (quintiles) and spearman correlation for daily intakes of selected nutrients measured by the orfr and the weighed record (wr) (n = 73) percent subjects in first quintile wr classified into first and second quintiles in orfr . Significance of the spearman correlation: * p <0.01; * * p <0.001 . All subjects (n = 73) collected a 24-h urine sample during the weighed food record period . As we considered urine samples with a recovery of less than 85% incomplete, only 59 samples were used for comparison with the respective dietary records . The daily protein intake estimated using orfr was 31% lower than the calculated values from the 24-h un (p <0.0667, r = 0.59), while that estimated using the wr was 22% lower (p <0.0001, r = 0.27) (table 5). Intake of protein (g / d) measured by orfr, wr and 24-h urinary nitrogen excretion (un). The figures are presented as mean and (sd) and their correlation (n = 59) significance difference from urinary protein: * p <0.0001 . The average daily consumption of foods (aggregated into major food groups) obtained by the two methods is shown in table 6 . The consumption of cheese, milk and other milk products as well as coffee, tea and alcohol was significantly higher when estimated using the orfr method than when using the wr method, while the opposite was seen for vegetables, meat and meat products, fish, bread, cereals and pasta, cakes and biscuits, jam, sweet drinks and desserts, chocolate and sugar (table 6). No differences were seen for spreads, potatoes, fruit, juice and eggs . Average consumed amounts, mean and (sd) of food among healthy, elderly men according to the optically readable food record (orfr) and the weighed record (wr) (n = 73) use of the cut - off point <1.27 (21) showed a majority of the men reported an ei below this value when using the orfr and wr, respectively . Further, it was found that a higher percentage had an implausibly low ei according to goldberg, i.e. Cut - off <1.1 (23), when using the orfr (26%) than when using the wr (12%). The main results from the present study show that estimates of average intakes of energy and several nutrients differed significantly between the two methods, higher figures being obtained when using the wr . However, when expressed as nutrient density, i.e. Per mj, only a few nutrients were still lower when using the orfr . The mean spearman correlation coefficients between the intakes of energy and energy - yielding nutrients between the two methods were moderate to high, i.e. 0.40.6, while figures for most micro - nutrients were in the range 0.30.5 . The majority of the subjects intakes were classified into the same or adjacent quintile . In a series of dietary surveys earlier performed in sweden, a pre - coded record book has been used to estimate nutrient and food intake in different groups (5, 15, 26). The record book has proven to be very useful in larger dietary investigations, but has also been found to have some limitations (5, 15). One important advantage of a pre - coded record book, compared with open - ended records, is that considerable time may be saved by not having to code and enter data . Becker et al . (15) suggested that the time reduction was almost 50%: coding and data entry from the pre - coded record took 1.3 h per person as compared to 2.5 h per person for the wr . In a study including 500 participants, this means a saving of four man - months of work (15, 27). In the present study, a pre - coded record book designed for optical scanning was used, which also considerably reduced the time - consuming step of entering the data manually (15). As reported by others, and shown here, underestimation of food intake seems to be a general problem, as shown by various assessment methods (1, 2, 4, 5, 14, 15, 23, 28, 29). The reported eis obtained here are comparable with those from a number of other large investigations in which seven - day food records were also employed (5, 15, 28). In the present study, the proportions of energy - providing nutrients did not differ significantly between the two methods, indicating that under - reporting of this aspect was not specific to the method used (table 3). This means that the fat used for cooking, i.e. Included in the recipes, is of the same quality / type (amounts of total fat and fatty acid composition). Thus, if the subject uses other cooking fats with a different fat content or with a different fatty acid composition, this will not be recorded or calculated . The true picture of the dietary fat intake is thus limited when using orfr . The same observations have been made earlier (4, 5, 15, 17). However, when using wr, the subjects are more likely to specify the type of cooking fat, as well as the true amounts of spreads they consume, because they actually weigh every single food item and the amount they eat . In the present investigation, calculations of the adequacy of the reported ei values were made on the basis of estimated bmr values as suggested by fao / who / unu (21, 22) and the cut - off points suggested by goldberg et al . Were categorised as under - reporters using both methods, though a larger number using the orfr method . The proportion of subjects with implausibly low reported eis corresponds well with the findings of other surveys in which a record book (pre - printed, but not optically readable) was used ((5), 15, 26). There is some uncertainty in the calculations of under - reporting because physical activity, specific to this study, was not assessed and cut - off limits for the ei: bmr ratio were based on those calculated by goldberg et al ., with a sedentary physical activity level of 1.55 for n = 1 (4, 23). It was further found that both assessment methods yielded lower estimates for protein intake compared to estimates based on un excretion (table 5). The lowest calculated intakes were obtained by the orfr . Despite that only a single 24-h urine was collected, correlation coefficient (spearman) for orfr was relatively high (0.59), and close to figures reported by bingham et al . (13) for estimated food records (0.600.70) using eight 24-h urine per subject . Further, when we checked our results using paba, we did not include those who had <85% completeness, as such results obviously indicate significant underestimation . (15), also based on comparison of an open wr with the pre - coded record book . Some of the portion sizes used in the orfr provide the most probable explanation for the low figures obtained (5, 15, 30). An analysis of the food consumption figures is very much in line with the lower energy findings, as well as with some lower nutrient intake figures, when using the orfr . Lower consumption of, e.g. Meat, milk and fat products can be associated with lower intakes of energy, as well as with lower intakes of the major energy - yielding nutrients such as total fat and protein, and perhaps also with some of the vitamins and minerals . Further, lower consumption of bread and cereal products, as well as vegetables, may explain lower figures for carbohydrates and dietary fibre . In general, the portion sizes used in our orfr may have been too small for various food items and dishes . Flat slope syndrome is another explanation, suggested by gibson (31), for the tendency to overestimate low intakes and underestimate high intakes, and this explanation may also apply to the subjects in the present investigation . In an evaluation of portion sizes, hglin and co - workers (30) showed that choosing incorrect pictures may result in both under- and over - estimations . Although most elderly people are thought to have a regular meal pattern and eat most of their food at home, they may forget to weigh everything . This could explain the low protein intakes estimated when using the wr . As stated by bingham (12), energy and protein intakes have been found to be under - estimated by as much as 20% according to various dietary surveys (12, 33). Our results, as well as those of others (32), suggest that improvement of methods for estimating portion sizes should be a priority in future dietary assessment methodology . After the present study was performed, the orfr was also investigated by rosell et al . The aim of their study was to investigate how much of the energy and nutrients contributed by foods that has to be reported in free text . The authors concluded that to reduce the level of under - reporting, emphasis should be placed on improving the recording of between - meal eating . However, we would like to emphasise the two important advantages of a pre - coded, orfr: the recorder and the experimenter save a considerable amount of time and the accuracy of coding and data entry is increased . In a pilot study comprising 35 subjects, time savings in data entering, data checks and nutrient analysis were on average 30 min for the orfr compared with the manual pre - printed record book (carlsson & johansson, unpublished data). Therefore, after the suggested adjustments especially in, portion sizes, the orfr could be seen as a useful tool in a variety of food and nutrition studies . The above findings indicate that when a pre - coded food record is used this is designed for scanning, the following advantages ensue: it is less time - consuming for subjects to record their food intake because there is no need for weighing and writing.it allows more efficient data processing, e.g. By limiting time spent on data entry.a pre - coded and orfr is less expensive to process than a manual version is.emphasis should be put on estimations of the type and amount of fat used in order to obtain a valid assessment of total fat and fat quality.the portion sizes should be estimated carefully measured to reflect the actual portion sizes in the study population . It is less time - consuming for subjects to record their food intake because there is no need for weighing and writing . It allows more efficient data processing, e.g. By limiting time spent on data entry . A pre - coded and orfr is less expensive to process than a manual version is . Emphasis should be put on estimations of the type and amount of fat used in order to obtain a valid assessment of total fat and fat quality . The portion sizes should be estimated carefully measured to reflect the actual portion sizes in the study population . The author has not received any funding or benefits from industry to conduct this study.
With the number of sequenced genomes at various stages of completion being in the tens of thousands and the number of genomic features (genes, rnas, etc .) Identified across these genomes in the millions, the need for accurate and consistent genomic annotation is paramount . The gene ontology (go) was created in 1998 by researchers at flybase, the saccharomyces genome database (sgd), and the mouse genome database as a collaborative effort to address the need for consistent descriptions of gene products across different databases . This group has since grown to include 26 consortium members and associates and the go is a key member of the open biological and biomedical ontologies (obo) community . In this context ontologies provide a controlled vocabulary for representing and communicating knowledge about a topic and a set of relationships that hold among the terms of the vocabulary . The topic for the go is genes and gene products such as transcripts, proteins, or rnas that are described in three related subontologies (also called namespaces), biological process, the broad biological system in which a gene product is involved; molecular function, the specific role a gene product has or potentially has within a biological process; and cellular component, the location in a cell where the gene product performs its molecular function . Each ontology is composed of nodes (terms) and edges (relationships) and is structured as a directed acyclic graph (dag). As one moves down the nodes of the graph from a parent to a child, terms become increasingly more specific . A dag allows for child terms to have more than one parent and this enables complex relationships to exist between them . The go currently contains approximately 38 thousand terms that have been used by consortium members to annotate almost 25 million gene products . Each annotation is accompanied by an evidence code to denote the method by which the annotation was made . Computationally sourced annotations, such as the evidence code iea (inferred from electronic annotation), are regarded as less confident annotations than those that are assigned on the strength of experimental evidence . All data is publicly available for download and the go team provides a number of entry points to query the data including an application programming interface (api) to permit custom queries to either a go database or an ontology flat file . The results of gene expression microarrays can be enriched with terms pertaining to biological process, which helps to discover if entire pathways are upregulated as opposed to simply individual genes . Subcellular location can be predicted for an unknown gene sequence by performing a similarity search such as blast and inferring from the cellular component go terms of top hits . One such limitation is that, by design, there is no link between the three subontologies . This means one cannot directly answer questions such as what are all the biological processes occurring in the cellular component mungall exploited the high degree of regularity in phrase structure of obo term definitions and converted tokenized definitions from the go, biochemical ontology, and the cell ontology into a language (obol) that can be parsed computationally with a reasoner . This was used, amongst other things, to find missing relationships within the go and between ontologies . Similarly, wroe et al . Converted the go to a daml + oil framework to enable reasoners to parse the ontology . Bada and hunter used regular expressions to find patterns in go definitions and create an assertional model to integrate the three go subontologies, the chemical entities of biological interest ontology (chebi), and the cell type ontology (cto). Interrogated the go, compiling groups of terms cooccurring in annotations of gene products, in order to suggest potential biologically relevant terms to annotators . Describe functionality within the quickgo tool from the go annotation (goa) group that provides information on how many times a query term cooccurs with other go terms specifically across the uniprot knowledgebase (uniprotkb) database . This allows researchers to input a query term such as nucleus and view all terms that are frequently annotated alongside this term . However, there are limitations to this method, such as not being able to explore custom subsets of data within the uniprotkb database and a hard limit on the number of terms returned to the user . This paper presents golink, an alternative tool for finding go terms from across the three go namespaces that cooccur with a given query term . Golink differs from existing tools by using the go api to mine the full complement of the go database, using methods that take into account the namespace of a query term when assessing cooccurrence . The key advantages of using this particular method and source data are described along with other practical uses for the tool . These include predicting the most likely biological process for genes by using just their assigned molecular function terms, where golink achieves levels of specificity, sensitivity, and accuracy above 80% . Golink is written in perl and requires the go database api (go - db - perl) module (go::apphandle), available from the comprehensive perl archive network (cpan). Golink requires access to a go database (ideally locally installed). For the analyses in this paper, the go database v201212 was downloaded from http://www.geneontology.org/go.downloads.database.shtml and installed using the instructions at http://archive.geneontology.org/latest-full/readme . All analyses were run under 64-bit linux on a single 2.3 ghz core of a multicore amd server; however, golink can be run on both windows and macos platforms provided perl, the required cpan modules, and a go database are installed . Golink is made freely available from the project website (http://bioinformatics.childhealthresearch.org.au/software/golink/) under the gnu general public license (gpl). All usage instructions are detailed in the software manual also available from the project website . A user provides golink with a go term such as go:0005634 representing nucleus, which is referred to as the query term, along with any desired filters (evidence codes or annotation source databases see below). Golink will then retrieve all genes that have been annotated with the query term or any of its child terms (figure 1(a)). Each gene is assessed in turn (figure 1(b)) and its full complement of go annotation is assigned to one or more terms list (figure 1(c)) based on whether or not the annotation fulfills the list's specific criteria as follows.all_list: stores all the gene's annotated go terms.query_list: stores all the gene's annotated go terms only if the query term is one of those terms . This method is similar to quickgo.parchild_list: stores all the gene's annotated go terms only if all terms within the same namespace as the query term are either one of the query term's parents or children . Query_list: stores all the gene's annotated go terms only if the query term is one of those terms . Parchild_list: stores all the gene's annotated go terms only if all terms within the same namespace as the query term are either one of the query term's parents or children . In an iterative process, each list is gradually formed of cooccurring terms from all namespaces that fit the criterion above and that can subsequently be confidently associated with the initial query term . Once the lists are compiled, scores are calculated for terms in each list to allow them to be ranked . Pr empirical probability ratio: (1)(t / q)(c / a). S%empirical probability similarity ratio: (2)(t(q+ct))100, where, given term x in the context of a single list, q = the number of gene products fulfilling the criteria of the list; t = the number of gene products fulfilling the criteria of the list that contain term x; c = the number of gene products annotated with term x in the entire go database, given any filters; and a = the number of gene products in the entire go database, given any filters . The empirical probability ratio is a measure of how similar t is with c. while being a useful score this penalises interesting candidates that have high c but a slightly lower value of t. this discrepancy is addressed in the empirical probability similarity ratio, which not only accounts for the relationship between t and c but gives higher scores where t and q are also similar in value . Users can explore subsets of go annotation data by optionally applying a choice of three filters . The namespace filter controls the nature of the terms output to the final terms list . Unfiltered, golink will return terms from all namespaces that cooccur with the query term . However, users can request that only terms from a limited set of the three namespaces are returned . When a go term is assigned to a gene product by a go consortium member (e.g., flybase, sgd) an evidence code golink provides a database filter to target annotations made by a particular consortium member and an evidence code filter to allow fine - grained control on the level of quality of terms returned . For convenience a can be specified to not include terms from a particular database or evidence code . Golink will only consider terms that are not attributed with the evidence code iea (inferred from electronic annotation). Given that the iea evidence code suggests a less confident term assignment, using! Golink terms lists can be ordered as required; however, it is recommended that results are sorted by descending empirical probability similarity ratio (s%) and then descending empirical probability ratio (pr). This serves to place the terms most strongly linked to the query term at the top of the list and also mirrors the ordering method used by quickgo . Golink outputs three terms lists, each with related yet contrasting results due to the differing criteria used to compile each list . When reviewing these lists it is important to take the initial query term used for the analysis into account . If the query term originates from towards the top of go dag, it will be more likely to have a large number of child terms, which inevitably results in many more terms being returned to all the golink terms lists than for more specific query terms at lower levels of the go graph . In this case conversely, lower level terms are more likely to not return any results to the more stringent golink terms lists due to there being fewer annotations where specific criteria are met . Here golink terms lists were generated using the biological process query term regulation of gene expression comparisons of golink terms lists were performed using a three - way venn diagram of all terms in each list using venny . A perl script was created that, given an initial go term, pulls all genes annotated with that term from the go database along with their other annotated go terms . Using this script a positive and a negative list of genes and their go annotation (go:0010468) for the positive list and a combination of lipid metabolic process (go:0006629) and cell motility (go:0048870) for the negative list . Genes were only included in each list if they had at least one molecular function term as part of their annotation . Golink terms lists were then generated using the biological process query term regulation of gene expression (go:0010468), opting to only output molecular function terms . In all cases the evidence code filter! A gene in the positive or negative list was deemed as having a role in the regulation of gene expression if any of its molecular function terms matched either the first 10, 20, 30, 40, or 50 (separately) molecular function terms in each of the three golink terms lists . Those genes in the negative list providing positive results were manually checked in uniprotkb to ensure that they did not also have a role in the regulation of gene expression as well as either lipid metabolic process or cell motility . A statistical assessment of performance was calculated as follows: (3)sensitivity = tp(tp+fn),specificity = tn(fp+tn),positive predictive value (ppv)=tp(tp+fp),negative predictive value (npv)=tn(tn+fn),accuracy=(tp+tn)(tp+tn+fp+fn), where tp = number of genes correctly predicted to be involved with the regulation of gene expression; tn = number of genes correctly predicted to be not involved with the regulation of gene expression; fp = number of genes incorrectly predicted to be involved with the regulation of gene expression; and fn = number of genes incorrectly predicted to not be involved with the regulation of gene expression . In this context, sensitivity refers to how likely golink predicts a role in the regulation of gene expression for those genes in the positive list (tp), specificity measures how well golink predicts no role in the regulation of gene expression for genes in the negative list (tn), ppv assesses the chance that a gene is involved with regulation of gene expression given a prediction that it is involved, and npv calculates the chance that a gene is not involved with regulation of gene expression given a prediction that it is not involved in that process . Mixed bar and line graphs displaying the results of this assessment were produced using r . (saccharomyces genome database), pombase (main resource for the fission yeast schizosaccharomyces pombe), mgi (mouse genome informatics), zfin (the zebrafish model organism database), and uniprotkb, separate golink terms lists were generated using the biological process query term regulation of gene expression using data from each respective all_list, pairwise comparisons were made between terms from uniprotkb and each of the other source databases using venny . A quickgo (released on friday, 04 january 2013) terms list was exported by using the query term regulation of gene expression and selecting from the! Iea an equivalent golink dataset was generated by using the query term regulation of gene expression and applying the! The position at which each of the 100 quickgo terms fell in the sorted golink list was then obtained . Terms list was used to compare against the quickgo terms list as the term assignment criteria for both lists are the most comparable . The exported quickgo terms list was also subjected to a separate similar statistical assessment to that described above, whereby a gene in the positive or negative list was deemed as having a role in the regulation of gene expression if any of its molecular function terms matched any of the molecular function terms in the quickgo terms list . Tables 1(a)1(c) show the top 10 golink cooccurring terms and their scores in each terms list generated using the query term regulation of gene expression (go:0010468) and applying the evidence code filter! The complete lists can be found in supplementary file 1 (see supplementary material available online at http://dx.doi.org/10.1155/2013/594528). Overall the all_list, query_list and parchild_list, returned 6513, 419, and 322 terms, respectively . As expected there are a large number of unique terms in the all_list given the comparatively large number of terms returned and there are no unique terms found within either the query_list or parchild_list . The 360 terms overlapping the all_list and query_list in the venn diagram (figure 2) represent terms from gene products that are annotated with the query term but have other terms in the biological process namespace that are not either a parent or a child of the query . The 263 terms (figure 2) overlapping the all_list and parchild_list represent terms from gene products that were not directly annotated with the query term but with one of its child terms . Overall, there were 59 consensus terms across the three lists, which are listed in supplementary table 1 . A useful application of the golink term lists is, for example, to use a gene's existing molecular function go terms to establish whether it has a role in a particular biological process, such as the regulation of gene expression . If a gene has been annotated with only molecular function terms, one can compare these terms to a golink terms list for a biological process query term of interest . If the molecular function terms are found in the golink terms list, one can infer that the gene's function may be associated with the initial biological process query term . Figure 3(a) and supplementary file 2 show the sensitivity, specificity, ppv, npv, and accuracy of such an assessment matching only the molecular function terms from the three golink terms lists with a positive and negative list of genes and their annotated go terms . They also show the proportion of genes found in each of the positive and negative lists by the three golink terms lists . The positive list contained 41 genes (1163 terms, 154 mf terms) annotated as having a role in the regulation of gene expression, while the negative list contained 154 individual genes (2723 terms, 503 molecular function terms). The genes contained in the positive and negative lists and their associated go annotation can be found in supplementary file 2 . The top 50 molecular function terms from each of the three golink terms lists that were used in the matching process are also shown in supplementary file 2 and a venn diagram showing their overlap can be found in supplementary figure 1 . It should be noted that the rationale behind using only up to the first 50 molecular function terms from the golink terms lists in the matching process was simply that each list contained at least 50 terms . Overall the query_list outperformed the other two golink terms lists in predicting genes with a role in the regulation of gene expression using only their assigned molecular function terms . For this list, sensitivity, specificity and accuracy all averaged between ~80% and 90% with the highest values occurring with comparisons using the top 30 golink molecular function terms . Using these top 30 terms in the query_list yielded 33/41 true positives and only 12/154 false positives giving a ppv of 73% and a npv of 94% . The all_list and parchild_list showed increasingly higher sensitivity as more terms were added in to the comparison, but this was at the expense of specificity, which remained above 50% but much lower than that of the query_list . Eleven of the 154 negative genes were deemed to be actually true positives (supplementary table 2) with a role in the regulation of gene expression as well as either cell motility or lipid metabolic process despite all but one not being directly annotated with these genes (such as sterol regulatory element - binding proteins 1 and 2 and coup transcription factor 2) were removed from their respective negative lists and the statistics recalculated, which served to increase values marginally across the board (table (a) in supplementary file 3). Most of the false positive genes predicted by the golink terms lists were annotated with the go term protein binding (go:0005515). This fairly generic term has relevance for both the negative and positive lists so users of the software are cautioned to consider this when performing analyses of this nature and may opt to remove terms from the golink lists where appropriate . Indeed removing both the false positives from the negative list and removing the go term protein binding from the golink terms lists have a substantial impact on the statistical assessment across the board as can be seen in figure 3(b). Figure 4 shows the overlap of golink terms obtained using four species specific annotation source databases (mgi, pombase, zfin, and sgd) in comparison with terms obtained from the more broad uniprotkb database, using the same query term (regulation of gene expression) and evidence code filter (! Iea). For each of the species specific databases figure 4 shows that, as expected, the majority of terms (between 53% and 87%) in their respective lists were also in the uniprotkb list . This group represents those terms that cooccur with the query term in annotation within both the species specific database and in annotation by uniprotkb for other species . The remaining terms are either unique to the species specific database or to uniprotkb . In the context of the species specific database these unique terms are either highly relevant to the species and only ever likely to have been used in annotation by the curators of the species specific database (e.g., ascospore formation within pombase and sgd, fin development within zfin) or represent terms that only cooccur with the query term in this database compared to uniprotkb . Conversely, those terms unique to uniprotkb represent terms highly irrelevant to the species specific database (e.g., viral reproduction in all cases) or similarly only cooccur with the query term within uniprotkb assigned annotation . Tables (a)(c) in supplementary file 5 show the full golink query_list and quickgo terms lists used in the comparison ((b) and (c)) and a combined summary detailing the positions of the top 100 quickgo terms as found in the golink query_list (a). Overall the vast majority (72%) of quickgo terms are confined to the top 200 golink terms and figure 5 shows that there is a 64% overlap between the top 100 quickgo and golink terms . While all 100 quickgo terms are represented somewhere in the golink query list, table (a) in supplementary file 5 shows that there are distinct differences in the ordering of the results within the more significant first 100 terms . This can be explained by the way protein identifiers are mapped within the database underlying quickgo . The database used by golink is maintained by the go consortium . Within this database, the database for quickgo is maintained by the goa team and provides high quality annotation for all proteins within uniprot . Here, single protein identifiers are first mapped to uniprot accessions, and in many cases one identifier can map to several uniprot accessions (since there is likely to be several matches in trembl, the nonreviewed section of uniprot). In these cases the same or similar annotation is assigned to each of the uniprot accessions . While appropriate in the context of the goa project this leads to more redundancy in annotation in the database used by quickgo than within that used by golink . Consequently, when considering cooccurring terms in quickgo, terms from those genes that have multiple entries in trembl will appear to cooccur more often than is the case within the go database, which may not be desired . For example, in table (a) in supplementary file 5, quickgo reports that the term forebrain development, which appears second in the quickgo terms list, has been used to annotate 308 proteins within the goa database . This term appears tenth in the golink list, where golink reports its assignment to 200 gene products within the go database . Furthermore quickgo and golink report that this term cooccurs in annotation with the query term on 56 and 26 occasions respectively . On closer examination, multiple uniprot accession numbers exist that map to single mgi identifiers annotated with the term forebrain development and this has the effect of inflating the number of cooccurrences with the query term, thus influencing the s% calculation and positioning it higher in the quickgo terms list . A more extreme example can be seen for the term spermatid differentiation which appears at position 11 in the quickgo list but position 531 in the golink list . In addition, the result of the pr calculation is altered as a consequence of the increased number of proteins within the goa database . As both quickgo and golink order their results by s% and then by pr and quickgo only provides the first 100 hits, this explains why equivalent search parameters on these similar but different source databases lead to similar but differing results for golink compared to quickgo . In order to test quickgo's ability to predict a gene's biological process from its molecular function a similar matching process to that undertaken for golink was performed with the top 100 cooccurring terms from quickgo . Only 9 of the 41 positive genes were found using these terms, which consequently gave lower comparative performance scores to golink (data not shown), however it should be noted that as seen in table (c) in supplementary file 5 this list of terms only contained 4 molecular function terms to use in the comparison process . The gene ontology is a dynamic, growing resource for the annotation of genes and gene products . It is organized into three subontologies describing the cellular location of action of a gene or gene product, the main process it is involved in, and its role in this process . In order for the three subontologies to remain orthogonal, there are no designed links between them . However, this prevents legitimate attempts to answer queries involving data from more than one of the subontologies . This paper discusses a new tool, golink, which uses the go perl api to explore the go database and implement three increasingly stringent methods to compile lists of go terms cooccurring in gene product annotations with a provided query term . When given an initial query term, the three methods store either all terms cooccurring with the query term or any of its child terms (all_list), only those terms cooccurring with query term (query_list) or only those terms cooccurring in annotations with the query term or its child terms where other annotated terms in the same namespace (cellular component, molecular function, and biological process) as the query term are limited to the query term and any of its parent or child terms (parchild_list). However, the key difference between the remaining golink lists and quickgo is that the latter uses a pool of terms from genes directly annotated with the query term, whereas the golink all_list and parchild_list use a pool derived from genes annotated with the query term and/or its child terms . The main reason golink uses this extended pool is to not penalize the partonomic / taxonomic relationship between a term and its child terms . Furthermore, this allows golink to apply further logical stringency in the parchild_list to specifically exclude occurrences where the query term is annotated together with terms in the same namespace that are neither a parent nor a child of the query term . For example, if a gene performs a particular function and operates both in the cytoplasm and the nucleus it may well be annotated with 2 cellular component terms (nucleus and cytoplasm) and at least 1 molecular function term . In this case the molecular function term cannot be exclusively linked with either of the cellular component terms as the molecular function term may only be relevant for the gene's function in the cytoplasm . If this distinction is required then the parchild_list will take this into account, whereas this functionality is not possible using quickgo . The other main advantages of golink over quickgo lie in the distinct differences between the underlying databases of the two software and the availability of flexible methods to filter the terms assessed and those returned . The process of identifier mapping in the goa database that provides the annotation used by quickgo leads to an increased redundancy of annotation across this database as single gene product identifiers commonly map to multiple uniprot accessions . This results in a potentially inflated cooccurrence of go terms as demonstrated in this paper . Quickgo provides two results sets; one unfiltered list of cooccurring terms and one with a! Filter applied to discount associations from purely computationally assigned terms . For both, only the first 100 cooccurring terms are returned . Golink is much more flexible in that not only does it produce multiple terms lists of varying stringency, but it also provides fine - grained customizable filtering . The database filter allows a user to identify cooccurring terms within the context of a particular species that could be diluted if searching within a broader context such as the full uniprot database . The evidence code filter gives precise control over the quality of annotation considered during an analysis using the wide variety of evidence codes provided within the go database . Finally, applying the golink namespace filter will only return results from a defined set of the three available namespaces . If an analysis only called for examining cooccurring molecular function terms then this assessment can only be confidently made using golink as the full complement of cooccurring molecular function terms may not be available within the static top 100 results gleaned from quickgo . This limitation of quickgo also hinders its use in the types of analyses undertaken in the statistical assessment of golink to predict, for example, a gene's biological process from its assigned molecular function terms, as there may not be sufficient molecular function terms returned . Quickgo cooccurring terms are immediately available via the quickgo web interface, whereas the golink algorithm can take a number of hours to run for some analyses . There are a number of sections of the golink algorithm; however, that lend themselves to parallel processing . This will be available in future versions of the software; however, its absence in the current version does not hinder the tool's utility . The golink method performs very well in predicting the biological process from a gene's assigned molecular function terms, showing both high sensitivity and specificity . This application of golink is equivalent to first predicting molecular function terms for an unknown gene using, for example, interproscan then using golink to generate terms lists for a biological process query term of interest and then predicting the likelihood of the unknown gene being involved in that particular biological process . Similarly using a cellular component query term and comparing the golink terms with interproscan or otherwise derived go terms, a researcher can predict the cellular component of a gene . In cases where a more direct prediction of, for example, biological process is required, multiple molecular function query terms could be provided to golink with the most likely associated biological process being the highest scoring consensus term found in all terms lists . In addition, as cellular component, molecular function, and biological process terms can be incorporated into the golink terms lists, in a similar method to bada et al ., golink can be used to help annotators choose potential terms to assign to a gene . Furthermore, it was discovered that a number of false positives found in the statistical analysis were not specifically assigned the term regulation of gene expression but clearly have a role in this process . Golink is a perl based tool that finds terms cooccurring with a given query term in annotation across the full complement of the gene ontology database . It has advantages over other existing tools and can be used in a variety of applications.
During 20042007, as part of a broader biodiversity survey and inventory program, we sampled birds from mostly forested sites in guangxi and guizhou provinces in the southern part of the people s republic of china (figure). Sampling was conducted by mist netting and selective harvesting with shotguns; all birds in the study were apparently healthy and behaving normally at the time of collection . Because initial sampling was focused on endoparasite communities, samples from 20042005 consisted of complete gastrointestinal tracts frozen in liquid nitrogen . In 20062007 southeastern asia, showing 5 sites in the people s republic of china where land birds were collected and tested for influenza a virus . Prevalence values were 4% (n = 103) in dashahe in 2006; 6% (n = 194) in kuan kuoshui in 2006; 0.3% (n = 328) in shuipu in 2007; 3% (n = 184) in jing xi, in 2004; and 0% (n = 130) in shiwandashan in 2005 . A total of 184 samples were collected from jing xi municipality in guangxi (21.122n, 105.964e) in 2004, 130 from shiwandashan nature reserve in guangxi (21.840n, 107.880e) in 2005, 103 from dashahe nature reserve in guizhou (29.167n, 107.575e) in 2006, 194 from kuan kuoshui nature reserve in guizhou (28.226n, 107.160e) in 2006, and 328 from shuipu village, guizhou province (25.485n, 107.882e) in 2007 (figure). Samples were tested for influenza a virus by real - time reverse transcription pcr (6) in 2 diagnostic laboratories (southeast poultry research laboratory, us department of agriculture, athens, ga, usa, and national wildlife health center, us geological survey, madison, wi, usa). Of 939 samples tested, 24 were positive for influenza a viruses (prevalence 2.3%, table; complete summary in appendix table). If migratory behavior (species classified as migratory or nonmigratory on the basis of descriptions by mackinnon and phillipps), was considered, 11 (4.8%) of 231 samples from species showing marked seasonal migrations were influenza positive . However, only 13 (1.8%) of 708 samples from nonmigratory species were positive . The cumulative binomial probability that such a high number (11) of positive samples would result among the 231 migratory - species samples, were the true prevalence to be 1.8%, is low (p = 0.0013). Thus, migratory species appear to have higher influenza infection rates . In terms of general habitat use (7), open - country species were slightly more prone to be influenza positive (8 [2.9%] of 274 samples) than forest species (16 [2.4%] of 665 samples), but the difference was not significant (cumulative binomial probability, p>0.05). Interactions between migratory behavior and habitat use were not significant (contingency test, p>0.05). Although all infections detected were among songbirds (passeriformes), the sampling also concentrated on songbirds (94.3%). Thus, we could not test adequately the hypothesis that influenza prevalence was equivalent between songbirds and other birds . An obvious question is whether the influenza a viruses we detected belong to the highly pathogenic subtype h5n1 strain currently circulating across much of asia . Pcr (6), although this result does not exclude the possibility that h5 viruses were among the positive samples . The preservation status of samples we tested prevented virus isolation or full, strain - level characterization of influenza viruses . The subtype h5n1 strain of influenza virus has spread rapidly and has been detected across much of central and southern eurasia . Although movements of wild birds have been implicated in this spread (8), other studies question (9,10) or contradict (11) this idea . An important part of the argument centers on the question of the occurrence of the virus in wild birds without obvious illness, which can be difficult to interpret given the low prevalence of influenza . For instance, a recent study based on sampling> 13,000 migratory birds in china detected the subtype h5n1 strain of influenza virus only 8 times (12), and similar results have been obtained elsewhere (2). Our study, although not successful in characterizing influenza viruses to specific strains, nonetheless shows that influenza a virus infection occurs in more bird species than previously assumed and that influenza a infections can be found in birds that behave normally and show no sign of illness . Although a review of avian influenza virus ecology (1) discussed the occurrence of influenza viruses across all groups of birds (and other vertebrates), subsequent studies have assumed that waterbirds are the primary reservoir (8,13,14). In this study, although waterbirds could have higher prevalences, we have demonstrated broad occurrence of influenza viruses in diverse taxa of passeriformes (songbirds) in southeast asia . This result suggests that land birds may also be a major reservoir of influenza viruses . We have taken a step toward a more complete understanding of influenza virus ecology among wild birds . Our partial survey of influenza virus distributions across the rich avifaunas of the southern region of china demonstrated frequent infections . We suggest that to be effective future surveillance efforts will need to include the full diversity of wild birds.
Research into the pathogenesis of vitiligo has shown a complex of genetic and environmental factors . Endothelin-1 (edn1) and edn receptor type a (ednra) are implicated in the inflammatory process . Vitiligo is an acquired cutaneous disorder with a 0.52% incidence worldwide, and is characterized by a loss of melanocytes from epidermis . Although the etiopathogenesis has not been clearly elucidated yet, it was suggested that this common skin disease is a consequence of genetic and environmental factors . In the etiopathogenesis of the disease several mechanisms including autoimmune, autotoxicity, and neural mechanisms induced by biochemical and neurochemical mediators have been proposed . Previously, keratinocyte - derived endothelin-1 (edn1) has been shown to control melanocyte growth and function . It is a potent stimulant on melanocyte proliferation, melanogenesis, and migration; implying a link with vitiligo . Polymorphisms of edn1 and ednra genes have been investigated in autoimmune diseases such as hashimoto's thyroiditis (ht), graves disease, scleroderma, primary biliary cirrhosis, and psoriasis as well . There are only two studies in the literature examining the relationship between edn1 gene polymorphisms and vitiligo with controversial results . Kim et al . Have found that some haplotypes (gt and ag haplotypes of intron 4 g / a and 5665 g / t polymorphic loci) of edn1 gene are associated with vitiligo, whereas lan et al . Therefore, the aim of this study was to investigate both edn1 gene polymorphisms (g5665 t [or lys198asn] and t-1370 g) and ednra gene polymorphisms (c + 70 g and g-231a), in the etiopathogenesis of vitiligo . In addition, genotypes and clinical types of vitiligo found in these patients were also evaluated . All the patients and controls were recruited from a single center and all provided written informed consent . A total of 100 patients with vitiligo (53 women/47 men) vitiligo patients older than 18 years, showing any clinical picture except segmentary type and who were not on systemic or topical therapy during previous 2 months were included in the study . The control group consisted of 185, age and sex - matched, dermatology outpatients (95 women/90 men), with no past history of any systemic, infectious, autoimmune, genetic, or atopic disease . Peripheral venous blood samples were taken in the morning subsequent to an overnight (12 h) fast in edta - k3 tubes for genotype analysis . Genomic dna was isolated from peripheral blood leukocytes by using high pure polymerase chain reaction template preparation kit (roche diagnostics gmbh, mannheim, germany). We examined the g5665 t and t-1370 g polymorphisms in edn1 gene as well as c + 70 g and g-231a polymorphisms in ednra gene in our dna samples . These polymorphisms were selected according to the following criteria: previous association for susceptibility to other diseases, and adequate frequency in caucasian populations to perform the evaluation . For detection of the mentioned polymorphisms, the detection of polymorphisms was performed in a lightcycler (roche diagnostics, mannheim, germany). Snp in the edn1 and ednra genes all statistical analyses were performed with spss 15.0 for windows (ibm spss statistics, chicago, il, usa). Differences in genotype distributions and allele frequencies in the cases and the controls were compared using the chi - square test . Bonferroni correction is an adjustment made to p values when several dependent or independent statistical tests are being performed . The statistical significance for deviations from hardy weinberg equilibrium (hwe) was determined using the pearson chi - square test . Odds ratios (ors) were calculated and given with 95% confidence intervals (cis). Linkage disequilibrium (ld) and haplotype frequencies were estimated using the haploview 4.2 software (haploview, mit / harvard broad institute, ma, usa) and compared between cases and controls using a contingency chi - square test . In addition, the ncss 2000 statistical software (ncss, lcc, kaysville, utah, usa) was used to evaluate the power analysis . A total of 285 subjects (100 vitiligo and 185 controls) were included in this case control study . Table 2 depicts the clinical characteristics of the vitiligo patients including the clinical type of disease, duration, family history, leukotrichia, stability within 1 year, and associated diseases . The mean ages were 38.6 12.0 years (range 1876) and 38.2 9.7 years (range 1872 years) for patients and controls, respectively . There was no significant difference between the study and control groups in terms of mean age and sex distribution . We had 87% power to detect an effect size (w) of 0.20 using a 2 degrees of freedom (= 0.05). The genotypic and allelic distributions of edn1 (g5665 t and t-1370 g) and ednra (c + 70 g and g-231a) polymorphisms for patients and controls are shown in table 3 . The allelic frequencies of the studied polymorphisms found in our control population were similar to the results in turkish, english, and german populations . However, when the relationship between these polymorphisms and clinical characteristics of patients were evaluated, we found that ednra + 70 variant g allele and genotypes containing g (cg, gg and cg + gg) showed protective effects from presence of associated disease in patients with vitiligo [table 4]. When the bonferroni correction was performed, the significance still existed . In our vitiligo group, we designated a large spectrum of autoimmune diseases as follows: ht, type 1 diabetes mellitus, rheumatoid arthritis, alopecia areata, idiopathic thrombocytopenic purpura, and multiple sclerosis . In addition, g allele seems to protect against generalized type of vitiligo when compared to focal type (or: 0.42, 95% ci = 0.210.86, pcorr = 0.03 [table 5]). In addition, frequency of gg genotype was marginally lower in generalized type of vitiligo when compared to focal type (or: 0.18, pcorr = 0.057 [table 5]). However, there is no significant difference in the distribution of this allele between generalized and acrofacial types [table 5]. Edn1 - 1370 gg genotype was 5-fold more in vitiligo patients with leukotrichia in comparison with healthy controls which may be seen in table 6 . Characteristics of the patients with vitiligo distribution of genotypes and allele frequencies for patients with vitiligo and control group distribution of ednra c + 70 g genotypes and allele frequencies for patients with vitiligo: impact of presence of associated disease distribution of ednra c + 70 g genotypes and allele frequencies for patients with focal, acrofacial and generalized type of vitiligo distribution of genotypes and allele frequencies for vitiligo patients with leukotrichia and control group levontin's standardized disequilibrium coefficient (d) was calculated as a measure for ld between the studied polymorphisms in the edn1 and ednra genes [figure 1]. Edn1 5665 and -1370 were found to be in strong ld (d = 0.73, r = 0.405). In addition, a weak ld between ednra 231 and + 70 was found (d = 0.22, r = 0.032). The most frequent haplotype among the patients and controls were edn1 5665/-1370 gt and ednra 231/+70 gg . The edn1 5665/-1330 tt haplotype was significantly overrepresented in controls than in patients (p = 0.04). There was strong linkage disequilibrium between endothelin-1 5665/-1370 (d = 0.73, r = 0.405); and weak linkage disequilibrium between endothelin receptor type a 231/+70 (d = 0.22, r = 0.032) haplotype analysis of the edn1 and ednra polymorphisms in patients with vitiligo and control subjects edn1 is a well - known peptide for its role in epidermal hyperpigmentation by signaling mechanisms of mitogenesis and melanogenesis . Previously, the keratinocyte - derived edn1 has been shown to be decreased in vitiligo . Furthermore, edn1 was found to be increased in patients undergoing puva treatment indicating a role of this peptide in vitiligo pathogenesis . Besides, edn1 is a strong vasoconstrictive agent which may contribute to controlling the inflammatory process seen in vitiligo . Indeed, blocking the edn1/ednra signaling resulted in the loss of pigmentation in vivo . However, the exact role of edn1 in vitiligo has not been clearly elucidated yet . Edn1 gene polymorphisms have been investigated and incriminated in the etiopathogenesis of various diseases before . The mutant t allele of a g5665 t polymorphism in edn1 gene was found as a risk factor for some diseases and responsible for disease progression in others . Some studies were shown that the presence of t allele was associated with higher edn1 levels, raised systolic blood pressure, and predisposed patients to preeclampsia, suggesting the functional importance of this polymorphism in pregnancy . Confirmed the impact of the g5665 t polymorphism on vascular reactivity . In the present study, the lack of association between g5665 t with ht is also noteworthy since ht is a close counterpart of vitiligo . Two former studies investigating the role of g5665 t polymorphism in vitiligo did not show a significant relationship as well . Considering the potential regulatory effects of promoter region polymorphisms on gene expression and plasma protein levels, we also studied t-1370 g polymorphism which is located in the promoter region of edn1 gene . Recently, it has been shown that the edn1 mrna and the secreted edn1 levels in primary osteosarcoma hcell culture obtained from tt homozygotes were significantly higher than those from gg homozygotes . Interestingly, the gg genotype frequency of t-1370 g polymorphism, related with the low production of edn1, was increased 5-fold in vitiligo patients with leukotrichia in comparison to controls . However, this subset of patients is relatively therapy resistant, and this information may be valuable in determining therapy options . However, in the present study we did not detect a relationship between this polymorphism and vitiligo in general . Lack of association between g5665 t and t-1370 g polymorphisms and vitiligo indicates that these polymorphisms alone may not play a major role in the pathogenesis of this autoimmune disease . On the other hand, our haplotype analysis revealed that there was strong ld between g5665 t and t-1370 g polymorphisms . The edn1 5665/-1370 tt haplotype was significantly less presented in vitiligo patients in comparison with controls . T alleles in both 5665 and -1370 are known to be related with the high production of edn1 protein which causes melanocyte proliferation and melanogenesis . Actually, many studies have shown that vitiligo skin expresses low levels of edn1 . Consequently, our haplotype results are in line with what is expected; namely, high levels of edn1 means increased amount of melanin protection against vitiligo . Lan et al . And kim et al . Also did haplotype analyses between g5665 t and two different intronic polymorphisms in edn1 gene (intron 2 a / g and intron 4 g / a, respectively). Lan et al . Could not find a significant difference between cases and controls in haplotype frequencies of g5665 t and intron 2 a / g . However, kim et al . Reported that gt and ag haplotype frequencies at intron 4g / a and g5665 t loci of edn1 gene in focal and segmental clinical types differed significantly from healthy controls . These findings suggest that edn1 polymorphisms may not be a direct risk factor alone, but their presence, especially with g5665 t may contribute as additional / cumulative factors in the development of vitiligo . Regarding polymorphisms of ednra gene, the functional consequence of ednra g-231a and c + 70 g polymorphisms are unknown, since the functional studies are not yet available . The c + 70 g and g-231a polymorphisms of edra gene are within the 3-untranslated region (utr) and the 5- utr of the gene, respectively . 3- utrs of eukaryotic mrnas are usually implicated in the posttranslational regulation and have a pivotal role in message stability . The functional consequences of 5- utr polymorphisms might be related to the regulatory sequences of gene transcriptions and associated with altered gene expression . In addition, such polymorphisms may create novel splice sites influencing the function of the receptor and receptor - ligand interactions . Therefore, these polymorphisms could have significant effects on mrna expression and message stability, and thus affecting the total amount and activity of the functional receptor . On the other hand our study showed the variant g allele of ednra + 70 polymorphism was protective against the generalized type of vitiligo . It was previously demonstrated that that carrying variant allele protects also from early onset disease of ht . We think it may be possible that + 70 wild c allele carrying receptors bind their ligands with decreased affinity, resulting in at least weakening of signals at the membrane level, or slowing internalization of signal in the melanocytes with subsequent ceasing of melanocyte proliferation or melanocyte destruction . In another study, examining the differences of edn1 and ednra expressions, aly et al . However, in that study the patients were few in numbers (n = 10) and we think a larger number of cases and advanced methods of evaluation could show different results . Altogether, it can be concluded that ednra does not contribute to vitiligo process, rather its + 70 g polymorphism is protective against both generalized type of vitiligo and associated diseases . Although most vitiligo patients are otherwise healthy, vitiligo may be associated with a large spectrum of autoimmune diseases . This is one of the few studies examining the edn1 and ednra polymorphisms in vitiligo . Our results suggest that edn1 (g5665 t and t-1370 g) and ednra (c+70 g and g-231a) polymorphisms may not have a causative effect in vitiligo . Furthermore, ednra + 70 polymorphism which is protective against generalized type of vitiligo and associated diseases, may indicate that there should be other causes, yet unknown, disrupting the normal function of keratinocyte - melanocyte interaction, in the course of vitiligo . The studied endothelin.1 (edn1) (g5665 t and t.1370 g) and edn receptor type a (c+70 g and g-231a) polymorphisms may not have a causative effect in vitiligo . In fact, haplotype analyses denoting protective effects against generalized vitiligo and associated diseases suggest that there should be other causes disrupting melanogenesis in the course of vitiligo . The studied endothelin.1 (edn1) (g5665 t and t.1370 g) and edn receptor type a (c+70 g and g-231a) polymorphisms may not have a causative effect in vitiligo . In fact, haplotype analyses denoting protective effects against generalized vitiligo and associated diseases suggest that there should be other causes disrupting melanogenesis in the course of vitiligo . There are no conflicts of interest . The studied endothelin.1 (edn1) (g5665 t and t.1370 g) and edn receptor type a (c+70 g and g-231a) polymorphisms may not have a causative effect in vitiligo . In fact, haplotype analyses denoting protective effects against generalized vitiligo and associated diseases suggest that there should be other causes disrupting melanogenesis in the course of vitiligo.
Papillary and follicular carcinomas of the thyroid gland, commonly referred to together as differentiated thyroid carcinoma (dtc), are characterized by a slowly progressive course, and a 10-year survival rate as high as 80 - 95% . Distant metastases are seen in a minority of the patients and the reported rates of occurrence range from 4% to 15% . The most common site of distant metastases is the lung, followed by the bone . We present a case of asymptomatic radioiodine - avid renal and brain metastases presenting 20 years after hemi - thyroidectomy for adenomatous goiter and identified with i - whole body scintigraphy (wbs) and i single photon emission computed tomography / computed tomography (i - spect / ct). A 65-year - old man, investigated at a different hospital for intermittent cough and white - colored expectoration for 4 months, was found to have multiple bilateral nodular lesions on a chest x - ray [figure 1a] and a hyperdense lesion (arrow) in the right posterior parietal lobe of the brain on ct [figure 1b]. He had undergone left hemi - thyroidectomy 20 years previously, reportedly for adenomatous goiter . Since serum thyroglobulin (tg) was> 3000 ng / ml, a provisional diagnosis of metastatic dtc was made . However, abdominal ultrasonography (usg) showed multiple echogenic round lesions with areas of necrosis and internal vascularity in both kidneys . (a) chest x - ray showing multiple nodular lesions in both lungs and (b) computerized tomography of the brain showing a hyperdense lesion (arrow) in the right posterior parietal lobe i wbs [figure 2a] and spect / ct [figure 2b and c] performed 48 h after oral administration of 2 mci i showed tracer uptake in the head, neck (residual thyroid), bilateral hilar lymph nodes, right third and fourth ribs with lytic changes, lung nodules, s1/s2 spinal canal, right femur, acetabulum and humerus, and multiple exophytic renal nodules . Usg - guided biopsy of a left renal nodule revealed only thyroid follicular cells (positive for tg immunostaining) with no pathological evidence of malignancy . Postoperative investigations after 6 weeks showed tg> 3000 ng / ml, anti - tg 30.1 iu / ml, and thyroid stimulating hormone 14.28 iu / ml . He was treated with 207 mci of i under steroid cover (prednisolone 40 mg / day). I whole body scan (a) 48 h after administration of 2 mci 131i showing increased tracer uptake at multiple sites in the neck, chest, abdomen, pelvis and right thigh . A small focus of radioiodine uptake is seen on the right side of the head posteriorly (arrow), adjacent to a known focus of tracer contamination (arrowhead). Single photon emission computed tomography / computed tomography of the neck and chest; (b) localizes tracer uptake to residual thyroid, bilateral hilar lymph nodes, right 3rd and 4 ribs with lytic changes and multiple lung nodules . Single photon emission computed tomography / computed tomography of the abdomen; (c) shows tracer uptake in multiple exophytic renal nodules a fifth - day posttherapy whole body planar and spect / ct scan [figure 3a and b] confirmed metastasis in the brain (arrow) and other sites seen in the pretherapy scan . After an uneventful hospital stay, he was discharged on a suppressive dose of thyroxin (225 g / day). Whole body planar (a) and single photon emission computed tomography / computed tomography (b) scans 5 days after 207 mci 131i showing increased tracer uptake in the brain (arrow), d11 vertebra, sacrum, and right acetabulum, in addition to residual thyroid and lungs distant metastases from dtc may present after a latent period of many years . Around 10% of the patients with dtc present with multiple sites of distant metastases other than lung, bone, and lymph nodes, 50% of which involve the brain, 25% the liver, and 25% other sites . The reported incidence of renal involvement from a primary thyroid malignancy is very rare, being only about 4.5 - 5.9%, whereas of all secondary metastases to the kidneys from all cancers, thyroid cancer constitutes only about 2.5 - 2.7% . So far, most of the subjects were female and older than 45 years of age, whereas our patient was an elderly male . Renal metastasis usually appears in the setting of multifocal metastases, as in the present case . However, in our patient, no pathological evidence of malignancy was identified in any of the surgically resected thyroid specimens . It is possible that malignancy (most likely follicular thyroid carcinoma) might have escaped detection in the left hemi - thyroidectomy specimen 20 years earlier; a similar case has been reported previously . Apart from a recent onset of cough, our patient was asymptomatic in the presence of renal as well as bone and brain metastases . Metastases from dtc may develop several years (occasionally decades) after the removal of the primary thyroid malignancy . Furthermore, they may remain undetected for a long time if no radioiodine intervention is done . Since multiple iodine avid lesions were seen throughout the body on planar scintigraphy, a whole body spect / ct scan was performed for lesion localization . This helped in precisely localizing abnormal radioiodine uptake in the hilar nodes, lung nodules, kidneys, bone, spine, and brain . . However, our patient showed radioiodine uptake in the brain metastasis in the presence of radioiodine - avid residual thyroid, renal, and other metastases, which is extremely uncommon . Metastases to the brain are observed in only about 1% of the thyroid carcinomas and, in one study, 23% of the brain metastases were discovered only at autopsy . The median interval between the diagnosis of dtc and that of brain metastasis was 6.5 years . However, the actual number of reported cases of brain metastases from thyroid carcinoma remains small, and there has been little evidence of benefit from any specific mode of therapy . The median disease - specific survival from diagnosis of brain metastases was 16.7 months for patients who underwent local excision of one or more brain metastases, compared with 3.4 months for those who did not (p <0.05). Our patient had metastatic involvement in multiple organs, which were not amenable to surgery . The patient's condition improved symptomatically with absence of any cough during the 2 months following iodine treatment; he is currently doing well under regular follow - up . To conclude, metastatic dtc should be suspected in asymptomatic patients with incidentally detected lesions, raised serum tg and history of thyroid lesions.
The prosite database uses two kinds of signatures or descriptors to identify conserved regions, i.e. Patterns and generalized profiles, which both have their own strengths and weaknesses defining their area of optimum application . Each prosite signature is linked to an annotation document where the user can find information on the protein family or domain detected by the signature: origin of its name, taxonomic occurrence, domain architecture, function, 3d structure, main characteristics of the sequence, domain size and some references . As a more detailed description of the prosite database has already been provided in previous publications (1,2), this paper will only focus on recent developments that have taken place during the last 2 years . Patterns or regular expressions are useful tools to identify short and well - conserved regions, such as catalytic sites, binding sites, post - transcriptional modifications (ptms) or zinc fingers . They are also easy to construct and to use by biologists that have no knowledge in bioinformatics . If a new sequence has an amino acid at a conserved position that was not present in the seed alignment used to construct the pattern, it will not be recognized . Thus, patterns need to be updated regularly to introduce this new variability in the regular expression . This tool uses the prosite match list, which stores true positives, false positives (fp), false negatives (fn), partial and unknown matches, to generate a new pattern that minimizes fp and fn . We first take care of fn in a three - step procedure: the patterns that can potentially be updated are selected . Updating a pattern to recover fn amounts to introduce more variability in the pattern, but it increases the risk of creating new fp . Hence the selection procedure consists of running all prosite patterns on a random database to keep only the ones that do not produce too many matches.mismatches produced by each fn are detected and the pattern is modified accordingly to accept the observed residues.the new pattern is tested on a random database to see whether it is still stringent enough . If it produces too many matches in a random database, the pattern is refined and some mismatch positions are removed . Updating a pattern to recover fn amounts to introduce more variability in the pattern, but it increases the risk of creating new fp . The selection procedure consists of running all prosite patterns on a random database to keep only the ones that do not produce too many matches . Mismatches produced by each fn are detected and the pattern is modified accordingly to accept the observed residues . The new pattern is tested on a random database to see whether it is still stringent enough . If it produces too many matches in a random database, the pattern is refined and some mismatch positions are removed . To remove false positives we check we look at these positions for amino acids that are only found in fp sequences . Forbidden ({} with the prosite syntax) at these positions in the new pattern . The new pattern is then used to scan swiss - prot and all new matches are checked manually . Only patterns that produce no new false positives are kept . This strategy has allowed the automatic update of 943 patterns (out of a total of 1322 patterns in prosite). 2661 fn (out of a total of 14 412) and 1927 fp (out of a total of 7446) were removed . We have also removed the less specific patterns that could not be updated and have replaced them by profiles . The application of these two strategies allowed a decrease of the number of fp and fn in the swiss - prot part of uniprot by 25% . When a signature identifies a conserved region in a given protein, it is important to know what functional information can be transferred to this new protein according to what is known about the function of the conserved region . If the information that is transferred is very general (name and position of a given domain in a sequence) only the occurrence of a match with a descriptor at a reliable score is enough . But descriptors can supply much more precise information . If one looks at the residue level, functional sites such as active sites, disulfide bridges or ptm sites can be identified . One can also look at the domain arrangement to discriminate between particular families or sub - families . Prosite has a long experience in documentation and detailed annotation of domains, families and functional sites . This information is mainly stored in free text and used by biologists who read the various documents and make their own decision on the function of their protein according to the prosite matches . But with the rapid growth of sequence databases, there is an increasing need for a reliable tool that can generate automatically precise and accurate functional annotation in standard format . We thus decided to group some functional information stored in prosite in a database of rules that can easily be read by a program and applied on proteins that are recognized by prosite profiles . Prorule generates annotation in swiss - prot format for de, cc, kw or ft lines . Two types of information are stored in prorule: general information: the occurrence of a match with a profile is enough to trigger this annotation . Usually, it is restricted to the name of the domain and the position of its boundaries.conditional information: this is dependent on the presence of given amino acids at precise positions, on the occurrence of other domains or on taxonomic specificity . For example, an enzymatic active site is annotated only if the correct amino acid is found at the required position . General information: the occurrence of a match with a profile is enough to trigger this annotation . Usually, it is restricted to the name of the domain and the position of its boundaries . Conditional information: this is dependent on the presence of given amino acids at precise positions, on the occurrence of other domains or on taxonomic specificity . For example, an enzymatic active site is annotated only if the correct amino acid is found at the required position . Prorule is extensively used by swiss - prot curators to facilitate the annotation work and to check the consistency of swiss - prot entries . But it can also be accessible for external users through the scanprosite web page (see below) or downloaded from the prosite ftp site under prosite license conditions . For more details on prorule and its range of application the prosite website was redesigned and new predictive tools were implemented to assign more detailed functional information to the scanned proteins . Users who want to scan their own proteins against all prosite entries or to scan a prosite entry against a protein database will find a new version of the scanprosite web page . Prosite matches on uniprot knowledgebase (uniprotkb) or pdb entries are now pre - calculated and stored in a relational database (postgresql) that is maintained in collaboration with swiss - prot (4). Each prorule associated with a prosite profile is also scanned, which allows the localization of interesting functional residues such as active sites, ptms and disulfide bridges . These features are only shown if the expected amino acid is found at the right position . But we also indicate missing features when we expect another amino acid at a given position . This tool can be used to identify divergent subfamilies of proteins like inactive enzymes . In figure 1a, we show the scanprosite output for the human ephrin b4 receptor, which is a functional kinase receptor (5), and its paralogue the ephrin b6 receptor, which is known to have an inactive kinase domain (6). The scanprosite output indicates that the expected asp residue was not found at the position of the active site in ephrin b6 receptor . To test the efficiency of the method we used scanprosite to identify all mammalian homologues of the ephrin b6 receptor and to construct a multiple sequence alignment (msa) of this subfamily (figure 1b). The msa also shows that the conserved asp residue of the active site is found in none of the ephrin b6 receptor orthologues . Scanprosite can also be used to identify new uncharacterized subfamilies of putatively inactive enzymes (figure 1c). From the scanprosite web page, we have searched with the kinase profile (ps50011) for plant proteins that have no detected active site and a common domain arrangement . We have identified an uncharacterized family of putatively inactive kinases, which is conserved in various plant genomes as it is shown in the msa . The documentation page has also been reorganized . It now contains three main sections: the description part that exposes the main characteristics of the domain or the family and a representative list of proteins that contain the domain or belong to the family.a technical section that refers to the descriptors used to identify the domain or family . For each descriptor, there is a link to a domain architecture view of uniprotkb proteins matched by the descriptor, an msa in different formats, a link to retrieve the list of proteins matched by the descriptor in various formats and a link to a taxonomy tree view of all entries containing the domain . There is also an external link to msdsite (7) to view ligand binding statistics of the domain and a link to 3d structures.the third section is the reference block where, for each reference, we added the pubmed i d and a direct link to the article . The description part that exposes the main characteristics of the domain or the family and a representative list of proteins that contain the domain or belong to the family . A technical section that refers to the descriptors used to identify the domain or family . For each descriptor, there is a link to a domain architecture view of uniprotkb proteins matched by the descriptor, an msa in different formats, a link to retrieve the list of proteins matched by the descriptor in various formats and a link to a taxonomy tree view of all entries containing the domain . There is also an external link to msdsite (7) to view ligand binding statistics of the domain and a link to 3d structures . The third section is the reference block where, for each reference, we added the pubmed i d and a direct link to the article . The architecture view of prosite profiles is now visible, from each uniprotkb entry on the expasy server, from the prosite documentation page and from the scanprosite web page . In each view, some interesting residues are tagged according to prorule predictions (see figure 1a). All these tools were designed to recover very divergent proteins (<20% of similarity). They were developed 10 years ago when protein databases were quite small and very few representative genomes were sequenced . There was thus a strong sample bias when constructing seed alignments and profile tools that used these seeds needed to be strongly predictive . Currently, proteins databases are 10 times bigger and thousands of genomes have been sequenced spanning the whole tree of life . It is now easier to have a seed alignment with representatives of all possible variability and descriptors can be more conservative . There is rather an increasing need for more specific descriptors in order to have more precise functional information . As we described previously, specific annotation can be assigned to a match with a profile when looking in the matched region at the residue level for the presence of particular amino acids at particular sites, such as enzymatic active sites, disulphide bridges, etc . We thus have developed a new strategy to annotate the msa at these particular sites and to transfer this information to the profile builder program . We have used this strategy to adjust specific parameters in a column - dependant manner . The tool aim is to be more stringent on specific columns and to produce a better local alignment, which then helps to re - localize the functional residues in sequences matched by the profile ., commercial users should contact: the swiss institute of bioinformatics by email: license@isb-sib.ch or its commercial representative: geneva bioinformatics (genebio) s.a, case postale 210, ch-1211 geneva 12, switzerland, tel: + 41 22 702 99 00, fax: + 41 22 702 99 99, email: info@genebio.com . Weekly updates of prosite are available on our ftp server: .prosite is also accessible from the hits page: .frame - tolerant scans can be performed at the following address: . The left protein is a classical ephrin receptor protein (ephrin b4 receptor protein) which is known to transduce a signal through its kinase domain (5). The right protein is also an ephrin receptor protein (human ephrin b6 receptor protein) but with an inactive kinase domain (6). The prorule associated with the kinase domain identifies an active site in ephrin b4 receptor but not in ephrin b6 receptor (absent feature: active site). (b) we used scanprosite to identify orthologues of the ephrin b6 receptor in mammals, searching for proteins that have the same domain arrangement and have a putative inactive kinase domain . (c) we also identified with scanprosite an uncharacterized plant subfamily of kinase receptors with a putative inactive kinase domain . Both multiple sequence alignments were generated on the scanprosite web page using the alignment with the kinase profile (ps50011).
Periodontitis (pd) is a chronic inflammatory disease where resident cells and preformed mediators induce leukocyte infiltration and progressive destruction of the tooth supporting tissues as a result of interaction between bacterial products, cell populations, and mediators in disease - susceptible individuals [1, 2]. This is also influenced by genetic and environmental risk factors and is characterized as a complex disease with multifactorial etiology [3, 4]. In this context, environmental factors, including oral hygiene / bacterial plaque, smoking, and stress, play an important role in the expression of pd . Furthermore, it has been evidenced by some authors that there is a joint influence of polymorphisms in multiple genes, such as the genes of il-10 and il-6 . Polymorphonuclear neutrophils (pmns) represent the first line of defense to protect the host from periodontal pathogens in the gingival sulcus and junctional epithelium . Pmns are a critical arm of defense against periodontitis, but bacterial evasion of the neutrophil microbicidal machinery coupled with delayed neutrophil apoptosis may transform the neutrophil from defender to perpetrator . Actually, these cells can release a variety of factors, such as reactive oxygen species, collagenases, and other proteases, [1, 9], such as stimulation from a wide range of cytokines . In this scenario, macrophages can act as antigen - presenting cells, promoting the activation of lymphocytes . Therefore, the cellular concentration of neutrophils in the inflammatory infiltrates decreases during the transition between gingivitis and periodontitis, in which there is a predominance of lymphocytes . It has been described that proinflammatory cytokines, prostaglandin e2, matrix metalloproteinase (mmp), nitric oxide (no), and other inflammatory mediators play a crucial role in the pathogenesis of pd [1012]. Moreover, an increase of tnf-, il-1, il-6, il-11, and il-17 can induce osteoclastogenesis by increasing the expression of receptor activator of nf-b ligand (rankl) and by reducing the osteoprotegerin (opg) production in osteoblasts and stromal cells . In fact, it was demonstrated that il-17 and rankl were overregulated and il-10, an anti - inflammatory cytokine, and tgf-1 were downregulated in active periodontal lesions compared with inactive lesions [14, 15] (figure 1(a)). Considering that an imbalance between bone formation and resorption is also linked to various diseases, studies suggest that pd may be a risk factor for other diseases, such as rheumatoid arthritis (ra), but without consensus . Although pathogenesis of ra is not completely understood, it is recognized that the activation of the complement system is important in disease development, the abnormal response of circulating lymphocytes from patients, and an alteration in the structure of these cells, which contribute to the autoimmunity, immunosuppression, and the genesis of the disease . Studies report there is a correlation between both pd and ra since the mechanisms for the development of ra have consonance with the pathogenesis of chronic pd . In fact, ra is defined as an inflammatory and autoimmune disease characterized by accumulation of leukocyte inflammatory infiltrate in the synovial membrane, as well as mediators such as pge2, tnf-, il-1, il-6, il-12, il-17, il-18, il-33, granulocyte macrophage colony - stimulating factor (gm - csf), monocyte colony - stimulating factor (m - csf), rankl, mmps, and no, all being found in the synovial fluid [2024], and leading to synovitis and joint architecture destruction . Some studies have suggested that the susceptibility of ra may be associated with genetic or environmental factors . One of the most important genetic factors is the human leukocyte antigen (hla) class ii . Certain alleles of this antigen are often associated with the development of rheumatoid arthritis (hla - drb10101, hla - drb10102, hla - drb10401, hla - drb10404, hla - drb10405, hla - drb10408, hla - drb10410, hla - drb11001, and hla - drb11402). Other factors include the allele of 620w of ptpn22 (protein tyrosine phosphatase nonreceptor type 22), a gene encoding tyrosine phosphatase that is involved in controlling the intracellular signaling triggered through t and b receptors; c5-traf1, which can interfere with disease susceptibility and severity of the alteration in the structure, function, and levels of complement component c5/factor 1 associated with the tnf receptor; gene encoding the ctla4 (cytotoxic t lymphocyte antigen-4), the protein responsible for the regulation of t lymphocyte activation; peptidylarginine deiminase (pad2), the enzyme responsible for the generation of citrullinated proteins, which are related to the formation of anticyclic citrullinated peptide autoantibodies (figure 1(b)). With regard to environmental factors, smoking is a risk factor that duplicates the risk of developing ra, but its effect is limited to those with antibodies to citrullinated peptides [30, 31]. Other factors refer to the excessive consumption of coffee (more than 10 cups daily) which can be related to the development of the disease and bacterial microbiota, including oral bacterial species which can participate in the etiopathogenesis of ra . On the other hand, the intake of alcohol may exert a protective effect in rheumatoid arthritis in a dose - dependent manner . The literature shows that the basic difference between both diseases is that ra is an inflammatory autoimmune disease, while pd is an immunoinflammatory disease of bacterial origin . However, it is noteworthy that many epidemiological studies seem to dilute the subtle differences expressed by some parameters, though clinically important . Indeed, analyses of inflammatory mediators and other molecular markers are examples where the differences found in a trial with few participants could disappear in a large and diverse sample . In this sense, this review is a critical appraisal of studies that address potential associations of periodontitis with ra and with an overall comprehensive approach . For this review, the us national library of medicine national institutes of health pubmed was searched by two independent researchers who agreed with the search criteria of studies with patients with both pd and ra and checked by a third researcher separately . The keywords periodontitis and rheumatoid arthritis were used and 367 articles published in english were found . The time period was limited from january 2012 to march 2015, and 162 references were found . Then, a critical reading based on titles and abstracts was made and 136 papers were excluded, such as reviews, assays in vitro and animal studies, articles that were not in english, studies not related to both pd and ra, case study, workshop, or unavailable and incomplete articles . Then, 26 articles were finally included for this review, which related to pd and ra, considering epidemiological aspects, mechanical periodontal treatment, mediators of inflammation, oral microbiota, and antibodies as seen in figure 2 . Table 1 shows demographic data, such as gender, age and habits, comorbidities and medications, and the relationship between both diseases investigated through clinical and epidemiological associations, presence of oral bacterial dna in patients with ra, proinflammatory mediators, antibodies against bacteria, and autoantibodies, as well as the effects of mechanical periodontal treatment, related to the 26 selected articles . In most articles (92.3%), the analyzed groups were mainly composed of women . Regarding age, most patients were 40 years old, except for the study of dev et al . (2013) and ranade and doiphode (2012), whose patients were above 20 and 30 years old, respectively . Among the 26 articles, 57.7% [3436, 38, 39, 42, 5057, 59] used samples with smoker patients, while 30.8% established smoking as criteria for excluding [37, 41, 43, 4549]. 11.5% did not mention smoking status of patients [40, 44, 58]. Comorbidities such as diabetes, sjgren's syndrome, hypertension, cardiovascular disease, hyperlipidemia, renal disease, and osteoporosis / osteopenia have only been reported in studies of mikuls et al . 50% [3537, 40, 41, 44, 45, 49, 50, 52, 53, 55, 58] of the articles did not specify the pharmacological treatment . In the remainder of the studies, the most frequently reported treatment for rheumatoid arthritis included disease - modifying antirheumatic drugs (methotrexate, sulfasalazine, and leflunomide) [38, 39, 42, 43, 46, 51, 54, 56, 57, 59], biologic therapy (anti - tnf-) [34, 38, 39, 42, 59], corticosteroids (prednisolone) [38, 42, 43, 46, 51, 54, 56, 59], and/or nonsteroidal anti - inflammatory drugs [43, 4648, 51, 54, 57]. Among the selected trials, eight studies broached the epidemiological and clinical relationship of patients with pd and ra [38, 41, 44, 45, 49, 54, 56, 59], indicating a higher prevalence of pd in patients with ra, which have worse periodontal parameters . The effect of mechanical removal of foci of infection in the oral cavity on the severity of ra and periodontal clinical parameters were shown by four studies [37, 42, 46, 51], which demonstrated the beneficial effects of the mechanical treatment in the improvement of clinical parameters of ra . Two studies were related to the oral bacteria influence of the pathogenesis of ra [40, 52]. Seven trials highlighted the presence of citrullinated proteins and their antibodies, antibodies to p. gingivalis in patients with ra and periodontitis, and also the association between anti - p . Gingivalis and periodontal parameters, and the titers of rheumatoid factor and antibodies anticyclic citrullinated peptide, which were also related to the severity of pd [35, 36, 39, 50, 53, 55, 57]. Regarding the inflammation in both diseases, five trials considered the mediators of inflammation to the pd and ra [34, 43, 47, 48, 58], such as mmp-9, tnf-, il-17, rankl, and opg . Considering the relationship between rheumatoid arthritis and periodontitis, only two articles showed no statistical significant association, while 24 studies have established this association, either by descriptive (3 studies) or statistical analysis (21 studies). In this review, demographic data and other aspects that can modify one or both diseases were presented, as well as the relationship between both diseases investigated through clinical and epidemiological associations, effects of mechanical periodontal treatment, presence of oral bacterial dna in patients with ra, proinflammatory mediators, antibodies against bacteria, and autoantibodies . This aspect was interesting, as a possible relationship between female sex hormones and susceptibility of rheumatoid arthritis had been reported in the literature, so that low levels of those hormones at menopause promote the risk of developing the disease early . However, a protective role of oral contraceptives on the risk for rheumatoid arthritis in women is still controversial [6163]. On the other hand, there is strong evidence that estrogen deficiency influences the severity of periodontitis, since worse periodontal parameters were observed as bleeding on probing, gingival recession, and clinical attachment loss in postmenopausal women with osteoporosis . Cigarette smoking is considered an important risk factor for the development of rheumatoid arthritis, since it was demonstrated that lifelong cigarette smoking was positively associated with the risk of ra even among smokers with a low lifelong exposure . Moreover, it has been related that smoking interacts with hla - dr se genes and increases the risk of anti - ccp antibodies in patients with rheumatoid arthritis . Regarding the periodontium, it was shown that smokers presented greater probing depths, when compared to the probing depths of patients who never smoked . The literature shows that pd does not usually require pharmacological treatment, except for mechanical periodontal treatment as routine . In this review, this fact was also observed, while half of the studies had shown that rheumatoid arthritis involved some pharmacological approach . The use of disease - modifying antirheumatic drugs (dmards) aims to reverse the symptoms of the disease, reduce the progression of joint damage, and consequently improve the quality of life of patients . The conventional synthetic dmards include methotrexate, sulfasalazine, and leflunomide; the available tumor necrosis factor inhibitors (adalimumab, etanercept, and infliximab), the t cell costimulation inhibitor (abatacept), the anti - b cell agent (rituximab), and the interleukin-6 receptor blocking monoclonal antibody are included in biological dmards . These medications may be associated with glucocorticoids (gc) or nonsteroidal anti - inflammatory drugs (nsaids). The long - term, low - dose glucocorticoid and nsaids therapy were shown to reduce joint symptoms, pain, and other systemic manifestations [70, 71]. Although these benefits are present, the long - time treatment with gc and methotrexate decreased immune response and promoted oral changes, such as candidiasis, periodontitis, and oral ulceration besides impaired saliva secretion . Indeed the literature demonstrated that patients on corticosteroids exhibited higher levels of candidiasis, clinical attachment loss, and probing pocket depth . These aspects, at least in part, may contribute to the worse periodontal status of ra patients when compared to healthy patients . Moreover, the use of medications referred to in half of the articles could compromise the evaluation of this review . However, it is noteworthy that the other half of the articles did not use any medication [3537, 40, 41, 44, 45, 49, 50, 52, 53, 55, 58]. Analysing the articles, it was observed that most patients with ra showed a significant increase in the incidence of pd as compared to healthy individuals, while only few articles concluded the opposite, probably due to the lack of standardization of parameters in evaluating the different types of periodontitis . Although epidemiological studies outlined by dev et al . (2013) have not observed a significant ra incidence in subjects with periodontitis where these authors suggested that periodontitis is an independent factor for ra, several other studies have shown that patients with ra were more susceptible to the development of periodontitis [38, 44], since these patients had worse periodontal parameters, such as clinical attachment level [37, 56], alveolar bone loss [56, 59], probing depth [37, 49], plaque index, and bleeding on probing [37, 41, 54]. Indeed, the mechanical periodontal treatment as scaling and root planning in the control of periodontal infection interfered not only with the severity of ra but also with the periodontal clinical parameters . This result can be explained by a reduction in the foci of oral bacteria, and therefore the low levels of inflammation demonstrated a decrease of das28 (disease activity score in 28 joints) and serum levels of il-1, tnf-, c - reactive protein, and erythrocyte rate sedimentation [37, 42, 46, 51]. In this sense, studies have defended the hypothesis that oral infections play an important role in the pathogenesis of ra, promoting the citrullination of proteins, which can be based on the detection of bacterial dna using the techniques of dna isolation (pcr and dna - dna hybridization) and high titers of antibodies against bacteria in synovial fluid and serum samples from patients with ra [40, 51, 52]. Most of the studies have shown the presence of oral bacteria in patients with ra, highlighting p. gingivalis and f. nucleatum [40, 52]. Markedly, p. gingivalis is the most elucidated in the development of ra, and studies using animal models have demonstrated the potential of this proinflammatory bacterium promoting the development of experimental arthritis and increased serum levels of c - reactive protein, tnf-, il-1, il-17, mmp-13, and rankl . Furthermore, ra is an autoimmune disease characterized by autoantibodies specific for citrullinated peptide antigen (anticyclic citrullinated peptide), which are synthetized by peptidylarginine deiminase and characterized as the most specific markers for the diagnosis of the disease [76, 77]. Considering that the p. gingivalis is regarded as being capable of expressing this enzyme (pad), it is suggested that infection with this microorganism could influence the pathogenesis of ra [78, 79]. These citrullinated proteins were also found in periodontal tissues, indicating a link between these peptides generated in the oral cavity and those observed in articular tissues [36, 80]. Additionally, the presence of antibodies to p. gingivalis was investigated . (2015) have not detected this, antibody titres significantly differ between early rheumatoid arthritis and healthy controls . Other studies observed the antibodies to p. gingivalis in patients with ra and severe periodontitis and were associated with probing depth and clinical attachment level and the titers of rheumatoid factor and anticyclic citrullinated peptide autoantibodies [35, 50], which may be found in patients with ra and related to the severity of periodontitis . In summary, the studies suggested that p. gingivalis might play a role in the pathogenesis of ra . The response in periodontitis was related to uncitrullinated peptide, suggesting that these peptides break tolerance and can be involved in pathogenesis of ra (figure 1(c)). Most of the studies have found high levels of proinflammatory cytokines and other mediators of inflammation, such as mmp-9, tnf-, il-17, rankl, and opg . Moreover, it was demonstrated that the hypomethylated status, a single region of the il-6, may contribute to elevated serum levels of this cytokine, implying a role in the pathogenesis of these diseases, while the anti - inflammatory cytokines in the gcf, such as il-4 and il-10, showed no consensus among studies regarding the differences observed among individuals with pd and ra . In addition, hypotheses have been proposed to explain the relationship between periodontitis and systemic diseases, such as rheumatoid arthritis . In the literature, studies have suggested that chronic periodontitis generates local constant high levels of microparticles, which have been considered inflammatory biomarkers or mediators responsible for distant cell signalling and regulation . Moreover, it has been reported that these microparticles play an important role in thrombosis and angiogenesis and mediate cellular communication by transferring mrnas and micrornas from the cell of origin to target cells . Thus, the microparticle participation and its spread into the bloodstream could constitute the explanation to the increased risk for systemic disease in patients with periodontitis . Despite these evidences showing a link between rheumatoid arthritis and periodontitis, the exact mechanisms involving this association thus, well - designed longitudinal multicentre clinical trials and further studies with sufficient sample sizes are required to determine the biochemical processes and clinical relationships between these chronic inflammatory conditions . Moreover, these studies should consider other potential confound factors such as the drugs administered for the treatment for each disease or differences in oral hygiene or smoking habits in these patients . The majority of the articles have confirmed that there is a correlation between pd and ra, since both disorders have characteristics in common and result from an imbalance in the immunoinflammatory response . Although it is necessary to highlight the importance of the mechanical treatment for periodontitis and pharmacological treatments mainly for ra patients, more research is needed to assess whether the coexistence of both diseases can affect the clinical signs of periodontitis and systemic markers of rheumatoid arthritis and strengthen the capacity of oral bacteria to stimulate an autoimmune response, thus establishing that cell constituents or mediators could share common pathophysiological pathways for both diseases and therefore define the best therapy.
Aids is a fatal disease that can affect human immune system and makes patients vulnerable to opportunistic infections . It is still one of the most important diseases in the world, which not only causes health problems, but also affects the political, social, and cultural aspects (1). The use of effective anti - viral therapy in the patients with delayed onset of aids and the increase of life expectancy and well - being of hiv - infected individuals reveal the need for effective prevention methods in this population (2). Advances improved the survival rates of the hiv - infected individuals, but not always with a good quality of life (qol) (3). Various studies conducted around the world have reported that as the hiv infection progresses, it affects the individuals' qol (4 - 6). According to the latest report by iran's ministry of health, by the end of february 2013, 26125 people will be identified with hiv in iran . Overall, the disease was transmitted by intravenous drug injection, sexual intercourse, and mother to child in 52.1%, 33.6%, and 3.2% of the cases, respectively . However, the transmission route is unknown in 11.1% of the patients (7). In iran, the first wave of aids occurred in 1987 due to transmission of agent is through blood as well as blood products and its second wave occurred in 1996 - 1997 because of intravenous drug abusers sharing needles . Now, at the third wave of aids, is through sexual relationships because of perversion which threatens several parts of the society (8). Health - related quality of life (hrqol) outcomes are of importance in study of persons with the relapsing and remitting disease and the need to evaluate effects of newer treatments on improving their health status (9). Moreover, effective anti - viral medication have delayed the onset of aids cycle and increased the patients life time . Nevertheless, from the psychological perspective, aids patients are faced with a large number of social and cultural limitations which affect different dimensions of their health and quality of life . In general, quality of life is defined as the individuals understanding of their life status in cultural as well as value systems fields, which is related to their goals, hopes, and standards . Thus, quality of life can be considered as the sum of physical, mental, and social welfare, including happiness, satisfaction, dignity, health, and economic status, perceived by the individuals (10). Meanwhile, the people s qol is affected by various factors, such as individual, economic, and social factors (sex, age, employment, marital status, etc . ), with stronger effects on those suffering from chronic disease . Therefore, the present study aims to investigate the qol based on some individual factors, such as age, sex, history of drug abuse, employment status, length of disease, marital status, transmission route, and the distance between the house and the service providers, in hiv - positive patients of shiraz behavioral counseling center, iran . The present study was a cross - sectional type in which the hiv - positive patients of shiraz behavioral counseling center with active profile were examined . According to the previous studies and the sample size formula, the study data were collected through 2 questionnaires, the first included the demographic information, such as age, sex, marital status, employment status, length of disease, level of education, history of drug abuse, and the distance between the house and the service providing center . The second questionnaire was sf-36, which is a 36-item self administered or interviewer - administered instrument with eight scales: physical functioning, role limitations caused by physical health problems, pain, general health perceptions, emotional well - being, role limitation caused by emotional problems, social functioning, and energy / fatigue . Reliability estimates for these eight scales was favorable in both general population and chronic diseases samples (11 - 19). A survey conducted in iran confirmed that, the persian version of this questionnaire was a reliable and valid instrument (20). Though the sf-36 has eight separate scales, factor analyses in previous studies have shown that these scales represent two underlying dimensions: physical and mental healths (16, 21 and 22). According to rand scoring system, the items of the questionnaire are scored from 0 to 100; and the closer to 100, indicates higher qol . Moreover, chi - square, t - test, and anova were used to determine the relationship between the variables and mean differences, and, p<0.05 considered statistically significant . According to rand scoring system, the items of the questionnaire are scored from 0 to 100; and the closer to 100, indicates higher qol . Moreover, chi - square, t - test, and anova were used to determine the relationship between the variables and mean differences, and, p<0.05 considered statistically significant . Of the 129 patients studied, 115 (89.8%) were male and 13 (10.2%) female . The patients age ranged of 25 - 58 years (mean + sd= 38.9 + 6.7). In addition, the mean age of the male and female subjects were 39.3 + 6.8 and 35.2 + 4.5 years, respectively and the highest frequency related to 35 - 44 years age group . In this study, 77 (61.1%), 26 (20.6%), and 23 (18.3%) patients in addition, 27 patients (21.3%) employed, while 99 (78.7%) were unemployed . Finally, 85.2% of the patients had a history of drug abuse, while 14.8% did not . 76 (61.8%), 26 (21.1%) and 21(17.1%) of the patients were infected through needle use, sexual and other route of transmission, respectively . The overall hrqol score in these patients was 48.8 + 17, and the mean for qol in physical and mental dimensions were 50.4 + 18.9 and 46.3 + 17.8, respectively with a statistically significant base on difference(p<0.05). The mean scores of the patients qol in each of the scales of sf-36 questionnaire are presented in table 1 . The difference between different age groups mean scores quality of life was not statistically significant (p>0.05). Investigation of the patients qol with regard to sex, employment status, history of drug abuse, age, level of education, length of disease, and the distance between the house and the service providing center revealed that this parameter for, female, employed, and non - addicted patients was significantly higher than that of the male, unemployed, and addicted ones, respectively (p<0.05). However, no significant relationship was found between the qol and age, level of education, length of disease, and the distance between the house and the service providing center (p>0.05). Finally, after obtaining significant results in anova regarding marital status and transmission routes, the schiff's post hoc test was used to determine the significant difference of qol in different levels of the above - mentioned variables . The results revealed significant relationships between married, single, and other groups of patients as well as intravenous drug abuse and other transmission routes (p<0.05)(table 3). The present study aimed to investigate the qol and its related factors in hiv patients of shiraz behavioral counseling center . In this study, the patients mean score of qol was 48.8 + 17 which was comparable with the study done by nojoumi et al . Reported the mean score of quality of life as 47 + 6.2 (23). Moreover, the mean scores of physical and mental dimensions were 50.4 + 18.9 and 46.3 + 17.8, respectively that was consistent with a great number of studies conducted in other countries, including imam s study in bangladesh and hay in the u.s ., as well as those performed in iran which have all revealed the hiv patients low qol in all the dimensions of sf-36 questionnaire, particularly in mental and social dimensions, compared to normal individuals and even those with chronic diseases (10, 24 - 29). In fact, biological treatments are not enough for treating such patients and, at the same time, getting familiar with and eliminating their mental problems can be a good prognosis for their treatment . Investigation of mean differences of qol relative to different levels of the variables revealed that women had a higher qol in comparison to men . Moreover, the hiv infection was mostly transmitted through intravenous drug abuse (61.8%) and sexual route (21.1%). Nevertheless, in comparison to the individuals infected through sexual relationships, those who infected by intravenous drug abuse had a lower qol . This is in contrast with the findings of the study conducted by hasanah in malaysia which reported a higher qol for the individuals who had been infected through intravenous drug abuse compared to those through sexual relationships (27). According to the study findings, qol of married subjects was significantly higher than that of the other participants, which might be due to the families and particularly the spouse s psychological supports which play a major role in improving the patients qol . Employed subjects had also a better qol compared to the jobless ones . In the studies conducted by nojomi in iran and eriksson in sweden, a significant relationship was found between the quality of life and sex, marital, and employment status which are in line with the findings of the present study (23,30). Application of the results in this study showed that the effects of different factors on the qol . Interventions, including resource allocation, better care for the patients, improving the relationship between the physicians and the patients, and increasing the trainings as well as consultations, can all be carried out . Creating appropriate job opportunities and employing the patients by the health system as well as the behavioral counseling centers are among the major supportive measures for these patients . In fact, employment are the most important concerns of such patients; therefore, a large number of financial and, consequently, psychological problems of the patients could be resolved by providing job opportunities . Finally, it is hoped that using the findings of this research, steps will be taken toward improving the hiv patients qol.
According to the who, over 3 000 000 children under the age of five die each year due to causes related to environmental risk factors . Identification of recognized environmental health risk factors is considered to be one of the most important objectives of health policy, as it relates to the everyday work of medical health - care professionals including family doctors, pediatricians, and nurses . Determination of mentioned risk factors will allow implementation of proper preventive measures, and a system of providing parents and teachers with information about health risks will, in turn, decrease the risks which may improve the effectiveness of health care . The who green page questionnaire was designed as a potential tool to determine and monitor children s environmental conditions in all places where they live and develop . It can be a valuable source of supplemental information acquired in the course of children s and parents appointments with family physicians, and it may also be a great support for doctors in determining children s environmental risks at home and school . The who green page questionnaire has not yet been analyzed in terms of its practical utility with respect to medical diagnostics . The expected goal of this research was to assess the possibility of implementation of the who green page as a tool to supplement basic medical interviews with potential environmental health risk factors for children and determination of real risk factors currently existing in home and school environments . The who green page questionnaire was implemented with parents of children from urban, suburban, and rural environments who visited a family practice doctor . Due to exclusion of questionnaires missing the majority of data (more than 50% of the answers were missing), the responses of 159 patients were analyzed . When we analyzed the questionnaires with less than 50% of the questions unanswered, the denominator for percentage calculation was lower than that for the 159 parents . Wilk test was carried out . For measurable (quantitative) variables, arithmetic means, and standard deviations were calculated, while for qualitative variables, the frequency (percentage) was determined . The analysis of qualitative variables was based on contingency tables and the test . To compare quantitative variables in two non - related and related groups, wallis test was conducted for means of variables that did not meet the criteria for variance analysis . In total 159 parents took part in the study, including 87 parents of girls (58% of the examined) and 63 parents of boys (42% of the examined). The average age of the children was 11.2 years (sd 6.2, median 11). The children mostly lived with both parents (91.3%, 136), while 6.7% (10) of them lived with their mothers only . The majority of the examined cases came from urban areas (56.2%, 87), while 38.3% (59) and 5.2% (8) were from rural and suburban areas respectively (p<0.05). Overpopulation at home was noted only among 4.4% (7) of the respondents . Domestic animals were present around the homes of 74.5% (117) of the respondents, and the distribution of answers was statistically significant (p<0.05), as it relates to the child s place of living . Contact with domestic animals was predominant in rural areas (animals were present in 91.53% of cases) as opposed to urban and suburban areas (63.95% and 57.14% respectively; p<0.05). It was noted that 24.3% (34) of caregivers expressed concern about their children s environment (vs 75.7%, 106), which did not maintain statistically significant dependency with the children s living environment (p>0.05). Knowledge and awareness of particular existing environmental risks were noted in 23.7% (32) of the subjects . Most of the respondents (96.2%, 153) were aware of the presence of disease - transmitting factors: 42.5% of the subjects knew disease - transmitting factors . The distribution of the answers to this question was dependent on the children s living environment it was distinctively different in rural areas (disease - transmitting factors were identified by 64.3% of the examined) as opposed to urban and suburban areas (correct answers were given by 31.4% and 33.3% the examined respectively), p<0.05 . In addition 7.0% (11) of the surveyed stated that their children had sustained injuries in connection with road traffic prior to the questionnaire study, and one child had sustained injuries in connection with fire . The distribution of the answers to those questions was not dependent on the children s living environment (p>0.05). Existing exposure to chemical substances (pesticides and detergents) was confirmed by 5.2% (8) of the respondents, and 6.4% (10) of the respondents reported that their children had been poisoned before the questionnaire as a result of contact with chemical substances . Furthermore, 9.6% (15) maintained that there existed a threat from poisonous animals . The distribution of the answers to the questions about contact with chemical substances and threat from poisonous animals was not dependent on the children s living environment (p>0.05). Following analysis of the respondents answers, it was determined that the examined children lived in a densely built - up area in 79.6% (125) of the cases and in a low - risk geographical zone in 76.3% (119) of the cases . The food they ate was of appropriate quality according to 94.9% (149) of the respondents, the indoor air quality was considered to be good by 81.5% (119) of the respondents, and the outdoor air quality was considered to be average by 51.0% (80) of the respondents . The land they lived on was seen as appropriate by 72.9% (113) of the respondents, and 92.3% (143) of the examined thought that sewage was disposed of in an appropriate way . Appropriate disposal of waste was confirmed by 87.9% (138) of the respondents, and the noise level was considered low by 68.2% (107) of the examined . Exposure to chemical substances was low according to 70.7% (111) of the respondents . Road traffic was seen as low by 44.6% (70) of the examined (table 1table 1abc of environmental conditions homeschoolabcabcbuilt - up environment79.6% (125)20.4% (32)0.0% (0)60.8% (90)38.5% (57)0.7% (1)geographical zone76.3% (119)23.1% (36)0.6% (1)71.3% (102)28.0% (40)0.7% (1)food94.9% (149)4.5% (7)0.6% (1)83.5% (111)15.8% (21)0.8% (1)indoor air quality81.5% (119)17.1% (25)1.4% (2)64.1% (84)34.4% (45)1.5% (2)outdoor air quality40.8% (64)51.0% (80)8.3% (13)35.0% (50)55.9% (80)9.1% (13)water - drinkable86.5% (128)13.5% (20)0.0% (0)82.1% (110)17.9% (24)0.0% (0)land / soil72.9% (113)25.8% (40)1.3% (2)63.2% (91)35.4% (51)1.4% (2)sewage disposal92.3% (143)7.7% (12)0.0% (0)88.0% (125)11.3% (16)0.7% (1)waste disposal87.9% (138)11.5% (18)0.6% (1)79.2% (114)20.1% (29)0.7% (1)noise68.2% (107)28.7% (45)3.2% (5)28.6% (42)51.7% (76)19.7% (29)exposure to chemical substances70.7% (111)28.7% (45)0.6% (1)65.3% (96)32.7% (48)2.0% (3)road traffic44.6% (70)43.9% (69)11.5% (18)25.2% (37)56.5% (83)18.4% (27)environmental risk factors: a, proper; b, average; c, potential . Differences between answers are statistically significant (p<0.05).). Environmental risk factors: a, proper; b, average; c, potential . (90) of cases, the children schools were in very well - built areas, and the schools were situated in low - risk geographical zones in 71.3% (102) of cases . The food offered at school was of proper quality according to 94.4% (149) of the examined . Indoor air quality at schools was good according to 64.1% (84) of the respondents, and outdoor air quality was considered to be average by 55.9% (80). The land where their schools stand was considered appropriate by 63.2% (91) of the examined, and 88.0% (125) of the examined thought that sewage was disposed of in an appropriate way . Appropriate waste disposal was confirmed by 79.2% (114) of the respondents, and the noise level was considered average by 51.7% (76) of the examined . Exposure to chemical substances was low according to 65.3% (96) of the respondents . Road traffic was seen as moderate by 56.5% (83) of the examined (table 1). The majorities of the children involved in the environmental assessment were of school age, lived with both parents, and came from urban areas . The caregivers expressed minor concerns about the children s living and study environments as well as low knowledge and awareness of existing environmental risks . A very low percentage of parents stated that their children sustained injuries in connection with road traffic, and in one case, a child sustained injuries in connection with fire; these were not statistically significantly dependent on where they lived, exposure to chemical substances, or threat from poisonous animals . Both the places they lived and studied were located in low - risk geographical zones . In the opinions of the majority of the respondents, the food, indoor and outdoor air, tap water, land, sewage and waste, noise level, exposure to chemical substances, and road traffic were appropriate quality in both places . This picture is positive and does not fully correspond with reality from the hygienic point of view and after performing an environmental interview, which emphasizes people s low awareness of environmental risks . The questionnaire may prove to be a useful tool in determining the kind of potential environmental risk factors that exist in children s living and study places . The who green page supplements the information of the basic medical interview by being a part the patient s medical history.
Skills) is one of 11 clinical data research networks (cdrns) funded by the patient centered outcomes research institute (pcori) in 2014 . Pcori, a non - governmental organization created under the patient affordable care act seeks to build an information technology (it) backbone to support comparative effectiveness research at a national scale across both cdrns and also patient powered research networks (pprns). Scilhs engages patients, clinicians, health systems leadership, and key healthcare stakeholders as collaborators to build on an existing network of hospitals and health systems that have already adopted a common clinical and translational research it and regulatory framework . Scilhs, comprising 10 health systems (box 1), is a step toward answering the institute of medicine's call for a learning healthcare system (lhs)1 2 to generate and apply the best evidence for the collaborative healthcare choices of each patient and provider; to drive the process of discovery as a natural outgrowth of patient care; and to ensure innovation, quality, safety, and value in health care. Beth israel deaconess medical center boston children's hospital boston health net (boston medical center and community health centers) cincinnati children's hospital medical center columbia university medical center and new york presbyterian hospital morehouse school of medicine / grady memorial hospital (research centers in minority institutions) partners healthcare system (includes massachusetts general and brigham & women's hospital) university mississippi medical center the university of texas health science center at houston wake forest baptist medical center fifteen years ago, scilhs informatics leaders began a quest to develop informatics infrastructure and regulatory innovation that would convert the emerging electronic health record (ehr) into a research tool for improving patient outcomes . All of our work and open source toolkits have been supported by grants from the national institutes of health, centers for disease control and prevention, and office of the national coordinator of health information technology (onc). First, we built indivo,3 4 the first personally controlled health record, which gave patients their data, and apps to make those data useful . Then, i2b2 (informatics for integrating biology and the bedside)57 created an open source analytic platform to the ehr, to fuse and analyze data produced by the delivery system, and identify research cohorts . Our next advance was shrine (shared health research information network),810 a tool enabling investigators to query i2b2 nodes in real time across multiple sites for collaborative population research . I2b2 has been successfully implemented at more than 100 sites across the usa, thereby enabling investigators to use delivery system data to identify patients with specific illnesses and clinical characteristics . A recent pcori survey of all pcornet sites revealed that 37% of the existing cdrn nodes and 31% of the pprn nodes already used i2b2 . Finally, we built smart (substitutable medical applications, reusable technologies)a platform to enable any developer to contribute to an app store for health and research compatible with i2b2-shrine instances or compliant ehrs.1113 these informatics tools and associated research policy advances have already contributed to transformation in the clinical research enterprise real - time, collaborative population health research is now enabled across shrine member sites distributed nationally but they have yet to yield substantial improvements in the health of our patients . Now, in establishing pcornet, pcori has catalyzed a new national research dialog to answer patient - oriented questions and improve human health . We directly address this challenge via a strategy intended to avoid prior mistakes of large - scale, top - down, costly software infrastructure efforts that failed to scale (eg, cabig14), instead building scilhs with open source, free, modular components5 15 with vibrant user and software developer communities that have already spread virally to scale across heterogeneous health systems . Here, we detail the informatics approaches taken by scilhs to identify large cohorts of patients and engage them for research . Our technology strategy links lockstep to processes for regulatory innovation, development of robust governance constructs and policies, and local adoption by hospital leadership and institutional review boards . Sidecar warehouse to the ehr, effectively leveraging existing data collected by ehrs during routine care while avoiding costly, time - consuming ehr integrations (figure 1). Developed intensively over the past 5 years at harvard medical school, this approach employs vendor agnostic, free, open source, scalable, and interoperable technologies to produce the only research - based, shared repository of ehr data that can be queried in real - time . Of already proven value in the research ecosystem, these components support a cost - effective and sustainable research network of> 8 million patients . Each site will install the scalable collaborative infrastructure for a learning healthcare system (scilhs) sidecar, for identifying and reviewing cohorts and the myscilhs suite to: (a) manage linkage of contact data to the de - identified patient cohort list produced by the multisite shared health research information network (shrine) query; (b) administer and store consent documents; (c) outreach to patients through web - based survey and telephony; and (d) promote ongoing patient engagement through outgoing messaging, including (in the future) return of research results to patients . The web - based survey will be administered using redcap and indivo technologies, and will be accessible either by patients at home, or at the point of care, through tablet / kiosk - based interaction . Once completed, patient - reported data will have subject identifiers encoded; its standardized survey metadata will then be loaded into the corresponding scilhs sidecar (i2b2 node), enabling semantic data linkage with electronic health record data via shrine / i2b2, while preserving subject confidentiality . These software platforms will be provided to sites as self - contained, pre - configured virtual machines, enabling rapid dissemination of these technologies while minimizing administrative and software development overhead at each site . We consider the heterogeneity of collaborating institutions to be a key measure of success; via adoption of the sidecar approach, we enable any institution to join our scilhs network . Specifically, a primary goal is inclusion of diverse populations within our cdrn network, thereby enabling capture of the genetic, genomic, and socioeconomic variation that exists beyond insured populations in managed care settings alone . Further, by freely sharing the processes and software that have been developed and supported by harvard, we hope to catalyze the formation of many other new networks across heterogeneous health systems and institutions, and involve new partners in improving our core components, common data models, and ontologies . The sidecar infrastructure is composed of the following: i2b2 (informatics for integrating biology and the bedside). Data analytic platform employed for ehr data analytics and clinical research at> 100 academic medical centers worldwide (nih funded).shrine (shared health research informatics network). Federated query and response system that enables investigators to discover ehr data housed in i2b2 nodes across multiple independent institutions (nih ctsa funded).smart platforms . First described in the new england journal of medicine,12 smart has programmatic interfaces and applications that transform both ehrs and their sidecars into platforms that run substitutable iphone - like apps.11 smart enables a national scale the original personally controlled health record3 4 16 17 links patients to clinical and research settings . Used by hundreds of thousands employees of dossia's founding companies (wal - mart, intel, and at&t), indivo was also the initial software codebase for microsoft's healthvault platform (nih, cdc, and onc funded).redcap (research electronic data capture). Electronic data capture tool18 19 with 757 institutional partners, used to survey patients online (nih ctsa funded). Data analytic platform employed for ehr data analytics and clinical research at> 100 academic medical centers worldwide (nih funded). Shrine (shared health research informatics network). Federated query and response system that enables investigators to discover ehr data housed in i2b2 nodes across multiple independent institutions (nih ctsa funded). Smart platforms . First described in the new england journal of medicine,12 smart has programmatic interfaces and applications that transform both ehrs and their sidecars into platforms that run substitutable iphone - like apps.11 smart enables a national scale app store for pcor for rapid cycle innovation of pcor methods (onc funded). The original personally controlled health record3 4 16 17 links patients to clinical and research settings . Used by hundreds of thousands employees of dossia's founding companies (wal - mart, intel, and at&t), indivo was also the initial software codebase for microsoft's healthvault platform (nih, cdc, and onc funded). Electronic data capture tool18 19 with 757 institutional partners, used to survey patients online (nih ctsa funded). Scilhs will combine ehr data with payer claims to facilitate longitudinal tracking of patients over time and across sites of care . The sidecar approach provides the capability to implement new data models without transforming all of the stored source data a key element in the scalability and interoperability of our platform (table 1). By enabling well - designed, cross - mapped ontologies that support a pcornet common data model, this approach incorporates otherwise disparate clinical data sources into an easily - queried system that stores data in a flexible format . Data are stored in i2b2 using an entity attribute value model,20 21 employing a central fact table based upon kimball's star schema22 wherein each row stores a flexibly defined, atomic fact or observation for a patient.5 much of i2b2's versatility arises from its focus on a semantic definition of patient observations that can represent various existing and newly defined data elements: claims, ehr, genetic and imaging data, as well as patient reported outcomes and demographics . Analogous to a capacious warehouse with adjustable shelves and bins, i2b2 accommodates various nomenclatures for data elements, and supports robust tags of associated modifiers and values . This approach enables database indexing of facts and observations to support high performance execution of expressive queries and filters . Approaches to scalability and interoperability ehr, electronic health record; omop, observational medical outcomes partnership; pcori, patient centered outcomes research institute; smart, substitutable medical applications, reusable technologies . I2b2 employs an ontology - based approach that supports flexible, on - the - fly incorporation of new data elements and coding systems . Terminologies such as icd, ndc, and loinc may be pre - loaded as hierarchical concept trees; new or ad - hoc terminologies including patient - reported outcome measures or locally defined data dictionaries readily coexist and may be cross - mapped in i2b2 . Concepts may be grouped using simple hierarchies and then optionally re - mapped into other reference coding systems and data models (eg, observational medical outcomes partnership (omop) data model).23 in this way, i2b2 accommodates diverse real - world coding systems while maintaining a straightforward query interface for its users . The shrine adaptor cell maps local i2b2 terminologies into a common, standards - based shrine ontology . This enables a common shared ontology for federated queries while allowing individual i2b2 instances within institutions to retain local hierarchies and terminologies . The adaptor transforms a federated shrine query into a query that runs on the local i2b2 database . The adaptor then converts the result of that query back into the common shrine message format, using well - maintained standards including rxnorm, icd9, and loinc . In addition, shrine includes tools for ontology mapping and ontology - based data mining . Simple shrine customizations enable use of other query systems, for example the queryhealth distributed query system (onc) uses popmednet to query i2b2.24 25 shrine and i2b2-based research includes characterization of rare morbidities of common diseases,26 very rare diseases such as peripartum cardiomyopathy (discovered in shrine and published in nature27), detections of drug drug interactions,28 and measures of quality and clinical efficacy across self - organized shrine networks in europe, the university of california healthcare systems, and a just - in - time network to study the prevalence of complication rates of type 1 and type 2 diabetes in hospitals across this country . Others have used shrine to characterize and track the rising incidence of colorectal cancer29 and further characterize it, and to identify and optimize practice variation in inflammatory bowel disease and intervene to change that practice.30 i2b2 and shrine have been implemented as the base infrastructure for a variety of enhanced chronic disease registry - based research efforts.31 the childhood arthritis and rheumatology research alliance uses the shrine / i2b2 registry framework to federate clinical care data and patient - reported data from 62 academic medical centers in the usa and canada32 33 and is currently piloting consensus treatment protocol trials.3437 the harvard inflammatory bowel disease (ibd) longitudinal data repository employs the same infrastructure.31 improvecarenow38 utilizes i2b2 as its centralized data warehouse for ibd - related quality improvement development at 50 centers . The health systems that have joined scilhs reflect the american demographic an essential requirement for reaching statistically valid, clinically meaningful, and patient - centric conclusions about therapies across the diverse spectrum of all healthcare consumers . In order to achieve the comprehensive, patient - centered outcomes infrastructure called for by pcori, we introduce a new, patient - centric platform (myscilhs) based on the indivo system and incorporating the redcap electronic data capture tool . Myscilhs will support the blue button rest api for standards - based interactions with pprns and other patient - selected tools . This api exposes up - to - date, structured clinical summary data for each participating patient . Via a consumer - friendly workflow based on web standards including oauth2, patients can authorize third - party apps and services, including pprns, to access their clinical data . Figure 2 shows the workflow from an initial query through the analytic phase in a comparative effectiveness study . Each node in the network maintains an instance of i2b2 containing claims and de - identified electronic medical record data . Scilhs is a true peer - to - peer network, meaning that any shrine - based node can initiate a query, using a common ontology, that aggregates results from all participating sites . After the initial query, the investigator can automatically pass the query to each site where duly authorized local site investigators may review individual subject data for study eligibility using i2b2 smart apps (figure 3). The myscilhs research contact management module links de - identified i2b2 records to patient demographics and contact information . Patients are engaged by web survey, telephony, or smart apps; patient - reported data are returned to i2b2 and are then transferred into a secure comparative effectiveness (ce) study environment for analyses . In the ce environment, we anticipate that pcornet - level queries, which may launch against the full complement of 11 cdrns and 18 pprns, will be initiated at the pcornet adapter . We anticipate that natural language processing (nlp) of provider notes will play an important role for adding complete longitudinal coded data to the hospital - based record.39 early findings demonstrate that nlp of hospital - based ehr notes provides quite complete longitudinal data even when compared with centers for medicare and medicaid services claims data (personal communication, katherine liao, brigham and women's hospital, 2014). Using nlp on hospital and clinic notes will complement our strategy of concatenating ehr data with external sources such as claims and pharmacy data . There will be important variations depending on the nature of the study, whether in - person consent is required, and whether patient identifiers are needed . Shared health research information network (shrine) architecture implemented as a modular framework . Using a mapper toolkit, each site exposes a common queryable data model, implemented in the ontology (ont) cell . The ont cell manages the vocabulary of the data model and is one of several cells in the i2b2 architecture, including the broadcaster - aggregator cell (agg, broadcasts the query across all i2b2 nodes in the shrine peer - to - peer network and aggregates the results), the identity management cell (i m, used for authentication), the clinical research chart (crc, manages the clinical data), the workplace cell (work, manages the workflow), and the substitutable medical applications, reusable technologies (smart) cell (manages the smart api). A query from a patient centered outcomes research institute (pcori) approved study is translated to a shrine central node query either manually, or by a pcornet adaptor, the specifications for which are still to be determined . The shrine central node broadcasts the query across the true peer - to - peer network (arrow 1). I2b2 nodes containing coded data are queried at each site to identify appropriate patients returning obfuscated, aggregate patient counts (arrow 2). Patient identifiable data remains at each site where investigators can use smart apps to review records prior to aggregation (arrow 3). Also, see figure 3 . The patient list is passed to myscilhs for outreach to patients via apps, survey, or telephony (arrow 4). Patient generated data are imported into i2b2 via simple input formats (csv, for example) and placed into the i2b2 data model in a flexible schema that allows these to become first - class queryable data objects (arrow 5). The adjudicated patient data (reviewed by investigators using smart apps and confirmed as valid) from each site, including patient - reported data can be added (arrow 6) to a research data mart in one of several analytic data models (including the pcori common data model) with a level of identifiers appropriate to the level of consent obtained . Additional, outside data such as centers for medicare and medicaid claims can be added in this step . A substitutable medical applications, reusable technologies (smart) platforms html5 app running on i2b2, providing a richly featured electronic health record - like view of the data . Scilhs includes 10 legally and financially independent institutions whose ceo or equivalent senior institutional official has committed to active participation in governance, policy development, data sharing, and sustainability planning . Each member has pledged to invest additional personnel and resources to ensure the network meets local patient and clinical stakeholder needs . By harmonizing informatics infrastructure, data models, regulatory processes and policies, and patient participation within and across member institutions, we anticipate that scilhs will achieve and remain a successful model for inter - institutional pcor . Utilizing the innovative scilhs sidecar it approach to ehr access, we minimize local informatics burden, further enabling a sustainable and adaptable pcor infrastructure.
Peutz - jeghers is a rare autosomal dominant disorder characterized by hamartomatous polyps and discoloration of mucosal membranes . The polyps can occur anywhere in the gastrointestinal tract and can grow large enough to cause bowel obstructions . A 16-year - old male presented to the emergency department with signs and symptoms of an acute bowel obstruction . He had a previous history of a colonoscopy with polypectomy at age 4, and hyperpigmentation of his mucous membranes . An exploratory laparoscopy found an intussusception of the mid jejunum . A laparoscopic - assisted small bowel resection was performed . Colonoscopy and upper endoscopy revealed 5 more polyps in the stomach and colon that were removed . The treatment of obstruction in these patients is to remove the offending hamartomatous polyp(s). The rest of the intestine needs to be examined and those polyps found should be removed . Peutz - jeghers syndrome (pjs) is a rare autosomal dominant condition characterized by hamartomatous polyps and mucocutaneous pigmentation of the lips, buccal mucosa, and digits . Polyps can occur anywhere in the gastrointestinal tract and can grow large enough to cause bowel obstructions . A 16-year - old male presented to the emergency department with signs and symptoms of an acute bowel obstruction . He had a previous history of a colonoscopy with polypectomy at age 4, had hyperpigmentation of his mucous membranes, and his mother and maternal grandfather had a history of gastrointestinal polyps . The patient underwent an exploratory laparoscopy and was found to have an intussusception of the mid jejunum (figure 1). Pathology showed a 5-cm polyp that had acted as a lead point for the intussusception (figure 2). The patient had an uncomplicated postoperative course and was discharged home on postoperative day 3 . He is one year out from surgery and has resumed his normal activities with no evidence of recurrence . Pjs was first described in 1921 by peutz and subsequently elaborated upon by jeghers in 1949 . Germline defects in the tumor suppressor gene serine / threonine kinase 11 (stk11) are implicated in this rare autosomal dominant inherited disease . Its incidence is calculated in 1 in 200,000 liveborns, and its mean age of onset is 25.2 years . The most common location of the hamartomatous polyps is the small bowel (78%), followed by the colon (42%), stomach (38%), and rectum (28%). These polyps can cause obstruction in up to 43% of cases and rectal bleeding in up to 14% of patients . The syndrome is associated with a 2% to 10% increased risk of cancer of the intestinal tract, from the stomach to the rectum . There is also an increased risk of extraintestinal malignancies, including cancer of the breast, ovary, cervix, fallopian tubes, thyroid, lung, gallbladder, bile ducts, pancreas, and testes . However, recurrence of intussusception episodes occurs in at least 10% of cases, resulting in repeated surgical intervention . Thus, the rest of the intestine needs to be examined, and those polyps found should be removed . Once the gastrointestinal tract has been cleared of polyps, the recommended interval of small bowel follow - through is from 2 years to 3 years . The presence of polyps larger than 1.5 cm in diameter mandates another complete gastrointestinal evaluation with endoscopic removal of polyps . Patients should also be screened periodically for malignancies of the breast, cervix, ovary, testis, stomach, and pancreas . To our knowledge, there are at least 2 other published case reports regarding the laparoscopic management of bowel obstructions in pjs . The ideal way to remove a pedunculated polyp acting as a lead point, laparoscopy offers a safe and effective method for surgical management with reduction of the intussusception and small bowel resection . The diagnosis of pjs should be considered in patients presenting with a clinical picture of bowel obstruction and mucocutaneous hyperpigmentation . If the diagnosis is made preoperatively, optimal management should include laparoscopic treatment of the bowel obstruction and intraoperative enteroscopy . If the diagnosis is made after the operation, the patient needs complete evaluation of their gastrointestinal tract.
Precise localization of the iac is important and successful implant placement or orthognathic surgery depends on the exact determination of the location of the inferior alveolar nerve (ian). Permanent or temporary ian damage can cause numbness of the lower lip and chin in orthognathic surgery [1]. Pressure on this nerve during implant placement is one of the common causes of treatment failure and postoperative pain . On some radiographs, the iac has a cortical boundary, but in others, the canal may be mistaken for bone marrow especially in osteoporotic patients [2]. In addition, the anatomical variation of the ian may be a factor that leads to the failure of block injections [3]. Recently, by use of cbct, high anatomical variability of this nerve was discovered [4]. Although the morphology and the position of the canal vary in different ethnic groups and in different types of jaws, these variations are ignored in many cases and cause problems in dental treatments [5]. A detailed understanding of the factors affecting the anatomical variations of canal shape and morphology can minimize this problem . Therefore, in this study, we investigated the relationship between the ga size and course of the iac in human dry mandibles using cbct . The results may be useful for more accurate localization of the iac on radiographs before dental treatment . In this in vitro study, we obtained dry mandibles from the anatomy department of mashhad university of medical sciences . Samples with no anomaly or bone defects were assessed, and those in primary or mixed dentition were excluded [3, 6 8]. A total of 25 dry adult human mandibles and 6 left and 5 right hemi mandibles (a total of 61 hemi mandibles) of unknown gender or origin were studied . To increase the accuracy and for easy tracing of the canal, a 0.5 mm diameter orthodontic wire was inserted into the iac before imaging [7]. The cbct scans were obtained (promax 3d, planmeca, helsinki, finland) with 8080 mm field of view . As the image field did not cover the entire mandibular bone, the stitch option of cbct was used, and an oral and maxillofacial radiologist, using romexis software 2.4.2.r (planmeca inc . Intra - observer error was calculated by re - measuring a random sample of 30 images after an interval of two weeks . Both readings were then analysed by paired t - test . In the sagittal view of each ramus, line b was traced tangential to the most prominent point on the posterior border of the ramus and condyle . The angle between lines b and d was measured as ga (fig . 1) [9 14]. Gonial angle measurements the scans were divided into the following two ga groups: low angle (125) and high angle (> 125) [15]. To evaluate the shape and position of the canal in coronal views, the distance between the mandibular foramen and the mental foramen was divided into three equal segments and the central cuts (c1, c2, and c3) were measured in each segment [16, 17]. In order to evaluate the canal shape, the superior - inferior and buccolingual diameters of the canal were measured in each slice . To evaluate the buccolingual position of the iac, we measured the distances from the center of the iac to the tangential lines with buccal and lingual borders and to the lowest point of the inferior border (fig . 2) [17, 18]. In each slice, the distance from the buccal plate to the lingual plate was measured as buccolingual width of the mandibular body [16, 19]. Consequently, in order to define the buccolingual position of the canal in each section, the ratio of the distances between the center of the iac and the buccal and lingual plates to the width of mandible in each section was calculated [16]. Evaluation of the buccolingual position of the iac by calculating the distance from the center of iac to buccal, lingual, and inferior borders in c1, c2, and c3 slices to investigate the total length of iac, the nerve was traced in coronal views . By using the serial measurements option, the total length of the nerve was exactly measured on the sagittal view (fig . 3). To investigate the canal course on the sagittal view, the nerve path was classified into three types of a, b, and c [7, 17]. In type a, the canal course had a straight path and was positioned at the same level of the mental foramen . In type b, the canal had a curved path, and in type c, the canal had a forward path and then ran superiorly to reach the mental foramen with a sharp ascent (fig . To evaluate the canal course on the axial view, the nerve path was traced and classified according to the mental foramen angle . If the canal course made an acute angle with mental foramen (90), it was defined as type a1; otherwise, it was type a2 (fig . 5) [7]. Different types of canal course on the sagittal view type a: the canal course has a straight path at the same level as the mental foramen . Type c: the canal has a forward path and then runs up to reach the mental foramen with a sharp ascent . The canal course forms an acute angle with the mental foramen on the right side (a1 type) and an obtuse angle on the left side (a2 type) statistical analysis was done using pasw version 18 (spss inc ., paired - sample t - test was used for right and left symmetry and calculation of intra - observer agreement between the two readings . Pearson s correlation coefficient was used to examine the correlation between the right and left gas . Also, independent - samples t - test was used for quantitative comparison between different canal types on different views . Pearson s chi - square test was applied for evaluation of the correlation of canal course in the sagittal plane and ga . A p - value less than 0.05 intra - observer reproducibility for all variables was calculated by re - examining 30 scans . The differences between measurements in the first and second readings were not significant (p=0.160.35). The mean size of ga was 121.87.05 at the right side and 123.86.32 at the left side (table 1). The results showed that there was a statistically significant correlation in the ga size at both sides (p=0.000, r=0.9); however, a significant difference was noted between them (p=0.03). The mean standard deviation of canal length at the right and left sides were 63.035.48 mm and 62.75.51 mm respectively . There was no significant correlation in the mean total canal length in the right and left sides . No statistically significant difference was found between the two sides in terms of canal lengths (p=0.53). Descriptive findings for gas sd: standard deviation after evaluating different sagittal views of the mandibular canal, it was obvious that the most common type of iac course on the sagittal view was type b (53.8%), followed by type c (26.2%) and type a (20%). In 70% of the cases, there was no right - left symmetry in the course of the canal on the sagittal view . In addition, it was clear that there was no significant difference between the canal length on the sagittal view, according to canal course (p=0.59). The samples were grouped according to ga size as high angle (> 125) and low angle (125). The gas were smaller than 125 and larger than 125 in 60.7% and 39.3% of samples, respectively . As shown in table 2, there was a significant relationship between different types of canal courses on the sagittal view and the different ga groups (p=0.04). Therefore, in the low ga group, type b was the more common canal course (73.5%), while type a was dominant in the high ga group (66.7%) as shown in table 2 . Cross tabulation between different canal course types and ga groups on the sagittal view . After evaluating the canal course on the axial view, type a1 was found to be more common (73.43%) than type a2 (26.56%). According to the data in 10.34% of the cases, the canal type was different at both sides of the mandible . There was no statistically significant relationship between the canal course on the axial view and ga group (p>0.05). The total canal length was 63.255.4 mm in type a1 and 60.744.7 mm in type a2 . No significant difference existed in the canal length in different types of canal course on the axial view (p=0.09). Evaluation of the iac course on the coronal view in segments c1, c2, and c3 showed that on the right side, the canal was closer to the lingual cortex in 86% of the cases in c1, 100% in c2, and 90% in c3; on the left side the corresponding percentages were 77%, 93%, and 93% of the cases, respectively . According to our results, the canal is positioned lingually throughout its path up to the mental foramen, and then reaches the mental foramen with an acute or an obtuse angle . Analysis of the diameters of iac showed that the greatest mean diameter in the superior - inferior and buccolingual dimensions was in c1 and the smallest was in c2 . In this study, the gender of the individuals from whom the dried human mandibles were obtained was unknown . Many previous studies, including those conducted by de oliveira - santos et al, [4] ozturk et al, [7] kisser et al, [8] liu et al, [17] apinhasmit et al, [20] angel et al, [21] and raustia and salonen [22] demonstrated that the position of some anatomic landmarks, such as iac and ga, is not related to gender or age . The mean size of ga in this study was similar to that found in earlier studies [9 13, 23]. The mean ga of the right side was 2 smaller than that in the left side, and this difference was statistically significant . The difference in the mean size of the right and left ga was mentioned in the study by raustia and salonen [22], who stated that the right ga was significantly smaller than the left one . When we analysed the canal shape in the coronal sections, we found that ovoid shape was more common than other shapes . Similar to ozturk et al, [7] we found that type b was the most common type of canal course on the sagittal view, followed by types c and a. liu et al . [17] classified the canal course into four groups (types 1, 2, 3, and 4) using panoramic radiography [17]. The shape of the canal in type 1 was similar to type a in the current study and the study carried out by ozturk et al [7]. Types 2 and 3, which had a catenary like path, were similar to type b, and type 4 was similar to type c. in the study by liu et al, [17] the frequency sum of types 2 and 3 was greater than that of type b, but the frequency of types 1 and 4 was lower than that of types a and c [17]. The difference in the prevalence rate of iac course shape in the study by liu et al, [17] compared to that observed by ozturk et al, [7] and in the current study, may be due to the difference in the type of imaging system and methodology of the studies, and may also be because of the difference in ga [7, 17]. There was no difference in the total canal length between types a and b; however, there was a high prevalence (66.7%) of type a in the high - angle group, while type b was dominant (73.5%) in the low - angle group . It is analogous to a situation in which you have two identical ropes; if you tie the ends of one of the ropes, its path will be bent, whereas the other rope would have a more direct route . There was no difference in the canal length observed in our study and that found by liu et al [17]. Although the method to obtain the total canal length used by liu et al . Was via the tracing cord with a 5 mm arch, while we calculated the length using a specific type of software; the average length found in both studies was very similar [17]. Introduced four types of canal course on the axial view, and two of these (types b and c) were introduced for the first time [7]. Similar to liu et al, [17] we did not observe these types in the current study, but type a1 was the most commonly found, which is in accordance with the results of ozturk et al [7]. In the current study, the iac was located near the lingual plate throughout its path, and then travelled to the buccal plate with a sharp ascent near the mental foramen in the majority of samples . Ozturk et al, [7] hwang et al, [19] som and curtain [24], kim et al, [25] and fabian [26] also found that the iac is close to the lingual plate, and then runs to the buccal plate near the mental foramen [7, 19, 24 hwang et al . Showed that the iac is close to the lingual plate in the posterior two thirds of the mandible and runs towards the buccal side in the anterior third [19]. In our study, the entire canal course was of a1 and a2 types on the axial view, which shows that the canal is in the lingual plate, then in front of the mental foramen, it reaches with a sharp turn to the buccal side [7, 19]. The canal was located almost 1 cm above the inferior border of the mandible in the second premolar and the first molar region, which appears to provide the greatest superior - inferior dimension for implant placement . Showed that the least distance to the lower border was almost 1 cm in the first molar region [7]. According to the results of the current study, the iac has the greatest diameter in the ramus region (behind the third molar) and the smallest diameter in the molar region . De oliveira - santos et al . Studied canal diameter in the first molar region and their conclusion was in accordance with our findings [4]. No other study has calculated iac diameter on the coronal view . In this study we were not able to identify the sex of subjects whose dry mandibles were evaluated; thus further studies on human mandibles are suggested taking into account the sex and age parameters . We concluded that: 1-the ga size has a correlation with the iac course . In subjects with small gas but in cases with large gas, the canal is more straight and at the same level as the mental foramen.2-the alveolar crest has the maximum distance from the iac in the second premolar and first molar region . The ga size has a correlation with the iac course . In subjects with small gas but in cases with large gas, the canal is more straight and at the same level as the mental foramen . The alveolar crest has the maximum distance from the iac in the second premolar and first molar region . Since the iac is located in the lingual side of the mandible, the safe zone for implant placement and pre - prosthetic surgery is believed to be the buccal side . In addition, the greatest height of bone can be obtained in the second premolar and first molar regions.
Worldwide, the prevalence of overweight and obesity has reached epidemic proportions and includes not only adults but also children and adolescents [2, 3]. For example, it has been reported that the worldwide prevalence of overweight and obesity includes approximately 110 million children while in the united states (us), an estimated 12.5 million children and adolescents are either overweight or obese . This is problematic because overweight and obese youth have been shown to be at an increased risk of becoming overweight and obese adults, and thus placing them at an increased risk for premature all - cause mortality . Based on 2005 data, overweight and obesity as well as physical inactivity in adults were reported to be the third leading causes of preventable death in the us (about 1 in 10 deaths each) behind cigarette smoking and high blood pressure . The issue of obesity has become so problematic that it has recently been recognized as a disease by the american medical association . Exercise, a nonpharmacologic intervention that is available to the vast majority of the general public, may play a pivotal role in the treatment of overweight and obese children and adolescents . Systematic reviews with meta - analysis, a quantitative approach for combining the results of different studies on the same topic, are considered by many to be the most important type of evidence for determining the efficacy and effectiveness of various treatments on selected outcomes [9, 10]. Unfortunately, with the proliferation of systematic reviews on the same topic, it becomes difficult to make informed decisions regarding the effects of various interventions on selected outcomes . For example, a recent systematic review identified 22 previous meta - analyses examining the effects of exercise on blood pressure . Given the proliferation of reviews, a need now exists to systematically review these previous reviews in order to provide decision - makers with the information they need to make evidence - based decisions regarding the efficacy and effectiveness of various interventions on selected outcomes as well as provide direction for future research . Given the former, the purpose of the current study was to conduct a systematic review of previous meta - analyses addressing the effects of exercise (aerobic, strength training, or both) in the treatment of overweight and obese children and adolescents . The a priori inclusion criteria for this study were as follows: (1) previous systematic reviews with meta - analysis of randomized controlled trials or data reported separately for randomized controlled trials, (2) children and adolescents 5 to 18 years of age, (3) aerobic exercise and/or progressive resistance training intervention(s) lasting for an average of at least 4 weeks, (4) published and unpublished (dissertations and master's theses) studies in any language from 1990 forward, and (5) exercise minus control group difference in one or more of the following variables that were primary outcomes in the original meta - analysis: body weight, body mass index, body mass index percentile, body mass index z - score, percent body fat, fat mass, and fat - free mass . Post hoc, percentage overweight, adjusted for height as well as waist - to - hip ratio were also included as outcomes . Meta - analyses were limited to randomized controlled trials because they are the only way to control for unknown confounders as well as the fact that nonrandomized controlled trials tend to overestimate the effects of treatment in healthcare interventions [13, 14]. Potentially eligible meta - analyses were also limited to those that included studies in which exercise was an intervention, defined here as planned, structured, and repetitive and purposive in the sense that the improvement or maintenance of one or more components of physical fitness is the objective . While somewhat arbitrary, 4 weeks was chosen as the minimum length of exercise since one should expect some type of change in overweight / obese outcomes during this period of time . Based on a pubmed search, 1990 was chosen as the starting point for searching because it was the first year in which a potentially eligible study was identified for review . Any studies that did not meet all of the above criteria were excluded from our review . Ineligible studies were broadly categorized as excluded based on one or more of the following reasons: (1) inappropriate population (adults, animals, etc . ), (2) inappropriate intervention (nutrition, pharmacologic, etc . ), (3) inappropriate comparison (exercise versus diet), (4) inappropriate outcome (blood pressure, lipids, etc . ), and (5) inappropriate study type (meta - analysis that included nonrandomized controlled trials, systematic review without meta - analysis, etc . ). Using the graphical - user interfaces for each database, the following electronic sources were searched: (1) pubmed, (2) sport discus, (3) web of science, (4) scopus, (5) proquest, (6) cochrane database of systematic reviews (cdsr), (7) physiotherapy evidence database (pedro), (8) database of abstract of reviews of effects (dare), and (9) health evidence canada (hec). All searches were conducted during the month of april, 2013 with the last searches conducted on april 20 . Scopus was included in our database searches because it has been reported to provide coverage of embase . With the exception of pubmed, which was searched from its inception in order to identify a starting year for searching, a list of all search strategies for each database is shown in supplementary file 1 (supplementary material available online at http://dx.doi.org/10.1155/2013/783103). In addition to electronic database searches, cross - referencing for potentially eligible meta - analyses from retrieved reviews was also conducted . The coding sheets could hold up to 253 items from each included meta - analysis . Both authors coded all studies independent of each other . Upon completion of coding, all coding sheets using cohen's kappa statistic (), the overall agreement rate prior to correcting discrepancies was 0.68 . Methodological quality for each included meta - analysis was assessed using the assessment of multiple systematic reviews (amstar) instrument [2225]. Amstar was chosen over other instruments [26, 27] because of its reported interrater reliability (= 0.70), construct validity (intraclass correlation coefficient = 0.84) and feasibility (average of 15 minutes per study to complete). No, can't answer, or not applicable . The response can't answer is chosen when an item is relevant but not described . The response not applicable is chosen when an item is not relevant (meta - analysis of data not possible, etc .) When summing responses, the following question was modified from was the status of publication (i.e. Grey literature) used as an inclusion criterion? To was the status of publication (i.e. Grey literature) as an inclusion criterion avoided? In addition, we considered the question regarding conflict of interest as adequately met if the authors of the systematic review provided a statement on conflict of interest versus the reporting of conflict of interest by both the authors of the systematic review and the original studies included in the meta - analysis . Using cohen's kappa statistic (), the overall agreement rate prior to correcting discrepancies was 0.82 . A priori, the overall results from each meta - analysis were extracted, with a focus on random effects models since they incorporate between - study heterogeneity into the model and should almost always be the model of choice regardless of whether or not significant heterogeneity exists [28, 29]. Overall point estimates and 95% confidence intervals (cis) along with the q statistic, a measure of heterogeneity, were extracted for each outcome . An alpha value 0.10 was considered to represent statistically significant heterogeneity . However, because of issues surrounding the power of the q statistic, the i statistic was also reported if it was provided in the meta - analysis . If it was not provided, it was calculated if sufficient data existed to do so . The i statistic = 100% (q df)/q, where q is cochran's heterogeneity statistic and df, the degrees of freedom . Negative values of i are set to zero (0) so that i falls between 0% and 100% . A value of 0% indicates no observed heterogeneity while larger values indicate increasing heterogeneity . While somewhat arbitrary, values of 25%, 50%, and 75% were considered to represent low, moderate, and high amounts of heterogeneity . Since it was assumed that none of the eligible meta - analyses would include 95% prediction intervals (pis), these were calculated if the overall findings were statistically significant and the results from each study included in each meta - analysis were provided [3234]. Prediction intervals are used to estimate the treatment effect in a new trial [3234] and are calculated as follows: (1)meantdf(se2 + 2), where t is the centile point (95%) of the t distribution with k 2 degrees of freedom, se is the squared standard error, and is the between - study variance [34, 35]. All pis were calculated using the user - written metan command in version 11.0 of stata . In order to enhance application, the number - needed - to treat (nnt) was calculated for any overall findings that were reported as statistically significant . In addition, the nnt was used to provide gross estimates of the number of obese children and adolescents in the us who could benefit from exercise, based on 12.5 million obese children and adolescents as well as the number of overweight and obese children worldwide who could benefit from exercise, based on 110 million overweight or obese children [2, 38]. It was assumed that none of the children and adolescents included in the original estimates were exercising regularly . Because neither of the included meta - analyses assessed publication bias or conducted influence analysis with each outcome deleted from the model once, a post - hoc decision was made to test for both if sufficient data were provided . Publication bias was assessed using the regression - intercept approach of egger et al . . Both publication bias and influence analysis were conducted using comprehensive meta - analysis (version 2.2). For those outcomes that were reported using the standardized mean difference (smd), values of 0.2, 0.5, and 0.80, were considered to represent small, medium, and large effects . With the exception of heterogeneity, tests with two - tailed alpha levels 0.05 were considered to be statistically significant . Two - tailed alpha levels> 0.05 but 0.10 were considered as a trend towards statistical significance . Precision of estimates was considered robust if 95% confidence intervals for continuous outcomes did not cross zero (0). Dispersion statistics were reported as either standard deviations (sd) or standard errors (se). With the exception of fat - free mass, negative values for all other outcomes were indicative of improvement . Of the 511 citations initially identified, 308 (60.3%) remained after removing duplicates . Of the 308 articles that were screened, the full text from 25 articles (8.1%) was retrieved and assessed for potential eligibility . Upon completion of the review, two aggregate data meta - analyses met the criteria for inclusion [42, 43]. The major reasons for exclusion of the other studies were an inappropriate study design (49.8%) followed by an inappropriate population (21.2%), outcome (15.9%), intervention (12.0%), and comparison (1.6%). A flow diagram that depicts the search process can be found in figure 1 while a list of excluded studies, including the reasons for exclusion, is shown in supplementary file 2 . For the two included meta - analyses [42, 43], one focused specifically on exercise while the other focused on nonsurgical interventions, including exercise . Both meta - analyses included overweight and obese children and adolescents according to the criteria described by each of the original studies they included [42, 43]. A general description of the characteristics of each meta - analysis is provided in table 1 . Satisfied 7 of the 11 amstar criteria (64%) while the study by mcgovern et al . Both meta - analyses were judged as (1) not avoiding the status of publication as an inclusion criterion, (2) not providing a list of excluded studies, and (3) not assessing for potential publication bias [42, 43]. In addition, the meta - analysis by atlantis et al . Was judged as not providing a conflict of interest statement . Amstar results for each question from each meta - analysis are shown in supplementary file 3 . A description of the overall findings from each meta - analysis is shown in table 2 . A statistically significant reduction in percent body fat along with nonoverlapping 95% cis was observed for both the atlantis et al . Meta - analysis while nonoverlapping 95% pis were observed for the mcgovern et al . An examination for publication bias indicated no statistically significant publication bias for either the atlantis et al . (b0, 0.32, 95% ci, 4.0 to 3.4, p = 0.84) or mcgovern et al . (b0, 1.09, 95% ci, 3.4 to 1.2, p = 0.27) meta - analyses . With each outcome in each meta - analysis deleted from the model once, results remained statistically significant or trended towards statistical significance (p <0.001 to 0.07) for both, ranging from an smd of 0.31 to 0.50 in the atlantis et al . No statistically significant changes in bmi or bmi - related outcomes were found for either the atlantis et al . In addition, overlapping confidence intervals were observed for both meta - analyses [42, 43]. For the mcgovern et al . Meta - analysis, no statistically significant heterogeneity or publication bias (b0, 0.62, 95% ci, 3.2 to 2.0, p = 0.60) was observed . With each outcome deleted from the model once, changes remained nonsignificant (p = 0.29 to 0.99), ranging from a smd of 0.11 to 0.02 . Insufficient bmi data were available to test for heterogeneity, publication bias, and influence analysis in the atlantis et al . Meta - analysis also reported outcome results for body weight and central obesity (waist circumference and waist - to - hip ratio). A trend for statistically significant reductions in body weight in addition, publication bias was also found to be statistically significant (b0, 1.7, 95% ci, 3.3 to 0.05, p = 0.05). With each outcome deleted from the model once, results were statistically significant, and all heterogeneity was removed when one outcome from one study was deleted from the model (x-se, 3.7 1.4 kg, 95% ci, 6.4 to 0.9, p = 0.009; q = 7.0, p = 0.63, i = 0%). For central obesity, a trend for statistical significance (p = 0.07) was reported but 95% cis were overlapping . No statistically significant heterogeneity or publication bias (b0, 0.90, 95% ci, 4.7 to 2.9, p = 0.41) was observed . Results were in the direction of benefit (smd, 0.19 to 0.29) but remained nonsignificant (p = 0.12 to 0.23) when each outcome was deleted from the model once . The atlantis et al . Meta - analysis conducted several additional analyses beyond the overall findings for percent body fat and body weight . For percent body fat, reductions were greater (smd, 0.6, 95% ci, 0.8 to 0.3, p <0.001) when studies were limited to higher (x-sd, 177 23 minutes per week) versus lower (x-sd, 153 25 minutes per week, <3 days per week) doses of exercise as well as when strength training studies were removed from the model (smd, 0.5, 95% ci, 0.8 to 0.1, p = 0.003). Results remained stable when separate analyses were conducted with studies that reported changes in dietary intake (p = 0.02) and exercise - only studies (p = 0.005) deleted from the models . Results were no longer statistically significant when studies that did not report exercise compliance or changes in exercise were deleted from the analysis (smd, 0.3, 95% ci, 0.7 to 0.2, p = 0.11). For changes in body weight, reductions were greater (x-, 4.9 kg, 95% ci, 9.1 to 0.7, p = 0.01) when studies were limited to higher (x-sd, 156 25 minutes per week) versus lower (x-sd, 117 46 minutes per week, <3 days per week) doses of exercise as well as when strength training studies were removed from the model (x-, 3.1 kg, 95% ci, 6.1 to 0.1, p = 0.02). With studies that reported dietary intake removed, reductions in body weight increased (x-, 5.1 kg, 95% ci, 8.6 to 1.6, p = 0.002). In contrast, results were no longer statistically significant when studies that did not report exercise compliance or changes in exercise were deleted from the analysis (x-, 2.3 kg, 95% ci, 6.8 to 2.1, p = 0.20). For dose - response, no statistically significant associations were observed for changes in percent body fat, body weight, and central obesity when correlated with the volume of prescribed exercise (minutes per week) and total dose (minutes per week length of study in weeks). A trend was observed for greater reductions in body weight and exercise interventions that occurred over a greater number of weeks . Meta - analysis, changes in percent body fat were reported to be greater than changes in bmi - related measures (p for interaction = 0.0007). However, when limited to trials that assessed both, results were no longer statistically significant (p = 0.28). The nnt and gross estimates of the number of overweight and obese children and adolescents who might reduce their percent body fat from participation in an exercise program are shown in table 3 . As can be seen, the 95% cis for the nnt and subsequent estimates from the atlantis et al . Depending on the meta - analysis, approximately 2.8 to 3.6 million of the 12.5 million overweight and obese children in the us could reduce their percent body fat (ideally) by participating in a regular exercise program . The purpose of the current study was to conduct a systematic review of previous meta - analyses addressing the effects of exercise (aerobic, strength training, or both) in the treatment of overweight and obesity in children and adolescents . Overall, it appears that exercise reduces percent body fat in overweight and obese children and adolescents . This interpretation is further supported by the robustness of results across both meta - analyses [42, 43] with respect to magnitude of effect, nonoverlapping confidence intervals, influence analysis (each study deleted from the model once) and publication bias . In contrast, heterogeneity and overlapping 95% pis were found for the atlantis et al . In addition, while the absolute nnt was similar across both meta - analyses [42, 43], the 95% cis were wider for the atlantis et al . Study . Given that the atlantis et al . Most notably, greater reductions in percent body fat were found with higher exercise doses as well as when strength training studies were deleted from the model . While the former results appear plausible, the latter may be questioned . However, it is feasible that the potentially increased caloric expenditure from aerobic exercise may have resulted in greater reductions in percent body fat . The former notwithstanding, these results need to be interpreted with caution for at least two reasons . First, given the large number of statistical tests conducted, these findings could have been nothing more than the play of chance . Second, because studies are not randomly assigned to covariates, they are considered to be observational in nature . Consequently, the results of moderator and regression analyses conducted in a meta - analysis do not support causal inferences . Nonetheless, these findings are probably important as they support the need for addressing these potential associations in future, well - designed, randomized controlled trials . While improvements in percent body fat were observed, there is currently insufficient evidence that exercise improves bmi - related measures, body weight, and central obesity in overweight and obese children and adolescents . However, it is important to understand that a lack of evidence of effect does not mean evidence of no effect . As additional evidence accumulates, one may gain a better understanding regarding the effects of exercise on these outcomes in overweight and obese children and adolescents . The results of the current systematic review of previous meta - analyses on the effects of exercise in the treatment of overweight and obese children and adolescents have several implications for future research . First, while the overall quality of the two meta - analyses was considered adequate, there are several areas that might be improved upon in future meta - analytic work . These include (1) avoiding the use of publication status as an inclusion criterion, (2) documenting and providing a list of not only included studies but also excluded studies, including the reasons for exclusion, and (3) assessing publication bias . The former notwithstanding, avoiding the use of publication status as an inclusion criterion could be questioned . For example, van driel et al . Concluded that (1) the difficulty in retrieving unpublished work could lead to selection bias, (2) many unpublished trials are eventually published, (3) the methodological quality of such studies is poorer than those that are published, and (4) the effort and resources required to obtain unpublished work may not be warranted . Second, both of the included studies were aggregate data meta - analyses [42, 43]. While this continues to be the most common type of meta - analysis, individual - participant data meta - analyses (ipd) are considered to be the gold standard when attempting to quantitatively thus, future meta - analysts may want to consider using the ipd approach when addressing the effects of exercise in the treatment of overweight and obese children and adolescents . However, the use of the ipd approach needs to be considered with respect to the ability to retrieve ipd from investigators as well as the increased costs associated with the conduct of such, although methods to address the former have recently been developed . Third, given the apparent lack of available data in the original studies included in the two meta - analyses [42, 43], there is a need for future randomized controlled trials to examine and report the safety and cost - effectiveness of their exercise intervention(s) in the treatment of overweight and obesity among children and adolescents . In addition, since the average length of studies in the included meta - analyses was only 16 and 23 weeks, a need exists for longer intervention studies, including follow - up studies, to more fully understand the longitudinal effects of exercise on adiposity . Similarly, the apparent focus on per - protocol versus intention - to - treat analyses in the original trials allows one to draw conclusions regarding the efficacy (does the treatment work?) But not the effectiveness (does the treatment work in the real world?) Of exercise in the treatment of overweight and obese children and adolescents . Fourth, the dose - response effects of exercise on measures of adiposity remain elusive . While the atlantis et al . Meta - analysis concluded that 155 to 180 minutes per week of moderate to high intensity exercise is effective for reducing body fat in overweight and obese children and adolescents, additional research on this topic is needed, especially with respect to body weight, bmi - related measures, and central obesity . Fifth, percent body fat, but not bmi, appeared to be a more sensitive indicator of exercise - induced changes in adiposity among overweight and obese children and adolescents . Thus, future researchers may want to focus on percent body fat as their primary outcome despite the finding that bmi has been shown to correlate well with body fatness in children and adolescents . However, while numerous methods exist for the assessment of percent body fat (skinfold calipers, hydrostatic weighing, whole body air - displacement plethysmography, dilution, dual energy x - ray absorptiometry, computerized tomography, magnetic resonance imaging) [45, 51], none may be practical in many settings, including the community - based setting . Thus, the use of bmi - related measures such as bmi z - score and bmi percentile may need to be considered but interpreted with the realization that they may not be very sensitive to change . In addition, given its simplicity, bmi is currently the universally accepted method for assessing adiposity in children and adolescents . Sixth, because neither meta - analysis reported nnt [42, 43], it is suggested that future meta - analytic work includes such . From the investigative team's perspective, the reporting of such information is important because it provides practically relevant information to decision - makers (practitioners, policy - makers, etc .) Along those lines, formulas now exist for calculating nnt from continuous data . Finally, the most recent meta - analysis that met our inclusion criteria, published in 2008, included studies published up to february 2006, more than 7 years ago . While there is no definitive consensus regarding when to update a systematic review, with or without a meta - analysis, recent research by pattanittum et al . Concluded that three practical statistical methods could be applied to examine the need to update systematic reviews, with or without meta - analysis . Such updated work is critical with respect to providing guidelines based on the most recent evidence available . First, while there is a lack of cost - effectiveness and safety data, the use of exercise appears to be efficacious for improving adiposity, specifically percent body fat, in overweight and obese children and adolescents . The relatively low nnt observed as well as the potential number of overweight and obese children and adolescents who may benefit lends further support for this recommendation . Second, while the dose - response effects of exercise in the treatment of overweight and obese children and adolescents have not been fully elucidated, it would appear prudent to recommend that practitioners follow the general recommendations for exercise in children and adolescents, that is, 60 minutes or more of physical activity each day . The majority of the 60 minutes should be comprised of moderate to vigorous aerobic activity (bicycling, running, etc .) As well as muscle strengthening (pushups, etc .) And bone strengthening (jumping rope, etc . ), 3 days per week . Given the initial difficulty that overweight and obese children and adolescents may have in meeting these requirements, an individual exercise prescription that gradually progresses them to this level of effort seems appropriate . Third, given the apparent lack of sensitivity of bmi, it is recommended that practitioners assess and track changes in adiposity using one of the numerous methods available for assessing percent body fat . If not possible, then the assessment of adiposity using bmi z - score or percentile can be used with the understanding that the true effects of exercise on adiposity in overweight and obese children and adolescents may not be fully realized with this approach . Finally, given the observed magnitude of response of exercise on percent body fat in overweight and obese children and adolescents, exercise combined with other lifestyle and/or pharmacological interventions may be necessary for eliciting a health - improving impact on percent body fat in overweight and obese children and adolescents . Along those lines, an evaluation and treatment algorithm currently exists for addressing this issue . First, to the best of the authors' knowledge, this is the first systematic review of previous meta - analyses that has examined the effects of exercise in the treatment of overweight and obese adolescents, an increasingly important approach for addressing the effects of various healthcare interventions . Second, the additional analyses conducted based on the available data (influence analysis, publication bias, etc .) Helped strengthen the validity and findings of the two included meta - analyses [42, 43]. For example, the finding of no apparent publication bias for those studies that examined changes in percent body fat helped to strengthen the conclusions regarding the effects of exercise on percent body fat in overweight and obese children and adolescents . While conducting additional analyses beyond those reported in an original meta - analysis does not appear to be common when conducting systematic reviews of previous meta - analyses, future investigators may want to incorporate this methodology into their reviews while at the same time considering the additional time and effort involved in such an endeavor . Third, the nnt and gross estimates of the absolute number of overweight and obese children and adolescents who might reduce their percent body fat by participating in a regular exercise program were provided . In the authors' opinion, fourth, the calculation and inclusion of pis for statistically significant outcomes in the current study provide investigators with information that can aid them in planning future randomized controlled trials . In addition to the strengths of the current study, there are several potential limitations . First, the investigative team established fairly strict eligibility criteria for the current systematic review . As a result, only two previous meta - analyses met all eligibility criteria [42, 43]. While more focused and applicable, other relevant issues such as the effects of exercise on quality - of - life in overweight and obese children and adolescents were not captured . Second, the gross population estimates for the number of children who could reduce their percent body fat by participating in an exercise program assumed that none of the overweight and obese children and adolescents were exercising regularly . Finally, as with any systematic review, many of the biases inherent in both the included meta - analyses as well as the original trials that comprise each meta - analysis may also be present in a systematic review of previous meta - analyses . The results of the current systematic review of previous meta - analyses suggest that exercise is efficacious for reducing percent body fat in overweight and obese children and adolescents . However, there is currently insufficient evidence to suggest that exercise reduces bmi - related measures, body weight, and central obesity in overweight and obese children and adolescents.
Mucormycosis is an aggressive invasive opportunistic fungal infection belonging to the order mucorales, usually found in immunocompromised individuals . It is a life and limb threatening fungal infection with high mortality rate of 40%.1 though mucormycosis presents as a spectrum of disease, it is very rare in the musculoskeletal system . Almost all the immunocompromised patients with osteomyelitis of long bones due to mucormycosis require aggressive debridement and may sometimes need amputation.2 we present a patient with acute myeloid leukemia, who was diagnosed to have mucormycosis osteomyelitis of right proximal femur and was treated with limb salvage surgery successfully . A 41-year - old gentleman presented with sudden onset of pain and swelling over his right groin and upper thigh for a period of 10 days . He was a known patient with type ii diabetes mellitus and acute myeloid leukemia m2 diagnosed on the basis of bone marrow morphology and immunophenotyping 2 years ago . He received induction chemotherapy with cytosine and daunorubicin, followed by consolidation chemotherapy and peripheral autologous bone marrow transplant . Fourteen months post bone marrow transplantation, he had remission and developed the above complaints . Examination revealed mild tenderness over the right proximal femur with no significant restriction of movements . Magnetic resonance imaging (mri) of the right hip revealed altered signal intensity in the right proximal femur with no cortical breach or evidence of abscess . His complaints gradually increased in the next 3 weeks and he had difficulty in weight bearing and walking . On clinical examination, he had severe tenderness over the right proximal femur with painful restriction of movements at the hip joint . Plain radiographs revealed a 6 3 cm, ill - defined osteolytic lesion in the proximal shaft of right femur extending into the greater trochanter with wide zone of transition and adjacent soft tissue swelling [figure 1a]. Mri showed altered signal intensity in proximal metadiaphysis of right femur, including greater trochanter, with focal collection of 5 2.6 cm with cortical breach . There were two small abscesses measuring 1.5 and 0.7 cm, respectively, in the soft tissues [figure 1b]. (a) plain radiograph revealing an osteolytic lesion involving the right proximal femur (b) mri showing altered signal intensity in the proximal metadiaphysis of right femur with abscesses and cortical breach (c) bone scan demonstrating increased tracer uptake in the metadiaphyseal region of right proximal femur bone scintigraphy with technetium 99 m methylene diphosphonate (tc 99 m mdp) demonstrated intense increase in tracer uptake in the proximal third of right femur with normal tracer distribution in rest of the skeleton, suggestive of an infective pathology [figure 1c]. Intraoperatively, black discoloration of the right proximal femoral metaphysis with foul smelling discharge was noticed . The tissue was smeared with 10% koh solution and calcofluor white solution initially and then cultured in sabraoud's dextrose agar (sda) and lactophenol cotton blue (lpcb) mount which revealed broad aseptate fungal hyphae suggestive of mucormycosis . Histopathology of the cancellous bone and soft tissue exhibited large areas of necrosis and hemorrhage, replaced by inflammatory granulation tissue with dense infiltrates of lymphocytes, plasma cells, histiocytes, neutrophils, and few multinucleate giant cells . There were many broad, twisted, and few branching fungal filaments suggestive of mucormycosis [figure 2]. Histopathology with broad, twisted, and few branching fungal elements after the diagnosis of mucormycosis was confirmed, patient was evaluated for primary infection . There was no history of gastrointestinal infections and previous biopsy from gastric mucosa was negative . Hence, primary source of infection could not be traced . In the immediate postoperative period, the patient developed pathological fracture at the same site and was put on skeletal traction for 19 days before definitive fixation . He was treated with liposomal amphotericin 3 mg / kg / day for 19 days, following which radical debridement of the lesion proceeded by skeletal stabilization with 95 condylar blade plate for the right proximal femur . Considering the location of the pathological fracture (proximal femur) and the need for aggressive debridement and stable fixation, a 95 condylar blade plate fixation was achieved after adequate debridement . To prevent the usual complications of external fixation instead of infection such as pin tract infection, difficulty in mobilization and also considering adequate preoperative antifungal therapy and aggressive debridement, internal fixation was decided . 300 mg of fungitericin (amphotericin b) in powdered form was mixed with 40 g of simplex cement polymer and the mixture was then added with monomer, and while setting, cement beads were made into chains using stainless steel wire (two strings with 15 beads each) [figure 3a]. (a) postoperative plain radiograph with proximal femur stabilized with condylar blade plate and augmented with antifungal cement beads (b) three years followup plain radiograph with no evidence of osseous lesion with abundant callus intraoperatively, he had torrential blood loss amounting to 2.53 litres and went into hypovolemic shock . He was resuscitated with five units of packed red cells, eight units of cryoprecipitate, and eight units of fresh frozen plasma . Radical debridement, inability to use the tourniquet, and difficulty in differentiating infected and uninfected marrow were the causes for unexpected blood loss . After aggressive debridement, bone to bone cortical contact was achieved for healing which led to loss of metaphyseal bone loss of 3 cm . Postoperatively, liposomal amphotericin 3 mg / kg / day (180 mg / day) was given for 3 weeks and downgraded to 1 mg / kg / day (60 mg / day) for the next 9 weeks . Patient had received a total dosage of 7560 mg of liposomal amphotericin for a total period of 12 weeks . He was advised postoperatively physiotherapy and toe - touch weight bearing crutch walking for 12 weeks . Bone grafting was done along with beads removal and the tissue sent during surgery for histopathology and microbiological review was negative for mucormycosis . At 3-year followup, there was 3 cm limb shortening noticed for which a shoe raise was given . Plain radiographs revealed healed fracture with no residual / recurrent lesion in the metadiaphysis region of right proximal femur [figure 3b]. The common predisposing factors are uncontrolled diabetes, hematological malignancies like leukemia, lymphoma, etc ., organ transplantation, severe burns, diseases like tuberculosis and aids, post bone marrow transplantation, neutropenia, renal failure, immunosuppressive medications, etc . Mucormycosis has been described in various sites like cranium, hands, and feet, humerus, tibiae, femur, vertebrae, and joints.1 the etiopathogenesis of mucormycosis follows after entry into the host via ingestion, inoculation, or inhalation . In the immunocompromised host, the spores undergo angioinvasion leading to local necrosis and necrotizing infection, ultimately leading to systemic inflammatory response syndrome, multiorgan dysfunction, and death . Surgery includes radical debridement, mostly amputation, followed by high doses of amphotericin b. role of antifungal cement beads is limited mainly due to its cement elution properties and high local concentration leading to toxicity . Multiple in vitro studies have reported amphotericin b impregnated pmma beads to have a successful role in the management of fungal infections.37 goss et al . In their study on elution and mechanical properties of amphotericin concluded that amphotericin was released from cement at a clinically insignificant level.8 in vitro studies on amphotericin beads have shown to provide adequate release of its concentration (1.75 - 2.0 microgm / ml) up to 110 days from all bone cements.3 excessive local concentration of amphotericin b above 100 microgm / ml is lethal and concentration between 5 - 10microgm / ml causes abnormal morphology and reduced proliferation.4 of the seven cases of mucormycosis of tibia described in the literature, five were immunocompromised and 60% of those patients underwent amputation.9 the causes of amputation may be related to delayed diagnosis of mucormycosis, compromised immune status, inadequate debridement, or inadequate antifungal therapy . We present this case in view of the unusual site of presentation, limb - threatening osteomyelitis which was treated with radical debridement, liposomal amphotericin therapy, and using antifungal beads to enable limb salvage . High index of clinical suspicion, early diagnosis, aggressive debridement, and adequate liposomal amphotericin b are the key treatment modalities in the successful management of musculoskeletal mucormycosis.
It has been projected that there will be 250,000 new cases per year by 2015 indicating 3% increase per year . However, with the number of increasing survivors worldwide due to improvements in the treatment modalities of breast cancer, several psychosocial issues have gained prominence . In india, one study has focused on the psychosocial impact of breast cancer treatment . The following article highlights the qualitative findings related to body image and sexuality which are from a larger doctoral study . The larger study had adopted a mixed methods approach in understanding the various quality of life factors that influence breast cancer survivorship trajectory . In the current article, the study adopted a mixed method design, was cross - sectional and exploratory in nature . The quantitative sample consisted of fifty survivors and the qualitative sample consisted of 15 from within the quantitative sample . The inclusion criteria of the study were (a) women who have undergone mastectomy / lumpectomy and those currently undergoing hormonal treatment as adjuvant therapy, (b) women in age group of 18 and above, (c) married women, (d) english or hindi speaking, (e) minimum education of high school completion, and (f) at least 6 months since administration of chemotherapy and radiation therapy . The exclusion criteria included (a) women currently undergoing chemotherapy or radiation treatment, (b) women with cognitive impairments, and (c) women with active or distant metastases . Data for in - depth interviews were collected from survivors associated with two nongovernmental organizations called (a) connect to heal based in bengaluru and (b) hitaishini based in kolkata, st . John's medical college and hospital, a private oncology clinic in bengaluru, and through snowball sampling . Institute's ethical committee board at national institute of mental health and neurosciences, bengaluru and st . Based on a review of literature, semi - structured interview schedules were developed by researchers, and the draft was given to seven experts for feedback . Participant information leaflets were provided, and informed consent was taken from survivors before the in - depth interviews were undertaken . The participants were informed about the duration of interviews occurring over 23 sessions, each lasting 1.52 h. these interviews were done at homes and offices of participants and audio recorded after their permissions had been sought . For one participant who refused consent to audio record the interview interviews were started after providing consent, filling out demographic and clinical data sheet, and quantitative measures . Interviews were done in english and conducted until no new phenomenological information emerged in the interviews . It utilizes the first person's point of view to focus and understand lived experiences . The following steps were used to analyze data: inq scribe (a free online software) was used to transcribe, proof - reading interviews, and re - read to acquire broad understanding of experiencesline - by - line reading was done to identify the areas of phenomena that had been capturedstatement pertaining to areas of phenomena were assigned meaningsclusters of categories, broader themes, and domains based on identified statementsexhaustive description of phenomena was done based on integrated findings and provided to coresearchers for reviewcoresearchers' feedback were incorporated to reflect the universal features of phenomena . Inq scribe (a free online software) was used to transcribe, proof - reading interviews, and re - read to acquire broad understanding of experiences line - by - line reading was done to identify the areas of phenomena that had been captured statement pertaining to areas of phenomena were assigned meanings clusters of categories, broader themes, and domains based on identified statements exhaustive description of phenomena was done based on integrated findings and provided to coresearchers for review coresearchers' feedback were incorporated to reflect the universal features of phenomena . It utilizes the first person's point of view to focus and understand lived experiences . The following steps were used to analyze data: inq scribe (a free online software) was used to transcribe, proof - reading interviews, and re - read to acquire broad understanding of experiencesline - by - line reading was done to identify the areas of phenomena that had been capturedstatement pertaining to areas of phenomena were assigned meaningsclusters of categories, broader themes, and domains based on identified statementsexhaustive description of phenomena was done based on integrated findings and provided to coresearchers for reviewcoresearchers' feedback were incorporated to reflect the universal features of phenomena . Inq scribe (a free online software) was used to transcribe, proof - reading interviews, and re - read to acquire broad understanding of experiences line - by - line reading was done to identify the areas of phenomena that had been captured statement pertaining to areas of phenomena were assigned meanings clusters of categories, broader themes, and domains based on identified statements exhaustive description of phenomena was done based on integrated findings and provided to coresearchers for review coresearchers' feedback were incorporated to reflect the universal features of phenomena . Sociodemographic characteristics clinical characteristics in the domain of body image, themes focused on impact on identity, surgery - related issues, hair loss, adjustments to clothing, and encountering difficult situations . Identity: womanhood, motherhood, and attractiveness the impact of breast cancer on identity as a woman manifested in several ways: fear of losing one's breast was common and maximal in days following surgery: what happens to us females is that it is so difficult when we don't have one breast . That thing can never be explained to anyone as treatment ended, use of prostheses or other adjustments made, continued to be a daily reminder about an empty space . Another concern expressed by one survivor was the slightest comment by one's spouse about the shape could be distressing . To some survivors, the event of hair loss was most distressing: okay hair loss fine one day it will just come back maybe we just use a couple of wigs . One survivor also expressed the cultural expectation that breasts are to be hidden or covered which thereby reduced impact of the loss: when we were young particularly the breast part maybe our community and as well as society at that time was such that you will conceal breast that's why always one dupatta (scarf) will be there so losing a breast was not really big thing for me with relation to motherhood, children of most survivors ranged in age from adolescent to adulthood years when they were diagnosed with the illness and it, therefore, did not affect them . However, it had been a matter of concern for those who were diagnosed with it at an early age in terms of breastfeeding, inability to conceive when they wanted more children, or losing all chances of fertility due to the treatment . In terms of attractiveness attractiveness is internal and it was defined as one's behavior and not appearance . However, few survivors themes suggested a sense of being cheated by life as due to cancer, they no longer received attention or i feel quite attractive that way in fact now many people say that after chemotherapy your skin is more clear and there is glow on your face in relation to one's appearance postsurgery, a typical theme voiced was the tendency to avoid looking at the mirror after the treatment had been long over: personally i stopped looking at myself in the mirror i've had the long full - length mirror i used to have in my room . Self - conscious and disfigurish even after more than two decades of the illness . A variant theme was related to the leukoderma that she developed as a reaction to chemotherapy: my body is like an atlas (laughs). In relation to the scar, the most common theme was of dissatisfaction either with the quality of the surgery in terms of scarring or persisting pain and itching in the site: i feel it's a cobbler's job . However, most of them also become habituated to the scar though they initially experienced distress when looking at it . I tell her she ate it up as a kid several survivors had had the experience of others wanting to see the scar . Few of them were not comfortable with it while few others were comfortable showing it to close relatives . As one survivor who also works as a cancer volunteer expressed, it was dependent on the context: some people have wanted to see it especially those who are very curious, i did not feel good at all about their curiosity as this is not a matter to be joked about, it is an illness . But when a patient has said, please show it to me . Whatever happened to you, is it same like mine? Then we show it to them we show it to them because their curiosity is different, they think, mine and hers are same in relation to the shape and size of the breast, there was a typical theme between those who had undergone mastectomy or lumpectomy . For those with lumpectomy, they expressed concern about breasts being lopsided and it was accentuated as the survivor aged: my left side looks young, and my right side due to age, length has become long . A variant theme was the presence of sensations in the area where the mastectomy had occurred: you may not touch your breast like that every day but now you're conscious . Now you get a scratchy or if you cut off a leg, don't they say they get sensation at the end of your toe even if your leg is cut off from here? Hair loss during chemotherapy was handled either through the use of scarves, wigs, hijab, or going bald . For one survivor, the bald head and the use of a scarf were particularly difficult for her young child: the shock was with my younger one because she couldn't see me that way and when i used to expose my head in front of her, i always had a dupatta (scarf) on my head while i was at home . Others either shaved their heads before the chemotherapy sessions began and had wigs made out of it or as one participant said: i bought a couple of wigs off the net and i would go around in my blonde avatar . Actually got a blonde (laughs) just to kind of cut off your nose to spite your face kind of thing one survivor who was of islamic faith would wear the religious headgear or the hijab to handle the situation . One survivor expressed the taboo associated with hair loss: and all those things unnecessarily traditionally or just it becomes a taboo or stigma also . Actually in islam you don't have but since we are in india, so those things matter a lot . Themes that emerged in relation to adjustments made for clothes have been divided on the basis of the breast, lymphedema, the chemo port scar, and leukoderma . Adjustments for those undergoing mastectomies involved using substitutes such as prosthesis, padded bras, and pads . The use of prosthesis on an everyday basis was convenient, but its weight was a concern and others would use pads made out of cloth . In those with lumpectomy, there was a tendency for breasts to be lopsided due to which they reported wearing baggy clothes with a scarf or a dupatta (scarf) and avoidance of white clothes . With a saree blouse, issues were regarding it's fit as it caused pain in the surgery site when worn for few hours and the neckline as there was a fear of the scar being exposed: when i go to my tailor i have to show him my scar because now when i see this scar on someone else then i know that person has had breast cancer or at least cancer of some sort to have this port cut . Because it's very obvious noh because it's got two lines so he has to make sure that the neckline comes this way right or a v? In relation to lymphedema, the need for different sleeve sizes was a matter of concern as they had to be stitched in different sizes . One survivor also expressed reduced interest in dressing style: now i feel i will never look good . So that parts gone away so now it's just dressing for activity which you do (laughs) but the other thing will also be that i am not as attractive as i used to be because i can't dress as attractively as i used to . In contrast, several survivors also reported taking more care than before about their appearance: so i have something new and just to feel good . Uncomfortable situations in relation to feeling uncomfortable in particular situations due to fears of exposure, it occurred when (a) going for swims, massages, changing clothes others presence, frisked by security personnel, (b) breast accidently collided with someone else in buses or crowded areas: when i would travel by train or bus and if someone collided, they would stare for a bit because it would feel different because i would wear a prosthesis and you could make out i always used to cover the area with my bag . Another concern was raised in terms of the prosthesis slipping out by few survivors: even for swimming it's not possible since it floated out once and i was able to put it back before anyone noticed when you are holding on top and travelling in a bus, one day the prosthesis came down, but i was able to put it back in its place without anyone seeing most participants were uncomfortable in sharing details regarding the physiological aspect of sexual functioning or sexual intimacy that are practiced in nonsexual behaviors . There is no sexual intimacy left or that there was initial difficulty after the treatment but it's all fine now . Therefore, some of the presented findings are based on the responses of few survivors . The themes related to sexuality have been divided on the basis of changes due to the treatment, partner's challenges and adjustments made, and attitudes held toward sexuality . Themes suggested immediately after surgery, few survivors reported that there was difficulty in communication, but spouses were empathetic: not because of anything else, he thought maybe you know i'm not ready or he didn't want to force anything on me . So there's lot of tension there in the beginning, but now i feel it's all well past gone . One survivor sensing her husband's difficulty consulted her physician before resuming sexual activity: after being cautious for good number of days, i felt he was finding it difficult as males get aroused easily as well as it diminishes fast . Then, i consulted my doctor, he said,' it is completely normal . Your sexual life is as normal as how it was before the operation . And your worry regarding hurting your breast, it doesn't get hurt . As treatment ended and several years had passed, few survivors voiced their concern about a lack of desire to engage in sexual activity and an inability to get aroused which was associated with vaginal dryness and pain . Moreover, one survivor with lumpectomy also expressed a change in her partner's overt sexual behavior in the form of preference for the normal breast . Few survivors mentioned that the spouse's health also played a role in the cessation of sexual life . Age was often voiced as a common factor for decrease in sexual activity by all older survivors . However, one survivor said they continue to be intimate through affection: somehow my husband also more into exercises this and that more into spirituality we have lot of sharing and bonding but not that physical urge to do sex, maybe we hold hand and things like that another participant had extreme views that sexual life could be equated to animal life and it serves the function of procreation purposes . In the domain of body image, themes focused on impact on identity, surgery - related issues, hair loss, adjustments to clothing, and encountering difficult situations . Identity: womanhood, motherhood, and attractiveness the impact of breast cancer on identity as a woman manifested in several ways: fear of losing one's breast was common and maximal in days following surgery: what happens to us females is that it is so difficult when we don't have one breast . That thing can never be explained to anyone as treatment ended, use of prostheses or other adjustments made, continued to be a daily reminder about an empty space . Another concern expressed by one survivor was the slightest comment by one's spouse about the shape could be distressing . Okay hair loss fine one day it will just come back maybe we just use a couple of wigs . One survivor also expressed the cultural expectation that breasts are to be hidden or covered which thereby reduced impact of the loss: when we were young particularly the breast part maybe our community and as well as society at that time was such that you will conceal breast that's why always one dupatta (scarf) will be there so losing a breast was not really big thing for me with relation to motherhood, children of most survivors ranged in age from adolescent to adulthood years when they were diagnosed with the illness and it, however, it had been a matter of concern for those who were diagnosed with it at an early age in terms of breastfeeding, inability to conceive when they wanted more children, or losing all chances of fertility due to the treatment . In terms of attractiveness attractiveness is internal and it was defined as one's behavior and not appearance . However, few survivors themes suggested a sense of being cheated by life as due to cancer, they no longer received attention or i feel quite attractive that way in fact now many people say that after chemotherapy your skin is more clear and there is glow on your face in relation to one's appearance postsurgery, a typical theme voiced was the tendency to avoid looking at the mirror after the treatment had been long over: personally i stopped looking at myself in the mirror i've had the long full - length mirror i used to have in my room . Self - conscious and disfigurish even after more than two decades of the illness . A variant theme was related to the leukoderma that she developed as a reaction to chemotherapy: my body is like an atlas (laughs). In relation to the scar, the most common theme was of dissatisfaction either with the quality of the surgery in terms of scarring or persisting pain and itching in the site: i feel it's a cobbler's job . However, most of them also become habituated to the scar though they initially experienced distress when looking at it . I tell her she ate it up as a kid several survivors had had the experience of others wanting to see the scar . Few of them were not comfortable with it while few others were comfortable showing it to close relatives . As one survivor who also works as a cancer volunteer expressed, it was dependent on the context: some people have wanted to see it especially those who are very curious, i did not feel good at all about their curiosity as this is not a matter to be joked about, it is an illness . Then we show it to them we show it to them because their curiosity is different, they think, mine and hers are same in relation to the shape and size of the breast, there was a typical theme between those who had undergone mastectomy or lumpectomy . For those with lumpectomy, they expressed concern about breasts being lopsided and it was accentuated as the survivor aged: my left side looks young, and my right side due to age, length has become long . A variant theme was the presence of sensations in the area where the mastectomy had occurred: you may not touch your breast like that every day but now you're conscious . Now you get a scratchy or if you cut off a leg, don't they say they get sensation at the end of your toe even if your leg is cut off from here? Hair loss during chemotherapy was handled either through the use of scarves, wigs, hijab, or going bald . For one survivor, the bald head and the use of a scarf were particularly difficult for her young child: the shock was with my younger one because she couldn't see me that way and when i used to expose my head in front of her, i always had a dupatta (scarf) on my head while i was at home . Others either shaved their heads before the chemotherapy sessions began and had wigs made out of it or as one participant said: i bought a couple of wigs off the net and i would go around in my blonde avatar . Actually got a blonde (laughs) just to kind of cut off your nose to spite your face kind of thing one survivor who was of islamic faith would wear the religious headgear or the hijab to handle the situation . One survivor expressed the taboo associated with hair loss: and all those things unnecessarily traditionally or just it becomes a taboo or stigma also . Actually in islam you don't have but since we are in india, so those things matter a lot . Themes that emerged in relation to adjustments made for clothes have been divided on the basis of the breast, lymphedema, the chemo port scar, and leukoderma . Adjustments for those undergoing mastectomies involved using substitutes such as prosthesis, padded bras, and pads . The use of prosthesis on an everyday basis was convenient, but its weight was a concern and others would use pads made out of cloth . In those with lumpectomy, there was a tendency for breasts to be lopsided due to which they reported wearing baggy clothes with a scarf or a dupatta (scarf) and avoidance of white clothes . With a saree blouse, pain in the surgery site when worn for few hours and the neckline as there was a fear of the scar being exposed: when i go to my tailor i have to show him my scar because now when i see this scar on someone else then i know that person has had breast cancer or at least cancer of some sort to have this port cut . So he has to make sure that the neckline comes this way right or a v? In relation to lymphedema, the need for different sleeve sizes was a matter of concern as they had to be stitched in different sizes . One survivor also expressed reduced interest in dressing style: now i feel i will never look good . Never look attractive to people, to men in general so that parts gone away so now it's just dressing for activity which you do (laughs) but the other thing will also be that i am not as attractive as i used to be because i can't dress as attractively as i used to . In contrast, several survivors also reported taking more care than before about their appearance: so i have something new and just to feel good . Uncomfortable situations in relation to feeling uncomfortable in particular situations due to fears of exposure, it occurred when (a) going for swims, massages, changing clothes others presence, frisked by security personnel, (b) breast accidently collided with someone else in buses or crowded areas: when i would travel by train or bus and if someone collided, they would stare for a bit because it would feel different because i would wear a prosthesis and you could make out i always used to cover the area with my bag . Another concern was raised in terms of the prosthesis slipping out by few survivors: even for swimming it's not possible since it floated out once and i was able to put it back before anyone noticed when you are holding on top and travelling in a bus, one day the prosthesis came down, but i was able to put it back in its place without anyone seeing most participants were uncomfortable in sharing details regarding the physiological aspect of sexual functioning or sexual intimacy that are practiced in nonsexual behaviors . There is no sexual intimacy left or that there was initial difficulty after the treatment but it's all fine now . Therefore, some of the presented findings are based on the responses of few survivors . The themes related to sexuality have been divided on the basis of changes due to the treatment, partner's challenges and adjustments made, and attitudes held toward sexuality . Themes suggested immediately after surgery, few survivors reported that there was difficulty in communication, but spouses were empathetic: my husband was scared to touch me even . Not because of anything else, he thought maybe you know i'm not ready or he didn't want to force anything on me . So there's lot of tension there in the beginning, but now i feel it's all well past gone . One survivor sensing her husband's difficulty consulted her physician before resuming sexual activity: after being cautious for good number of days, i felt he was finding it difficult as males get aroused easily as well as it diminishes fast . Then, i consulted my doctor, he said,' it is completely normal . Your sexual life is as normal as how it was before the operation . And your worry regarding hurting your breast, it doesn't get hurt .' After that, we had sexual activity, though it was very less . As treatment ended and several years had passed, few survivors voiced their concern about a lack of desire to engage in sexual activity and an inability to get aroused which was associated with vaginal dryness and pain . Moreover, one survivor with lumpectomy also expressed a change in her partner's overt sexual behavior in the form of preference for the normal breast . Few survivors mentioned that the spouse's health also played a role in the cessation of sexual life . Age was often voiced as a common factor for decrease in sexual activity by all older survivors . However, one survivor said they continue to be intimate through affection: somehow my husband also more into exercises this and that more into spirituality we have lot of sharing and bonding but not that physical urge to do sex, maybe we hold hand and things like that another participant had extreme views that sexual life could be equated to animal life and it serves the function of procreation purposes . The aim of the current study is to understand breast cancer survivorship trajectory from an indian perspective . In the current article, there has been an attempt to understand the impact of the diagnosis of breast cancer and its treatment on body image and sexuality issues using qualitative findings of a larger doctoral study . Qualitative findings of the study provide an understanding about how one experiences challenge on an everyday basis in dealing with body image alterations . The studies with similar findings have shown affects one's identity through its impact on sense of attractiveness, womanhood, and motherhood . However, in the india context, it is further complicated due to stigma often attached to the diagnosis of cancer and loss of a breast in a culture where sexuality is generally repressed . Varying degrees of habituation to the scar, discomfort in looking at oneself in the mirror and the shape of the breast which affected clothing habits were similar to findings in another study . However, in the indian context, adjustments to the dressing style with stitching of saree blouse's neckline and the sleeve sizes using a known tailor was associated with less threat of exposure, and saree was preferred as it helped camouflage the breast better . This finding was similar to the study wherein the saree was the preferred choice of clothing . In terms of womanhood, older women experienced a sense of loss of an organ that had nurtured their children, but it was not significant . Similar findings were made in a study done in iraq which found that older women believed that it had served its purpose and therefore, in terms of motherhood it did not signify an important loss . The process of body hair loss during chemotherapy has been considered a traumatic experience even though it is temporary in nature . Similar findings have been found in different ethnicities in the west . In india, as based on a survivor's belief, hair loss can induce extreme distress due to the notion that long hair is associated with femininity . In relation to understanding sexuality, the researcher encountered difficulties in eliciting in - depth data . Research has also consistently established the same in terms of diminishing sexual functioning with age due to both biological and psychosocial factors . An interesting psychosocial belief which manifested in the present study sex life is animal life, and family tradition of spouses sleeping separately (apart from age being a factor) again alludes to the tendency to repress sexuality by equating it to the function of procreation . Belief about how engaging in sexual activity could cause endometriosis also hints at the lack of awareness . A similar finding was reported in china, wherein survivors were asked by others not to engage in sex due fears of contracting endometriosis . These findings could be explained by the fact that along with cultural repression of sexuality, the free expression, or discussion about sexuality could suggest defying of standard sexual norms of the society thereby, alluding to a sense of immorality within the individual . Limitations of the study are (a) sample consisted of survivors from urban, educated, and middle socioeconomic level with at least 12 years of formal education . Survivors of rural background, low - income families, and less education levels could have had different concerns, (b) it was a cross - sectional study design with purposive sampling; therefore, self - selection bias could have been present . It does not provide scope to include perspectives of survivors experiences who refused consent, and (c) there was difficulty in understanding sexuality in - depth due to taboos attached to it, ethical considerations, and the fear of rupture of alliance between the researcher and the participant . Strengths of the study include (a) it is one of the first indian studies to focus on body image, and sexuality in breast cancer, (b) all interviews (except one) was audio taped after the consent of the participants, and (c) the study illustrates certain similarities in findings with western literature and the role of indian culture in understanding sexuality . The findings provide directions to help formulate interventions designed to address body image issues of breast cancer survivorship . Research implications include replicability of the study to rural contexts with survivors from low - income and less education backgrounds and including spouses to gain understanding about its impact on them . The findings highlighted above suggest breast cancer survivors can experience various forms of body image issues and difficulties in sexuality as a consequence of breast cancer treatment . Overtime, most survivors tend to find various ways to deal with body image related issues though improvements can be made in services geared towards helping them cope with treatment related changes . However, in terms of sexuality, due to the taboo attached to it, it will require innovative ways to firstly understand the difficulties experienced followed by designing interventions to address them.
In europe, prostate cancer (pca) is the most common malignancy in males and the third most common cause of cancer mortality . Currently, more extensive and earlier diagnosis has led to a decrease in pca related mortality [1, 2]. Despite an extended search for a more specific biomarker than prostate specific antigen (psa), psa still remains the only widely employed diagnostic and follow - up marker for pca . The predictive capability of the 4 ng / ml psa threshold as a biopsy indicator is deficient since 2040% of pca cases are thereby missed . Thus, other accurate diagnostic pca biomarkers, especially for aggressive and potentially life - threatening pca, are needed . Since increased aerobic glycolysis or the warburg effect has been identified as common to neoplastic cells, this mechanism promises potential for diagnostic and therapeutic targets . The enzyme transketolase - like 1 (tktl1), therefore, comes into investigational focus since it is a crucial enzyme for sugar fermentation, linking glucose and fat metabolism without pyruvate dehydrogenase [6, 7]. When overexpressed, tktl1 activates the pentose phosphate pathway (ppp), accelerating tumor cell growth and supporting tumor survival and systemic dissemination . Accordingly, an assay was developed to evaluate tktl1 based on the fluorometric epitope detection of specific antibodies in cd14/cd16 positive monocytes, following tumor cell phagocytosis and digestion (edim - test). Still, the role of tktl1 as a cancer biomarker is controversial [10, 11] and the edim - test has not been approved for routine clinical application . Nevertheless, though tktl1 does not play a role in conventional medicine, it has gained high popularity in alternative / complementary medicine, not only as a diagnostic but also as a prediction marker to assess the risk of metastatic progression . The goal of the current investigation was to compare the tktl1 serum level in patients with a clinically localized pca to that in healthy controls . Furthermore, the investigation was directed towards establishing whether the tktl1 serum level correlates with clinical and histologic parameters of the tumor, thus facilitating identification of patients harboring life - threatening disease requiring definitive treatment . For this purpose, the serum concentration of tktl1 in pca patients and healthy controls was analyzed by means of an enzyme - linked immunosorbent assay (elisa), which is a highly standardized detection system, and correlated to clinical and histologic parameters . Patients (n = 66) undergoing curative radical prostatectomy (rpe) for biopsy - proven pca in the department of urology, goethe - university, frankfurt am main, germany, were included in the investigation . Controls (n = 10) were healthy, age - matched, male volunteers . Firstly, 10 ml peripheral blood was drawn from patients several days before surgery and from controls . Blood samples were allowed to coagulate and then centrifuged at 3000 rpm at + 4c for 10 minutes . The concentration of tktl1 was determined in pca patient and control serum using a commercially available elisa kit (seh018hu, cloud - clone corp, houston, tx, usa; sensitivity: <0.055 ng / ml with no significant cross - reactivity or interference between tktl1 and analogues). All assays were done in duplicate and the concentration was calculated from a standard curve using a 4-parameter curve fit (magellan software, tecan). Univariate analysis was performed by the wilcoxon - man - whitney - test for comparison between two groups and the kruskal - wallis - test with the iman - conover - method (bonferroni - holm - corrected) for more than two groups . Hanns ackermann, epsilon - publishers, frankfurt, germany). The null hypothesis (tktl1 concentration in serum of pca patients does not differ from that of healthy volunteers) was rejected if p - values were less than 0.05 . The clinical tumor stage was classified according to the 7 edition of the ajcc and the pathologic tumor stage was determined according to the 6 edition of the tnm classification . Imaging was carried out according to the currently valid guidelines of the european association of urology . The median age at tumor diagnosis was 66 years (range 4688) and the median serum psa was 8.0 ng / ml (range 1.857.0) (median age control group: 60 years (5572); control psa: 2.8 ng / ml (2.04.0). Nearly all pcas submitted to surgery were clinically significant . None of the patients had evident clinical signs of infection or acute or chronic inflammation at surgery . Clinical and histopathological demographics of 66 pca patients values expressed as median with range or number (%). Rpe radical prostatectomy; dre digital rectal examination, psad psa density univariate analysis of the elisa investigation demonstrated that serum tktl1 was significantly lower in the serum of pca patients, compared to healthy controls (p=0.0001, effect size indicator r = z / sqr(n) = 0.4179, figure 1). However, correlation between serum tktl1 and serum psa (p = 0.38), biopsy and prostatectomy gleason sum (p = 0.79 and 0.89, respectively), prostate volume (p = 0.23), psa density (p = 0.80), clinical and pathologic t - stage (p = 0.66 and 0.65), highest gleason pattern in the biopsy and prostatectomy specimen (p = 0.83 and 0.74, respectively), upgrade of gleason sum from biopsy to prostatectomy (p = 0.86), pn-, l-, v-, n- and r status (p = 0.32, 0.88, 0.30, 0.90 and 0.32, respectively), extracapsular extension, seminal vesicle invasion as well as d`amico classification (p = 0.75, 0.89 and 0.34, respectively) did not reach statistical significance . Tktl1 serum concentration (ng / ml) in pca patients and controls . Box: lower line quartile q1 (25% quantile); middle line tktl1, as evaluated by the edim - test, has been propagated as a reliable cancer biomarker, since a positive correlation between tktl1 expression and tumor progression has been reported . However, investigations negating the reliability of tktl1 have also been published [11, 13], making a definitive assessment regarding tktl1 reliability as a biomarker questionable . Indeed, the decreased serum tktl1 found in pca patients in the present investigation stands in opposition to the increase in monocyte associated tktl1 claimed by the proponents of the edim - test . However, the different methods and localities, where tktl1 was measured, in serum and in macrophages, could account for the differing results . Speculatively, assuming the accuracy of the edim test together with the theoretical background proposed by coy and colleagues, an inverse correlation between tktl1 detected in macrophages and extracellular tktl1 in serum could occur, since tumor cells undergo phagocytosis . Tktl1 could therefore be sequestered in macrophages and the tktl1 level in serum be reduced . Still, this hypothesis is speculative and requires further evaluation . Although, in the present investigation, serum tktl1 levels differed in cancer patients and healthy persons, no correlation was apparent between serum tktl1 and clinical or pathologic parameters . Conflicting reports about tktl1 expression associated with various cancer entities and clinical outcome have been published by several investigators [10, 13, 14]. Tktl1, determined with the same immunohistochemical method in colorectal cancer tissues, has been positively associated with tumor progression and tktl1-expression level in one study, whereas another study pointed to a significant decrease in tktl1-expression associated with metastasis . Recently, the dna demethylating agent 5-aza-2'-deoxycytidine (5-aza) has been reported to augment expression of tktl1 in melanoma cells and was associated with enhanced invasion of the tumor cells, whereas others have demonstrated reduced invasion of melanoma cells under 5-aza treatment . Grimm et al . Correlated an increase of edim scores with a metabolic shift from aerobic to anaerobic conditions, whereas this correlation could not be confirmed by others . Possibly, these inconsistencies have led to the recommendation of combining tktl1 quantification with a standardized panel of established blood biomarkers . The ambivalence of tktl1 expression in so many different investigations shows that the role of tktl1 may be more complex than initially thought . This study was designed as a pilot investigation and thus includes a limited number of patients . Since patients only underwent a general health check, this could contribute to a potential, though unlikely, bias for the high serum tktl1 expression found in this cohort . The study was designed to assess the diagnostic potential of tktl1, not its prognostic ability . Since pca, in most cases, is associated with slow progression, long - term follow - up would be required to follow the course of serum tktl1 during the course of the disease . Serum tktl1 was decreased in patients with clinically localized pca, but failed to facilitate identification of patients with aggressive disease, who might particularly benefit from definitive cancer treatment . Based on these results, we cannot currently advise introducing serum tktl1 into clinical practice . Further long - term studies including larger patient cohorts and simultaneous measurement of serum tktl1 and macrophage sequestered tktl1 are warranted to clarify the role of tktl1 in pca and resolve its applicability as a pca biomarker.
Recurrent aphthous stomatitis (ras) is one of the most frequently encountered oral mucosal disorders . Despite extensive amount of research, the etiology of ras remains unclear . The aim of the study was to assess the levels of anxiety and salivary cortisol levels in patients with ras and also to determine the association and relationship of salivary cortisol levels to variations of stress . A total of 30 patients suffering with ras, along with the same number of age and sex matched healthy controls were included in the study . Saliva was collected from all the subjects at 9.00 am to avoid diurnal variations of cortisol levels . Student's t - test was used to compare the anxiety and salivary cortisol levels between both groups . The mean salivary cortisol level of the ras group showed a very highly significant difference (p = 0.000) from the controls . The mean anxiety scores of the ras group showed a very highly significant difference (p = 0.000) from the controls . The values of pearson correlation coefficient between anxiety and salivary cortisol was 0.980 and one with a p value of 0.000 showing that there is a highly positive correlation between anxiety and salivary cortisol . Thus besides traditional treatment of ras patients, our findings suggest that psychological support is also needed . Recurrent aphthous stomatitis (ras) is the most common type of ulcerative disease of the oral mucosa, and it affects approximately 20% of the general population with a range from 5% to 66% . Highest prevalence of 66% was found by ship et al . On dental and medical students . Minor ras, which makes up more than 80% of all ras cases, is a small (up to 1 cm in diameter), shallow, painful, well - circumscribed, and round - shaped ulceration that is covered with a yellow - grayish pseudo membrane and surrounded by an erythematous halo . Major ras is characterized by ulcers that are typically larger and deeper than minor ras . Herpetiform ulcers manifest as multiple recurrent clusters of small ulcers (less than 4 mm in diameter) that are scattered throughout the oral mucosa . Previous studies have suggested that psychological disturbances such as stress and anxiety could play a role in the onset and recurrence of ras lesions . The worldwide distribution, high frequency and decreased quality - of - life generated by ras have resulted in a great deal of research into the etiology and efficient therapy of this disease . However, the etiology of ras still remains unclear, and the currently available therapy remains inadequate . Several studies have reported a relationship between ras and various causes, but the results are conflicting . It is suggested that stress with its presumed effects on the immune system, constitutes one of the major causative agents of ras . An insight into a patient's psychological status can be estimated from both serum free and salivary cortisol levels . Cortisol, also called as a stress hormone, has been used as an indicator in the stress evaluation studies . Salivary cortisol may actually provide a better measure than serum cortisol of the stress response as it more accurately measures the amount of unbound cortisol compared to serum measures . Salivary cortisol exhibits a clear diurnal variation and circadian rhythmicity with a time course closely parallel to that of plasma cortisol . Various scales were used in the psychological assessment of patients in previous studies, some were self - reported and others assessed by a psychiatrist . Though stress and anxiety have been mentioned as possible factors related to the development of ras; this association somewhat remains controversial . The aim of this cross sectional study was to conduct an investigation in assessing the relationship between anxiety and salivary cortisol in patients with ras by using both psychological testing instrument (hamilton's anxiety scale [has]) and physiological testing instrument (salivary cortisol). The study was conducted in the department of oral medicine and radiology, kamineni institute of dental sciences, india, after approval by institutional ethics committee . Thirty patients suffering from ras (17 females 57%, 13 males 43%) were taken for the study after an informed consent . The inclusion criteria for patients were as follows: minor form of ras, non - smokers, and a minimum of 2 years of ras history to rule out from other types of acute recurrent ulcers like recurrent herpes . Patients suffering from systemic diseases including endocrine and metabolic diseases, hematinic deficiencies, patients using steroids / oral contraceptive pills, pregnant patients, smokers were excluded from the study . Saliva samples were obtained from the study group when aphthous stomatitis was absent after healing . Control and study group samples were collected between 9 and 9:15 am, before a meal without stimulation by passive drooling directly into a sterile glass tube until 5 ml is collected . The collected salivary samples were centrifuged for 15 min at 3000 rpm and frozen at -20c until shortly before assay . During assay, salivary cortisol was measured by competitive enzyme linked immunosorbent assay method, by using cortisol eia (diametra kit, korea). The normal cortisol concentrations that were given as a guide line according to the kit are in the ranges from 8.2 to 27.59 nmol / l (0.3 - 1 g / dl) at morning time and 1.65 - 6.89 nmol / l (0.06 - 0.25 g / dl) at evening collected samples . After saliva collection anxiety levels were measured by using has that provides the measures of overall anxiety, psychic anxiety (mental agitation and psychological distress) and somatic anxiety (physical complaints related to anxiety). This scale consists of 14 questions, among which seven address the psychic and the remaining seven addresses the somatic anxiety . The individuals are rated on a five point scale for each of the 14 items ranging from 0 to 4 . The total anxiety score ranges from 0 to 56, where <17 indicates mild severity, 18 - 24 mild to moderate severity and 25 - 30 moderate to severe . T - test was used to compare the anxiety and salivary cortisol levels between patients with ras and the control group as the base line data was normally distributed . Pearson's correlation analysis was used to study the correlation among anxiety and salivary cortisol levels in patients with ras . Logistic regression analysis was used for calculation of each variable's independent contribution to dependent variable . Ras was a dependent variable, and salivary cortisol and anxiety levels were independent variables in this model . For this trail 60 patients were enrolled . The ras group comprised of 13 males and 17 females and the mean age was 29.2 years (range 18 - 60 years). The control group comprised of the same number of age and sex matched individuals (mean age 29.2 years). The mean salivary cortisol levels were 47.73 8.80 nmol / l (1.73 0.319 g / dl) in ras patients and 13.905 3.55 nmol / l (0.504 0.129 g / dl) in controls with a p value of 0.000 . The mean anxiety levels in ras group were 27 4.76 and 10.2 3.27 in the control group (p = 0.000) as indicated in table 1 . Comparison of salivary cortisol levels and anxiety scores (meansd) of patients we found that salivary cortisol and anxiety levels were significantly higher in ras group as compared to control group . There was a highly significant positive correlation (p = 0.000) between salivary cortisol levels and anxiety . A logistic regression model in which the ras was taken as dependent variable and salivary cortisol and anxiety levels were taken as independent variables were performed . Salivary cortisol levels and anxiety scores were found significantly related with ras [table 2]. Logistic regression analysis, in which the group membership (ras or control) was dependent variable the impact of oral disorders on quality of life has been increasingly recognized as an important outcome measure for clinical trials, especially since oral disorders frequently have detrimental effects on speech, nutrition, physical appearance, self - esteem and social interaction . Ras frequently affects patient quality of life as a result of long lasting and recurrent episodes of burning pain . The etiopathogenesis of ras appears to be complex; interactions with genetic, nutritional and hematological factors are reported . Much has now been clarified about the mechanisms involved, interesting new associations such as the involvement of t - cell mediated immunologic reaction have emerged . Several reviews have, however, reported little objective evidence to support such an association . There is good evidence that stress and anxiety are related to increased resting levels of cortisol . The present study was undertaken in an attempt to gain a better understanding of the role that has been attributed to stress, anxiety and salivary cortisol in the development of ras and also to assess the relationship of salivary cortisol levels with stress and anxiety in patients with ras . A previous study mccartan et al . Investigated the possible association between anxiety, measured by hospital anxiety and depression scale, and salivary cortisol in patients with ras; and concluded that stress may play a role in the etiology of ras . Albanidou - farmaki et al . Conducted a case control study and compared the association between state and trait anxiety, measured by spielberger's state - trait anxiety inventory; and serum and salivary cortisol levels in patients with ras; concluding that stress may be involved in the pathogenesis of ras . Conducted a case control study to assess the influence of psychological stress on manifestations of ras; by means of a questionnaire developed by the psychology institute of sao paulo university (symptoms of stress list; vas visual analog scale questionnaire) and concluded that stress may play a role in the manifestation of ras . Present study results also showed statistically significant increase in salivary cortisol and anxiety levels in ras patients during inactive stage compared to control group . Our study in contrast to previous studies (2 and 5) used has, which provides the measures of overall anxiety, psychic anxiety (mental agitation and psychological distress) and somatic anxiety (physical complaints related to anxiety). Stress and anxiety may play a significant role in the onset and recurrence of ras lesions . Ras is typically observed during stressful situations such as school exam periods, dental treatments and periods of significant changes in life . Stress alters the regulation of both the sympathetic and parasympathetic branches of the nervous system, with consequential alterations in hpa - hypothalamic - pituitary - adrenal axis . Autonomic activation and elevation of hormones, including those produced by hpa axis play pivotal roles in regulating immune surveillance mechanisms . This immune regulatory activity with an increased number of leukocytes at the sites of inflammation induced by psychological stress is characteristic, often observed during the pathogenesis of ras . In conclusion, the present study showed a positive association between salivary cortisol levels and anxiety in ras patients during inactive stage . Therefore, measurement of the salivary cortisol and anxiety which reflect response to stress seems a promising parameter in the investigation of ras . In conclusion, we suggest that stress management interventions may be beneficial along with conventional treatment methods in ras patients.
Animal models utilizing human recombinant activated factor vii (rfviia; novoseven; novo nordisk a / s, bagsvrd, denmark) are limited by interspecies differences in terms of coagulation molecule interactions . The interaction between tissue factor and factor human factor vii has been shown to have reduced activity when it is exposed to porcine thromboplastin . These considerations are compounded by the fact that the majority of standard in vitro coagulation assays are performed with rabbit brain thromboplastin . Both rabbit and human thromboplastin induce coagulation more efficiently in human plasma than in swine plasma . Therefore, changes in coagulation parameters seen with the use of human rfviia in animals may not correlate with hemostatic end - points . Because of these interspecies differences with regard to human rfviia activity, minimal conclusions concerning optimal dosing in humans can be drawn from animal experiments . In pharmacologic models the dose of rfviia needed for effect varies substantially between animal species, including the dog (60 g / kg), rabbit (2 mg / kg), and mouse (10 mg / kg). Animals given 2 mg / kg rfviia had decreased time of bleeding and blood loss compared with control animals . The efficacy of reversing a bolus of low - molecular - weight heparin (lmwh) was studied in a rabbit ear puncture model . Animals were given 1800 anti - factor x units / kg of lmwh, which raised the primary bleeding time approximately fourfold . The absence of efficacy was hypothesized to be secondary to very high anti - factor xa levels produced by bolus dosing of lmwh . The efficacy of rfviia in reversing the effects of coumadin and the direct thrombin inhibitor melagatran has also been studied in a rat tail bleeding model . The use of rfviia resulted in decreased blood loss in animals given warfarin once but it did not reduce blood loss significantly in animals given warfarin twice . In rats given melagatran, 1 mg rfviia resulted in a decreased bleeding time but no significant reduction in blood loss . The effect of rfviia administration on dissemination of clotting and thrombogenicity was investigated in the rabbit . In a model of stasis - induced thrombosis, administration of rfviia at a dose that enhanced clot formation at the site of injury resulted in no change in platelet count or the plasma concentration of antithrombin, indicating that rfviia was not associated with systemic activation of coagulation . In an experimental model of arterial thrombosis, rfviia given in concentrations that decreased ear bleeding time and blood loss from incision sites in the liver and spleen did not affect arterial thrombosis . Currently, three swine trauma models exist in the literature for the study of the efficacy and safety of rfviia . These models include a grade v liver injury model, a liver avulsion model, and an infrarenal aortotomy model . In addition to differences in the mechanism and site of injury, these models also differ with respect to animal temperature and coagulation status, timing and rate of resuscitation, timing and dose of therapy, and use of conventional therapies . The first of these studies, reported by martinowitz and coworkers, utilized a specially designed clamp to create a grade v liver injury based on american association for the surgery of trauma organ injury scaling criteria (fig . 10 animals underwent a 60% isovolemic exchange transfusion with 6% hydroxyethyl starch and cooling to 33c . Thirty seconds after injury, blinded therapy, consisting of either 180 g / kg rfviia or saline, was given and liver injuries were packed with gauze . Resuscitation with 40c lactated ringer's was initiated at 250 ml / min 5.5 min after injuries . Animals were resuscitated and maintained at their baseline blood pressure for the 1-hour study period . Treatment with rfviia resulted in a statistically significant reduction in blood loss from 976 cc to 527 cc . Treatment also resulted in significant reduction in prothrombin time, and elevation in the fvii clotting activity (fvii: c) was observed . Postmortem analysis revealed no evidence of large clots in the hepatic veins or inferior vena cava and no evidence of microthrombosis . Clamp designed to create a grade v liver injury based on organ injury scaling criteria from the american association for the surgery of trauma [12, 13]. In a follow - up study, schreiber and coworkers repeated the analysis with minor differences in the model used . The 60% isovolemic hemodilution was performed with 5% albumin instead of hydroxylethyl starch to avoid the potential coagulopathic effects of starch solutions . The creation of the dilutional coagulopathy resulted in a hypotensive state with a starting mean arterial pressure of 45 mmhg . Blinded therapy, packing, and initiation of resuscitation were performed simultaneously at 30 s and animals were resuscitated with lactated ringer's at 100 ml / min . Three groups of 10 animals were given either 180 g / kg rfviia, 720 g / kg rfviia, or an equivalent amount of buffer solution . Similar to the study conducted by martinowitz and coworkers, animals treated with rfviia had significantly less blood loss than did controls (control animals 2187 ml, 180 g / kg group 1085 ml, and 720 g / kg group 1086 ml). Nadir blood pressures following injury were significantly lower in control animals than in treated animals . Treated groups also had significantly lower prothrombin times and higher thrombin antithrombin complexes, suggesting activation of thrombin . Death occurred in four out of 10 controls, three out of 10 in the 180 g / kg group, and two out of 10 in the 720 g / kg group . These differences did not achieve statistical significance . The only difference between the animals that received 720 g / kg rfviia and 180 g / kg rfviia was a dose - dependent increase in fvii: c levels following delivery of drug . The same investigators also tested the efficacy of rfviia as sole therapy in noncoagulopathic, normothermic animals . In this study, 30 animals were randomly assigned to receive either 150 g / kg rfviia or an equivalent volume of buffer solution 30 s after injury . Animals were resuscitated with warmed lactated ringer's solution at 100 ml / min initiated 15 min after injury to maintain their baseline blood pressure and normothermia (38c) for the 2-hour study period . Liver injuries were not treated in this model . Despite documentation of a significant increase in fvii: c and decrease in prothrombin time, there was no difference in blood loss between groups . Blood pressure curves were similar and thrombin antithrombin complexes became equally elevated between groups throughout the study . Potential explanations for the failure of rfviia to reduce blood loss in this model included the use of the drug as a sole agent in this large venous injury model and the normotensive state of the animals at the start of the study compared with the hypotensive state produced in the dilutional coagulopathy model . Lynn and coworkers described a reproducible grade iv liver injury whereby a clamp was utilized to crush and avulse the left median liver lobe and major vessels of the left lateral lobe . In the first study using this model, 13 animals were randomly assigned to receive 180 g / kg rfviia or blinded placebo given when the mean arterial blood pressure dropped by 10% of baseline . Blood loss in the treatment group was 33 ml / kg versus 27 ml / kg in the control group (p = 0.2). Mortality was 43% in the control group and 0% in the treatment group (p = 0.08). These investigators used the same model to study 24 animals divided into three groups: control (group 1); 180 g / kg rfviia (group 2); and 720 g / kg rfviia (group 3). The study drug was again given following a 10% drop in mean arterial pressure following grade iv liver injury . Death occurred in four out of eight in group 1, two out of eight in group 2, and none of eight in group 3 during the first hour (p = 0.02 for control versus 720 g / kg). All deaths in the first two groups occurred within 22 min and the only death in the third group occurred at 116 min . There was no evidence of microthrombosis or macrothrombosis within viewed sections of kidney, mesentery of small intestine, small intestine wall, lung, heart, or brain in the treated groups . The final animal study described in this review was designed by sondeen and coworkers to test the efficacy of rfviia in producing stronger clots and elevating the blood pressure at which rebleeding occurs in a pig aortotomy model . By elevating the blood pressure at which rebleeding occurs, rfviia would theoretically permit more effective resuscitation without increasing blood loss . Thirty pigs were randomly assigned to a control group, a 180 g / kg group, or a 720 g / kg group . Blinded treatment was administered 5 min before injury . A skin biopsy punch was used to make a 2 mm hole in the infrarenal aorta . Resuscitation was initiated 10 min after injury with lactated ringer's at 100 ml / min . If rebleeding did not occur before the mean arterial pressure achieved a plateau after administration of 4 l resuscitation fluid, animals were given epinephrine (adrenaline) to raise the blood pressure to a maximum of 200 mmhg . The rebleeding blood pressure in the control group was significantly lower (45 mmhg) than that in the 180 g / kg group (69 mmhg) and the 720 g / kg group (66 mmhg). There was a trend toward greater rebleed volume in the control group (39 ml / kg in controls versus 22 ml / kg and 26 ml / kg for the 180 g / kg and 720 g / kg groups, respectively). Four of 10 animals required epinephrine in the high - dose group versus one out of 10 in each of the other two groups . Significantly more animals in the control group (nine out of 10 animals) rebled at mean arterial pressures below baseline than in the rfviia groups (two out of 10 animals). Rfviia - treated animals received significantly more fluid resuscitation before rebleeding than did control animals . Similar to prior studies, fvii: c exhibited a dose - related increase in the two treatment groups . Cohort studies in humans have suggested that there is a population of patients who do not respond to treatment with rfviia . This group of patients includes those extremely ill individuals with the lethal triad of hypothermia, coagulopathy, and acidosis who are on pressor therapy . It also includes those with irreversible hemorrhagic shock, who are unlikely to respond to any therapy . Based on these findings, meng and coworkers attempted to determine the effects of temperature and acidosis on factor viia activity utilizing blood from healthy adults . Factor viia activity on phospholipid vesicles and on platelets was measured by determining factor xa generation in the presence and absence of tissue factor . Tissue factor dependent factor xa generation increased with an increase in temperature from 24c to 37c, whereas tissue factor - independent factor xa generation decreased to an equal degree . Alternatively, reduction in ph resulted in marked diminution in factor viia activity from both tissue factor dependent and independent mechanisms . The authors concluded that acidosis effects overall factor viia activity but that hypothermia does not . The efficacy and safety of rfviia has been demonstrated in multiple small and large animal models . Because of species specific differences in the interaction between human rfviia and animal tissue factor, comments regarding dosing remain guarded . However, there does appear to be a maximum dose above which no additional benefit is achieved . Published ex vivo data suggest that rfviia may not be effective in severely acidotic environments . Preclinical data fvii: c = factor vii clotting activity; lmwh = low - molecular - weight heparin; rfviia = recombinant activated factor vii.
When a limb muscle is stretched, changes in the expression of myosin heavy chain (myhc), the protein primarily responsible for the contractile speed and metabolism type of muscle fibers, are observable within days.1 when stretched, muscle fibers tend to change from a fast phenotype (mainly comprising myhc iib) to a slow phenotype (consisting mainly of myhc i or rarely i).1 - 6 these changes, although controversial, occur along a continuum of changes in the expression of myhcs from isoform iib to iso form i.7 in addition, the expression of less common isoforms, such as embryonic or neonatal myhcs (developmental myhcs), may also be increased.8 the same responses might occur in the masticatory muscles after an increase in occlusal vertical dimension and/or mandibular advancement.9,10 in animal models, functional appliances commonly used for orthodontic treatment induce a phenotypic change in masticatory muscle fibers, with a tendency toward transition to a slow phenotype.11,12 slow fibers are fatigue resistant and easily convertible to fast fibers; in comparison, fast fibers are fatigable but difficult to convert to slow fibers . Therefore, in a masticatory muscle stretched by a functional appliance, the fiber composition would gradually balance as the orofacial structures grow and then, treatment effects would be sustained without relapse provided that the new fiber proportion is maintained . However, a phenotypic change appears to occur only within the fast fiber population, and a definite molecular change among slow fibers remains controversial.9,10,13 the aims of this study were to examine whether a passive stretch stimulus by means of a functional appliance induces changes in the fiber composition of masticatory muscles and whether these changes are similar to the changes in stretched leg muscle fibers by using rt - pcr, western blot, and immunohistochemical assays . Eight male growing new zealand white rabbits with an average weight of 2.5 kg and average age of 11 weeks were divided into control and experimental groups, consisting of 3 and 5 rabbits, respectively . Under the assumption that the myhc composition in the masticatory muscles of the control animals would vary less than that in the experimental animals, the rabbits were brought to the institutional animal facility and allowed to acclimate for 2 weeks . In the experimental group, each animal was sedated with ketamine (40 mg / kg i.m .) And xylazine (5 mg / kg i.m . ), the maxillary central incisors were etched with 37% phosphoric acid, and a prefabricated nickel - chromium inclined plane was fitted on these teeth (figure 1) by using glass ionomer cement (principle; dentsply caulk, milford, de, usa). This inclined - plane appliance was designed to force the mandible approximately 2 mm forward and approximately 4 mm downward . The control group did not receive this appliance.11 while sedated, the experimental and control animals also received a cast (vetcast; 3 m, st . Paul, mn, usa) to stretch 1 hind limb as described by yang et al.,5 the limb was extended at an angle of 110 before casting as previously described.14,15 consequently, the extensor digitorum longus (edl) was stretched when the animal walked on this limb . The rabbits had free access to pellet food and water . At the end of the 1-week experimental period, they were sacrificed with an overdose injection of ketamine - xylazine along with placement in a co2 chamber . The masseter (superficial and deep parts), lateral pterygoid, and edl of both hind limbs were dissected immediately after the animals were sacrificed . Three representative regions from the mid - belly of each muscle were sectioned into 1-cm square pieces, flash - frozen in isopentane, cooled in liquid nitrogen, and stored at -80 for later sectioning . Each sample was serially sectioned transversely at 8 m on a cryostat at -20. twenty - four adjacent serial sections of each muscle were divided in 4 sets of 3 slides . Immunostaining was accomplished as previously described16 with mouse monoclonal antibodies against slow, fast, embryonic, and neonatal myhcs (leica bio systems, newcastle, uk) on 4 consecutive sections to identify the expression of different myhcs . For fiber composition analysis, the slides were visualized under a light microscope (eclipse, e600; nikon, tokyo, japan). One section per muscle from each animal for each antibody was photographed under the same magnification by using a microscope digital camera (spot rt; spot imaging soultions, diagnositc instruments inc ., sterling heights, mi, usa). Images were enhanced for contrast by using adobe photoshop, and a single random area with approximately 150 fibers from each slide was selected and the percentage of immunopositive fibers was determined . Rt - pcr assay was performed on extracted total rna by using trizol (invitrogen, life technologies, carlsbad, ca, usa) to investigate the mrna level differences between the control and the stretched muscles . Sequences for oligonucleotide primers were adapted from previous publications.17 western blot assay was performed to confirm the phenotypic changes in the muscle fibers by using mouse monoclonal antibodies to sarcoplasmic reticulum ca atpase 1 (serca1; enzo life sciences inc ., farmingdale, ny, usa), which is exclusively expressed in fast fibers, and serca2a (enzo life sciences), which is exclusively expressed in slow fibers.18 the rt - pcr and western blot assays were repeated thrice by using muscle blocks obtained from 3 different rabbits . Only immunohistochemical data were statistically analyzed . Inference on comparisons (8 extended vs. 8 control hind limbs) was tested by using student's t - test with the spss 12.0 software program (ibm - spss, chicago, il, usa). The kruskal - wallis test was used for comparisons of the masticatory muscles (5 experimental vs. 3 control animals). Any measurement could not assume a distribution of measurements, for instance, embryonic myhc concentration on the edl, kruskal - wallis test was used for this variable . Eight male growing new zealand white rabbits with an average weight of 2.5 kg and average age of 11 weeks were divided into control and experimental groups, consisting of 3 and 5 rabbits, respectively . Under the assumption that the myhc composition in the masticatory muscles of the control animals would vary less than that in the experimental animals, the rabbits were brought to the institutional animal facility and allowed to acclimate for 2 weeks . In the experimental group, each animal was sedated with ketamine (40 mg / kg i.m .) And xylazine (5 mg / kg i.m . ), the maxillary central incisors were etched with 37% phosphoric acid, and a prefabricated nickel - chromium inclined plane was fitted on these teeth (figure 1) by using glass ionomer cement (principle; dentsply caulk, milford, de, usa). This inclined - plane appliance was designed to force the mandible approximately 2 mm forward and approximately 4 mm downward . The control group did not receive this appliance.11 while sedated, the experimental and control animals also received a cast (vetcast; 3 m, st . Paul, mn, usa) to stretch 1 hind limb as described by yang et al.,5 the limb was extended at an angle of 110 before casting as previously described.14,15 consequently, the extensor digitorum longus (edl) was stretched when the animal walked on this limb . The rabbits had free access to pellet food and water . At the end of the 1-week experimental period, they were sacrificed with an overdose injection of ketamine - xylazine along with placement in a co2 chamber . The masseter (superficial and deep parts), lateral pterygoid, and edl of both hind limbs were dissected immediately after the animals were sacrificed . Three representative regions from the mid - belly of each muscle were sectioned into 1-cm square pieces, flash - frozen in isopentane, cooled in liquid nitrogen, and stored at -80 for later sectioning . Each sample was serially sectioned transversely at 8 m on a cryostat at -20. twenty - four adjacent serial sections of each muscle were divided in 4 sets of 3 slides . Immunostaining was accomplished as previously described16 with mouse monoclonal antibodies against slow, fast, embryonic, and neonatal myhcs (leica bio systems, newcastle, uk) on 4 consecutive sections to identify the expression of different myhcs . For fiber composition analysis, the slides were visualized under a light microscope (eclipse, e600; nikon, tokyo, japan). One section per muscle from each animal for each antibody was photographed under the same magnification by using a microscope digital camera (spot rt; spot imaging soultions, diagnositc instruments inc ., sterling heights, mi, usa). Images were enhanced for contrast by using adobe photoshop, and a single random area with approximately 150 fibers from each slide was selected and the percentage of immunopositive fibers was determined . Rt - pcr assay was performed on extracted total rna by using trizol (invitrogen, life technologies, carlsbad, ca, usa) to investigate the mrna level differences between the control and the stretched muscles . Sequences for oligonucleotide primers were adapted from previous publications.17 western blot assay was performed to confirm the phenotypic changes in the muscle fibers by using mouse monoclonal antibodies to sarcoplasmic reticulum ca atpase 1 (serca1; enzo life sciences inc ., farmingdale, ny, usa), which is exclusively expressed in fast fibers, and serca2a (enzo life sciences), which is exclusively expressed in slow fibers.18 the rt - pcr and western blot assays were repeated thrice by using muscle blocks obtained from 3 different rabbits . Inference on comparisons (8 extended vs. 8 control hind limbs) was tested by using student's t - test with the spss 12.0 software program (ibm - spss, chicago, il, usa). The kruskal - wallis test was used for comparisons of the masticatory muscles (5 experimental vs. 3 control animals). Any measurement could not assume a distribution of measurements, for instance, embryonic myhc concentration on the edl, kruskal - wallis test was used for this variable . Despite the presence of the intraoral appliance, no significant difference in body weight was observed between the control and the experimental groups during the experimental period . Both groups of rabbits lost less than 0.22 kg on the first day after the appliance and/or cast placement . By the third day the rt - pcr assay showed marked differences in the mrna expression of myhc i and iib in the stretched edl (figure 2a). The stretched edl had approximately double the amount of fibers expressing myhc i compared with the control edl (24.7 3.54% vs. 11.7 2.12%; p <0.004; figure 3a). A significant increase in the percentage of fibers stained positively for neonatal myhc was also observed (20.7 2.33% vs. 3.7 1.46%; p <0.001; figure 3b). Moreover, a small but nonsignificant increase in the percentage of fibers expressing embryonic myhc was detected in the stretched edl (3.7% vs. 0%). The superficial and deep parts of the stretched masseter had increased mrna expression of myhc iib and i, respectively (figure 2a), but similar proportions of slow and fast fibers (figure 3c and d). Most massetric fibers in the control group stained positively for fast myhc (figure 3a). In the experimental group, a slight decrease in the percentage of fibers stained positively for slow myhc was observed in the superficial (36.7% experimental vs. 40.6% control) but not the deep (44.7% experimental vs. 38.7% control) massetric part . Further, a very small percentage of fibers in the superficial (1.7%) and deep (0%) parts of the stretched masseter stained positively for embryonic myhc; the superficial and deep parts of the control masseter had about 9.3% and 4.3% positively stained fibers, respectively, which were not significantly different from the percentages in the experimental animals . Moreover, the superficial and deep massetric parts in the experimental and control groups did not have significantly different percentages of fibers stained positively for neonatal myhc . As in the case of the deep massetric part, myhc i mrna expression increased in the stretched lateral pterygoid (figure 2a). This observation was supported by a significant increase in serca2a expression in the deep massetric part and lateral pterygoid (figure 2b). The lateral pterygoid had mostly fast fibers: the percentages in the experimental and control groups were 84.5% and 77.1%, respectively, which were not significantly different (figure 3e). Both groups had approximately 25% slow fibers in this muscle, without a significant difference . Because only the experimental group had lateral pterygoid fibers stained positively for neonatal myhc (19.1%), the difference could not be tested statistically . Despite the presence of the intraoral appliance, no significant difference in body weight was observed between the control and the experimental groups during the experimental period . Both groups of rabbits lost less than 0.22 kg on the first day after the appliance and/or cast placement . By the third day the rt - pcr assay showed marked differences in the mrna expression of myhc i and iib in the stretched edl (figure 2a). The stretched edl had approximately double the amount of fibers expressing myhc i compared with the control edl (24.7 3.54% vs. 11.7 2.12%; p <0.004; figure 3a). A significant increase in the percentage of fibers stained positively for neonatal myhc was also observed (20.7 2.33% vs. 3.7 1.46%; p <0.001; figure 3b). Moreover, a small but nonsignificant increase in the percentage of fibers expressing embryonic myhc was detected in the stretched edl (3.7% vs. 0%). The superficial and deep parts of the stretched masseter had increased mrna expression of myhc iib and i, respectively (figure 2a), but similar proportions of slow and fast fibers (figure 3c and d). Most massetric fibers in the control group stained positively for fast myhc (figure 3a). In the experimental group, a slight decrease in the percentage of fibers stained positively for slow myhc was observed in the superficial (36.7% experimental vs. 40.6% control) but not the deep (44.7% experimental vs. 38.7% control) massetric part . Further, a very small percentage of fibers in the superficial (1.7%) and deep (0%) parts of the stretched masseter stained positively for embryonic myhc; the superficial and deep parts of the control masseter had about 9.3% and 4.3% positively stained fibers, respectively, which were not significantly different from the percentages in the experimental animals . Moreover, the superficial and deep massetric parts in the experimental and control groups did not have significantly different percentages of fibers stained positively for neonatal myhc . As in the case of the deep massetric part, myhc i mrna expression increased in the stretched lateral pterygoid (figure 2a). This observation was supported by a significant increase in serca2a expression in the deep massetric part and lateral pterygoid (figure 2b). The lateral pterygoid had mostly fast fibers: the percentages in the experimental and control groups were 84.5% and 77.1%, respectively, which were not significantly different (figure 3e). Both groups had approximately 25% slow fibers in this muscle, without a significant difference . Because only the experimental group had lateral pterygoid fibers stained positively for neonatal myhc (19.1%), the difference could not be tested statistically . The western blot findings regarding the phenotypic changes in fibers from the stretched edl are in agreement with those of previous studies,1,4,5 thus validating the immunohistochemical and rt - pcr assays . In particular, the significant increase in the percentage of myhc i - expressing fibers in the stretched edl well concerts with the rt - pcr result of decreased mrna expression of myhc iib, indicating reduced transcriptional activity of myhc iib . The extended hind limb had a twofold increase in the percentage of fibers stained positively for slow myhc, which was probably primarily myhc i, implying a change from fast fibers to slow fibers in the stretched edl . A previous study19 suggested an increase in the number of hybrid fibers expressing a combination of different myhcs; however, the present results can be explained by an increase in the percentage of myhc i - expressing fibers among hybrid fibers as the percentage of myhc ii - expressing fibers remained unchanged . Another explanation is that the increase or decrease in mrna expression of myhcs may not be sufficient to reflect changes in protein levels . Although the molecular mechanism underlying fiber transition from a fast to a slow phenotype is presently not fully understood, a change in the synthesis or degradation of contractile proteins is a possible mechanism . According to loughna et al.,15 passive stretch increases protein synthesis and has little or no effect on protein degradation . This may explain the lack of a change in the percentage of fibers expressing myhc ii and increase in the percentage of fibers expressing myhc i. developmental (embryonic and neonatal) myhcs usually emerge during the first postnatal week in many fibers.20 as the rabbit grows, these fibers are replaced with a definite myhc - expressing fiber type in accordance with the muscle function, and are almost absent in the adult rabbit.21 however, the percentage of neonatal myhc - expressing fibers increased when the rabbit edl was stretched for a week in a previous study as well as in the present one . According to yang et al.,5 this might be the result of stretch - induced muscle damage; he suggested that new muscle cells, generated from satellite cells, re - express their developmental program, including the expression of neonatal myhc, which will later be replaced by the adult slow type . Jacobs - el et al.21 also suggested that the phenotypic change from fast to slow fibers involves the re - induction of the development program . Contraction of muscle in the stretched state, namely lengthening (or eccentric) contraction, often results in tissue damage . Satellite cells22,23 participate when postmitotic muscle tissue is injured . In the current study, a significant increase in the percentage of neonatal myhc - expressing fibers was observed in the stretched edl, which may indicate the involvement of satellite cells in a repair process . Damage from mild eccentric contractions, however, tends to recover in 2 weeks.24 therefore, despite potential damage to the masticatory muscles, orthodontists may prefer to use functional appliances for gradual advancement and increase in the vertical dimension of the mandible to pro mote clinical outcomes.25 numerous studies have shown differences between the limb and the masticatory muscles.26 - 28 one of the most studied masticatory muscles is the masseter . Unlike the limb muscles, the masseter has many hybrid fibers, which makes classification difficult.28,29 the rabbit masseter is divided into 3 compartments: superficial, intermediate, and deep . These compartments are easily distinguishable by their fiber orientation; however, tissue harvested from both the superficial and the deep parts was used in this study to avoid potential ambiguity . The masseter of adults in many species is composed of slow, alpha cardiac, iia, iix, and iib fibers as well as developmental fibers such as embryonic and neonatal myhc - expressing fibers.27,30 widmer et al.31 demonstrated that myhc subtypes vary ac cording to the location as follows: myhc iia - containing fibers are found mostly in the anterior and ventral regions, and myhc iib - expressing fibers are located in the posterior and dorsal regions of the masseter in mice . Both the superficial and the deep parts of the masseter had similar changes in the present study . The experimental and control groups showed no significant differences in the percentages of slow and fast fibers in the masticatory muscles . This is in agreement with the study by ohnuki et al.,9 who used a bite appliance to increase the vertical dimension in guinea pigs and found no difference in the expression of myhc subtypes in the superficial part of the masseter between the control and the experimental groups . However, direct comparison of the data from the ohnuki et al.9 study with those of the present study is difficult because their appliance was designed only to open and not protrude the mandible . In addition, the diet of the guinea pigs was changed to a soft diet, which may itself be a confounding factor . In a different study, ohnuki et al.32 used a similar appliance to the present one in rats and found a significant difference in mrna levels of myhc in the superficial part of the masseter between the experimental and the control groups . In particular, they found an increase in the mrna expression of myhc iia compared with myhc iib in the rats with the bite - opening appliance . This finding is consistent with the findings of goldspink et al . And other groups,1,4,5 who found that stretch stimuli induce changes in myhc expression along a continuum from fast fibers to slow fibers (i.e., iib iix iia i). 's most recent report33 attempted to correlate the results of electromyograms (emg) with sodium dodecyl sulfate polyacrylamide gel electrophoresis results that were not precisely quantitative . Moreover, they did not compare the masticatory muscles with the edl in their studies on both rats and guinea pigs . Although the present study using rabbits was not performed for a sufficiently long time to assert the lack of changes in the masticatory muscles, as already discussed, significant changes in the edl occurred in 7 days . . Which factors would be responsible for the differences in the response to stretch between the limb and the masticatory muscles? One could speculate that the changes in the edl were induced by compensatory hyperactivity in the control limb following immobilization of the extended limb . However, disuse often results in a transition from slow fibers to fast fibers, which would reduce the differences between the experimental and the control animals . Other reasons for the lack of significant changes in the stretched masticatory muscles could be the small sample size and the variation in the masticatory muscles did not allow detection of a significant increase in the percentage of fibers expressing slow myhc . In addition, the appliance may have been placed for an insufficient duration to induce a change to a higher percentage of fibers expressing myhc i. only one study has shown a change from type iia to type i fibers in the masseter after 28 days of mandibular protrusion in pigs.11 the present findings very likely reflect fundamental temporal differences in the response to stretch of masticatory muscles compared with limb muscles . This view is supported in a recent review article stating that masticatory muscles do not seem to follow this strict pattern of fiber transition.34 nevertheless, the rt - pcr and western blot results, although qualitative, suggest that the stretch stimulus by using the inclined - plane appliance may have altered the concentration of ca - regulatory membrane proteins and myhc mrnas toward slow fiber phenotypes; however, the actual translation process for myhcs did not occur in the deep part of the masseter and lateral pterygoid . Contrary to limb muscles, where developmental myhc - expressing fibers disappear in the adult, widmer et al.31 localized these fibers in the masseter, albeit in a small number (<1%). In the present study, a few developmental myhc - expressing fibers were found in the masseter and most of these were of the neonatal type, although some embryonic myhc - expressing fibers were also found . To date, no other animal study has demonstrated changes in these fiber types in the masticatory muscles following the use of a bite - opening or protrusive appliance . Easton and carlson11 reported that the percentage of slow fibers in this muscle is 1% in rats . They also reported that the ratio of iia - to - iib fibers is 60:40 . In the present study, 77% of the fibers expressed myhc ii and 25% showed myhc i staining in the control rabbits . Further, they found an increase in the number of type i fibers and a 25% reduction in the number of iib fibers after using a mandibular protrusive appliance in rats.11 the shortening of the lateral pterygoid potentially reverses the transition of fiber types observed in the passive stretch model,14 because shortening or contraction of the lateral pterygoid would occur as the mandible is protruded downward and forward . In the present study, a significant difference in fiber types was not found between the experimental and the control groups except for the neonatal myhc - expressing fibers, which increased in percentage (from 0% to 19%) in the experimental group . However, increased mrna expression of myhc i and i suggests hyperactivity of the corresponding muscle and increased serca2a expression suggest a behavior in which calcium ions favor slow fibers . Fast - to - slow fiber transition in the stretched masticatory muscles was not observed in the present study . Although others have suggested that this change also occurs in the masticatory muscles, the current findings do not support this view completely . Changes in the masseter and lateral pterygoid are possibly more subtle than those in the limb muscles or take longer time to occur because of anatomical and functional differences . One of the reasons perhaps is that the masticatory muscles are predominantly fast muscles but contain a greater number of hybrid fibers than the edl.35 functional appliances for correction of malocclusions invariably stretch jaw closers; however, their efficacy remains highly controversial . The slow transition of fiber types in the masticatory muscles may be a reason why orthodontists favor the long - term use of functional appliances . The results of this study confirm the findings of others suggesting a change in the myhc composition of fibers in stretched limb muscles, providing confidence in the present methodology . On the other hand, the insufficient sample size does not eliminate type 2 errors . An increase in the amount of fibers expressing neonatal myhc in stretched limb muscles may also suggest reprogramming of new satellite cells that respond to local tissue damage, which may occur because of the stretch.22 the same fiber changes in the stretched masticatory muscles were not observed in this study . Considering the results of the rt - pcr and western blot assays, however, a prolonged stretch stimulus would induce a detectable change to a slow fiber phenotype in the deep part of the masseter and lateral pterygoid . 1 . Changes in fiber composition of masticatory muscles may take longer time to occur compared to those of limb muscles . This slow conversion speed may be a reason for the long - term use of a functional appliance that is favored by orthodontists . May be an adaptive response for homeostasis frequently observed in a skeletal muscle with a more complex structure such as masticatory muscles.
Fish viscera are one of the sources of digestive enzymes that may have some unique properties of fascinate with both basic research and industrial applications . Their survival in waters required adaptation of their enzyme activity to low temperatures of their habitats . That is to say, fish proteinases have higher catalytic efficiency at low temperatures than those from warm - blooded animals [1, 2]. In addition, the strong positive correlation between the habitat temperature of marine fish and the thermostability of its trypsin has been demonstrated [311]. High activity at low temperatures and instability against heat, low ph, and autolysis of fish proteinases are interesting for some industrial applications . Cod trypsin is already practically used in food production and cosmetics [13, 14]. Furthermore, pacific cod and atlantic cod trypsins were utilized as catalyst of enzymatic peptide synthesis [9, 15]. On the other hand, lipids in the tissue conventional methods for the removal of lipids from materials involve cooking, pressing, and liquid extraction . On liquid extraction for enzyme preparation, it is usually used with organic solvents, such as hexane, ethanol, and acetone, and so forth [16, 17]. However, the removal of lipids with organic solvents causes protein denaturation and/or loss of functional properties . Organic solvents are also highly flammable and are toxic for human health . Consideration of such factors has led investigators to apply supercritical fluid extraction techniques to the lipid separation . Carbon dioxide (co2) is a popular supercritical extractant particularly in food processing, flavor and aroma isolation, and pharmaceuticals manufacture, because co2 is nontoxic and does not leave a residue . Supercritical co2 (sco2) has been used for extraction of oils from some marine organisms [2022]. But, the aims of these studies were mainly the gain of oils rich in polyunsaturated fatty acids, especially epa and dha . So, the application of sco2 for isolation of enzymes and production of quality protein meal from different sources should be examined . Recently, we prepared a defatted powder of squid viscera treating with sco2 and detected protease, lipase, and amylase activities in crude extract from the powder . Next, we purified a phospholipase a2 from the starfish pyloric ceca defatted by sco2 extraction process . In this study, with the aim of utilization of fish trypsin for food industry, we purified a trypsin (sc - t) from the mackerel viscera powder treated by sco2 defatting process and compared its enzymatic properties with those of other fish trypsins purified from the viscera defatted by acetone . Mackerel (scomber sp .) Were caught off busan, republic of korea . The mackerel viscera were collected from f & f co., busan, republic of korea, and the visceral waste was brought to the laboratory in iced condition . The co2 (99.99% pure) was supplied by kosem, korea . N - p - tosyl - l - arginine methyl ester hydrochloride (tame) and ethylenediaminetetraacetic acid (edta) were obtained from wako pure chemicals (osaka, japan). 1-(l - trans - epoxysuccinyl - leucylamino)-4-guanidinobutane (e-64), soybean trypsin inhibitor, n - p - tosyl - l - lysine chloromethyl ketone (tlck), and pepstatin a were purchased from sigma chemical co. (st . Louis, mo, usa). The defatted powder of mackerel viscera was prepared as described by chun et al . The lipid extraction by sco2 was performed at temperature of 45c and pressure of 25 mpa . The total extraction time was 2.5 h. the sco2 defatted powder was stored at 60c until further analysis . Trypsin was extracted by stirring from 10.0 g of defatted powder in 50 volumes of 10 mm tris - hcl buffer (ph 8.0) containing 1 mm cacl2 at 5c for 3 h. the extract was centrifuged (h-200, kokusan, tokyo, japan) at 10,000 xg for 10 min, and then the supernatant was concentrated by lyophilization and used as crude trypsin (50 ml). Ten milliliters of crude trypsin was applied for four times to a column of sephacryl s-200 (3.9 64 cm) pre - equilibrated with 10 mm tris - hcl buffer (ph 8.0) containing 1 mm cacl2,and proteins were eluted (0.8 ml / min) with the same buffer . Each main trypsin fractions were gathered and concentrated by lyophilization . Then the concentrated fraction (10 ml) was applied to a sephadex g-50 column (3.9 64 cm) pre - equilibrated with the above buffer, and proteins were eluted (0.7 ml / min) with the same buffer . A single trypsin fraction was pooled and used as purified trypsin (sc - t). Trypsin activity was measured by the method of hummel using tame as a substrate . One unit of enzyme activity was defined as the amount of the enzyme hydrolyzing one micromole of tame in a minute . The effect of inhibitors on trypsin was determined by incubating trypsin with an equal volume of proteinase inhibitor solution to obtain the final concentration designated (0.1 mm e-64, 1 mg / ml soybean trypsin inhibitor, 5 mm tlck, 1 mm pepstatin a and 2 mm edta). After incubation of the mixture at 25c for 15 min, the remaining activity was measured, and percent inhibition was then calculated . The ph dependencies of trypsin were determined in 50 mm buffer solutions [acetic acid - sodium acetate (ph 4.07.0), tris - hcl (ph 7.09.0), and glycine - naoh (ph 9.011.0)] at 30c . The temperature dependencies of trypsin were determined at ph 8.0 and at various temperatures . The temperature and ph stabilities of trypsin were found by incubating enzyme at ph 8.0 for 15 min at a range of 2080c and by incubating the enzyme at 30c for 30 min at a range of ph 4.011.0, respectively . The effect of cacl2 on trypsin activity was found by incubating the enzyme at 30c and at ph 8.0 in the presence of 10 mm edta or 10 mm cacl2 . Sds - page was carried out using a 0.1% sds-13.75% polyacrylamide slab gel by the method of laemmli . The gel was stained with 0.1% coomassie brilliant blue r-250 in 50% methanol-7% acetic acid, and the background of the gel was destained with 7% acetic acid . To analyze the n - terminal amino acid sequence of sc - t, the enzyme was electroblotted to a polyvinylidene difluoride (pvdf) membrane after sds - page . The amino acid sequence of the enzyme was analyzed by using a protein sequencer, procise 492 (perkin elmer, foster city, calif, usa). The viscera of mackerel (scomber sp .) Were treated by sco2 to separate lipids on the condition of 40c, 25 mpa, and 2.5 h. since sco2 extracted almost all oil from the squid viscera in the previous study, we adopted the condition to remove lipids from the mackerel viscera . Trypsin was then extracted from 10.0 g of defatted powder by sco2, and the crude enzyme was prepared . As shown in table 1, the crude enzyme contained 1,390 mg of total protein and 1,049 u of total trypsin activity . Previously, we extracted trypsin from the pyloric ceca powder (13.9 g) of spotted mackerel defatted by acetone, and 3,633 mg of total protein and 3,270 u of total trypsin activity were detected in the crude enzyme solution . Although these data were not compared directly, the yields of protein and trypsin activity per weight of acetone powder were approximately two times higher than those of sco2 powder . However, the specific activity (0.8 u / mg) of crude enzyme in this study is almost the same as that (0.90 u / mg) in the previous study . So, it is thought that the difference of total activity might come from the variation of specimen, and the defatting condition with sco2 in this study would not cause significant denaturation of fish trypsin . The sc - t was purified from the crude enzyme solution by two steps of chromatographies including sephacryl s-200 and sephadex g-50, which is the same purification procedure as that in the previous study . The sc - t was consequently purified 48-fold with a high recovery (50%) from the crude enzyme solution (table 1) and had a specific activity of 36 u / mg which is fairly higher than that of spotted mackerel trypsin . In addition, the sc - t was found nearly homogeneous on sds - page (figure 1), and its molecular weight was estimated as approximately 24,000, which is similar to that of spotted mackerel trypsin . Furthermore, the n - terminal twenty amino acids sequence of sc - t was analyzed to be ivggyectpysqpwtvslns that accords with that of spotted mackerel trypsin . These results also show that the sco2 defatting process in this study removes lipids in fish viscera as effectively as acetone defatting process for preparation of fish trypsin . Crude enzyme extract usually contains various proteins, and sometimes fish trypsin is composed of some isozymes . In general, the purification of fish trypsin was carried out by the combination of some types of chromatography [2830]. However, we achieved a high purification of a trypsin from the mackerel viscera powder defatted by acetone using only two steps of gel filtration . So, in this study, we purified the sc - t by gel filtration according to the previous study . The sc - t was strongly inhibited by specific trypsin inhibitors (soybean trypsin inhibitor and tlck), but e-64 (cysteine proteinase inhibitor), pepstatin a (aspartic proteinase inhibitor), and edta (metalloproteinase inhibitor) had no inhibitory effect on the activity of sc - t (table 2). The influence of ph on the sc - t activity is shown in figure 2(a). The enzyme hydrolyzed tame substrate effectively between ph 7.0 and 9.0, with an optimum around ph 8.0 . The optimum ph of sc - t was the same as those of other fish trypsins [311, 3138], but lower than those of bluefish (ph 9.5) and atlantic bonito (ph 9.0). The sc - t was active over a broad temperature range (2070c) with the optimum at about 60c . Because mackerel is a temperate - zone fish, the sc - t possesses similar optimum temperature with other trypsins from temperate - zone fish, such as anchovy, true sardine, yellow tail, and jacopever . The optimum temperature sc - t is slightly lower than those of tropical - zone fish (around 65c) [3335, 39, 40] but is evidently higher than those of frigid - zone fish trypsins (around 50c) [4, 610]. The ph stability of sc - t is shown in figure 3(a). The sc - t was stable at 30c for 30 min in the ph range from ph 6.0 to 11.0 . Unlike mammalian trypsins, diminished stability of the trypsin was more pronounced after exposure at acidic ph . Instability at acidic ph was also observed for other fish trypsins [1, 311, 3235, 37, 39, 40]. For temperature stability, the sc - t was stable below 40c, but the activity quickly fell over 50c (figure 3(b)). While the sc - t and other temperate - zone fish trypsins are relatively less stable than tropical - zone fish trypsins, they are obviously stable than frigid - zone fish trypsins [8, 10]. As described previously, there is a strong relationship between habitat temperature of marine fish and thermostability of their trypsins [8, 10]. The effect of calcium ion on the stability of sc - t was then investigated . The stability of sc - t was enhanced by calcium ion (figure 4). Similar results have been reported for various fish trypsins [1, 311, 3335, 39, 40]. Bovine trypsinogen has two calcium - binding sites, and the primary site, with a higher affinity for calcium ions, is common in trypsinogen and trypsin and the secondary site is only in the zymogen [41, 42]. Occupancy of the primary calcium - binding site stabilizes bovine trypsin toward thermal denaturation or autolysis [41, 42]. In the previous paper, we described trypsin of arabesque greenling which also has the primary calcium - binding site . The sc - t was stabilized by calcium ion from denaturation in this study . So, the result suggests that the sc - t may possess the primary calcium - binding site like bovine and arabesque greenling trypsins . With the aim of utilization of fish trypsin for food industry, we purified a trypsin (sc - t) from mackerel (scomber sp .) Viscera powder treated by the sco2 defatting process and compared its enzymatic properties with those of other fish trypsins purified from the viscera defatted by acetone . In this study, we adopted the condition of 40c, 25 mpa, and 2.5 h to separate lipids from the viscera . Consequently, we could remove most of the lipids from the viscera and could extract considerable amount of trypsin from the defatted powder . The characteristics of purified sc - t were nearly the same as those of other fish trypsins, especially spotted mackerel trypsin . Therefore, we concluded that the sco2 defatting process is useful as a substitute for the organic solvent defatting process.
Twenty - one animals (16 cattle and 5 sheep) showing clinical signs suggestive of bluetongue were sampled by the federal agency for the safety of the food chain on august 18, 2006, at 11 farms in northeastern belgium . Two serologic tests that detect antibodies against the major serogroup antigen vp7 (bluetongue virus antibody competitive elisa [celisa]; veterinary medical research and development inc ., pullman, wa, usa and competitive vp7 bluetongue kit; idvet, montpellier, france) identified 21 virus - positive animals . Two newly developed and validated reverse transcription quantitative pcrs (rt - qpcrs) that detected btv strains representing the 24 serotypes (4) were then conducted to determine whether these seropositive animals also had viral rna . The first assay (rt - qpcr_s1), which amplified a 357-nt fragment in segment 1, detected virus in erythrocytes of the 21 seropositive animals (mean cycle threshold [ct] value 29.0). The second assay (rt - qpcr_s5), which amplified a 94-nt fragment in segment 5, detected virus in the same 21 seropositive animals (mean ct value 26.5). The 2 serologic tests and the 2 molecular assays detected btv in 21 animals from 11 belgian farms within 14 hours . Virus isolation was conducted on august 18, 2006, by injection of blood from infected sheep into 11-day - old embryonated chicken eggs, followed by passage on bhk-21 cells (atcc - ccl10) as previously described (5). The specificity of the cytopathic effect observed 48 hours after passage on bhk-21 cells was confirmed by rt - qpcr and electron microscopy (figure 1) after fixation and negative staining as previously described (6). Two virus neutralization tests were conducted on 2 virus strains isolated by the belgian and the french reference laboratories at 2 belgian farms 30 km apart . Each strain was also partially neutralized by reference serum against serotype 18, which confirmed cross - neutralization between serotypes 8 and 18 (7). To september 14, 2006, the study farms were screened for animals with clinical signs of bluetongue . Blood samples were tested by serologic tests or rt - qpcr . For cattle, 97 (68%) of 142 samples had antibodies to btv and 32 (78%) of 41 samples contained viral rna (table 1). However, for sheep, only 23 (29%) of 79 samples had antibodies to btv and 15 (45%) of 33 samples contained viral rna . Other diseases that cause similar signs might explain this lower frequency in sheep . Contagious ecthyma was diagnosed by using pcr and electron microscopy for several sheep that showed bluetonguelike signs but did not have antibodies to btv or viral rna . Agreement between celisa and rt - qpcr results was analyzed for 124 animals (table 2). One sample negative by rt - qpcr_s1 and rt - qpcr_s5 was positive by celisa . Although this result might reflect lack of specificity of the celisa, elimination of btv rna from the animal several weeks after being infected cannot be ruled out . A false - negative result in the rt - qpcr is unlikely because 1) 2 different rt - qpcrs that amplified 2 different segments were used, 2) the quality of the rna was confirmed by a third rt - qpcr that quantified mrna of -actin, and 3) both rt - qpcrs are highly sensitive (they can detect 0.01 infectious doses of virus) (4). False - positive results were not observed with the idvet celisa when we analyzed 650 negative serum samples from artificial insemination centers and field samples collected from belgian livestock in 2004 and 2005 . Seven animals with bluetonguelike clinical signs were positive according to each rt - qpcr but negative according to the celisa (table 2). These results support the finding that rt - qpcr can be used to detect viral rna in infected animals before antibodies are detectable . Celisa, competititve elisa; rt - qpcr, reverse transcription quantitative pcr . Samples with different results in celisa and rt - qpcr_s5 were retested with rt qpcr_s1 (4). This latter test always confirmed the result of the rt - qpcr_s5 . On september 14, 2006 (4 weeks after the first identification of btv in belgium) as many as 84 belgian farms had at least 1 btv - infected animal . The maximal distance between herds in this study was 110 km (figure 2a). Most outbreaks were confirmed in the area where the disease was initially detected (area i, figure 2). Most (64%) infected animals showed a high virus load with individual ct values <30 . Of the remaining animals, 30% had moderate virus loads (ct values 3035) and 6% had ct values> 35 . The high ct values for the latter animals might have remained undetected had pooled blood samples been analyzed . Thus, results of pooled samples need to be validated before being used for diagnosis . None of the animals from zones iii and iv showed a ct value <30, whereas all animals from zone ii showed low ct values, which are indicative of high virus loads . Lower virus loads and acute clinical signs in animals from zones iii and iv might indicate onset of infection . However, we cannot rule out decreased infection in these animals because they also had positive serologic results that indicated infection for at least 45 days . Further epidemiologic studies are required before conclusions can be drawn on the evolution of these epidemics . A) distribution of outbreaks of bluetongue (shaded areas) reported in belgium from august 18 through september 14, 2006 . B) cycle threshold (ct) values observed in different zones as a result of conducting reverse transcription btv has been detected and its isolation and characterization have considerably progressed in the first weeks of the epidemics . Results of virus neutralization tests for 2 belgian isolates and molecular characterization of the dutch btv strain by the community reference laboratory (8) indicate that btv serotype 8 is present in belgium and the netherlands . Although this observation suggests 1 serotype circulating in northern europe after a common virus introduction, it must be confirmed by detailed epidemiologic studies . Northward spread of bluetongue in europe has been correlated with climate warming (9). However, one characteristic of the current epidemics of bluetongue is the severity of clinical signs reported in cattle (10). The present results also demonstrate that clinical signs observed in cattle are more specific than those observed in sheep . Confusing clinical signs in sheep underline the need for developing diagnostic tests to discriminate between bluetongue and other confounding diseases such as contagious ecthyma, border disease, and foot - and - mouth disease . Our results also indicate the usefulness of rt - qpcr, which detected viral rna in recently infected animals with clinical signs of bluetongue but no detectable antibodies to btv . The rt - qpcr and elisa are independent but complementary tests because they detect viral rna and virus - specific antibodies, respectively . These tests indicated that an outbreak of bluetongue was occurring in belgium . Despite high sensitivity of rt - qpcr (4), our results suggest that using this test with pooled samples might not detect animals with low viral loads . Rt - pcr positive results in animals that are no longer infectious (11) should also be considered before deciding whether pooled samples are acceptable.
Within the last half century, there has been remarkable progress in the treatment of psychiatric disease . The first chemical antidepressants were discovered fortuitously in clinical trials designed to treat tuberculosis.1 since then, researchers in both academia and industry have developed an impressive array of related compounds, which work through similar mechanisms but treat a host of different psychiatric conditions.2 these developments have led to an abundance of pharmacotherapies that have dramatically increased the number of available treatment options for psychiatrists . Unfortunately, currently available drugs still produce numerous deleterious side effects and, for a substantial number of patients, are largely ineffective in alleviating their disease symptoms . Consequently, there remains a strong need for more effective, targeted pharmacotherapies to treat psychiatric disease . A better understanding of the neurobiological processes that underlie psychiatric disease states is expected to help drug development efforts . Accordingly, there has been a considerable effort to identify biological variables that consistently differ between healthy individuals and those with disease.3 studies in humans have implicated various circuits in specific psychiatric disease states using neuroimaging, genomic, and post mortem tissue analyses.4 however, data produced from these studies are largely post hoc in nature and cannot be used to conclusively infer cause and effect . The attribution of causality to specific neuronal circuitry or reliable biomarkers has been hampered by technological limitations and overall etiological complexities.5 in order to determine if a specific biological process is integral to a psychiatric condition, it must be experimentally manipulated in preclinical models of the disease . To this end, numerous animal models of psychiatric disease states have been developed.6 one approach to modeling psychiatric disease in an animal is to use a behavioral task in which an animal s performance is sensitive to drug treatments that have already proved to be effective in humans.7 when new drugs produce behavioral effects that are similar to those elicited with proven pharmacotherapies, they are likely to have similar mechanisms of action to the old drugs and be similarly effective in treating the disease . This strategy provides strong predictive validity in modeling treatment of disease states and has been successfully used to screen novel compounds . However, its utility may be limited to compounds that act via the same mechanisms as those used to initially develop the behavioral model . An additional caveat to this line of research is that predictive validity in modeling treatment does not necessarily mean that the etiology or mechanisms underlying the disease in humans are relevant to the given model . Thus, while animal models of psychiatric disease offer a powerful approach to understand symptomatology, they may be less able to provide information about the neural processes that are ultimately responsible for the initiation of disease states . The unclear neuronal substrates of psychiatric diseases and correspondingly slow progress of novel drug development have resulted in pharmaceutical companies recently abandoning development of psychiatric compounds.8 to reinvigorate research, it may be necessary to take a more refined technological approach to the study of disease symptoms and treatment . The maladaptive behaviors associated with complex psychiatric disorders are likely the result of specific aberrant processes in discrete, albeit multiple, neural circuits . The development of optogenetics has provided researchers with a powerful set of tools to manipulate neural activity in genetically defined populations of cells in a pathway - specific manner.9 in applying optogenetic strategies to established animal models of psychiatric disease, investigators have made rapid progress in defining the neural circuitry responsible for both psychiatric disease and symptom relief . Optogenetic approaches enable researchers to activate or inhibit groups of neurons, which can be defined by genetic identity and/or projection target . This is made possible through the targeted expression of light - sensitive proteins known as rhodopsins, which are responsive to specific wavelengths of light . Rhopdopsins are protein complexes that contain an opsin (channel) and a light - sensitive cofactor (retinal).10 the opsins used in optogenetic strategies are generally derived from the type 1 class of prokaryotic rhodopsin proteins that support a covalent bond with an internalized retinal molecule.11 the ionic environment around this covalent bond influences the spectral sensitivity of the protein, whereas the amino acid residues around it play a significant role in channel kinetics.12 photon absorption causes a conformational change in the rhodopsin protein, via retinal isomerization, which allows for ion translocation across the channel . Opsins are broadly categorized based on their depolarizing or hyperpolarizing effects . Depolarizing opsins, or channelrhodopsins (chrs), permit the translocation of cations into the intracellular space upon photon absorption and retinal isomerization . Channelrhodopsin-1 (chr1) was the first of this type to be characterized, but it produces relatively small photocurrents and is not widely used.13 channelrhodopsin-2 (chr2) has a higher photocurrent than its predecessor and has proved to be quite effective at altering neuronal firing patterns in mammalian tissue.14,15 it is maximally activated by 470 nm wavelength light and can sustain reliable firing up to 40 hz . Multiple variants of chr1 and chr2 have recently been developed through point mutations and chimeric complexes in an attempt to improve or alter ion permeability (catch), channel kinetics (cheta), and wavelength selectivity (c1v1)12,16 (see table 1 for a list of notable variants). The creation of red - shifted opsins, such as c1v1, has enabled independent excitation of two depolarizing opsins in the same tissue.17,18 in addition, longer wavelengths mitigate light scatter and absorption, thus promoting deeper tissue penetration.19 in general, the photocurrent generated from opsin activation decays quickly following light cessation . This feature is an asset for most experimental designs, but certain behavioral paradigms benefit from prolonged manipulations . Variants known as stabilized - step function opsins significantly extend the open - state of the channel from a single pulse of blue light, which promotes a sustained depolarized state . This state increases the likelihood of firing in response to endogenous input, and can be inactivated with a single pulse of yellow light.20 recent step function opsin variants can induce depolarized states that last as long as 30 minutes following a single activating pulse of light.21 hyperpolarizing opsins, in contrast, are more limiting in both their overall characteristics and variety . Most hyperpolarizing opsins have an activation profile that is significantly red - shifted in comparison with the standard depolarizing chrs . Potent inhibition can be achieved through the pumping of chloride ions into the neuron through halorhodopsin (eg, enphr3.022) or via expelling protons from the intracellular space using archaerhodopsin (eg, earch3.016). However, despite their effectiveness, pumps are not energetically efficient, owing to their one proton to one ion photocycle,23 and do not alter the cell s input resistance.24 additionally, sustained halorhodopsin activation can disrupt chloride gradients, leading to a change in gamma - aminobutyric acid type a (gabaa) receptor reversal potential, thus eliciting a post - inhibitory excitatory state.25 recently, however, it has been shown that point mutations in a chimeric chr protein can convert it into a chloride conducting channel.26,27 the slow mutant variant, known as slow chloc, possess a 10-second long open - state during which neurons are effectively silenced.26 longer periods of neuronal inhibition can be achieved with a pharmacogenetic approach that utilizes designer receptors that are exclusively activated by designer drugs.28 in addition to the advances in opsin - mediated excitatory and inhibitory neuronal activity, there has been success in engineering chimeric opsin proteins that directly alter intracellular signaling cascades through g - protein coupled receptors.29 this class of proteins are known as optoxrs, and they have been designed to incorporate bovine rhodopsin (type ii opsin) into endogenously expressed adrenergic30 and serotonergic31 receptors . As a result of these developments, optogenetics provides a temporally refined and multifaceted toolkit of opsins allowing various controls on neuronal activity within genetically defined neuronal cell types . To achieve selective opsin expression, dna constructs encoding the opsins, often fused with a fluorescent protein marker (eg, yellow fluorescent protein), are introduced into neurons in an anatomically restricted manner, commonly by transgenic introduction, viral injection, or in vivo electroporation.3235 expression of opsins within neurons can be further restricted with the use of selective promoters or dna recombination systems . For example, one popular expression protocol involves the use of cre - recombinase, for its ability to invert gene orientations based on cre - identifiable tags (lox sites) that flank opsin genes . Thus, cell - specific opsin expression can be achieved using transgenic animals that express cre under a cell - specific gene promoter (eg, tyrosine hydroxylase), followed by viral infection carrying a payload with the opsin gene in an inverted orientation . As a result, only neurons expressing cre will be able express the opsin gene since it will have been inverted to its correct orientation . Once the proteins are expressed, they will diffuse within the membranes of cells and traffic to distal neural processes . This allows for the direct manipulation of spiking activity, either through stimulation of cell bodies or their distal processes . Thus, there are multiple dimensions by which photostimulation can be restricted, ie, anatomical location of dna delivery, cell - type specific expression, and localized light delivery . When these approaches are used together, anatomically localized, genetically defined neural pathways can be repeatedly stimulated or inhibited, in vitro and in vivo . Current treatments for clinical depression include pharmacotherapies that directly increase synaptic levels of serotonin and/or norepinephrine via reuptake inhibition . These two neurotransmitter systems have diffuse projections throughout the brain and are involved in a variety of functions, which may be the reason why individual patients respond to different antidepressant drugs in a highly variable manner . While antidepressant drugs act on these entire systems, it is presumed that only a subset of these projections are ultimately responsible for the therapeutic actions.36,37 this appears to be an area ripe for optogenetic analyses . However, antidepressant drugs typically require weeks of medication before symptoms alleviate, which complicates experimental designs and raises questions about the direct role of serotonin / norepinephrine in the therapeutic effects of antidepressant medications . The initial optogenetic explorations of depression have instead attempted to dissect a nonpharmacological treatment for depression known as deep brain stimulation, a treatment that has been explored as an alternative therapeutic option for severely depressed individuals . Deep brain stimulation via implanted electrodes that target frontal cortical areas and the nucleus accumbens has been demonstrated to relieve symptoms of intractable depression in human subjects.3842 it is unclear if there is a common neurobiological substrate mediating the therapeutic effects of electrical brain stimulation and antidepressant medication . Unlike drug - induced antidepressant effects, symptom relief following deep brain stimulation has a rapid onset . However, interpretation of the immediate effects of electrical stimulation is problematic, because it is not obvious how neural activity is altered by this manipulation . Electrical stimulation elicits nonspecific activation of all neuronal cell types and processes in a small area around the electrode . Since frontal cortical areas integrate information from throughout the brain and have projections that are similarly diffuse, it is difficult to attribute therapeutic effects to specific neural pathways . As such, electrical stimulation lacks the resolution needed to precisely define which local networks and cell types are responsible for the observed therapeutic changes . Researchers attempts to dissect stimulation - induced antidepressant effects with optogenetic tools have focused on alleviating depression in animal models of the disease . One of these animal models is known as the forced swim test, where laboratory rodents are placed in tanks of water and scored for time spent actively struggling versus passively floating.43,44 antidepressant treatments increase the amount of time that animals spend struggling . Other models of interest expose animals to stressful events, which creates a behavioral phenotype that is sensitive to chemical antidepressant treatments.45 optogenetic studies employing these animal models (summarized in table 2) demonstrate that photostimulation of neurons in the medial prefrontal cortex can elicit antidepressant - like behavior in both forced swim and chronic social defeat models of depression.4649 additional research has demonstrated that optogenetic stimulation directed to the specific medial prefrontal cortex projections that innervate the dorsal raphe nucleus is sufficient to produce antidepressant - like effects.48 the dorsal raphe nucleus is the principal source of ascending serotonin projections and the neurotransmitter system that most antidepressant pharmacological treatments act upon.50 thus, convergent lines of evidence implicate the dorsal raphe nucleus and its regulation by the medial prefrontal cortex in the treatment of depression . Despite the strong evidence implicating the medial prefrontal cortex - dorsal raphe nucleus circuit in treating symptoms of depression, many ambiguities remain regarding its precise role in this process . This pathway appears to preferentially excite gabaergic interneurons in the dorsal raphe nucleus,46,5153 suggesting that excitatory medial prefrontal cortex input to the dorsal raphe nucleus may act to depress serotonin levels via feed - forward inhibition . Consistent with this idea, pharmacological inhibition of the medial prefrontal cortex has been shown to enhance both stress - induced serotonin activity and learned helplessness behavior.54 collectively, these optogenetic and pharmacological studies support the provocative conclusion that acute decreases in serotonin function are capable of producing antidepressant - like effects . Similar behavioral effects have been demonstrated by reducing serotonin function via dorsal raphe nucleus negative - feedback autoreceptor activation55 or overexpression.56 these findings go against studies in humans in which acute depletion of serotonin synthesis decreases mood in people with a history of major depressive disorder . Responses in some behavioral models might be produced by any perturbation of serotonin signaling, regardless of directionality of effect . In contrast with the forced swim test, optogenetic research exploiting the chronic social defeat model of depression has supported the more conventional conclusion that enhancing serotonin neuron activity in the dorsal raphe nucleus reduces depression - like behavior . Specifically, social defeat experiences have been demonstrated to sensitize gabaergic interneuron function in the dorsal raphe nucleus, and photoinhibition of this population of neurons prevents the acquisition of defeat - induced social avoidance.57 further, bidirectional manipulations of medial prefrontal cortex - dorsal raphe nucleus circuitry can modulate defeat behavior in positive or negative ways, accordingly.46 a greater understanding of these behavioral models and the microcircuitry of the dorsal raphe nucleus will help explain how different neural inputs are integrated in this region of the brain . Other medial prefrontal cortex projection targets, in addition to the dorsal raphe nucleus, have recently been explored as well . Optogenetic stimulation to the medial prefrontal cortex projections to the nucleus accumbens have been shown to produce antidepressant - like effects in mice that exhibited depressive symptomatology following cholecystokinin infusions into the medial prefrontal cortex.58 this work highlights a potential role for the nucleus accumbens in depression, which is a region that has been somewhat overlooked in relation to this disease state . While the nucleus accumbens does receive serotonin and norepinephrine input,59,60 it is most prominently innervated by dopaminergic fibers . Photoactivation of midbrain dopamine neurons that innervate the nucleus accumbens has been shown to elicit antidepressant - like effects in both the tail suspension and forced swim tests.61 on the other hand, activation of accumbens - projecting dopamine cell bodies exacerbates the effects of social defeat stress,62 so this system may not have a simple role in depressive symptomatology . Indeed, midbrain dopamine neuron stimulation has been reported to produce opposite effects on sucrose preference in different studies, which suggests that specific conclusions may be more attributable to experimental parameters than depression in general.6163 more research is needed to clarify how dopamine and its downstream effectors regulate mood in relation to chronic depression . Anxiety disorders are generally characterized by excessive feelings of apprehension in the absence of any immediate threat . This category of disorders is broad, as it includes generalized anxiety, obsessive - compulsive, and post - traumatic stress disorders.64 modern medications used to treat anxiety disorders largely target serotonin and gabaergic neurotransmitter systems; however, drugs that block norepinephrine function appear to have particular utility in post - traumatic stress disorder.65,66 research into the neurobiology of anxiety has largely focused on the amygdala and its extended compartment in the bed nucleus of the stria terminalis (bnst), as these regions are critical for the manifestation of fear . Early studies in laboratory animals found that electrical stimulation of the amygdala produced behavioral and autonomic responses that resemble a state of fear, whereas lesion or inactivation of this region inhibited the expression of experimentally induced anxiety and conditioned fear.67 the basolateral amygdala, where multiple pathways converge, appears to be the locus of associative fear learning . Neural activity here is acutely sensitive to the presentation of previously learned, fear - evoking stimuli.68 neurons in the basolateral complex innervate the central nucleus of the amygdala, which is the primary output structure of the amygdala nuclei . This region regulates the behavioral and physiological manifestations of fear.69,70 the bnst, which primarily receives input from within the amygdala, serves as an additional output relay station.71 recent optogenetic studies have extended this model of amygdala circuitry and highlighted a role for this structure in behavioral measures of anxiety (figure 1). Photostimulation of the basolateral amygdala is sufficient to induce fear, and any stimuli paired with this stimulation can become fear - inducing.72 in addition, activity in this region is critical for the consolidation of fear memories.73 these studies have supported the classical view of the amygdala as a critical site of fear learning . Other optogenetic studies have broadened this view by showing that neighboring projections out of the basolateral amygdala can mediate opposing effects on fear and anxiety . Photostimulation of basolateral complex projections to the central lateral amygdala reduces anxiety in the elevated plus maze,74 whereas projections to the ca1 region of the hippocampus increase these and other measures of anxiety.75,76 this work underscores the importance of characterizing the role of adjacent structures and discrete projections . Indeed, the hippocampus is similarly nonuniform, as photostimulation of the ventral, but not dorsal, dentate gyrus area elicits a robust anxiolytic effect.77,78 anxiety regulation within the extended amygdala is also complex . The bnst contains two parallel output pathways to the ventral tegmental area (vta), ie, a gabaergic pathway that is anxiolytic and a glutamatergic pathway that is anxiogenic.79 the behavioral and physiological components of anxiety can be dissociated via other bnst projections as well . Stimulation of bnst to lateral hypothalamus projections produces avoidance of open arms in an elevated plus maze, but has no effect on respiration rate . Conversely, stimulation of bnst to parabrachial nucleus projections increases respiratory rate without altering plus maze behavior.80 stimulation of bnst cell bodies directly can produce various responses that depend on the exact location and genetic cell type targeted by the stimulation, factors that likely influence which output pathways are preferentially recruited . Beyond the extended amygdala, a role for the lateral septum in anxiety has also been demonstrated with optogenetics . Although this structure has been classically associated with anxiolytic responses,81 optogenetic stimulation of cells expressing corticotropin - releasing factor receptor type 2 produces anxiogenic effects.82 this effect was demonstrated to occur via gabaergic projections to the anterior hypothalamus . The hypothalamus contains oxytocin - producing neurons that project to the amygdala and participate in the regulation of anxiety . Selective stimulation of this pathway reduced expression of conditioned fear by directly activating gabaergic neurons in the central lateral amygdala.83 obsessive - compulsive (ocd) disorder is a specific subset of anxiety disorders that is characterized by repetitive behaviors aimed at relieving anxiety related to intrusive, irrational thoughts . As with general anxiety disorders, research into ocd has revealed a role of striatal circuitry in the expression of stereotyped, repetitive actions . A genetic mouse model of ocd has been developed in which mice lack the postsynaptic scaffolding protein sapap3.84 this gene is expressed strongly in the striatum, and its deletion results in defective corticostriatal circuitry . Mice lacking the gene exhibit high levels of anxiety and excessive grooming behavior, both of which are reversed with chronic antidepressant treatment . Dorsal striatal medium spiny neurons in these mutant mice exhibit exaggerated responses to the presentation of cues associated with grooming.85 photostimulation of lateral orbitofrontal cortical projections to the dorsal striatum attenuated this striatal neuron hyperactivity by activating fast - spiking interneurons in the striatum . Other recent studies that have examined the orbitofrontal projections to the ventral striatum in wild - type mice found that photostimulation of this pathway can increase grooming.86 while this phenomenon was only produced following chronic stimulation, it persisted for 2 weeks and could be reversed by chronic antidepressant treatment . These studies highlight the importance of corticostriatal circuitry in ocd, but future studies need to clarify where stimulation could be targeted to ultimately alleviate symptoms of the disorder . Indeed, a recent study implicates the more lateral orbitofrontal cortex in controlling the shift from habitual to goal - directed actions.87 drug abuse disorders are characterized by compulsive drug use in the face of adverse consequences . Highly addictive substances seem to disrupt the processes of self - control associated with normal reward - seeking behavior . Initial research mapping out regions of the brain involved in reward processing and motivation began over a half century ago with electrical intracranial self - stimulation experiments.88 recent studies that have used optogenetic techniques to extend this research have already confirmed that stimulation of midbrain dopamine neurons in the vta and substantia nigra pars compacta is highly reinforcing.8992 although the predominant effect of vta dopamine neuron stimulation is to reinforce and motivate operant behavior, an aversive subcircuit has also been identified, that consists of vta dopaminergic neurons receiving input from the lateral habenula and projecting to the medial prefrontal cortex.93 in addition, photoactivation of gabaergic interneurons in the vta has proved to be aversive.94,95 this research has largely confirmed ideas that have been developed over the preceding years, but a new level of detail is starting to emerge . The reinforcing effects of midbrain dopamine neuron activity are believed to be mediated primarily via projections to the striatum,9698 although it has been recently shown that vta - habenula projections also contribute to this effect.99 within the striatum, multiple glutamatergic inputs, in addition to the dopaminergic pathway, can reinforce instrumental behavior.100102 projections out of the striatum are segregated into two populations based on the expression of certain receptor proteins in the efferent neurons as well as projection targets.103105 optogenetic activation of these neuronal populations produces opposing behavioral effects, as photostimulation of dopamine d1 receptor - expressing neurons (direct pathway neurons) produces reward - related behavior and photostimulation of dopamine d2 receptor - expressing neurons (indirect pathway neurons) produces aversion.106 more refined behavioral studies are necessary to expand upon this simple reward / aversion dichotomy . As individuals repeatedly abuse addictive substances, addictive behaviors develop that are marked by a loss of self - control . Research into the mechanisms of this process has focused on the neural plasticity that addictive drugs produce within reward circuitry.107110 within the striatum, drug exposure appears to preferentially potentiate glutamatergic inputs onto direct pathway striatal neurons.109,111 individual animals with greater potentiation onto the other projection neurons in the striatum, ie, the indirect pathway neurons, show resistance to compulsive drug use.111 following prolonged withdrawal, potentiation of formerly silent basolateral amygdala - striatum synapses is prominent.112 pharmacological reversal of this potentiation reverses behavioral manifestation of incubation of drug craving . Numerous pathways, however, appear to be sensitive to drug exposure . Within the dorsomedial nucleus accumbens, drug - induced synaptic potentiation has also been shown to occur within the glutamatergic hippocampus - striatum pathway.101 in addition, direct pathway striatal neurons (dopamine d1 receptor - expressing), which project back to the vta and target gabaergic interneurons, are also potentiated by repeated drug use.113 several studies have focused on plasticity occurring within the medial prefrontal cortex . Individual rats that exhibit compulsive drug - seeking show intrinsic hypoactivity of medial prefrontal cortex neurons,114 while presynaptic enhancement occurs in the prefrontal cortex - accumbens projection.115 the causal role of cortical areas in addiction appears to be highly complex . Whereas 1 hz stimulation of prefrontal cortex cell bodies inhibits compulsive drug - seeking,114 inhibition of the prefrontal cortex - accumbens pathway reduces both reinstatement of cocaine - seeking116 and compulsive alcohol self - administration.117 furthermore, depotentiation of the prefrontal cortex - accumbens pathway reverses cocaine - induced psychomotor sensitization.109 thus, while optogenetics have allowed for the ability to study pathway specificity in drug - induced plasticity, future studies are crucial for determining the role of parameters such as drug type and schedule in mediating this plasticity . Schizophrenia and autism involve a constellation of symptoms that are not easy to model in rodents . One avenue of research into schizophrenia has focused on neurobiological markers seen within human populations, rather than attempting to recapitulate behavioral models of specific symptoms.118 for example, cortical gamma oscillations (3080 hz), which are altered in schizophrenic patients, can be driven by optogenetic stimulation of fast - spiking cortical interneurons.119,120 this manipulation alters the gating of sensory information, which is a common symptom of schizophrenia . A related line of research examines how disruptions in the cellular balance of excitation and inhibition within neural networks underlie deficits in social behavior.121 recent research has found that increasing excitatory, but not inhibitory, input to pyramidal neurons of the prefrontal cortex causes social and cognitive disturbances in rodents.21 this experiment utilized a step function opsin in conjunction with red - shifted opsins to simultaneously alter excitatory and inhibitory components in cortical microcircuits . Autism spectrum disorders can manifest as repetitive actions and reduced social interaction, which are symptoms that animal studies have tried to address in relation to ocd8587 and depression.4649 as the symptomatology of many psychiatric diseases overlap, some rodent behavioral studies have the potential to address common underlying circuit disturbances . However, there are numerous and obvious differences between people suffering from autism spectrum disorders and those with ocd . For example, people with autism are generally not bothered by their repetitive behaviors, whereas compulsions in ocd are a significant source of anxiety . Additional studies are needed to delineate the neural circuit disruptions that may be common across disorders versus those that are unique to specific psychiatric diseases . Significant progress has also been made with optogenetic tools in counteracting neural circuit disruptions that underlie certain neurological disorders, such epilepsy and parkinson s disease, which are comparatively easy to model in animals . Epileptiform activity can be completely shut down with photoinhibitions targeted to excitatory neurons as well as with photostimulations targeted to inhibitory neurons.122124 this work is comparable with the advances seen in parkinson s disease, in which movement deficits have been minimized in animal models through photostimulations and inhibitions targeted to discrete pathways and groups of neurons.125127 the research on neurological disorders benefits from having straightforward behavioral assays that are directly relevant to the treatment of human disease . The use of genetically - encoded, light - sensitive proteins, which can activate and inhibit discrete neural circuits, is revolutionizing behavioral neuroscience . When integrated with established techniques, ie, brain imaging, genetic manipulations, behavioral assays, and electrophysiology, optogenetics can help uncover the circuitry responsible for complex psychiatric disease states . To date, these tools have been used preclinically to advance our understanding of these phenomena in both rodent models and in nonhuman primates . There has been early success with this research in delineating previously overlooked neural pathways that should be a target in future drug discovery efforts . However, a great deal of clarification is still needed in this work, particularly in the cases where different publications have supported disparate conclusions . As our understanding of the neurobiology of various psychiatric disease states advances, optogenetic tools may eventually be used as therapeutic agents in human patients . This research promises to advance therapeutic drug development and to help refine the groupings of diverse symptomatology present within many psychiatric diseases.
Lagophthalmos, which is the inability to close the eyelids completely, is a serious condition caused by orbicularis oculi muscle paresis or paralysis . Paresis of the orbicularis oculi muscle leads to diminished blinking, lagophthalmos, and impairment of the nasolacrimal pump . With diminished orbicularis tone, the lower lid becomes lax, and the resting position of the lower eyelid drops . Corneal surface disruption results from both increased surface exposure and from the disruption of tear film caused by inadequate blinking mechanisms . Alteration in viith nerve function may also affect the secretion of tears and result in an absent bell s phenomenon . Associated loss of vth nerve function further increases the risk of surface disruption caused by loss of the protective mechanism of corneal sensation and by loss of its protective neurotropic effect on growth of the corneal epithelium.1 the resulting corneal problems include epithelial defects, stromal thinning, exposure keratitis, bacterial infection, perforation, and blindness . Lagophthalmos may be the result of the residual effect of viith cranial nerve damage, which can be congenital (moebius syndrome), acquired (bell s palsy, vascular lesions), or iatrogenic (during surgery), or arise from trauma, infection, or degenerative diseases.2,3 treatment of lagophthalmos may be conservative and symptomatic, such as eye ointments, drops, lubricants, taping and moisture chambers, soft contact lenses, and scleral shells, with or without the need for surgical intervention . Surgical intervention may be required in patients who have failed medical therapy or in whom facial paralysis is not expected to improve.4 surgical techniques include simple tarsorrhaphy, which may be temporal, medial, or both,5 canthoplasty, palpebral springs, silicone slings, eyelid magnets, temporalis muscle transfers, and nerve grafts . These methods can result in narrowing of the lid aperture, increase the risk of extrusion, and have the disadvantage of a very high rate of mechanical failure . A gold weight placed on the upper eyelid on the affected side enables eyelid closure by gravity, with relaxation of the levator muscle of the upper eyelid . It is considered to be a physiological method which does not interfere with esthetic eyelid opening and preserves the field of vision . Another advantage is that the procedure can be reversed without changing eyelid anatomy.5,6 several recent modifications to the implant as well as the surgical technique have been reported to minimize complications.710 in this study, we evaluated the different ocular results and complications, as well as esthetic satisfaction, in patients who underwent gold weight implantation 2 mm or 5 mm from the lid margin . Sixty - one eyes of 60 patients (32 male and 28 female) were enrolled in this study . Sixty - one gold plates were used to treat lagophthalmos in 60 patients with facial nerve paralysis (one patient had moebius syndrome with bilateral complete facial nerve paralysis). Other etiologies included congenital facial nerve paralysis, cerebellopontine angle tumor excision, bell s palsy, fractured base of the skull, and iatrogenic facial nerve injury . In addition, a series of preoperative and postoperative measurements of the ipsilateral and contralateral eyelids was performed . These included palpebral fissure height (distance between eyelid margins in primary gaze), lagophthalmos (distance between eyelid margins with gentle closure), inferior scleral show (amount of sclera visible beneath the lower limbus with gentle closure, mrd1 [marginal superior reflex distance 1, ie, distance between upper eyelid margin and corneal light reflex in primary gaze] and mrd2 [marginal inferior reflex distance 2, ie, distance between lower eyelid margin and corneal light reflex in primary gaze]). Visual acuity was recorded, and examination of the cornea and ophthalmic sensation was done preoperatively and postoperatively . Different ocular symptoms, eg, burning and tearing, and the need for utilization of artificial tears were noted . Preoperative and postoperative photographs were taken, and the patients were followed up monthly for at least one year . At the start of the study, we placed the gold plates at a low level of insertion (first technique, n = 38 eyes), but during the course of the study, we observed an increased incidence of noticeable bulge and ptosis, so we shifted to placing the gold plate at a higher level to overcome these two problems (second technique, n = 23 eyes). The custom - made weights were made of 24-carat gold, and were 1618 mm long, 5 mm in height, and fashioned as a rectangle with round borders . The body of the lid load had three to five holes to facilitate suspension (figure 1). Each gold weight had a smooth surface and weighed 0.91.5 g. the correct weight was selected preoperatively by taping different weights to the upper eyelid . The correct weight should close the eye on levator relaxation and not cause ptosis on levator contraction . Intraoperatively, the malleable gold plate was shaped with convexity on its anterior surface and concavity on its posterior surface, which allowed the gold weight inside the eyelid to rest smoothly along the globe . Both techniques were performed in the same fashion with one exception, ie, the distance of the pocket from the lid margin . The surgical procedure was done either with local infiltrative anesthesia and intravenous sedation or under general anesthesia . An incision about 1 cm long was made in the skin crease in the upper eyelid centered over the pupil . The orbicularis oculi was then slit open down to the tarsal plate and a pocket was created beneath the muscle over the middle third of the upper eyelid . The orbital septum and the levator aponeurosis were always posterior to the gold plate in both techniques, and care was taken to avoid injury to either of these structures . Using the first technique, the lower edge of the pocket was kept at a distance of 2 mm from the lid margin, and using the second technique it was kept at a distance of 5 mm from the lid margin . The gold plate was inserted into the pocket and fixed in the tarsal plate through three holes with 60 prolene . The decision to make extra holes in the gold plate the orbicularis oculi was closed using 60 vicryl and the skin was closed with 70 prolene . An eye dressing was used for 24 hours and the patient was discharged with oral and topical antibiotics on the first postoperative day . Figures 2 and 3 show patients with gold plates inserted using the first and second techniques, respectively . The custom - made weights were made of 24-carat gold, and were 1618 mm long, 5 mm in height, and fashioned as a rectangle with round borders . The body of the lid load had three to five holes to facilitate suspension (figure 1). Each gold weight had a smooth surface and weighed 0.91.5 g. the correct weight was selected preoperatively by taping different weights to the upper eyelid . The correct weight should close the eye on levator relaxation and not cause ptosis on levator contraction . Intraoperatively, the malleable gold plate was shaped with convexity on its anterior surface and concavity on its posterior surface, which allowed the gold weight inside the eyelid to rest smoothly along the globe . Both techniques were performed in the same fashion with one exception, ie, the distance of the pocket from the lid margin . The surgical procedure was done either with local infiltrative anesthesia and intravenous sedation or under general anesthesia . An incision about 1 cm long was made in the skin crease in the upper eyelid centered over the pupil . The orbicularis oculi was then slit open down to the tarsal plate and a pocket was created beneath the muscle over the middle third of the upper eyelid . The orbital septum and the levator aponeurosis were always posterior to the gold plate in both techniques, and care was taken to avoid injury to either of these structures . Using the first technique, the lower edge of the pocket was kept at a distance of 2 mm from the lid margin, and using the second technique it was kept at a distance of 5 mm from the lid margin . The gold plate was inserted into the pocket and fixed in the tarsal plate through three holes with 60 prolene . The decision to make extra holes in the gold plate the orbicularis oculi was closed using 60 vicryl and the skin was closed with 70 prolene . An eye dressing was used for 24 hours and the patient was discharged with oral and topical antibiotics on the first postoperative day . Figures 2 and 3 show patients with gold plates inserted using the first and second techniques, respectively . Thirty - eight eyes (62.3%) underwent gold plate placement at a low level (2 mm from the eyelid margin, first technique) and 23 eyes (37.7%) underwent gold plate placement at a high level (5 mm from the lid margin, second technique). Average time from the onset of facial paralysis to the lid loading procedure was 2.5 years (range 20 days to 5 years). The most commonly weight used with the first technique was 1.1 g in 14 eyes followed by 1.2 g in another five eyes . The most commonly used weight in the second technique was 1.2 g in seven eyes followed by 1.3 g in another six eyes . The gold weights used in all other eyes weighed 0.91.5 g. exposure keratitis was graded preoperatively and postoperatively on a scale of 04 . No keratopathy was rated 0, and mild superficial punctate keratopathy anywhere over the cornea was rated 1 . If the superficial punctate keratopathy was located in the inferior quarter of the cornea, the rating was 2, in the inferior one - third to one - half of the cornea, the rating was 3, and if it covered more than half of the cornea or if the erosion existed anywhere over the cornea, the rating was 4 . For the first technique, the average preoperative and postoperative ratings were 1.20 and 0.30, respectively, with an average improvement of visual acuity from 6/36 to 6/6 . There were no significant differences in any of the parameters evaluated between the first and the second techniques, and the degree of improvement in eyelid closure, keratopathy, and visual acuity was the same . The mean preoperative palpebral fissure height was 11.7 (range 814) mm and the mean preoperative lagophthalmos was 8 (range 511) mm . Mean postoperative lagophthalmos was reduced to 2.6 (range 06) mm at 8 weeks following surgery . In three patients, a slight scleral show was present during forward gaze, which was assumed to be due to associated lower lid laxity . No extrusion or infection was observed during the study, although seven patients who underwent the first technique had noticeable bulge in the upper eyelid and one patient had implant migration causing disfigurement of the upper eyelid . Exploration of the previously placed gold implant was done with no further difficulty than the original procedure . Using the second technique, three patients had overcorrection (defined as ptosis> 2 mm) after gold plate insertion using the first technique and only one using the second technique . The rate of ocular inflammation was 36 episodes per month, which reduced to 01 per month using both techniques . One case of conjunctival perforation in one eye (1.6% of all eyes) occurred using the second technique . Complete resolution of symptoms was observed in 24 eyes using the first technique (63.1%), and in 15 eyes using the second technique (65.2%). Patients with facial nerve palsy suffer from a range of eye problems, including eye dryness and repeated eye irritation and infection.11 these patients need eye protection using either a medical (eye lubricants, eye drops and occlusive dressings) or a surgical strategy . Gold plate placement can be used to treat lagophthalmos either temporarily until recovery of the paresis of the orbicularis muscle, or permanently if recovery does not occur . When there is improvement and complete recovery of the paresis, the gold weights can be removed . However, if there is partial recovery, the plates can be removed and replaced by smaller weights.12 lower lid repositioning can also be done to treat lower lid laxity . Esthetic appearance is an important issue for patients, and the weights seem to mimic normal blinking of the eyes.13 in this study, the surgical techniques used to implant the gold plates, either at a high level or a low level, were the same, but the height from the lid margin was different . The standard technique in the literature is to place the gold plate 23 mm from the lid margin . In our early cases, we started by placing the gold plates at 2 mm from the lid margin, but in later cases, we tried to elevate the level to 5 mm from the lid margin, with the aim of hiding the plate behind the upper lid fold and decreasing the upper lid bulge . A preoperative trial of placing gold weights on the outer surface is important to assess the weight of the gold plates intraoperatively . In our study, when placing the gold plate at a high level, heavier gold plates were needed to achieve the desired level of the lids . This gives a better esthetic look, but needs a heavier weight to obtain lid closure.14 in eyes with lower level placement of the gold plates, the incidence of upper eyelid ptosis was higher, and there was a higher incidence of obvious upper eyelid bulge, which was annoying for the patients . According to the mathematical equation of work = force s, where in our study s represents the distance that can be moved by the gold plate on lid opening, and according to figure 4, if we are using the same gold weight (force), we notice that the exerted work is more for the first technique, because ab gold plate weight is less than cd same gold plate weight . No significant differences existed for any of the evaluated parameters between the first and the second techniques, and the degree of improvement in eyelid closure, keratopathy, and visual acuity was the same . Patients with high - level placement would need 0.2 g heavier gold plates than the lower - level placement to achieve sufficient load on the lids . Placement of gold plates at a higher level could avoid some of the drawbacks of lower - level plate placement, including upper eyelid bulge and ptosis, especially given that there is thinning of the eyelids and orbicularis muscle in facial palsy . This study reports that a higher level of insertion of the gold plate achieves better esthetic results with less noticeable bulge in the upper eyelid and with acceptable functional results . However, we believe that a comparative study in a larger series is required to investigate further the most appropriate technique for gold plate insertion . Until then, case by case analysis and surgical decision - making is recommended.
Diabetes mellitus is one of the most challenging chronic diseases (king et al 1998; lipscombe and hux 2007) associated with significant morbidity and mortality (baker et al 2008; pinto et al 2008). In order to avoid or delay microvascular complications, a strict metabolic control of blood glucose is recommended (ukpds 1998; leroith and smith 2005; vermeire et al 2005). Measure of glycosylated hemoglobin a (hba1c) is used to evaluate glycemia control . For the majority of diabetics, glycemia control is considered attained when hba1c levels are less than 7% (harris and lank 2004; anonymous 2008). Many patients do not achieve optimal glycemia control (saaddine et al 2002; putzer et al 2004). Longer diabetes duration and presence of complications are both associated with poor glycemia control (druss et al 2001; putzer et al 2004; weiner and long 2004; egede 2005; kerr et al 2007; kivimaki et al 2007; parchman et al 2007; suh et al 2008). The majority of diabetic patients have at least one comorbid chronic condition (druss et al 2001; egede 2005; kivimaki et al 2007), a problem that may also have an impact on glycemia control . Indeed, competing situations may occur in the treatment of patients (parchman et al 2007), and the presence of several symptomatic comorbidities may influence patients self management (kerr et al 2007). However, reports on the relationship between comorbidity and glycemia control are conflicting . In some cases, evidence suggests that comorbidity is associated with lower glycemia control (zhang et al 2000; weiner and long 2004; suh et al 2008), whereas other data argue against any association (el - kebbi et al 2001). These conflicting results may be explained by the use of different methods to measure comorbidity . In two studies, the charlson comorbidity index was used (zhang et al 2000; weiner and long 2004). However, this index was originally developed to predict mortality risk and includes a limited list of diseases . Another study used the chronic disease score (cds) (el - kebbi et al 2001), a comorbidity measure based on sex, age, and drugs prescribed to the patient . The remaining study took into account a limited number of conditions traditionally known as diabetes complications (suh et al 2008). Uncontrolled diabetes may result in complications such as nephropathy, renal failure, hypertension, congestive heart failure and coronary disease (ada 2008). Therefore, any study on a possible association between glycemia control and comorbidity, in which the measure of comorbidity would only include such chronic conditions would be biased towards finding an association . Furthermore, in addition to the type of comorbidity, its severity may either cause poor glycemia control or be a consequence of it . Since many diabetic patients have comorbidity (druss et al 2001; egede 2005; kivimaki et al 2007), it is important to determine if comorbidity increases the difficulty to manage the disease and to achieve good glycemia control . Therefore, the aim of the present study was to evaluate the relationship between diabetes control and comorbidity using a comprehensive comorbidity index in a primary care setting . We performed a retrospective descriptive study based on chart review at the family medicine unit (fmu) of the centre de sant et de services sociaux de chicoutimi, quebec, canada . The study was approved by the institutional ethics committee . Using the electronic laboratory database available, we performed a random selection of patients with type 2 diabetes who had at least two measurements of hba1c levels between january 1st, 2004 and september 1st, 2006 . Patients diagnosed with diabetes less than a year before the last measurement of hba1c and those under the care of the researchers involved in this study were excluded . Data extracted from the charts included sex, age, the last two measures of hba1c, the presence or absence of diabetes - related complications, time elapsed since the diagnosis of diabetes (diabetes duration at the moment of the second measurement of hba1c) as well as information about the patient s chronic conditions necessary to score the cumulative illness rating scale (cirs) (linn et al 1968; fortin et al 2005b, 2006a, 2006b). The three nurses, blinded to the objectives of the study, had received an half - day training in cirs scoring prior to the study . The cirs is a measure of comorbidity that takes into account all medical conditions and includes the notion of severity (linn et al 1968; fortin et al 2005b, 2006a, 2006b). It has been validated in primary care as a tool for quantifying comorbidity (hudon et al 2005, 2007). The cirs uses a scoring system that encompasses 14 anatomical domains (cardiac, vascular, hematological, respiratory, ophthalmologic otorhinolaryngologic, upper gastrointestinal, lower gastrointestinal, hepatic pancreatic, renal, genitourinary, musculoskeletal tegument, neurological, endocrine metabolic breast, and psychiatric) and assigns a value from 0 (no condition in this domain) to 4 (extremely severe problem) to determine a severity score for each domain . In the case of multiple conditions affecting a particular domain, the global score is the sum of each domain s score . To use the cirs as a measure of comorbidity for diabetes, diabetes was considered as controlled if the mean value of the two measurements of hba1c levels was less than 7%; mean values equal or greater than 7% were considered as uncontrolled diabetes (harris and lank 2004; anonymous 2008). The differences between patients with controlled and uncontrolled diabetes were tested using the t - test for continuous variables such as cirs score, age and diabetes duration, and the chi - square test for categorical variables like sex and diabetes - related complications . Then, variables with p <0.1 were used as covariables in a multivariable logistic regression model to measure the association between diabetes control (dependant variable) and the cirs score (independent variable). According to various studies, the percentage of patients controlling their diabetes varies from 33% to 55% (weiner and long 2004; meduru et al 2007; suh et al 2008). When we estimated the sample size, we used the worst case scenario and anticipated a 2-to-1 ratio when comparing subjects with uncontrolled diabetes to subjects with controlled diabetes . A total sample size of 75 subjects was needed in order to achieve 80% statistical power, to detect a difference of 4 points on the cirs with a standard deviation of 5.64 (based on a previous study) (fortin et al 2005a), with a two - sided t - test at the 5% significance level (nquery advisor 6.01). We increased this sample size by 30% to account for possible missing data in the charts and to allow controlling for 2 to 3 variables in the logistic regression model (requiring at least 30 subjects in the smallest group). Analyses were made with spss software (version 13.0; spss inc ., chicago, il) and a p value <0.05 was considered statistically significant . The differences between patients with controlled and uncontrolled diabetes were tested using the t - test for continuous variables such as cirs score, age and diabetes duration, and the chi - square test for categorical variables like sex and diabetes - related complications . Then, variables with p <0.1 were used as covariables in a multivariable logistic regression model to measure the association between diabetes control (dependant variable) and the cirs score (independent variable). According to various studies, the percentage of patients controlling their diabetes varies from 33% to 55% (weiner and long 2004; meduru et al 2007; suh et al 2008). When we estimated the sample size, we used the worst case scenario and anticipated a 2-to-1 ratio when comparing subjects with uncontrolled diabetes to subjects with controlled diabetes . A total sample size of 75 subjects was needed in order to achieve 80% statistical power, to detect a difference of 4 points on the cirs with a standard deviation of 5.64 (based on a previous study) (fortin et al 2005a), with a two - sided t - test at the 5% significance level (nquery advisor 6.01). We increased this sample size by 30% to account for possible missing data in the charts and to allow controlling for 2 to 3 variables in the logistic regression model (requiring at least 30 subjects in the smallest group). Analyses were made with spss software (version 13.0; spss inc ., chicago, il) and a p value <0.05 was considered statistically significant . Statistical results from the univariate analyses performed to measure the associations between diabetes control and the patients characteristics are indicated in table 1 . Diabetes control was not significantly related with the cirs score, age, sex, or diabetes - related complications . A logistic regression model was thus used to measure the association between diabetes control and the cirs, by including the diabetes duration as a covariable . The relation was still not significant (adjusted odds ratio = 0.889; 95% confidence interval: 0.7361.074; p = 0.223). The results of this study suggest that in patients with type 2 diabetes mellitus, the presence of comorbidity measured with the cirs, is not related to glycemia control defined by hba1c levels . The finding that glycemia control is associated with diabetes duration is similar to that of previous reports (leelawattana et al 2006; shim et al 2006; tascona et al 2006; heisler et al 2007; lopez stewart et al 2007; tien et al 2008; tikellis et al 2008). To our knowledge, this is the first study that used the cirs to measure comorbidity in order to assess the relationship between comorbidity and glycemia control in type 2 diabetic patients . The cirs score takes into account all comorbidities as well as their severity, and provides a good assessment of other chronic diseases that may coexist with diabetes mellitus, being or not etiologically associated with it . Compared to our comorbidity measuring method, previous studies on a possible association between comorbidity condition and glycemia control included a limited number of diseases (zhang et al 2000; weiner and long 2004; suh et al 2008) or diseases associated to diabetes exclusively (suh et al 2008). In these studies, our finding that the presence of comorbidities is not associated with glycemia control supports the conclusion of another study which also used a more comprehensive measurement of comorbidity, the chronic disease score (cds) (el - kebbi et al 2001). It has been reported that depending on the type of comorbid condition they have, diabetic patients are more or less likely to achieve hba1c levels under 7% (meduru et al 2007). Therefore, when hba1c less than 7% threshold is used as a performance measure for diabetes quality of care, assessment of an association between glycemia control and comorbidity may be influenced by the type of comorbid conditions . In fact, the likelihood of finding an association between poor glycemia control and comorbidity in patients with diabetes - related complications would be higher than in patients without this type of complications (meduru et al 2007). However, our study suggests that the presence of multiple chronic conditions is not a factor limiting the achievement of a good control of glycemia, even after taking into account the severity of the coexistent diseases . It has been reported that services received by diabetic patients do not differ based on comorbid illness burden (halanych et al 2007), however diabetic patients with elevated hba1c receive a closer attention in primary care encounters (parchman et al 2007). Therefore, in the presence of multiple comorbid conditions, diabetic patients may receive better care . As a matter of fact, it has been reported that vulnerable elders with multiple chronic conditions, including diabetes, receive better overall quality of care (min et al 2007), and that the quality of care improves as a patient s number of chronic conditions increases (higashi et al 2007). It could have been preferable to use a mean value of all hba1c over two years rather than using only the most recent two hba1c measures . However, given data abstraction techniques, it was not feasible to utilize additional values . Since we performed a retrospective study, one limitation of our method is that different variables such as the time elapsed between the two measurements of hba1c could not be controlled . Furthermore, in our sample, 69% of the patients had a good glycemia control (table 1), a higher proportion compared to that in other studies (33%55%) (weiner and long 2004; meduru et al 2007; suh et al 2008). The fact that our institution is a teaching hospital with a fmu may have influenced the quality of care provided, and biased the results towards a better glycemia control . There is also the possibility that a number of patients with more severe diabetes are followed up in specialized clinics and were therefore underrepresented in our sample . Another limitation of the study is that the patients were recruited from only one setting and therefore extrapolation of our results to other settings is more difficult to make . Lack of statistical power could be an issue in the assessment of the relationship between diabetes - related complications and glycemia control . Given that the proportion of complications is around 50% when diabetes is not controlled, the two group chi - square test at the 0.05 level had a 34% statistical power to detect an odds ratio of 0.5 with sample sizes of 30 (uncontrolled diabetes) and 66 (controlled diabetes) (nquery advisor 6.01). Despite these limitations, our study is strengthened by the use of a comprehensive comorbidity index to measure comorbidity that takes into account disease severity, a random patients selection and the mean of two hba1c measurements instead of a single measurement . It could have been preferable to use a mean value of all hba1c over two years rather than using only the most recent two hba1c measures . However, given data abstraction techniques, it was not feasible to utilize additional values . Since we performed a retrospective study, one limitation of our method is that different variables such as the time elapsed between the two measurements of hba1c could not be controlled . Furthermore, in our sample, 69% of the patients had a good glycemia control (table 1), a higher proportion compared to that in other studies (33%55%) (weiner and long 2004; meduru et al 2007; suh et al 2008). The fact that our institution is a teaching hospital with a fmu may have influenced the quality of care provided, and biased the results towards a better glycemia control . There is also the possibility that a number of patients with more severe diabetes are followed up in specialized clinics and were therefore underrepresented in our sample . Another limitation of the study is that the patients were recruited from only one setting and therefore extrapolation of our results to other settings is more difficult to make . Lack of statistical power could be an issue in the assessment of the relationship between diabetes - related complications and glycemia control . Given that the proportion of complications is around 50% when diabetes is not controlled, the two group chi - square test at the 0.05 level had a 34% statistical power to detect an odds ratio of 0.5 with sample sizes of 30 (uncontrolled diabetes) and 66 (controlled diabetes) (nquery advisor 6.01). Despite these limitations, our study is strengthened by the use of a comprehensive comorbidity index to measure comorbidity that takes into account disease severity, a random patients selection and the mean of two hba1c measurements instead of a single measurement . Our study suggests that comorbidity, measured with an exhaustive index, is not a factor that prevents the achievement of a good glycemia control in patients with type 2 diabetes . Further studies are needed to extend these results to other settings and to evaluate the impact of different types of medical conditions on glycemia control.
The fundamental principles of pay - for - performance include rewarding quality health care and aligning physicians financial incentives with the best interests of patients.9 although this inherent appeal to physician self - interest might be in tension with professional ideals of altruism and beneficence,1013 the principles that inform pay - for - performance are not inherently unethical . It seems just, for example, to financially reward physicians who demonstrate outstanding levels of patient - centered and evidence - based care . Nevertheless, systems intending to improve medical care must be guided by evidence and a precise definition of health - care quality to ensure that they are effective, valid, and fair . We define health - care quality in a manner that prioritizes patient - centered care14 while recognizing the importance of population - level health improvement:15health - care quality is the degree to which physicians and health - care institutions fulfill their care obligations to individual patients and the degree to which patients, physicians, and health - care institutions enable these obligations to be fulfilled justly across the population.16 health - care quality is the degree to which physicians and health - care institutions fulfill their care obligations to individual patients and the degree to which patients, physicians, and health - care institutions enable these obligations to be fulfilled justly across the population.16 this understanding of health - care quality informs our criticisms of current pay - for - performance arrangements and provides a roadmap to high quality care and ethical performance - based physician compensation . In light of these principles, we see the following potential ethical problems in the implementation of pay - for - performance systems . Lack of proven safety and benefit for patients: studies of performance - based physician compensation have generally shown scant evidence of quality improvement.27 implementation without proof of safety and effectiveness is ethically problematic . It is unclear, for instance, why a new drug to be used by several dozen individuals requires proof of safety and efficacy, while policy changes affecting millions do not . From an ethical perspective, pay - for - performance is a potentially risky experiment in health - care delivery.17 further, current pay - for - performance systems generally lack key safeguards against readily anticipated adverse effects 1 (discussed below), and we are concerned that negative outcomes may already be unfolding.inadequate definitions of quality: although commentators have proposed many definitions of health - care quality,1827 none are universally accepted, and they provide little guidance regarding accountability or how quality can be validly measured . Furthermore, current pay - for - performance arrangements are guided by a highly incomplete understanding of quality that does not resemble any published or well - reasoned definition . This understanding typically equates quality with the achievement of non - individualized, pre - determined health goals for broad populations and fails to consider contributions from stakeholders other than physician entities1 (such as health plans) that also have partial responsibility for ensuring quality . This approach has severely limited our ability to capture the myriad of elements comprising quality care, let alone the most complex but essential feature of a praxis like medicine the exercise of correct judgment which is only readily assessed by peers.28inadequate measures of quality: because they are based on inadequate definitions, existing pay - for - performance measures lack validity and comprehensiveness in assessing health - care quality . Measures typically cover only isolated and readily quantifiable aspects of physician clinical performance and fail to assess crucial realms such as judgment, compassion, and communication skills . Lack of proven safety and benefit for patients: studies of performance - based physician compensation have generally shown scant evidence of quality improvement.27 implementation without proof of safety and effectiveness is ethically problematic . It is unclear, for instance, why a new drug to be used by several dozen individuals requires proof of safety and efficacy, while policy changes affecting millions do not . From an ethical perspective, pay - for - performance is a potentially risky experiment in health - care delivery.17 further, current pay - for - performance systems generally lack key safeguards against readily anticipated adverse effects 1 (discussed below), and we are concerned that negative outcomes may already be unfolding . Inadequate definitions of quality: although commentators have proposed many definitions of health - care quality,1827 none are universally accepted, and they provide little guidance regarding accountability or how quality can be validly measured . Furthermore, current pay - for - performance arrangements are guided by a highly incomplete understanding of quality that does not resemble any published or well - reasoned definition . This understanding typically equates quality with the achievement of non - individualized, pre - determined health goals for broad populations and fails to consider contributions from stakeholders other than physician entities1 (such as health plans) that also have partial responsibility for ensuring quality . This approach has severely limited our ability to capture the myriad of elements comprising quality care, let alone the most complex but essential feature of a praxis like medicine the exercise of correct judgment which is only readily assessed by peers.28 inadequate measures of quality: because they are based on inadequate definitions, existing pay - for - performance measures lack validity and comprehensiveness in assessing health - care quality . Measures typically cover only isolated and readily quantifiable aspects of physician clinical performance and fail to assess crucial realms such as judgment, compassion, and communication skills . Quantifying health - care quality is notoriously difficult, and basing payment incentives upon inadequate measures and definitions of quality will make consequences difficult to control . Unfortunately, this approach is often used to make judgments about individual practitioners when variability in case mix and patient preferences precludes making valid judgments . For example, in a patient with difficult - to - control diabetes, a decline in hemoglobin a1c from 10.0 to 8.0 might be a remarkable achievement and more validly represent high quality care than a decline from 7.3 to 6.9 in another patient . Misallocating the locus of accountability for quality improvement: many pay - for - performance measures hold physicians accountable for aspects of quality beyond their control, such as health - care delivery problems, lack of incentives for coordinated care, and even social determinants of health . Some may hold physicians accountable for the care of patients with whom they have not had a continuous relationship.potential for adverse effects on patients and vulnerable populations: performance targets used today may have detrimental effects on quality . For example, it may seem reasonable to require that diabetic patients achieve hemoglobin a1c levels below 7.0 . In patients with previous hypoglycemic episodes, however, this target might be life - threatening . Misallocating the locus of accountability for quality improvement: many pay - for - performance measures hold physicians accountable for aspects of quality beyond their control, such as health - care delivery problems, lack of incentives for coordinated care, and even social determinants of health . Some may hold physicians accountable for the care of patients with whom they have not had a continuous relationship . Potential for adverse effects on patients and vulnerable populations: performance targets used today may have detrimental effects on quality . For example, it may seem reasonable to require that diabetic patients achieve hemoglobin a1c levels below 7.0 . In patients with previous hypoglycemic episodes, however, this target might be life - threatening . Or, consider a patient with a hemoglobin a1c of 7.5 who frequently skips preventive visits but happens to present with back pain . If bonuses are provided for reducing glucose levels, a physician might prefer to discuss diabetes control rather than ruling out life - threatening causes of back pain . Pre - determined population - centered measures might also induce physicians to avoid patients who are less likely to meet targets . Such patients are often society s most vulnerable members those with multiple chronic conditions, the poor, the educationally disadvantaged, those with limited english proficiency, and members of racial minority groups . Because physicians serving disadvantaged patients might receive lower compensation, less well - off practices would be left with fewer resources to improve care . Poorly designed pay - for - performance systems could therefore limit access to care for vulnerable populations, worsen health - care quality, and erode patient trust.2938potential for adverse effects on physicians: in systems using a limited set of population - level measures, physician professionalism and morale could decline . Some clinicians might pay - for - performance is also likely to increase the complexity of the reimbursement system, and metrics might be used against physicians for legal, credentialing, or recertification purposes . Such changes would decrease physician job satisfaction, with detrimental effects on patient care and the attractiveness of medicine (especially primary care) as a profession.potential for adverse effects on society: the potential detrimental effects above would have broader implications for society . Truly valid and comprehensive measurements might require overly burdensome or expensive systems, and could make the marginal value of performance - based compensation negligible . Deteriorating value could also result if physicians drive up expenses by ordering unnecessary tests or referrals to specialists.36 potential for adverse effects on physicians: in systems using a limited set of population - level measures, physician professionalism and morale could decline . Some clinicians might treat the measure or select the pay - for - performance is also likely to increase the complexity of the reimbursement system, and metrics might be used against physicians for legal, credentialing, or recertification purposes . Such changes would decrease physician job satisfaction, with detrimental effects on patient care and the attractiveness of medicine (especially primary care) as a profession . Potential for adverse effects on society: the potential detrimental effects above would have broader implications for society . Truly valid and comprehensive measurements might require overly burdensome or expensive systems, and could make the marginal value of performance - based compensation negligible . Deteriorating value could also result if physicians drive up expenses by ordering unnecessary tests or referrals to specialists.36 ultimately, insurers could face a backlash by patients and physicians against an effort that might be viewed cynically as another cost - containment attempt, offered disingenuously as quality improvement . Lack of structured monitoring for adverse outcomes: a substantial literature advocates structured oversight of any risky intervention not meant to directly benefit individuals.3953 although pay - for - performance is intended to improve patient care, some would argue that it is primarily a population - centered cost control measure with unclear effectiveness and a substantial risk - benefit ratio for certain populations.33 we believe the risks from pay - for - performance outlined above are serious enough and have a high enough probability of occurring to engender an ethical obligation for structured monitoring of key outcomes (discussed below). Lack of structured monitoring for adverse outcomes: a substantial literature advocates structured oversight of any risky intervention not meant to directly benefit individuals.3953 although pay - for - performance is intended to improve patient care, some would argue that it is primarily a population - centered cost control measure with unclear effectiveness and a substantial risk - benefit ratio for certain populations.33 we believe the risks from pay - for - performance outlined above are serious enough and have a high enough probability of occurring to engender an ethical obligation for structured monitoring of key outcomes (discussed below). In light of these principles, we see the following potential ethical problems in the implementation of pay - for - performance systems . Lack of proven safety and benefit for patients: studies of performance - based physician compensation have generally shown scant evidence of quality improvement.27 implementation without proof of safety and effectiveness is ethically problematic . It is unclear, for instance, why a new drug to be used by several dozen individuals requires proof of safety and efficacy, while policy changes affecting millions do not . From an ethical perspective, pay - for - performance is a potentially risky experiment in health - care delivery.17 further, current pay - for - performance systems generally lack key safeguards against readily anticipated adverse effects 1 (discussed below), and we are concerned that negative outcomes may already be unfolding.inadequate definitions of quality: although commentators have proposed many definitions of health - care quality,1827 none are universally accepted, and they provide little guidance regarding accountability or how quality can be validly measured . Furthermore, current pay - for - performance arrangements are guided by a highly incomplete understanding of quality that does not resemble any published or well - reasoned definition . This understanding typically equates quality with the achievement of non - individualized, pre - determined health goals for broad populations and fails to consider contributions from stakeholders other than physician entities1 (such as health plans) that also have partial responsibility for ensuring quality . This approach has severely limited our ability to capture the myriad of elements comprising quality care, let alone the most complex but essential feature of a praxis like medicine the exercise of correct judgment which is only readily assessed by peers.28inadequate measures of quality: because they are based on inadequate definitions, existing pay - for - performance measures lack validity and comprehensiveness in assessing health - care quality . Measures typically cover only isolated and readily quantifiable aspects of physician clinical performance and fail to assess crucial realms such as judgment, compassion, and communication skills . Lack of proven safety and benefit for patients: studies of performance - based physician compensation have generally shown scant evidence of quality improvement.27 implementation without proof of safety and effectiveness is ethically problematic . It is unclear, for instance, why a new drug to be used by several dozen individuals requires proof of safety and efficacy, while policy changes affecting millions do not . From an ethical perspective, pay - for - performance is a potentially risky experiment in health - care delivery.17 further, current pay - for - performance systems generally lack key safeguards against readily anticipated adverse effects 1 (discussed below), and we are concerned that negative outcomes may already be unfolding . Inadequate definitions of quality: although commentators have proposed many definitions of health - care quality,1827 none are universally accepted, and they provide little guidance regarding accountability or how quality can be validly measured . Furthermore, current pay - for - performance arrangements are guided by a highly incomplete understanding of quality that does not resemble any published or well - reasoned definition . This understanding typically equates quality with the achievement of non - individualized, pre - determined health goals for broad populations and fails to consider contributions from stakeholders other than physician entities1 (such as health plans) that also have partial responsibility for ensuring quality . This approach has severely limited our ability to capture the myriad of elements comprising quality care, let alone the most complex but essential feature of a praxis like medicine the exercise of correct judgment which is only readily assessed by peers.28 inadequate measures of quality: because they are based on inadequate definitions, existing pay - for - performance measures lack validity and comprehensiveness in assessing health - care quality . Measures typically cover only isolated and readily quantifiable aspects of physician clinical performance and fail to assess crucial realms such as judgment, compassion, and communication skills . Quantifying health - care quality is notoriously difficult, and basing payment incentives upon inadequate measures and definitions of quality will make consequences difficult to control . Unfortunately, this approach is often used to make judgments about individual practitioners when variability in case mix and patient preferences precludes making valid judgments . For example, in a patient with difficult - to - control diabetes, a decline in hemoglobin a1c from 10.0 to 8.0 might be a remarkable achievement and more validly represent high quality care than a decline from 7.3 to 6.9 in another patient . Misallocating the locus of accountability for quality improvement: many pay - for - performance measures hold physicians accountable for aspects of quality beyond their control, such as health - care delivery problems, lack of incentives for coordinated care, and even social determinants of health . Some may hold physicians accountable for the care of patients with whom they have not had a continuous relationship.potential for adverse effects on patients and vulnerable populations: performance targets used today may have detrimental effects on quality . For example, it may seem reasonable to require that diabetic patients achieve hemoglobin a1c levels below 7.0 . In patients with previous hypoglycemic episodes, however, this target might be life - threatening . Misallocating the locus of accountability for quality improvement: many pay - for - performance measures hold physicians accountable for aspects of quality beyond their control, such as health - care delivery problems, lack of incentives for coordinated care, and even social determinants of health . Some may hold physicians accountable for the care of patients with whom they have not had a continuous relationship . Potential for adverse effects on patients and vulnerable populations: performance targets used today may have detrimental effects on quality . For example, it may seem reasonable to require that diabetic patients achieve hemoglobin a1c levels below 7.0 . In patients with previous hypoglycemic episodes, however, this target might be life - threatening . Or, consider a patient with a hemoglobin a1c of 7.5 who frequently skips preventive visits but happens to present with back pain . If bonuses are provided for reducing glucose levels, a physician might prefer to discuss diabetes control rather than ruling out life - threatening causes of back pain . Pre - determined population - centered measures might also induce physicians to avoid patients who are less likely to meet targets . Such patients are often society s most vulnerable members those with multiple chronic conditions, the poor, the educationally disadvantaged, those with limited english proficiency, and members of racial minority groups . Because physicians serving disadvantaged patients might receive lower compensation, less well - off practices would be left with fewer resources to improve care . Poorly designed pay - for - performance systems could therefore limit access to care for vulnerable populations, worsen health - care quality, and erode patient trust.2938potential for adverse effects on physicians: in systems using a limited set of population - level measures, physician professionalism and morale could decline . Some clinicians might pay - for - performance is also likely to increase the complexity of the reimbursement system, and metrics might be used against physicians for legal, credentialing, or recertification purposes . Such changes would decrease physician job satisfaction, with detrimental effects on patient care and the attractiveness of medicine (especially primary care) as a profession.potential for adverse effects on society: the potential detrimental effects above would have broader implications for society . Truly valid and comprehensive measurements might require overly burdensome or expensive systems, and could make the marginal value of performance - based compensation negligible . Deteriorating value could also result if physicians drive up expenses by ordering unnecessary tests or referrals to specialists.36 potential for adverse effects on physicians: in systems using a limited set of population - level measures, physician professionalism and morale could decline . Pay - for - performance is also likely to increase the complexity of the reimbursement system, and metrics might be used against physicians for legal, credentialing, or recertification purposes . Such changes would decrease physician job satisfaction, with detrimental effects on patient care and the attractiveness of medicine (especially primary care) as a profession . Potential for adverse effects on society: the potential detrimental effects above would have broader implications for society . A decreasing supply of primary care physicians would exacerbate problems in access and quality . Truly valid and comprehensive measurements might require overly burdensome or expensive systems, and could make the marginal value of performance - based compensation negligible . Deteriorating value could also result if physicians drive up expenses by ordering unnecessary tests or referrals to specialists.36 ultimately, insurers could face a backlash by patients and physicians against an effort that might be viewed cynically as another cost - containment attempt, offered disingenuously as quality improvement . Lack of structured monitoring for adverse outcomes: a substantial literature advocates structured oversight of any risky intervention not meant to directly benefit individuals.3953 although pay - for - performance is intended to improve patient care, some would argue that it is primarily a population - centered cost control measure with unclear effectiveness and a substantial risk - benefit ratio for certain populations.33 we believe the risks from pay - for - performance outlined above are serious enough and have a high enough probability of occurring to engender an ethical obligation for structured monitoring of key outcomes (discussed below). Lack of structured monitoring for adverse outcomes: a substantial literature advocates structured oversight of any risky intervention not meant to directly benefit individuals.3953 although pay - for - performance is intended to improve patient care, some would argue that it is primarily a population - centered cost control measure with unclear effectiveness and a substantial risk - benefit ratio for certain populations.33 we believe the risks from pay - for - performance outlined above are serious enough and have a high enough probability of occurring to engender an ethical obligation for structured monitoring of key outcomes (discussed below). Sgim supports evidence - based, ethical, and comprehensive efforts to improve health - care quality and physician compensation . While carefully designed pay - for - performance systems could be a component of such an approach, current iterations fail to reach acceptable ethical standards for the reasons above . We therefore advocate the following four major strategies to achieve high quality health care and ethical performance - based physician compensation (tables 1, 2, 3). Table 1potential ethical problems in the implementation of pay - for - performancei . Lack of structured monitoring for adverse outcomestable 2major strategies to achieve high quality health care and ethical performance - based physician compensationi . Current pay - for - performance systems should rapidly adopt safeguards to protect vulnerable populations (see table 3)ii . Researchers and policy makers should develop valid and comprehensive quality measures for use in the next generation of compensation systems that reward genuine qualityiv . Researchers and policy makers should use a cautious evaluative approach to long - term development of compensation systems that reward qualitytable 3recommended safeguards to protect vulnerable populations and prevent unintended consequences within current pay - for - performance systems1 . Balance current population - level measurements with the best available measures of quality from the patient perspective2 . Reduce or stabilize the percentage of physicians salaries at stake population - level measures should: a. be evidence - based and clearly linked to valued patient outcomes b. assess domains clearly within the influence of the physician or physician group, especially for complex patients c. assess quality at the level of large physician practices rather than individual physicians d. assess improvement toward goals in addition to achievement of cut - points5 . If systems utilize population - level outcomes measures, they should: a. explicitly assess patient complexity and vulnerability b. carefully adjust for case - mix based on relevant patient factors c. carefully adjust for the manner in which responsibility for patient outcomes is shared between physicians, patients, health plans, and other health - care institutions6 . Monitoring should assess: a. patient satisfaction, access, continuity, and coordination of care; effects on vulnerable patients as a particularly important focus b. physician satisfaction and professionalism, administrative burden, effects on the patient - physician relationship c. effects on disparities between physician practices serving vulnerable and non - vulnerable populations d. payer satisfaction and value for health - care expenditures potential ethical problems in the implementation of pay - for - performance major strategies to achieve high quality health care and ethical performance - based physician compensation recommended safeguards to protect vulnerable populations and prevent unintended consequences within current pay - for - performance systems until researchers develop valid and comprehensive quality measures, pay - for - performance systems must prioritize the protection of vulnerable populations and minimize readily anticipated adverse consequences (table 3). Pay - for - performance leaders should institute the following safeguards to achieve these aims: balance current population - level measurements with the best available measures of quality from the patient perspective . The non - patient - centered nature of current pay - for - performance systems could be partially remedied by appropriate measures . For example, the consumer assessment of healthcare providers54 places a strong emphasis on measuring how well health - care providers communicate with patients . A growing body of research55,56 could inform the development of valid measures in the outpatient setting.reduce or stabilize the percentage of physicians salaries at stake . Policy makers should limit bonus amounts to reduce temptations to game the system, especially in arrangements that do not adjust for case mix . Current levels of approximately 5% of physicians salaries seem reasonable in systems that adjust for case mix, while lower levels would be appropriate for those that do not.provide adequate off - setting compensation to physicians serving vulnerable patients . For example, the 2006 massachusetts health - care reform legislation included provisions to base medicaid hospital rate increases on quality improvement, including the reduction of health - care disparities.57 if such provisions are designed meticulously and fairly,58 financial incentives could encourage and reward physicians for serving patients with low levels of expendable income, complex medical conditions, non - adherence to recommended treatments, or limited health literacy.recommendations regarding population - level measures . Pre - determined population - level measures of quality must be instituted carefully because they are inherently non - patient - centered . Because such measures are pervasive in modern pay - for - performance systems, we recommend several strategies to maximize the protection of vulnerable patients: utilize population - level measures that are evidence - based and clearly linked to valued patient outcomes . For example, pneumonia and influenza immunizations have been proven to prevent potentially debilitating illnesses while having minimal adverse effects . Other commonly utilized measures may fail to reach these standards; hemoglobin a1c targets are based on evidence from randomized control trials, but the applicability to individual patients on real - life physician panels is often unclear.35,59population - level measures should assess domains clearly within the influence of the physician or physician group, especially for complex patients . Basic process measures, such as vaccination rates and the frequency of diabetic eye exams, are imperfect measures of quality, but are more within the influence of physicians and practice groups than outcomes measures . Process measures seem less likely than outcomes measures to cause avoidance of vulnerable patients and physician frustration.measures should assess quality at the level of large physician practices rather than individual physicians . Experts skilled in statistical analysis should determine minimum patient population sizes for each measure to provide optimal data and avoid statistical error . Only practice groups with sufficient numbers of patients should initially be measured.measures should assess improvement toward goals in addition to achievement of cut - points . For example, physician groups could be rewarded both for achieving vaccination rates at a pre - determined level as well as for annual improvements toward the target.recommendations regarding population - level outcomes measures . Population - level outcomes measures are methodologically complex, and the validity of current measures is uncertain . This will likely preclude their use in an ethically defensible manner in the short - term unless provisions that maximize validity are closely followed, including: explicitly assess patient complexity and vulnerability . This would require integrating patient survey data and medical record data regarding sociodemographic characteristics and medical comorbidities.carefully adjust for case - mix based on relevant patient factors . For example, it would be inappropriate to reduce systolic blood pressure levels below 140 mmhg in an 85-year - old diabetic patient with multiple co - morbidities taking three antihypertensive medications . Proper case - mix adjustment might allow this patient s physician to prioritize other care, while a lack of adjustment could induce either dangerous efforts to lower blood pressure or substantial physician frustration.carefully adjust for the manner in which responsibility for patient outcomes is shared between physicians, patients, health plans, and other health - care institutions . For example, consider two physicians who must eventually prescribe three hypoglycemic medications to similar diabetic patients whose initial hemoglobin a1c levels were 9.5 . The first patient has generous health insurance, enabling him to purchase all three medications and lower his hemoglobin a1c to 6.5 . The second patient must pay the full cost of medications, and she can only afford two . A proper system would adjust for health insurance status.pay-for-performance leaders should initiate monitoring before and after implementing the above changes . Monitoring should assess important patient outcomes not often included in pay - for performance studies, such as satisfaction, access, continuity, and coordination of care . Studies should also assess physician satisfaction and professionalism, administrative burden, effects on the patient - physician relationship, and the impact on disparities between physician practices serving more vulnerable and less vulnerable populations . Balance current population - level measurements with the best available measures of quality from the patient perspective . The non - patient - centered nature of current pay - for - performance systems could be partially remedied by appropriate measures . For example, the consumer assessment of healthcare providers54 places a strong emphasis on measuring how well health - care providers communicate with patients . A growing body of research55,56 could inform the development of valid measures in the outpatient setting . Policy makers should limit bonus amounts to reduce temptations to game the system, especially in arrangements that do not adjust for case mix . Current levels of approximately 5% of physicians salaries seem reasonable in systems that adjust for case mix, while lower levels would be appropriate for those that do not . For example, the 2006 massachusetts health - care reform legislation included provisions to base medicaid hospital rate increases on quality improvement, including the reduction of health - care disparities.57 if such provisions are designed meticulously and fairly,58 financial incentives could encourage and reward physicians for serving patients with low levels of expendable income, complex medical conditions, non - adherence to recommended treatments, or limited health literacy . Pre - determined population - level measures of quality must be instituted carefully because they are inherently non - patient - centered . Because such measures are pervasive in modern pay - for - performance systems, we recommend several strategies to maximize the protection of vulnerable patients: utilize population - level measures that are evidence - based and clearly linked to valued patient outcomes . For example, pneumonia and influenza immunizations have been proven to prevent potentially debilitating illnesses while having minimal adverse effects . Other commonly utilized measures may fail to reach these standards; hemoglobin a1c targets are based on evidence from randomized control trials, but the applicability to individual patients on real - life physician panels is often unclear.35,59population - level measures should assess domains clearly within the influence of the physician or physician group, especially for complex patients . Basic process measures, such as vaccination rates and the frequency of diabetic eye exams, are imperfect measures of quality, but are more within the influence of physicians and practice groups than outcomes measures . Process measures seem less likely than outcomes measures to cause avoidance of vulnerable patients and physician frustration.measures should assess quality at the level of large physician practices rather than individual physicians . Experts skilled in statistical analysis should determine minimum patient population sizes for each measure to provide optimal data and avoid statistical error . Only practice groups with sufficient numbers of patients should initially be measured.measures should assess improvement toward goals in addition to achievement of cut - points . This could apply to both process and outcomes measures . For example, physician groups could be rewarded both for achieving vaccination rates at a pre - determined level as well as for annual improvements toward the target . Utilize population - level measures that are evidence - based and clearly linked to valued patient outcomes . For example, pneumonia and influenza immunizations have been proven to prevent potentially debilitating illnesses while having minimal adverse effects . Other commonly utilized measures may fail to reach these standards; hemoglobin a1c targets are based on evidence from randomized control trials, but the applicability to individual patients on real - life physician panels is often unclear.35,59 population - level measures should assess domains clearly within the influence of the physician or physician group, especially for complex patients . Basic process measures, such as vaccination rates and the frequency of diabetic eye exams, are imperfect measures of quality, but are more within the influence of physicians and practice groups than outcomes measures . Process measures seem less likely than outcomes measures to cause avoidance of vulnerable patients and physician frustration . Measures should assess quality at the level of large physician practices rather than individual physicians . Experts skilled in statistical analysis should determine minimum patient population sizes for each measure to provide optimal data and avoid statistical error . For example, physician groups could be rewarded both for achieving vaccination rates at a pre - determined level as well as for annual improvements toward the target . Population - level outcomes measures are methodologically complex, and the validity of current measures is uncertain . This will likely preclude their use in an ethically defensible manner in the short - term unless provisions that maximize validity are closely followed, including: explicitly assess patient complexity and vulnerability . This would require integrating patient survey data and medical record data regarding sociodemographic characteristics and medical comorbidities.carefully adjust for case - mix based on relevant patient factors . For example, it would be inappropriate to reduce systolic blood pressure levels below 140 mmhg in an 85-year - old diabetic patient with multiple co - morbidities taking three antihypertensive medications . Proper case - mix adjustment might allow this patient s physician to prioritize other care, while a lack of adjustment could induce either dangerous efforts to lower blood pressure or substantial physician frustration.carefully adjust for the manner in which responsibility for patient outcomes is shared between physicians, patients, health plans, and other health - care institutions . For example, consider two physicians who must eventually prescribe three hypoglycemic medications to similar diabetic patients whose initial hemoglobin a1c levels were 9.5 . The first patient has generous health insurance, enabling him to purchase all three medications and lower his hemoglobin a1c to 6.5 . The second patient must pay the full cost of medications, and she can only afford two . This would require integrating patient survey data and medical record data regarding sociodemographic characteristics and medical comorbidities . For example, it would be inappropriate to reduce systolic blood pressure levels below 140 mmhg in an 85-year - old diabetic patient with multiple co - morbidities taking three antihypertensive medications . Proper case - mix adjustment might allow this patient s physician to prioritize other care, while a lack of adjustment could induce either dangerous efforts to lower blood pressure or substantial physician frustration . Carefully adjust for the manner in which responsibility for patient outcomes is shared between physicians, patients, health plans, and other health - care institutions . For example, consider two physicians who must eventually prescribe three hypoglycemic medications to similar diabetic patients whose initial hemoglobin a1c levels were 9.5 . The first patient has generous health insurance, enabling him to purchase all three medications and lower his hemoglobin a1c to 6.5 . The second patient must pay the full cost of medications, and she can only afford two . Pay - for - performance leaders should initiate monitoring before and after implementing the above changes . Monitoring should assess important patient outcomes not often included in pay - for performance studies, such as satisfaction, access, continuity, and coordination of care . Studies should also assess physician satisfaction and professionalism, administrative burden, effects on the patient - physician relationship, and the impact on disparities between physician practices serving more vulnerable and less vulnerable populations . Monitoring should examine payer satisfaction and value for health - care expenditures . A crucial first step in achieving ethically defensible health - care quality improvement will be for key stakeholders to develop consensus regarding their shared and unique obligations to individual patients and patient populations . For example, to improve blood glucose control among diabetic patients, physicians must recommend evidence - based, patient - centered management strategies, practice groups must provide access to testing facilities, health insurers must facilitate receipt of affordable medications and testing, and patients must adhere to therapeutic plans . Bringing health insurers, patients, employers, and physicians to the table would highlight opportunities to improve coordination and continuity of care; new paradigms for quality improvement that integrate assessment at the individual physician level and institution level could emerge . A long - term strategy for quality improvement will be guided by a framework of accountability in which physicians, practice groups, health plans, and public payers are measured based on how well they fulfill well - defined obligations to individual patients and populations . For example, measures of physician quality should assess multiple domains, such as accessibility, adherence to evidence - based but patient - centered care, and communication skills . Appropriate measures would account for individualized patient - physician goals, be based on the best available evidence, and minimize administrative burden and expense . Measures of health - care institution quality (e.g., physician groups, hospitals, and public and private payers) should assess domains such as how well these groups foster teamwork, facilitate achievement of patient goals, strengthen the doctor - patient relationship, and improve access, coordination, and continuity of care for individual patients . Equally important will be development of valid population - level health - care quality measures . In addition to measuring how well physicians and health - care institutions fulfill obligations to individual patients, comprehensive quality measures would assess the degree to which patients, physicians, and health - care institutions maximize health - care resources available to the population, distribute them fairly,60 and fulfill their obligations justly . For example, all persons involved in creating new measures should, at minimum, be required to state potential conflicts of interest . After developing evidence - based measures of physician, health - care institution, and population - level quality, policy makers should implement carefully planned, small - scale pilot programs that reward physician and health - care institution quality . Benefits and adverse effects should be monitored . Those entities implementing innovations in payment and quality improvement should take the lead in funding these studies . Even with results from well - designed studies, judgments about the ethics of pay - for - performance will remain challenging . We base our suggestion to begin with pilot programs upon an ethical principle of precaution . However, efforts should be scaled up if benefits prove sufficient, health disparities are reduced and adverse outcomes are minimized . Until researchers develop valid and comprehensive quality measures, pay - for - performance systems must prioritize the protection of vulnerable populations and minimize readily anticipated adverse consequences (table 3). Pay - for - performance leaders should institute the following safeguards to achieve these aims: balance current population - level measurements with the best available measures of quality from the patient perspective . The non - patient - centered nature of current pay - for - performance systems could be partially remedied by appropriate measures . For example, the consumer assessment of healthcare providers54 places a strong emphasis on measuring how well health - care providers communicate with patients . A growing body of research55,56 could inform the development of valid measures in the outpatient setting.reduce or stabilize the percentage of physicians salaries at stake . Policy makers should limit bonus amounts to reduce temptations to game the system, especially in arrangements that do not adjust for case mix . Current levels of approximately 5% of physicians salaries seem reasonable in systems that adjust for case mix, while lower levels would be appropriate for those that do not.provide adequate off - setting compensation to physicians serving vulnerable patients . For example, the 2006 massachusetts health - care reform legislation included provisions to base medicaid hospital rate increases on quality improvement, including the reduction of health - care disparities.57 if such provisions are designed meticulously and fairly,58 financial incentives could encourage and reward physicians for serving patients with low levels of expendable income, complex medical conditions, non - adherence to recommended treatments, or limited health literacy.recommendations regarding population - level measures . Pre - determined population - level measures of quality must be instituted carefully because they are inherently non - patient - centered . Because such measures are pervasive in modern pay - for - performance systems, we recommend several strategies to maximize the protection of vulnerable patients: utilize population - level measures that are evidence - based and clearly linked to valued patient outcomes . For example, pneumonia and influenza immunizations have been proven to prevent potentially debilitating illnesses while having minimal adverse effects . Other commonly utilized measures may fail to reach these standards; hemoglobin a1c targets are based on evidence from randomized control trials, but the applicability to individual patients on real - life physician panels is often unclear.35,59population - level measures should assess domains clearly within the influence of the physician or physician group, especially for complex patients . Basic process measures, such as vaccination rates and the frequency of diabetic eye exams, are imperfect measures of quality, but are more within the influence of physicians and practice groups than outcomes measures . Process measures seem less likely than outcomes measures to cause avoidance of vulnerable patients and physician frustration.measures should assess quality at the level of large physician practices rather than individual physicians . Experts skilled in statistical analysis should determine minimum patient population sizes for each measure to provide optimal data and avoid statistical error . Only practice groups with sufficient numbers of patients should initially be measured.measures should assess improvement toward goals in addition to achievement of cut - points . This could apply to both process and outcomes measures . For example, physician groups could be rewarded both for achieving vaccination rates at a pre - determined level as well as for annual improvements toward the target.recommendations regarding population - level outcomes measures . Population - level outcomes measures are methodologically complex, and the validity of current measures is uncertain . This will likely preclude their use in an ethically defensible manner in the short - term unless provisions that maximize validity are closely followed, including: explicitly assess patient complexity and vulnerability . This would require integrating patient survey data and medical record data regarding sociodemographic characteristics and medical comorbidities.carefully adjust for case - mix based on relevant patient factors . For example, it would be inappropriate to reduce systolic blood pressure levels below 140 mmhg in an 85-year - old diabetic patient with multiple co - morbidities taking three antihypertensive medications . Proper case - mix adjustment might allow this patient s physician to prioritize other care, while a lack of adjustment could induce either dangerous efforts to lower blood pressure or substantial physician frustration.carefully adjust for the manner in which responsibility for patient outcomes is shared between physicians, patients, health plans, and other health - care institutions . For example, consider two physicians who must eventually prescribe three hypoglycemic medications to similar diabetic patients whose initial hemoglobin a1c levels were 9.5 . The first patient has generous health insurance, enabling him to purchase all three medications and lower his hemoglobin a1c to 6.5 . The second patient must pay the full cost of medications, and she can only afford two . A proper system would adjust for health insurance status.pay-for-performance leaders should initiate monitoring before and after implementing the above changes . Monitoring should assess important patient outcomes not often included in pay - for performance studies, such as satisfaction, access, continuity, and coordination of care . Studies should also assess physician satisfaction and professionalism, administrative burden, effects on the patient - physician relationship, and the impact on disparities between physician practices serving more vulnerable and less vulnerable populations . Balance current population - level measurements with the best available measures of quality from the patient perspective . The non - patient - centered nature of current pay - for - performance systems could be partially remedied by appropriate measures . For example, the consumer assessment of healthcare providers54 places a strong emphasis on measuring how well health - care providers communicate with patients . A growing body of research55,56 could inform the development of valid measures in the outpatient setting . Policy makers should limit bonus amounts to reduce temptations to game the system, especially in arrangements that do not adjust for case mix . Current levels of approximately 5% of physicians salaries seem reasonable in systems that adjust for case mix, while lower levels would be appropriate for those that do not . For example, the 2006 massachusetts health - care reform legislation included provisions to base medicaid hospital rate increases on quality improvement, including the reduction of health - care disparities.57 if such provisions are designed meticulously and fairly,58 financial incentives could encourage and reward physicians for serving patients with low levels of expendable income, complex medical conditions, non - adherence to recommended treatments, or limited health literacy . Pre - determined population - level measures of quality must be instituted carefully because they are inherently non - patient - centered . Because such measures are pervasive in modern pay - for - performance systems, we recommend several strategies to maximize the protection of vulnerable patients: utilize population - level measures that are evidence - based and clearly linked to valued patient outcomes . For example, pneumonia and influenza immunizations have been proven to prevent potentially debilitating illnesses while having minimal adverse effects . Other commonly utilized measures may fail to reach these standards; hemoglobin a1c targets are based on evidence from randomized control trials, but the applicability to individual patients on real - life physician panels is often unclear.35,59population - level measures should assess domains clearly within the influence of the physician or physician group, especially for complex patients . Basic process measures, such as vaccination rates and the frequency of diabetic eye exams, are imperfect measures of quality, but are more within the influence of physicians and practice groups than outcomes measures . Process measures seem less likely than outcomes measures to cause avoidance of vulnerable patients and physician frustration.measures should assess quality at the level of large physician practices rather than individual physicians . Experts skilled in statistical analysis should determine minimum patient population sizes for each measure to provide optimal data and avoid statistical error . Only practice groups with sufficient numbers of patients should initially be measured.measures should assess improvement toward goals in addition to achievement of cut - points . For example, physician groups could be rewarded both for achieving vaccination rates at a pre - determined level as well as for annual improvements toward the target . Utilize population - level measures that are evidence - based and clearly linked to valued patient outcomes . For example, pneumonia and influenza immunizations have been proven to prevent potentially debilitating illnesses while having minimal adverse effects . Other commonly utilized measures may fail to reach these standards; hemoglobin a1c targets are based on evidence from randomized control trials, but the applicability to individual patients on real - life physician panels is often unclear.35,59 population - level measures should assess domains clearly within the influence of the physician or physician group, especially for complex patients . Basic process measures, such as vaccination rates and the frequency of diabetic eye exams, are imperfect measures of quality, but are more within the influence of physicians and practice groups than outcomes measures . Process measures seem less likely than outcomes measures to cause avoidance of vulnerable patients and physician frustration . Measures should assess quality at the level of large physician practices rather than individual physicians . Experts skilled in statistical analysis should determine minimum patient population sizes for each measure to provide optimal data and avoid statistical error . For example, physician groups could be rewarded both for achieving vaccination rates at a pre - determined level as well as for annual improvements toward the target . Population - level outcomes measures are methodologically complex, and the validity of current measures is uncertain . This will likely preclude their use in an ethically defensible manner in the short - term unless provisions that maximize validity are closely followed, including: explicitly assess patient complexity and vulnerability . This would require integrating patient survey data and medical record data regarding sociodemographic characteristics and medical comorbidities.carefully adjust for case - mix based on relevant patient factors . For example, it would be inappropriate to reduce systolic blood pressure levels below 140 mmhg in an 85-year - old diabetic patient with multiple co - morbidities taking three antihypertensive medications . Proper case - mix adjustment might allow this patient s physician to prioritize other care, while a lack of adjustment could induce either dangerous efforts to lower blood pressure or substantial physician frustration.carefully adjust for the manner in which responsibility for patient outcomes is shared between physicians, patients, health plans, and other health - care institutions . For example, consider two physicians who must eventually prescribe three hypoglycemic medications to similar diabetic patients whose initial hemoglobin a1c levels were 9.5 . The first patient has generous health insurance, enabling him to purchase all three medications and lower his hemoglobin a1c to 6.5 . The second patient must pay the full cost of medications, and she can only afford two . This would require integrating patient survey data and medical record data regarding sociodemographic characteristics and medical comorbidities . For example, it would be inappropriate to reduce systolic blood pressure levels below 140 mmhg in an 85-year - old diabetic patient with multiple co - morbidities taking three antihypertensive medications . Proper case - mix adjustment might allow this patient s physician to prioritize other care, while a lack of adjustment could induce either dangerous efforts to lower blood pressure or substantial physician frustration . Carefully adjust for the manner in which responsibility for patient outcomes is shared between physicians, patients, health plans, and other health - care institutions . For example, consider two physicians who must eventually prescribe three hypoglycemic medications to similar diabetic patients whose initial hemoglobin a1c levels were 9.5 . The first patient has generous health insurance, enabling him to purchase all three medications and lower his hemoglobin a1c to 6.5 . The second patient must pay the full cost of medications, and she can only afford two . Pay - for - performance leaders should initiate monitoring before and after implementing the above changes . Monitoring should assess important patient outcomes not often included in pay - for performance studies, such as satisfaction, access, continuity, and coordination of care . Studies should also assess physician satisfaction and professionalism, administrative burden, effects on the patient - physician relationship, and the impact on disparities between physician practices serving more vulnerable and less vulnerable populations . A crucial first step in achieving ethically defensible health - care quality improvement will be for key stakeholders to develop consensus regarding their shared and unique obligations to individual patients and patient populations . For example, to improve blood glucose control among diabetic patients, physicians must recommend evidence - based, patient - centered management strategies, practice groups must provide access to testing facilities, health insurers must facilitate receipt of affordable medications and testing, and patients must adhere to therapeutic plans . Bringing health insurers, patients, employers, and physicians to the table would highlight opportunities to improve coordination and continuity of care; new paradigms for quality improvement that integrate assessment at the individual physician level and institution level could emerge . A long - term strategy for quality improvement will be guided by a framework of accountability in which physicians, practice groups, health plans, and public payers are measured based on how well they fulfill well - defined obligations to individual patients and populations . For example, measures of physician quality should assess multiple domains, such as accessibility, adherence to evidence - based but patient - centered care, and communication skills . Appropriate measures would account for individualized patient - physician goals, be based on the best available evidence, and minimize administrative burden and expense . Measures of health - care institution quality (e.g., physician groups, hospitals, and public and private payers) should assess domains such as how well these groups foster teamwork, facilitate achievement of patient goals, strengthen the doctor - patient relationship, and improve access, coordination, and continuity of care for individual patients . Equally important will be development of valid population - level health - care quality measures . In addition to measuring how well physicians and health - care institutions fulfill obligations to individual patients, comprehensive quality measures would assess the degree to which patients, physicians, and health - care institutions maximize health - care resources available to the population, distribute them fairly,60 and fulfill their obligations justly . For example, all persons involved in creating new measures should, at minimum, be required to state potential conflicts of interest . After developing evidence - based measures of physician, health - care institution, and population - level quality, policy makers should implement carefully planned, small - scale pilot programs that reward physician and health - care institution quality . Benefits and adverse effects should be monitored . Those entities implementing innovations in payment and quality improvement should take the lead in funding these studies . Even with results from well - designed studies, judgments about the ethics of pay - for - performance will remain challenging . We base our suggestion to begin with pilot programs upon an ethical principle of precaution . However, efforts should be scaled up if benefits prove sufficient, health disparities are reduced and adverse outcomes are minimized . In order to aid in the above processes, sgim is committed to having general internists participate in articulating the quality - related obligations that physicians and health - care institutions have to patients and the population . Sgim encourages its members to take the following actions: (1) help develop measures of physician, health - care institution, and population - level health - care quality, (2) evaluate pay - for - performance measures and programs, and (3) participate in the ongoing monitoring of effects of pay - for - performance on vulnerable populations and physicians . Sgim will continue to develop collaborative alliances with other key national organizations to ensure fair, valid, and comprehensive measures and to promote ethical compensation reform . Performance - based physician compensation, if carefully guided by a comprehensive understanding of health - care quality and evidence - based evaluations, might improve patient care, narrow health disparities, and promote fair physician compensation while increasing health - care value . If research and monitoring determine that improved payment systems can benefit patients, physicians, and payers while minimizing risks, they could be ethical arrangements . However, until such data are available, widespread expansion of untested pay - for - performance systems poses substantive ethical issues associated with potential harm to patients, clinicians, and organizations.
In recent years, various strategies have been adopted for specific drug delivery to well - defined sites of the gastrointestinal (gi) tract, the colon being the most important one [15]. Enteric polymers are used for this purpose, as they are able to release the drug at a particular ph . The ph - sensitive copolymers, such as methacrylic acid / methyl methacrylate copolymers and eudragit types l and s, dissolve in aqueous media at ph 6 and 7, respectively, which may be equivalent to drug release in the distal ileum . Similarly, chitosan - based polyelectrolyte complexes have been employed as potential carrier materials in drug delivery systems . Furthermore, a growing interest in polyelectrolyte complexes has led to the formulation and characterization of systems involving a variety of anionic and cationic polymers: eudragit l 30 d-55 and gelatin, eudragit l 100-eudragit s 100, eudragit e - eudragit l [10, 11], eudragit e - sodium alginate, chitosan - alginate / chitosan - carrageenan (mainly kappa - carrageenan with low amounts of lambda - carrageenan), chitosan - polygalacturonic acid, chitosan - carboxymethylcellulose, and chitosan - alginate . Conventional drug delivery is unfavourable to special cases where drug targeting is applied, that is, when avoidance of gastric dissolution or targeting to the colon is desirable . Colon - targeted drug delivery differs from ordinary enteric coatings (that are designed to merely avoid drug release in the stomach) in that the tablet or capsule is specially formulated to channel greater quantity of drug release to the colonic compartment, thus preventing or highly reducing drug release until the dosage form reaches the colon . Although the large intestine is difficult to access through peroral delivery, it is still favoured as the appropriate site to tackle local colon - related diseases . Colon - targeted delivery could be achieved by the use of ph - dependent systems, time - dependent systems, colonic microflora - activated systems and use of prodrugs . Anti - inflammatory, antibacterial, antiamebic, protein drugs, are a few out of other drugs that can be targeted for site - specific delivery to the colon . Ibuprofen is a nonsteroidal anti - inflammatory agent belonging to the group of propionic acid derivatives; it presents a plasmatic half - life of 1.82.0 h; as a result, it has to be administered three to six times a day, making this drug a suitable candidate for a controlled release formulation . The swellability properties of ipecs prepared from chitosan (cs) and eudragit l 100 - 55 (l 100 - 55) have been evaluated for their possible pharmaceutical application as new carrier for oral colon - specific drug delivery systems (ddss). Similarly, a comparative study of ipecs of chitosan with eudragit l 100 and eudragit l 100 - 55 as potential carriers for controlled oral delivery of diclofenac sodium has been undertaken . However, to the best of our knowledge, there is no scientifically reported study on chitosan - eudragit rl-100 (cs - el) polyelectrolyte complexes of ibuprofen . Thus, this study was designed to investigate the formation of ipec between cs and el, to characterize the product formed, and to evaluate its performance as a matrix for controlled release of drugs, using ibuprofen (ibf) as a model . Ibuprofen (basf, germany), acetic acid, acetone, ammonium acetate, maize starch, magnesium stearate, lactose, concentrated hydrochloric acid (bdh, england), sodium hydroxide (merck, germany), and monobasic potassium phosphate (sigma chemical co., usa) were used as purchased from the manufacturers without further purification . Chitosan of low viscosity nd was fines were retaine (fluka, switzerland) and eudragit rl 100 (mw 135,000) (rohm pharma, germany) were preliminarily dried at 40c under vacuum for two days . The ipec of cs and el was prepared following the standard procedures with slight modifications [12, 2327]. Chitosan 300 mg was accurately weighed and dissolved in 15 ml of 3% v / v acetic acid followed by the addition of 8 ml volume of 5 m ammonium acetate . Similarly, eudragit rl 100 (300 mg) was separately dissolved in 7 ml ethanol and was covered to prevent evaporation . The mixture was poured in a petri plate and was dried at 50c for 48 h. films with a total polymer content of 2.5% w / v containing 60: 40, 50: 50, and 40: 60 (i.e., 3: 2, 1: 1, and 2: 3) ratios of chitosan: eudragit rl 100 were prepared using this method . A control batch (el) containing only eudragit rl 100 was also prepared . Ibf granules (average weight 297.3 mg) containing 200 mg of ibf were prepared by wet granulation technique using cs: el interpolymer complexes (50: 50 w / w) as binder . 10 (1.7 mm mesh) and was dried at 50c for about 1 h until all the moisture was removed . 16 (1.0 mm mesh) and was stored in a desiccator until used . Magnesium stearate (1% w / w) (lubricant) and lactose (bulking agent) were added to the granules . Tablets were compressed using 4 mm biconvex punches in a single station tablet compression machine (manesty, england) at a pressure of 50 kg / cm . The formulated ibf tablets containing cs: el (50: 50 w / w) as binder were coated with aqueous solutions containing (50: 50, 60: 40, and 40: 60 w / w) of cs: el ratio as ipecs films . The coating solution was sprayed at a rate of 5 ml / min with the help of peristaltic pump using a spray gun of 1 mm nozzle in a coating pan (12 diameter) being rotated at 18 rpm . The inner surface of coating pan was modified by attaching inert tubes (8 mm diameter) from the centre to the periphery for easy rolling of tablets, thereby ensuring efficient mass transfer of polymer . A control batch coated with eudragit rl 100 was also prepared . The degree of swelling of films of the ipec was investigated simulating the physiological conditions of the gastrointestinal tract [2327]. For this purpose, the films were placed in a preweighted basket of the dissolution equipment and immersed for 2 h in 30 ml of 0.1 m hydrochloric acid, then 10 ml of 0.20 m tribasic sodium phosphate was added to ph of 6.8 0.05, and after additional 3 h, another 10 ml of phosphate buffer ph 7.4 was added and the experiment was allowed to continue for another 19 h, giving a total of 24 h. the temperature of the medium was 37 0.5c . The measurements consisted in removing the basket from the medium, blot - drying by filter paper, and weighing in an analytical balance (mettler al 204, mettler - toledo int . Inc . The degree of swelling was calculated using the formula (1)h(%)=(m2m1)m1100, where m1 is the initial weight of the film (g) and m2 is the final weight of the swollen film (g). Twenty tablets from each batch were weighed together and individually, and the mean weight and percentage deviation were calculated according to british pharmacopoeia . The tablets were set to rotate at 25 rpm for 10 min in an erweka friabilator . The friability was calculated according to the formula (2)friability=(w1w2)w1, where w1 is the initial weight and w2 is the final weight . The force required to break each tablet was determined using a monsanto - stokes tablet tester . The average force of the ten tablets was taken as the crushing strength (kgf). Three tablets were randomly selected from each batch and were placed in the inner compartment of a disintegration apparatus (which was tied with a thermoresistant thread to the clamp of a retort stand) of the disintegrating apparatus containing 500 ml of distilled water maintained at 37 1c . The medium was stirred at 150 rpm and the time taken for the tablets to disintegrate was recorded . The test was performed in triplicate for each batch, and the average time for each batch was calculated . In vitro release of ibuprofen from the tablets was performed using usp (dissolution apparatus 1-basket method) at 37 0.5c and 100 rpm in three release media (ph 1.2, 6.8, and 7.4). Each tablet was placed in the cylindrical basket of a dissolution apparatus (veego, india) attached to the rotating spindle suspended in the dissolution medium of volume of 900 ml (ph 1.2). The rectangular glass container into which the one - litre cylindrical plastic container was immersed was filled with sufficient water to get more than half of the cylindrical container immersed in the water . The heating element in it the equipment was switched on to rotate at a speed of 100 rpm . At predetermined time intervals, 5 ml samples of the dissolution medium were withdrawn and were assayed spectrophotometrically (uv / vis, unico, usa) after appropriate dilution and filtration . Meanwhile, two hours were chosen to mimic the average gastric emptying time . At the end of the 2 h, the equipment was switched off the rotating spindle attached to the basket - bearing tablet was unscrewed out and properly rinsed of the previous medium after carefully removing the tablet . The cylindrical plastic material containing the dissolution medium was also disposed of the ph 1.2 medium and adequately rinsed with purified water . Then, 900 ml of a second dissolution medium, ph 6.8, was emptied into the 900 ml plastic container and the temperature allowed to attain 37 0.5c . The spindle was screwed back in place and dissolution run as before but for 3 h. the average time for change of dissolution medium was about 20 min . Three hours was chosen because the reported average intestinal transit time is 3 - 4 h . At the end of 3 h, the medium was again removed and replaced with a third medium of ph 7.4 to mimic the ileocecal ph and the same process was repeated but this time until the tablet released all or nearly all the drug . Previous studies indicate that polymers did not interfere with the determination of the model drug, ibf [2327]. The withdrawn samples were immediately analyzed using a spectrophotometer at 221 nm, 272 nm, and 281 nm for the release study in the ph 1.2, 6.8, and 7.4 medium, respectively . Administration of nsaids such as ibuprofen is usually associated with gastrointestinal disturbances [12, 2327]. Thus, research efforts have been directed to solve, or at least improve, this inconveniences, through various techniques of protection of the gastric mucosa or alternatively of preventing the release of nsaids in the gastric region . The site - specific delivery of drugs to the colon can be highly advantageous for various applications including the local treatment of inflammatory bowel diseases (ibds). In this study, various ipecs, formed between el and cs, were obtained and evaluated as potential colon - targeted oral controlled release matrices for ibf, a model nsaid . The ipecs films were formulated by nonstoichiometric method, and tablets containing ibf and ipecs were prepared by wet granulation technique . The formulations were evaluated in terms of friability, hardness, disintegration, swellability, and drug dissolution . Here, the liquid ethanol was employed for dissolving the el so as to enable its proper incorporation into the cs to form the ipecs . Lactose was selected as the bulking agent, maize starch as the disintegrant, and magnesium stearate as the lubricant . The mean weight of the various batches of the tablets (table 1) ranged from 296.32 0.30 mg to 301.57 0.93 mg . This shows that all the batches met compendial requirement for weight variation [28, 29], implying that these tablets were uniform in weight . Table 1 equally indicates that average times of 55.97 2.84, 70.25 1.63 and 60.81 3.87 min each was required for tablets containing respectively 1: 1, 2: 3 and 3: 2 ratios of cs and el, and 35.79 2.45 min for el only - based tablets to disintegrate at the experimental conditions . This (disintegration time) test was performed to determine the ease with which ibf is released from the tablets at a controlled temperature of 37 1c . The results indicate that the adhesive force existing between the components of the tablets of batch 2: 3 is more than that in the tablets of batch 3: 2 and lowest in the tablets of batch 1: 1 . The reason for this is uncertain, but may be attributed to greater concentration of the el on the tablets of batch 2: 3 than tablets of batch 3: 2 . This implies that el exerted significant effect on the force of adhesion of the tablet ingredients, thereby increasing the disintegration time . The data equally revealed that the tablets of batches 2: 3 and 3: 2 demonstrated greater sustained release effect than tablets of batch 1: 1 . It is also likely that high concentration of el and cs in the tablets of batch 2: 3 and 3: 2, respectively, was responsible for this . More so, the low disintegration time of the tablets of batch 3: 2 suggests that these tablets have prospects of dose dumping . Furthermore, the friability test was carried out to determine the ability of the tablets to withstand mechanical shock or abrasion . Low values of friability indicate high resistance to abrasion and good binding / adhesion properties [28, 29]. The friability test result revealed that all the batches met compendial requirement for resistance to abrasion, with tablets of batch cs: el (2: 3) and batch el having the greatest (0.95 0.01%) and least (0.72 0.03%) resistance to abrasion, respectively . In addition, the crushing strength test was undertaken to determine the level of resilience of the tablets to crushing when a force is applied . The crushing strength results show that tablets of batch cs: el (2: 3) possessed the highest mean crushing strength of 4.71 0.32 kgf followed by tablets of batch cs: el (1: 1), which is 4.62 0.09 kgf . The lowest crushing strength of 4.15 0.27 kgf was observed in tablets of batch el . The implication is that tablets of batch cs: el (2: 3) have higher adhesive force than tablets of batch cs: el (1: 1), and this force holds the components of these tablets together such that they are not easily broken . Tablets of batch el have the least force of adhesion, and thus these tablets are easily broken . For compressed tablets, a crushing strength 5 kgf is considered the upper limit of acceptance and since none of the batches of the tablets exceeded this value; then they are acceptable . It is well known that the potential of polymeric carriers to be used as controlled release materials can be predicted by determination of their swelling characteristics . In a previous study, a group of researchers evaluated the swelling behavior of polycomplex matrices made from cs and el 100 in simulated gastro - intestinal tract (git) and all systems used were stable in ph 1.2 (1 h) and ph 6.8 (2 h). According to the specifications of degussa, the dissolution of el depends on the copolymer structure and is well regulated by the ratio between methyl methacrylate or ethyl acrylate and methacrylic acid . Figure 1 shows degree of swelling at equilibrium and time of swelling for the different ipec films . In figure 1, h1.2 and t1.2 represent the degree of swelling at equilibrium at ph 1.2 and the time of swelling, respectively . Similarly h6.8 and t6.8 also represent the degree of swelling at equilibrium at ph 6.8 and the time of swelling, respectively . The swelling profiles are similar: increasing degree of swelling in acidic medium due to a progressively increasing number of ionized nh3 groups of cs and decreased swellability for systems containing el, probably due to leaching of undissolved particles of el . The swelling behavior of ipecs films is completely different from that of the el only - based films (figure 1). In these systems, the electrostatic repulsion of free ionized amino groups is responsible for swelling . In case of ipec made up of cs: el 2: 3, the degree of swelling was 150% at ph 1.2, but afterwards a two - fold increase in swelling at ph 6.8 could be observed . On immersing the polycomplex matrix into the acidic medium (ph 6.8), free amino groups got protonated and their hydration increased the degree of swelling within the first part of the experiment . Later, full ionization of all amino groups turned it into a polyelectrolyte with a relatively high charge density . As a result, the structure of the ipec is changed because the ionic bonds are not fixed and they could move from one electrostatic site to another [30, 31]. The protonated carboxylic acid groups of el (weak polyacid) became charged by ionized amino groups of cs to form new interpolymer contacts . Comparable observations were made with ipec prepared from two types of eudragit [911] and eudragit e 100 and alginate sodium . After transferring the matrix to the second medium of ph 6.8, carboxylic groups of el became more ionized giving rise to an increase in the degree of swelling . However, previously protonated amino groups began to lose their charge and may be responsible for the increase in the hydrophobic units in the ipec structure . As a result, the swelling slightly decreased at the end of the second medium (ph 6.8) but began to increase in the third buffer (ph 7.4) due to a progressive increase in the number of carboxylate group, in spite of the solubility of cs which decreased at higher ph values . The formulated ipecs, as many of the investigated stoichiometric polycomplexes, would have a more or less homogenous network structure in the swollen state, which could be changed during swelling . Completely different changes were observed in the cs: el (3: 2) system . The polycomplexes showed the highest degree of swelling; increasing the cs content led to an increase in the swellability of the ipecs . This system is stable in the first acidic medium, but with a relatively low degree of complexation, and completely destructive to individual polymers afterwards . This system is very sensitive to ph and is not stable in simulated intestinal tract (sit) conditions . The reason is that polycomplexes with participation of el (consisting of more hydrophobic methacrylate chains) are simply destroyed in neutral media . Similar results of high ph sensitivity were observed in polycomplex systems made up of cs - pectin and cs - dextran sulfate . In order to assess the potential of the ipecs to be used in matrix controlled drug delivery systems, we evaluated the release of the model drug (ibf) from all investigated matrix systems . Based on the results of the previous studies from dissolution behavior of ibf, as a model drug, from the polycomplex matrix systems based on cs and el in gastrointestinal simulated conditions, we decided to use three release media (ph 1.2, 6.8, and 7.4) in the present study . D1.2 and t1.2 represent the cumulative amount of drug released at ph 1.2 and the time of release respectively . Similarly, d6.8 and t6.8 also represent the cumulative amount of drug released at ph 6.8 and the time of release, respectively . As expected, very low ibf release occurred in a ph - gradient (from 6.8 to 7.4) medium showing that, below solubility of the enteric copolymers, no drug release occurred (el). As shown in figure 2, polycomplex matrices made up of cs and el showed a release behavior that is somehow slower than that of the tablets coated with only el . The reason is that due to high swelling properties at all ph values, these polycomplexes form gel - like matrices, which can sustain ibf release . In case of cs: el (3: 2) polycomplex, the release of ibf was slowest, with the most constant drug release rate as well as swelling properties when compared to all the other systems . This means that an excess of cs in the ipec structure led to formation of a well - equilibrated polycomplex (with a high degree of complexation) which is not so ph sensitive and stable in sit conditions . It is evident that general retardation and low amount of drug release took place at ph 1.2 and 6.8, respectively; that is, all the ipecs batches released negligible quantity of drug in the first two dissolution media when compared to ph 7.4 . It has been reported that a successful colon - targeted delivery system should be able to retard or withhold drug release in the upper part of the gastrointestinal region but release the drug promptly on entry into the colon, since the ph gradient ranges from 1.2 in the stomach through 6.6 in the proximal small intestine to a peak of up to 7.5 in the distal small intestine . The control batch (el) coated with only el recorded the lowest cumulative drug release at both ph 1.2 and ph 6.8 . However, a closer look showed that the cumulative percent drug release (d7.4) at ph 7.4 for the three (ipec) batches were between 80 and 95% . This means that it has the potential of making sufficient quantity of drug available in the colon, but then how long (t7.4) it would take the drug to be released in the colon is more important . Although it is good for a colon - specific delivery system to withhold drug release at both ph 1.2 and 6.8 for some reasonable hours, but being able to promptly release drug at the ascending colon, it is much better if release spreads throughout the colon . The longer the time (t7.4) the higher the probability that release would continue all through the colonic transit period . The ipecs presented the possibility of having a greater contact time in the colon, greater duration of action, and larger area of action . It has been reported that gastrointestinal (gi) absorption of orally administered drugs is determined by not only the permeability of gi mucosa but also the transit rate in the gi tract . This envisaged that improved drug release by the ipecs may likely cause the ibf to impinge on infected cells as in colitis and colorectal cancer, consistent with an earlier report . Overall, the release profiles of the tablets based on the ipecs are characterized by a constant and slow release behavior (sustained - release systems). More so, the release profiles are in agreement with the results obtained in the swelling studies . It is pertinent to draw attention to some advantages of our coated tablets, which have sustained release property may have in common with multiparticulate dosage forms . Actually some reporters have favoured multiparticulate dosage forms as presenting better advantages over single dosage forms . This is because the use of single unit dosage forms for colon - targeted delivery has been found to be fraught with some shortcomings such as premature disintegration due to production flaws or sudden change in git physiology, which could lead to reduced bioavailability or therapeutic efficacy . On the other hand, some advantages of multiparticulate dosage forms for colon targeting include reduced risk of systemic toxicity, increased bioavailability, low propensity to cause local irritation, and predictable gastric emptying . Our dosage form design is composed of multiparticulates within a unit dosage form from where gradual release took place . This may likely enable the coated tablets to enjoy many if not all the advantages of multiparticulates enumerated previously . In order to understand the mechanism and kinetics of drug release, the results of the in vitro drug release study were fitted into various kinetic equations like zero order (cumulative percent drug released versus time), first order (log cumulative percent drug retained versus time), higuchi (cumulative percent released versus t), and peppas (log of cumulative percent drug released versus log time) as depicted in table 2 . The kinetic model that best fits the dissolution data was evaluated by comparing the coefficient of determination (r) values obtained in various models . In the peppas (fickian diffusion) model, mechanisms of drug release are characterized using the release exponent (n value) n value of 1 corresponds to zero - order release kinetics (case ii transport); 0.5 <n <1 means an anomalous (non - fickian) diffusion release model; n = 0.5 indicates fickian diffusion, and n> 1 indicates a super case ii transport relaxational release . Results of the kinetic analysis of drug release (table 2) indicates that the most predominant release mechanism was zero order . N values of between 1.00 and 1.16 which implies super case ii release kinetics (a strong indication of zero order). Zero - order release is the ideal in controlled drug release and has been reported not to be common with matrix systems, this being attributed to time - dependant changes in drug depleted matrix surface area and diffusional path length . Therefore, to achieve linear or zero - order release with matrix systems, several manipulative strategies would be inevitably required to impart geometric and structural adjustments on the tablets [4143]. Zero - order release has a lot of advantages including ability to deliver drug at a constant rate, thus providing a predictable bioavailability status . The differences between the different ipecs that were observed during the swelling experiments as well as during the drug release studies show that drug release could be tuned based on the composition of the ipec, with cs: el (3: 2) ipecs as the best formulation . This study has shown that ipecs based on cs and el could be exploited successfully for colon - targeted delivery of ibf in the treatment of ibds.
Ethical discussions between caregivers affect the quality of the older person's care, and gren bolmsj et al . Associate the discussion of ethical values with a deeper level of communication, and in order to achieve depth in such a dialogue, an ethical code and a set of ethical values which penetrate caring are needed . Awareness of such ethical values equips caregivers with a freedom and strength to make conscious decisions to do well and to do right in a given care situation . A caregiver's ability to do well and do right is strengthened in the dialogue between caregivers and other health care professionals . In this study, we use bayesian belief networks [4, 5] (bbns) to analyse ethical values (ethos) and ethical manners in daily work with older people . The advantage with bbns is the possibility to use and compute with symbolic (symbolic data has no per se measurable or comparable values), as opposed to numeric or nominal (1,2,3,4,5 are nominal not to be seen as numerical), data . Data used in this study are nominal in the answers to questions in the questionnaire, but inherently symbolic when arriving at ethical data and classifications of ethical manner . Further, bbns are able to manage stochasticity and uncertainty and can work simultaneously with objective and subjective probabilities in one and the same model . Material is based on questionnaire data collected by the instrument for the self - assessment of individual ethos in the care of older people (isaec) in spring 2007 in a municipality in western finland . The study is based on a caring science perspective, and caregivers' ethical values and ethical manner which are evaluated in the study have been interpreted to the theory of caritative caring ethics and to previous research on ethics in the care of older people [813]. The caring science perspective appears in the statements of the questionnaire, and in the concept, which are given the clusters and nodes, generated with bbn . Biostatistics or, generally speaking, statistics as used in the care domain is indeed strictly statistics . Statistic inference is not logic inference but ad hoc conclusions derived from statistical observations and analysis . Such conclusions are not expressed in any logical language but still within the statistical machinery . However, health and social care involving observations, assessments, and decision - making mean that somewhere along the line statistics moves over to logics . It is syntactic reasoning as related to its semantic counterpart, namely, logical satisfaction . We will illuminate our epistemology with syntactic entailments, where the choice of a specific logic, first - order or otherwise, is not relevant as we are providing a complete ethical ontology in this paper . In the theory of caritative caring ethics and in the previous research on ethics in caring, related to the care of older people can we see, among other, values as dignity, [10, 1517] integrity, [11, 16, 1820] autonomy and participation, [10, 16, 17, 21], respect and safety [13, 20, 22, 23]. We can also find different explanations about caregivers' possibilities to act in an ethical manner in the daily work with the older persons . It is not self - evident that ethical values of the caregivers turn into ethical manners in the daily work, and we have to state that a good intention goes wrong, and the caregiver encounters different ethical problems and challenges that need to be resolved . Often there is not one solution to the problem, rather, many different solutions . The essence of caring is to alleviate the patient's suffering and promote health and wellbeing . Eriksson described caring ethics in terms like love and mercy, caring relationship, human dignity and respect, which accordingly affects human beings' decisions and choices in a specific manner [7, 2426]. A professional caring relationship implies a responsibility of caregiver vis - - vis the patient he / she takes care of . An ethically aware caregiver strives to invite the patient into a caring relation that mediates strength as well as respect for the integrity and wholeness of the human being . Do right, and take responsibility, and he / she wanted to show the patient respect . To act ethically in an ontological sense acting ethically exists in the moment when goodness becomes a conscious choice for the caregiver . To act ethically in the daily work requires a professional freedom, enabling caregiver to choose and decide just in the moment when caregiver and patient meet each other . This kind of freedom goes behind routines and stereotypical behaviours and thereby promotes unique meetings . The aim of this study is to establish structured clusters and well - defined ontological entities (nodes) describing ethical values as both ideal and opportunity for ethical manner as perceived by the caregiver . This additionally provides an enlargement and enrichment of the underlying ethical assumptions about ethical values and the dynamics in spectra of caregiver ethical manner . An additional objective is to evaluate the effect of fixing nodes to certain assessments levels, in order to see how other nodes are affected in themselves and from the viewpoint of the entire cluster . This in turn contributes to knowledge elicitation and epistemological enhancement with respect to the ontological framework . This paper focuses on the following questions: (ontological question) which are the main patterns involving ethical value and ethical manner emerging from this study, given the underlying structural entities and dynamical ethical values and manners? (complementary epistemological question) which are the various types of conditional changes of ethical values and their related ethical manners that appear when fixing nodes to particular values and thereby clusters to specific characters? (societal impact) how will this elicited knowledge in the end affect daily care of older people and as viewed from an ethical perspective? (ontological question) which are the main patterns involving ethical value and ethical manner emerging from this study, given the underlying structural entities and dynamical ethical values and manners? (complementary epistemological question) which are the various types of conditional changes of ethical values and their related ethical manners that appear when fixing nodes to particular values and thereby clusters to specific characters? (societal impact) how will this elicited knowledge in the end affect daily care of older people and as viewed from an ethical perspective? Caregivers from 10 units in the care of older persons were invited to participate in the study . Three units represented home care, four units nursing home care, and three units long - term care . A majority (n = 80) of the informants worked within nursing home care or within long - term care, whereas the remaining informants (n = 25) worked within home care . A majority of the informants had a vocational degree, for instance, registered nurses and practical nurses . Totally, 24 caregivers had attended shorter courses according to older educational programs, such as courses for care assistants, and six of the informants lacked formal competence for their work . Data were collected with an instrument called the instrument for self - assessment of individual ethos in care of older persons and redact isaec . Twenty - eight statements refer to ethical values as ideals, and 30 statements refer to the possibilities to act in an ethical manner, in the daily work with the older person . Statements in isaec instruments were allocated into five groups, as follows: group i = individual care, group ii = dignified care, group iii = safety care, group iv = caring communion, and group v = closeness or / and distance . The participants were asked to answer the questionnaires by choosing the alternative which best responded to their opinion . The alternatives were stated as follows: not at all agree = 1, partly agree = 2, sometimes = 3, nearly agree = 4, and totally agree = 5 (ethical values as ideals) and never = 1, nearly never = 2, sometimes = 3, mostly = 4, and always = 5 (ethical manners). These alternatives were textually presented so that the attached numbering was only intended as an index for that particular alternative and not a gradation . In other words, the instrument aims at presenting the alternatives as symbols and not as numerical values . However, as numbers 15 were visible in the instrument, it can be expected that the set of alternatives was seen as an ordinal scale, so that, for example, sometimes is before mostly, this is indeed the main reason why we cannot compute with 15 as numbers, but rather as symbols in an ordinal scale, and this is why computing with conditional probabilities in bayesian networks is very suitable . Learning from data by hugin creates a network of nodes connected according to respective conditionalities between nodes . For a higher level of information the structure learning algorithms in hugin are based on making dependence tests that calculate a test statistic which is asymptotically chi - squared distributed assuming (conditional) independence . If the test statistic is large for a given independence hypothesis, the hypothesis is rejected; otherwise, it is accepted . The probability of rejecting a true independence hypothesis is given by the level of significance, which was selected to be 0.05 . Several methods, including numerical, logical, and probabilistic ones, have been proposed to manage uncertainty in decision - support systems . The probabilistic approach with bayesian networks are appealing as they capture a computational view of conditional probabilities, which is particularly useful in presence of questionnaires with interdependent questions and using symbolic or ordinal values . Let p(a, b) be the joint probability for a and b. then, the conditional probability is defined as p(a \| b) = p(a, b)/p(b). This then gives the expression p(a, b) = p(a \| b)p(b) for joint probabilities with dependent variables . The event a is said to be conditionally independent of event b if p(a \| b) = p(a), that is, whenever p(a, b) = p(a)p(b). The previous formulas are used to arrive at bayes' rule p(a \| b) = p(b \| a)p(a)/p(b) which is the most important rule used and manipulated in bayesian networks . Bayes' rule makes it possible to calculate conditional probabilities p(a \| b), once the opposite conditional probability p(b \| a) is known together with the probabilities for the individual events a and b. the bayesian network notation for the probability situation p(a, b) = p(a \| b)p(b) is depicted as indicating that b is conditional to a. similarly, p(a, b) = p(b \| a)p(a) is depicted as indicating a conditional to b. given bayes' rule, it is then clear that the direction of the arrow is interchangeable depending on the conditional context . For several nodes, we then need to consider all pairs of conditional probabilities, that is, for the joint probability (1)p(a, b, c, d)=p(a)p(b a)p(c a, b)p(d a, b, c). We have the depiction in the most simple cases, events are two valued, and we may, for example, write either a = 0 or a = 1 . A probability like p(c = 1) is then computed as (2)p(c=1)=a{0,1}b{0,1}d{0,1}p(a = a, b = b, c=1,d = d), and a conditional probability like p(a = 1 \| c = 1) is computed as (3)p(a=1 c=1)=p(a=1,c=1)p(c=1)=b{0,1}d{0,1}p(a=1,b = b, c=1,d = d)p(c=1). In the aforementioned we depict the use of binary data only . Clearly, we can work with more than just two classes of events . In this study, we work mainly with the alternative set {0, 1, 2, 3, 4, 5}, and conditionalities like p(a {3,4, 5} \| b = 4) can be computed for tables in the results section . The learning algorithm, as implemented in hugin, finds the appropriate and correct conditionalities given data . This then creates the bayesian belief network (bbn), where the interconnection defines the structure of the network . This structure and network identification capability is one of the significant advantages of bbn developments . A cluster is a subset of entities, so that, on the one hand, no entity in this subset is conditionally dependent with any other entity outside that cluster, and, on the other hand, there are no further subclusters within that cluster . We may also speak of independent clusters, to further underline that such clusters with no conditional dependency to their outside world are molecular . Results are polarized, on the one hand, by ethical values in the daily care with older people, and, on the other hand, by possibilities to act in an ethical manner in the daily care with older people . However, ethical value and possibility are not complementary or mutually exclusive but rather appear as valuation domains for enabling projection and transformation of given value criteria . From ethical point of view, good care refers in particular to dignity, participation, safety, caring community, and closeness and distance, where the latter is concerned mostly with aspects of possibility and the others project to both ethical value and possibilities . This enables, for example, the concept of dignity to be seen as fundamentally ethical at the same time as consideration of dignity becomes a possibility . Similar multimodality with respect to ethical value and possibility can be said about the other concepts, respectively, participation and safety and caring community . Concepts for ethical value and possibility are further characterized by underlying nodes or entities in clusters related to these concepts, thereby also the entities being members of specific clusters, and conditionalities between these entities . Among all 58 statements, it turned out that ten statements became one - node clusters; that is, these statements did not show any conditionality with any other statements . These statements were left out of further analysis, since no matter how the dynamics within such a statement is further analysed, it has no effect on any other statement . Indeed, a main objective of this paper is to analyse situations within clusters, where fixation of particular ordinal values for one statement may affect dynamics concerning sibling statements in that cluster . Note that we may speak of sibling nodes, even if it is seldom clear where parent nodes reside in clusters . Directed, and changing directions of conditionalities is done by bayes' rules . Interesting among these clusters were that clashing and enforcing them into one node and restructuring all the remaining nodes provided such two - node clusters to became singleton clusters, in all cases expect for one case where that singleton node become integrated into a larger cluster . The number of remaining clusters is 9, and the number of nodes in those respective clusters varies from 3 to 7 . Among these 9 clusters, the interesting next step is now the semantics of these 9 clusters and their nodes . What is the name of the cluster, and what is the interpretation of that name? A major part of this results section is to analyse what happens when certain nodes in clusters are frozen to particular ordinal values . The bayesian network then recomputes the distribution functions for the other nodes, and the recomputation is enabled by the conditional probabilities . This enables a number of interesting what - if analyses, like if e2, e4, and c2 are fixed at 4, that is, the distribution functions for all these nodes become 100% at ordinal value 4, how does the move or shift of the function is dynamic for the other nodes? About 8594 percent of the informants totally agree with all seven statements . In table 1 nodes where level 4 responses in percentage exceed 10 are selected for fixation to a response rate of 100% . These respective fixations are then compared with the shift in response rates for the other nodes, as well as the way they shift in response rates according to the underlying conditional probabilities model of the network . In order to see this more precisely, let node e2, to see the needs of the older person, be fixed at response level 4 the influence on node e4, caring community, is a 17% shift of level 5 responses to level 4 responses, and on node c2, encourage the older persons to utilize their own resources, the effect is an increase of level 4 responses by 18% and level 3 responses by 16% . = 100%, node f4, human love and mercy, decreases at level 5 by 10% to level 3, node c2, encourage the older persons to utilize their own resources, decreases at level 5 by 10% and at level 4 by 8% with an increase at level 3 by 12% and increase on missing data, that is, cannot say data, by 6%, and for a2, to respect the equal value of each older person, there is decrease of 13% from level 5, and as this node had a rather low occurrence of missing data, it is notable how the portion of missing data increases as much as 13% (see table 2). Concerning node c2, encourage the older persons to utilize their own resources, changes due to conditionalities for node f4 and e4 are seen in table 3 . The following clusters are named community (cluster ii), safety (cluster iii), and integrity (cluster iv). In table 6, we find the clusters and the named nodes for each cluster . We did not find any dynamical shift in the distribution functions after conditionalities for the nodes had been fixed in level 4 to 100% . Based on these results, we can state that caregivers, participating in the study, agree that dignity, community, safety, and integrity are important ethical values, in order to guarantee an ethically defensible care, in care with older people . The last cluster of ethical ideal was named integrity . The distribution function for this cluster five clusters describe the caregivers' possibilities to act in an ethical manner, within their daily work with the older persons . The clusters are named as follows: possibility for closeness and distance, community, dignity, safety, and participation . The degree of coherence between the statements and the own opinions varies significantly between the informants . A dynamical shift in the distribution functions, when conditionalities for respective node have been changed, can be found within all clusters . It is apparent that the shape of the clusters is affected as the underlying conditions and properties change, for example, with respect to change of caregivers, as new personnel enter the unit, and the condition spectra change at the unit either for particular individuals or by individuals leaving and new patients entering . Further, organizational and administrative aspects and/or changes may also have effect on ethical manner . Cluster v consists of three nodes: sensitivity, professional approach, and a genuine interest in the quality of life of the older person . The nodes are closely intertwined, and dynamics of the conditionality in respective nodes affects each other within the cluster . Fixation in one node implies adjustments of frequencies in the other nodes . Thereby changes like fixations at level 4 will imply downshifts in the other nodes towards levels 4 and 3 . Thereby, ethical manners, as caregivers' professional approach and interest for the older person, will be important entities for upholding closeness and/or distance in the daily work with the older person (table 7). Cluster vi consists of six nodes (the nodes and the distribution function for the cluster are presented in table 8). For upholding dignified care, entities as respecting the philosophy of life, creating a meaningful life, and continuously keeping the older patients informed on the phenomena of significance for their health and wellbeing, as many as 18 percent feel that their possibilities to continuously inform the older about their health situation remain at level 3, and 10 percent of the informants state that they sometimes have possibilities to respect the philosophy of life of the older person . About 16 percent of the informants feel they have limited possibilities to create a meaningful life for the older patients . We shall see what most likely happens inside the cluster if we fix some of the entities at level 4 to be 100 percent . A dignified care seems to be dependent on caregivers' attitude to the older patients . A fixation at level 4 for node b21 (treating the older as an adult person) will imply downshifts in nodes f21 (to continuously inform the older person), c21 (to respect the philosophy of the life of the older), and e21 (to create a meaningful life for the older). The following example also reinforces the attitude of the caregivers as an important factor for opportunities to enable dignified care for the older persons . A fixation at level 4 for node e21 (to create a meaningful life for the older) downshifted nodes d21, a21, and b21, at level 3 about 419 percent (table 10). These two examples show how the attitude of caregivers makes distinctions about upholding the dignity of the older person in the daily work, and we can see the same tendencies with fixation at level 3 or 4 for the other nodes within the cluster . The cluster consists of seven nodes, where the nodes including freedom and believing the older person are entities that informants perceive as important entities to facilitate patient involvement in their own care . The distribution functions show that ability for the patients to participate in their own care is limited . The caregivers' desire to care for the same patient during a longer period, sharing moods with the older, and being humble when facing the older person provide further dimensions for evaluations and enrichment as related to patients themselves given possibilities for participation . Making use of the older person's resources and capacities is a real challenge, and on the basis of the results, it appears that caring is more about doing for than doing with the patient, that is, indeed being participative in these respects . What happens within the cluster if a fixation at level 4 to 100% is done for some of the nodes in the cluster? Nodes a41 (promote continuity and preservation in the care process as a whole), f41 (being emphatic and sharing the moods with the older person), a11 (support resources of the older patient), g41 (being humble while caring for the older person), and d41 (encourage participation) being affected both upwards and downwards in the set of alternatives, as well as participation of the patient, are dependent on caregiver attitudes . The effects on distributions for respective nodes are described in tables 11, 12, 13, and 14 . The way caregivers feel about encourage participation has effect on each of the nodes within table 12 . Cluster viii consists of three nodes, respectively, to be responsible for the inner safety of older persons, to create a safe situation for the older person, and protect against mistreatment of the older person (see table 16). The final cluster a caring communion will be established when the older person is in charge of his / her own life . In these situations, caregivers really want to fulfil the needs of the older person and thereby also show respect for the older person, that is, show respect for the older person, be flexible, and encourage participation (table 17). The potential for a caring communion to reach care relationships is dependent on the desire to listen to the older person, monitoring of older person's satisfaction about care, flexibility, and respect for the older person's own decisions . Node c11 (flexibility) has the greatest ability to affect other nodes within the cluster . Fixing node c11 level to 4, corresponding to an improvement of the original result, the number of observations for each node increases (table 18). In the study of ethical values and ethical manners, the bayesian approach, represented by the use of bayesian belief networks (bbns), has been a useful method, because of the ability to compute with symbolic data . In particular, the ability to show the effects of using extended conditionalities, involving both original nodes as well as clusters of nodes, has been a useful insight . The computing process, as enabled by the structure identification capability of bbns, generated clusters of network structures and ended up in a total of nine clusters . Four clusters described ethical values (ethos), and five clusters explained possibilities as experienced by caregivers and how to act in an ethical manner in the daily work with the older people . Clusters consist of a bbn of nodes (three to seven nodes per cluster), and the relation between nodes within a cluster forms the specific character of the cluster . The comprehension of clusters is therefore given by the statements, in form of conditional probabilities, within that specific bnn . We can state that the bayesian approach had possibilities to generate clusters and underlying structural entities of relevance for the aim of the study . The structure identification capabilities enabled to find distinct dynamics within the clusters, as soon as the conditions of individual nodes were changed from the initial conditions . What happens if the conditions of one or several specified nodes within a cluster are changed? The dynamics of fixation of one or more entities to a given level can be seen in the cluster dignity consists of seven nodes or entities . About 8594 percent of the informants totally agree with all seven statements . In table 1 tables 1, 4, 5, and 6 represent changes between different assessment levels . We have to note that the material for the study was limited, and far - reaching conclusions cannot be made, but the results of the study point at entities possess power to change ethical ideals and manners in a remarkable way . This insight is of vital importance for the development of ethical manners in daily nursing care . Ethical values (ideals) such as integrity, dignity, safety, and caring community are embraced by the majority of respondents . However, we still have to note that cluster dignity has three percent (n = 735) of the observations at level 3 (sometimes) or lower, while within cluster integrity four percent (n = 315). Within this group of clusters, dignity was the only cluster which showed significant effects when the conditions for any of the nodes were changed . Clusters describing ethical manners were the following: closeness, distance, dignity, safety, participation, and caring communion . Within these clusters, we can see numbers of significant effects if the conditionalities for given node were changed (tables 9, 10, 12, and 15). Powerful negative effects on the entities probably change the opportunities for the older person to have good ethical care . However, earlier studies highlight the importance to develop an ethical culture at the unit . Without ethical discussions and models, the way to act in ethically critical situations, each caregiver acts in their own way, and it will not be possible to guarantee the older person ethical good care, because the quality of the care is depending on the individual caregiver's attitudes and manners [27, 28]. Promoting ethical good care is the responsibility of the whole work team . Caregivers participating in the study indeed approved ethos as it was expressed in the clusters of dignity, community, security, and integrity . A certain dynamics can be seen within cluster dignity, that is, coherence between the statement and the opinion of the informant . This was clear particularly in relation to basic care needs and in care situations with older people who generally suffer from frailty and increasing degree of cognitive decline . The study shows that the caregivers' attitudes to entities like compassion and mercy, moments of calm, respect, and compliance with the wishes and needs of the older person are of major importance in maintaining a dignified care . The perception and comprehension of the older person, as a person who lived a rich and meaningful life and where disease and illness changed that person's life, is important for the formation of ethical manner . Positive approaches to the older person, seeing the person behind the illness and suffering, are basic prerequisites for ethical manners in the daily care . Based on earlier research and results from this study, we know that the view of the older person and knowledge about ageing processes are some of the most important entities in the daily care of older persons [2931]. Respect and dignity of the older person were also in earlier research proven to constitute one of the major caring challenges . The views of the older person link to numerous ethical challenges, and therefore greater attention should be considered in both education and training of caregivers . Tornstam shows in his theory of gerotranscendence how the ageing process results in a value of displacement in the elderly [29, 32]. The older persons experience about their life as meaningful, having the right to control their lives regardless of the health and functional status, and feeling respected is important entity for the experience of dignity . Wadensten and carlsson [31, 33, 34] showed in their studies of the previous phenomena, that the caregivers did not properly perceive and comprehend the older person . In order to guarantee ethical care for the elderly, it seems likely that the view of the elderly needs to be changed, and the elderly should be seen as an adult with her own life to live, regardless of health or illness . Wadensten and carlsson [31, 33] state that caregivers have not observed the value shifts as tornstam explained in his study . Earlier research and findings [810, 12, 20] from this study opportunities for unethical manners and ethical challenges are in previous research described in a rather context dependent and descriptive way, and often without prospective and prognostic aspects . This study aims to include foresightedness and to highlight the probable effects on and prospects of ethical care . If we do not take these negative attitudes seriously, as we can see in the findings section, good ethical care will be at risk . On the basis of the present study, we can only state that there are differences in caregivers attitudes . Possible to act in an ethical manner, while others did not feel they had sufficient possibilities to act ethically . The reasons for those possibilities being limited is not confirmed in the present study but appear in some previous research [13, 35, 36]. In addition to the views of the elderly and knowledge about the ageing process, we should further include observations about situations where the total resources are not in balance to meet the needs of the elderly [37, 38]. Different opinions within the care unit about the caregivers ability to provide care in accordance with the ideals, related to good ethical care, moral anxiety, and guilt, are further circumstances to be considered beyond the scope of this study . Earlier research reports about serious medical errors and even death, illness, indifference, and arrogance [3537]. We cannot overlook leader's responsibilities, as leadership sets the norms and upholds a culture . The leaders support and create conditions for caregivers to act in accordance with the collective agreements about ethical good care, but they also explicitly provide and disseminate the ethical care criteria for and within the organisation . Leaders also take questions about resource allocation seriously, by creating human and material conditions for caregivers to act in accordance with their ethical ideals . This study is unique in its kind, both concerning the selected approach and methodological questions . Bbns have not been used significantly in nursing research, nor are there any studies that examine the ethical possibilities with focus on the probable effects upon changing conditions . An important point to make is that results must be understood given the translation of symbolic data from numbers to logical concepts and statements . Ethical ideals and ethical manners are phenomena that indeed make no sense to be explained exclusively by numeric data . The data represented and presented in the study are seen as symbolic data and appear, for example, as clusters of ethical ideals and attitudes which obtain their special character of the entities (nodes) and forms the network within the cluster . The nine clusters within the study are interpreted from caring science perspective [7, 2426] and earlier research about ethical questions in the daily work with the older person . In view of this transformation from numeric to statements, the study has opened up new opportunities to prevent services from becoming increasingly impersonal and stereotypical, and instead becoming care - based concerning ethos with ethical manners . The nodes that describe the cluster's character may in the future not only serve as a basis for ethical discussions and decision - making, but also be starting points for caregiver's individual development . The structure of the cluster with the underlying entities, that the study generated, seems to be an interesting development of the isaec instrument . The clusters with underlying nodes could be used as a framework for the continued development of instruments to identify caregiver attitudes to ethical values and ethical approach . In summary, we can state that the study has enriched the ethical discussion and opened up new what - if questions.
Af is associated with 5-fold increased stroke and 2-fold increased mortality risk.1) thromboprophylaxis is the cornerstone of treatment in these patients . Dabigatran etexilate is one of the new oral anticoagulants shown to reduce the risk for stroke in patients with af . It is an oral direct thrombin (factor iia) inhibitor approved by united states food and drug administration (fda) in 2010.2) dabigatran etexilate is a prodrug that is excreted mainly by the kidneys.2) it is superior to warfarin for prevention of stroke or systemic emboli without increasing bleeding in patients with non - valvular af.3) various bleeding complications associated with dabigatran were previously reported.4)5) however, there are very few cases of hemopericardium associated with dabigatran use.6)7) in this case, we described a patient with af who suffered from hemopericardium under dabigatran treatment . A 66 year - old female patient was admitted to the emergency room for progressive shortness of breath and poor health condition for 1 week . Her blood pressure was 80/50 mmhg and arterial blood gas analysis was ph; 6.98, pco2; 99 mmhg, po2; 66 mmhg . Echocardiography revealed massive pericardial effusion with cardiac tamponade (posteriorly 3 cm, anteriorly 2.5 cm, laterally 2 cm and 2.7 cm in adjacency with the right ventricle). The patient had non valvular af and been receiving dabigatran etexilate 150 mg twice daily for one year . In addition to dabigatran, the other medications included verapamil, budesonide, valsartan, and hydrochlorotiazide . She had a history of chronic obstructive pulmonary disease, hyperthyroidism, hypertension and gastroesophageal reflux disease . Her blood tests performed 1 year ago showed serum creatinine 0.5 mg / dl (normal range 0.5 - 0.9 mg / dl) and estimated creatinine clearance 136 ml / minute (using the cock - croft - gault equation). On admission her blood tests showed blood urea nitrogen 163.9 mg / dl (normal range 16.6 - 48.5 mg / dl), creatinine 3.99 mg / dl (normal range 0.5 - 0.9 mg / dl), estimated creatinine clearance 16 ml / minute (using the cock - croft - gault equation), fasting glucose 83.9 mg / dl (normal range 74 - 109 mg / dl), sodium 130 mmol / l (normal range 136 - 145 mmol / l), potassium 5.3 mmol / l (normal range 3.5 - 5.1 mmol / l), prothrombin time (pt) 44.5 s (normal range 11.5 - 15 s), activated partial thromboplastin time (aptt) 123.7 s (normal range 26 - 32 s), international normalized ratio (inr) 4.79, white blood cell count 10.59 10/ul (normal range 4.4 - 11.3 10/ul), hemoglobin 7.7 g / dl (normal range 11.7 - 16.1 g / dl), hematocrit 25.7% (normal range 35 - 47%), and platelet count 183 10/ul (normal range 152 - 396 10/ul). Urgent pericardiocentesis was performed with echocardiographic guidance and 1500 ml of hemorrhagic fluid was removed . Pericardial fluid analysis showed hemoglobin 7 g / dl and plasma hemoglobin level of the patient was 7.7 g / dl . Biochemical tests showed total protein 5.4 mg / dl, lactate dehydrogenase 707 mg / dl and albumin 2.98 mg / dl . Six hours after pericardiocentesis, the patients' blood pressure was over 100 mmhg systolic and she began to urinate . Red blood cell and fresh frozen plasma transfusions were made and post - transfusion hemoglobin level was 12 g / dl, pt 36.4 s (normal range 11.5 - 15 s), aptt 100.5 s (normal range 26 - 32 s), and inr 4.1 . On the second day after pericardiocentesis, hemorrhagic fluid flow via drainage catheter persisted and additional fresh frozen plasma transfusion was administered . Pericardial drainage was terminated after achievement of no flow through catheter and no pericardial effusion on control echocardiography . Laboratory tests repeated on the fourth day of admission were as follows: blood urea nitrogen 109 mg / dl, creatinine 1.4 mg / dl, inr 1.4 and hemoglobin 12 g / dl . It has constant bioabsorption and limited individual variability, hence, it does not require monitoring.8)9) since it is not metabolized by cytochrome p450 enzyme, drug interactions are scarce.9) bleeding rate associated with dabigatran was not higher than vkas in relevant trials.8) however, bleeding with dabigatran is a considerable problem because there is no direct antidote or blood product to reverse the anticoagulant effect entirely . The thrombin clotting time is not useful for coagulaopathy with dabigatran since it is highly sensitive . Ecarin clotting time is a sensitive test providing dose - dependent response; however, it is not yet approved for routine coagulation test and not approved by the fda for monitoring of dabigatran etexilate.10) there are a small number of case reports about hemopericardium associated with dabigatran use.6)7) our patient had been using dabigatran etexilate for 1 year before she suffered hemopericardium and cardiac tamponade . Acute renal failure was attributed to hypotension due to dehydration and hemodynamic compromise secondary to af with rapid ventricular rate . Impaired hemodynamics triggered by hemopericardium and impaired renal perfusion might have caused acute renal failure; however, dabigatran overdose due to acute renal failure might have induced hemopericardium as well . Patients receiving dabigatran treatment are exposed to increased bleeding risks secondary to dabigatran overdose in cases of renal failure since more than 80% of dabigatran etexilate is excreted via the renal pathway.8) detection of aptt>90 s and inr>2 was shown to be associated with dabigatran overdose in several trials.11)12) dabigatran inhibits thrombin that helps conversion of fibrinogen to fibrin therefore it effects all routine coagulation tests to a certain extent . Since inr measures prothrombin time of the extrinsic coagulation cascade, therapeutic concentrations of dabigatran cause only mild elevation of inr levels . However, increased plasma levels of dabigatran exhibit a linear relationship with inr.12) epistaxis and elevated inr levels (8.8) were reported in a patient with chronic renal failure, receiving both hemodialysis and dabigatran treatment.13) similarly, our patient had elevated inr levels (4.8) on admission . Meanwhile, many other studies demonstrated insensitivity of aptt and inr at therapeutic doses of dabigatran and found no linear relationship between.14) further studies are needed to investigate the relationship between inr and bleeding risk of dabigatran etexilate . Further evidence is required to evaluate the relationship between inr and bleeding risk of dabigatran etexilate.
Two representative patients who had different aaa geometries and who were to undergo fenestrated stent graft repair were selected for inclusion in the study . The pre- and post - operative ct datasets were obtained with using a 64-detector row scanner (beam collimation 640.5 mm, toshiba medical systems, kingsbury, uk) with the following parameters: section thickness 0.5 mm, pitch 1.0, a reconstruction interval of 0.5 mm, 120 kv and 140 mas . The fenestrated stent graft that was used in the study was a zenith aaa endovascular graft (william cook, brisbane, australia). The type of fenestration implanted in our patients involved small fenestrations (width and height: 66 mm or 68 mm) in the renal arteries . The fenestrated renal stents were successfully deployed into the bilateral renal arteries with an intra - aortic protrusion that measured between 4.4 mm and 5.8 mm . A type i endoleak (arising from proximal fixation of the stent graft) developed in one of the patients . The regions of interest (aortic branches, the aneurysm and the stent - graft lumen) were identified using ct number thresholding (20), and segmentation was performed with a semi - automatic technique, seeded region growing and the creation and separation of objects . For generating 3d realistic aaa models, the ct volume data was postprocessed with commercially available software analyze v 7.0 (analyzedirect, inc ., figure 1 shows the segmented aortic branches and an aneurysm from a sample of the ct volume data . Following segmentation of the volume data, an unstructured surface mesh of triangles was created over the segmented volume by using the marching cube algorithm . The geometric information was saved in the' stl (stereolithography)', which is a common format for computer - aided design (cad) and rapid prototyping . The' stl' file was converted into the cad model files by using catia v5 r17 (dassault systems, inc . The aorta mesh model consists of 2 parts: part 1 refers to the artery wall model of the pre- and post - stent grafting, which was generated by tetrahedral volume meshes with using ansys meshing 11 (ansys, inc ., part 2 is the blood flow model of the pre- and post - stent grating with insertion of the simulated fenestrated stent wires, and this was generated by tetrahedral and hexahedral volume meshes, respectively, with using ansys icem cfd 11 (ansys, inc ., figure 2 shows the segmented aorta models based on the pre- and post - stent grafting ct data in patient 2, while figure 3 demonstrates examples of the aaa mesh models of the pre- and post - fenestrated stent grafting in the same patient . Although the segmented post - stent grafting aaa models were generated with ct number thresholding and other postprocessing methods (objection creation and separation), which focus on the high - density stent wires, a detailed configuration of the fenestrated renal stents inside the renal arteries could not be displayed in the final mesh models . To achieve this goal, we simulated the fenestrated stent structures that were later inserted into the aorta models to reflect the actual patient treatment . The models of the fenestrated stent wires were created by taking a reference from the intraluminal appearance of a fenestrated stent inside the renal artery that was visualized with 3d virtual endoscopy (fig . First, we measured the renal artery diameter and we used it as the baseline for constructing the scaffolding of the stent wires . We then generated the structure profile of the stent wires to produce the surface and solid models (fig . 4b). Finally, we inserted the simulated model into the renal artery with an intro - aortic protrusion of 5.0 - 7.0 mm, as is shown in figure 4c . The thickness of the stent wires is about 0.4 mm in diameter, and the fenestrated renal stents consist of 6 - 8 v - shaped metal wires protruding into the abdominal aorta with a length of less than 7 mm, according to our previous experience (15), and so the simulated renal stents were generated and these reflected the realistic treatment of the patients . In summary, there were a total of 4 entire aorta models (both pre- and post - stent grafting) that comprised the abdominal aorta, the aortic aneurysm, the renal arteries and the common iliac arteries . In addition, another two juxtarenal models were generated that focused on only the fenestrated renal stents to specifically study the flow changes to the renal arteries . Therefore, a total of 6 models were tested in our study . As the study mainly deals with the renal artery and fenestrated renal stents, we kept only the renal arteries, the main abdominal aorta and aneurysm, as well as iliac artery branches, in the segmented models, while we remove the celiac axis and the superior mesenteric artery branches . However, the ostium of these two branches still remained patent, thus allowing calculation of the flow velocity to these main branches . In order to satisfy the criteria for mesh convergence, the meshes for both the fluid and solid domains were refined until we achieved mesh - density independence of the results . The maximum number of nodes per element was 18,020 and 71,921 for the artery wall mesh model and the blood flow mesh model, respectively . A coupled fluid - structure simulation was performed at a variable time step with different cardiac cycles so that the fluid forces and velocities across the fluid - solid interface could be demonstrated and calculated in our analysis . In order to ensure that our analysis reflects the realistic environment of human blood vessels, this allows studying the aneurysmal fluid mechanics by taking into account the instantaneous fluid forces acting on the wall and the effect of the wall motion on the fluid dynamic field . The fluid and materials properties for different entities were referenced from a previous study (23). The boundary conditions are time - dependent (24). The velocity inlet (the abdominal aorta at the level of celiac axis) boundary conditions are taken from the referenced value that shows measurement of the aortic blood velocity and reynold's number (fig . 5). A time - dependent pressure is also imposed at the outlets (fig . 6). The fluid (blood) is assumed to behave as a newtonian fluid, as this was known to be true for the larger vessels of the human body . The fenestrated stent within the blood is set as a non - fluid material because it is solid and non - elastic . The fluid density was set to 1,060 kg / m and the viscosity was set at 0.0027 pas, which correspond to the standard values cited in the literature (24). The flow was assumed to be incompressible and laminar . Given these assumptions, the fluid dynamics of the system is fully governed by the navier - stokes equations, which are shown as follows: where v is the blood velocity vector, p is the blood pressure, is the blood density, is the blood viscosity, f is the body force at time t acting on the fluid per unit mass, is the gradient operator and (t) is the fluid domain at time t. the solid (blood wall) is assumed to be elastic material and isotropic . The wall is set at 1.0 mm thick in both the pre- and post - stenting aaa models . The solid density was set to be 1,120 kg / m with a poisson ratio of 0.49 and a young's modulus of 1.2 mpa, and these correspond to the standard values cited in the literature (25). From these assumptions, the blood wall is governed by the following constitutive equation: where ij is the stress tensor, cijkl is elastic constant tensor, kl is the strain tensor and (t) is the structural domain at time t. the convergence of residual target 110 for the governing equations of the fluid domain was solved using ansys cfx 11 (ansys, inc ., canonsburg, pa). The residual target 110 for the governing equations of the structural domain the two - way fluid - structure interaction (fsi) calculations were used in the transient simulation, and the transfer forces with the coupling time steps were set at 0.025 s with a total duration of 0.9 s. the meshes are deformable during the computational fluid dynamic (cfd) analysis . Based on the above parameters, the cfd analysis was performed with the blood flow simulated at different cardiac phases (the systolic and diastolic cycles). The blood flow was calculated in the aortic aneurysm, the renal arteries and the common iliac arteries in terms of the flow pattern, the wall pressure and the wall shear stress at pre- and post - fenestration by using ansys multiphysic (ansys, inc ., canonsburg, pa) two representative patients who had different aaa geometries and who were to undergo fenestrated stent graft repair were selected for inclusion in the study . The pre- and post - operative ct datasets were obtained with using a 64-detector row scanner (beam collimation 640.5 mm, toshiba medical systems, kingsbury, uk) with the following parameters: section thickness 0.5 mm, pitch 1.0, a reconstruction interval of 0.5 mm, 120 kv and 140 mas . The fenestrated stent graft that was used in the study was a zenith aaa endovascular graft (william cook, brisbane, australia). The type of fenestration implanted in our patients involved small fenestrations (width and height: 66 mm or 68 mm) in the renal arteries . The fenestrated renal stents were successfully deployed into the bilateral renal arteries with an intra - aortic protrusion that measured between 4.4 mm and 5.8 mm . A type i endoleak (arising from proximal fixation of the stent graft) developed in one of the patients . The regions of interest (aortic branches, the aneurysm and the stent - graft lumen) were identified using ct number thresholding (20), and segmentation was performed with a semi - automatic technique, seeded region growing and the creation and separation of objects . For generating 3d realistic aaa models, the ct volume data was postprocessed with commercially available software analyze v 7.0 (analyzedirect, inc ., figure 1 shows the segmented aortic branches and an aneurysm from a sample of the ct volume data . Following segmentation of the volume data, an unstructured surface mesh of triangles was created over the segmented volume by using the marching cube algorithm . The geometric information was saved in the' stl (stereolithography)', which is a common format for computer - aided design (cad) and rapid prototyping . The' stl' file was converted into the cad model files by using catia v5 r17 (dassault systems, inc . The aorta mesh model consists of 2 parts: part 1 refers to the artery wall model of the pre- and post - stent grafting, which was generated by tetrahedral volume meshes with using ansys meshing 11 (ansys, inc ., part 2 is the blood flow model of the pre- and post - stent grating with insertion of the simulated fenestrated stent wires, and this was generated by tetrahedral and hexahedral volume meshes, respectively, with using ansys icem cfd 11 (ansys, inc . Figure 2 shows the segmented aorta models based on the pre- and post - stent grafting ct data in patient 2, while figure 3 demonstrates examples of the aaa mesh models of the pre- and post - fenestrated stent grafting in the same patient . Although the segmented post - stent grafting aaa models were generated with ct number thresholding and other postprocessing methods (objection creation and separation), which focus on the high - density stent wires, a detailed configuration of the fenestrated renal stents inside the renal arteries could not be displayed in the final mesh models . To achieve this goal, we simulated the fenestrated stent structures that were later inserted into the aorta models to reflect the actual patient treatment . The models of the fenestrated stent wires were created by taking a reference from the intraluminal appearance of a fenestrated stent inside the renal artery that was visualized with 3d virtual endoscopy (fig . First, we measured the renal artery diameter and we used it as the baseline for constructing the scaffolding of the stent wires . We then generated the structure profile of the stent wires to produce the surface and solid models (fig . 4b). Finally, we inserted the simulated model into the renal artery with an intro - aortic protrusion of 5.0 - 7.0 mm, as is shown in figure 4c . The thickness of the stent wires is about 0.4 mm in diameter, and the fenestrated renal stents consist of 6 - 8 v - shaped metal wires protruding into the abdominal aorta with a length of less than 7 mm, according to our previous experience (15), and so the simulated renal stents were generated and these reflected the realistic treatment of the patients . In summary, there were a total of 4 entire aorta models (both pre- and post - stent grafting) that comprised the abdominal aorta, the aortic aneurysm, the renal arteries and the common iliac arteries . In addition, another two juxtarenal models were generated that focused on only the fenestrated renal stents to specifically study the flow changes to the renal arteries . Therefore, a total of 6 models were tested in our study . As the study mainly deals with the renal artery and fenestrated renal stents, we kept only the renal arteries, the main abdominal aorta and aneurysm, as well as iliac artery branches, in the segmented models, while we remove the celiac axis and the superior mesenteric artery branches . However, the ostium of these two branches still remained patent, thus allowing calculation of the flow velocity to these main branches . In order to satisfy the criteria for mesh convergence, the meshes for both the fluid and solid domains were refined until we achieved mesh - density independence of the results . The maximum number of nodes per element was 18,020 and 71,921 for the artery wall mesh model and the blood flow mesh model, respectively . A coupled fluid - structure simulation was performed at a variable time step with different cardiac cycles so that the fluid forces and velocities across the fluid - solid interface could be demonstrated and calculated in our analysis . In order to ensure that our analysis reflects the realistic environment of human blood vessels, the normal physiological hemodynamics should be considered for the 3d numerical simulations . This allows studying the aneurysmal fluid mechanics by taking into account the instantaneous fluid forces acting on the wall and the effect of the wall motion on the fluid dynamic field . The fluid and materials properties for different entities were referenced from a previous study (23). The boundary conditions are time - dependent (24). The velocity inlet (the abdominal aorta at the level of celiac axis) boundary conditions are taken from the referenced value that shows measurement of the aortic blood velocity and reynold's number (fig . 5). A time - dependent pressure is also imposed at the outlets (fig . The fluid (blood) is assumed to behave as a newtonian fluid, as this was known to be true for the larger vessels of the human body . The fenestrated stent within the blood is set as a non - fluid material because it is solid and non - elastic . The fluid density was set to 1,060 kg / m and the viscosity was set at 0.0027 pas, which correspond to the standard values cited in the literature (24). The flow was assumed to be incompressible and laminar . Given these assumptions, the fluid dynamics of the system is fully governed by the navier - stokes equations, which are shown as follows: where v is the blood velocity vector, p is the blood pressure, is the blood density, is the blood viscosity, f is the body force at time t acting on the fluid per unit mass, is the gradient operator and (t) is the fluid domain at time t. the solid (blood wall) is assumed to be elastic material and isotropic . The wall is set at 1.0 mm thick in both the pre- and post - stenting aaa models . The solid density was set to be 1,120 kg / m with a poisson ratio of 0.49 and a young's modulus of 1.2 mpa, and these correspond to the standard values cited in the literature (25). From these assumptions, the blood wall is governed by the following constitutive equation: where ij is the stress tensor, cijkl is elastic constant tensor, kl is the strain tensor and (t) is the structural domain at time t. the convergence of residual target 110 for the governing equations of the fluid domain was solved using ansys cfx 11 (ansys, inc ., canonsburg, pa). The residual target 110 for the governing equations of the structural domain was solved using ansys simulation 11 (ansys, inc . The two - way fluid - structure interaction (fsi) calculations were used in the transient simulation, and the transfer forces with the coupling time steps were set at 0.025 s with a total duration of 0.9 s. the meshes are deformable during the computational fluid dynamic (cfd) analysis . Based on the above parameters, the cfd analysis was performed with the blood flow simulated at different cardiac phases (the systolic and diastolic cycles). The blood flow was calculated in the aortic aneurysm, the renal arteries and the common iliac arteries in terms of the flow pattern, the wall pressure and the wall shear stress at pre- and post - fenestration by using ansys multiphysic (ansys, inc . Changes of the aortic flow pattern were noted with placement of the fenestrated stent grafts and these changes were consistent with those reported in the literature (23 - 25). Based on assessing the streamline in the pre- and post - fenestrated geometries, flow recirculation patterns were observed in the pre - operative geometry that were not seen in the post - graft implantation where the flow was mostly attached to the graft . Figure 7 is the time - dependent velocity profile calculated at these main abdominal branches and the aneurysm . The apparent change of the velocity profile was noticed in the aneurysm with a more uniform flow pattern being observed after fenestrated stent grafting, as compared to the pre - stent grafting . The flow rate profile of the renal and common iliac arteries showed that the flow rate to the renal arteries was slower than that observed in the common iliac arteries, and this was especially apparent in the systolic phase . Figure 8 is an example showing the change of the flow pattern in patient 1, who was treated with a fenestrated stent graft . The blood flow became smoother and more laminar after fenestration (t = 0.1 - 0.9 s, top row images), when compared to the turbulent appearance observed at pre - fenestration (t = 0.1 - 0.9 s, bottom row images), and this is especially obvious in the diastolic phase for the pre - fenestrated flow analysis . The flow velocity was significantly increased inside the aortic aneurysm at the early systolic phase, as compared to that calculated at pre - fenestration . This indicates that the blood flowed through the new conduit formed by the stent graft instead of the dilated aorta . An endoleak was present in patient 2, with a similar flow pattern to that observed in the abdominal aorta, indicating there was a type i endoleak due to communication between the aneurysm sac and the systemic circulation . Figure 9 shows the flow pattern present in the aneurysm sac just below the right renal artery at a systolic phase of 0.2 s, which is the result of failure of proximal fixation of the stent grafts . The flow velocities to the renal arteries at pre- and post - fenestration were calculated and compared between the two cases, and our analysis showed there was no significant interference with the renal hemodynamics in the presence of stent protrusion . With the simulated fenestrated stents protruding into the abdominal aorta, flow recirculation patterns were observed in the proximal part of the renal arteries when compared to that seen at the time of pre - operative graft implantation, although this did not lead to significant changes of the flow velocity . Figure 10a demonstrates the flow effect in patient 1 after fenestrated stent implantation with the recirculation patterns being observed in the fenestrated renal arteries, with a slight decrease in blood velocity to the renal arteries (fig . While figure 11a shows another example of the flow effect in patient 2 following fenestrated stent implantation with obvious recirculation patterns being observed in the bilateral renal arteries, and there is a slight decrease of the flow velocity to the renal arteries (fig . Changes of the wall pressure following implantation of a fenestrated stent graft were observed in the simulation, as is shown in figure 12 . It was observed that high pressure was seen within the aneurysm sac prior to fenestration . After implantation of the stent - graft, the maximum wall pressure was much lower inside the aneurysm sac . As shown in figure 12, the wall pressure in the proximal renal arteries was similar to that observed in the common iliac arteries, but the wall pressure in the distal renal arteries was much lower than that observed in the common iliac arteries . The areas of high wall shear stress were mainly situated in the regions of enhanced recirculation or vortices . This was apparently observed at the level of the renal arteries because of the vortices caused by the protruded renal stents, which were implanted in the renal arteries . After stent - graft implantation, the maximum shear stress was significantly increased inside the aneurysm, and this was because of the laminar blood flow through the stent graft when compared to the turbulent pattern in the dilated aorta aneurysm . Although the shear stress was reduced to some extent at the proximal aneurysm neck when compared to that calculated for the pre - stent grafting (fig . 13), the difference was insignificant . A reduction of the shear stress at the renal arteries is most likely caused by the presence of stent wires inserted into the renal arteries, as is shown in figure 13 . Changes of the aortic flow pattern were noted with placement of the fenestrated stent grafts and these changes were consistent with those reported in the literature (23 - 25). Based on assessing the streamline in the pre- and post - fenestrated geometries, flow recirculation patterns were observed in the pre - operative geometry that were not seen in the post - graft implantation where the flow was mostly attached to the graft . Figure 7 is the time - dependent velocity profile calculated at these main abdominal branches and the aneurysm . The apparent change of the velocity profile was noticed in the aneurysm with a more uniform flow pattern being observed after fenestrated stent grafting, as compared to the pre - stent grafting . The flow rate profile of the renal and common iliac arteries showed that the flow rate to the renal arteries was slower than that observed in the common iliac arteries, and this was especially apparent in the systolic phase . Figure 8 is an example showing the change of the flow pattern in patient 1, who was treated with a fenestrated stent graft . The blood flow became smoother and more laminar after fenestration (t = 0.1 - 0.9 s, top row images), when compared to the turbulent appearance observed at pre - fenestration (t = 0.1 - 0.9 s, bottom row images), and this is especially obvious in the diastolic phase for the pre - fenestrated flow analysis . The flow velocity was significantly increased inside the aortic aneurysm at the early systolic phase, as compared to that calculated at pre - fenestration . This indicates that the blood flowed through the new conduit formed by the stent graft instead of the dilated aorta . An endoleak was present in patient 2, with a similar flow pattern to that observed in the abdominal aorta, indicating there was a type i endoleak due to communication between the aneurysm sac and the systemic circulation . Figure 9 shows the flow pattern present in the aneurysm sac just below the right renal artery at a systolic phase of 0.2 s, which is the result of failure of proximal fixation of the stent grafts . The flow velocities to the renal arteries at pre- and post - fenestration were calculated and compared between the two cases, and our analysis showed there was no significant interference with the renal hemodynamics in the presence of stent protrusion . With the simulated fenestrated stents protruding into the abdominal aorta, flow recirculation patterns were observed in the proximal part of the renal arteries when compared to that seen at the time of pre - operative graft implantation, although this did not lead to significant changes of the flow velocity . Figure 10a demonstrates the flow effect in patient 1 after fenestrated stent implantation with the recirculation patterns being observed in the fenestrated renal arteries, with a slight decrease in blood velocity to the renal arteries (fig . While figure 11a shows another example of the flow effect in patient 2 following fenestrated stent implantation with obvious recirculation patterns being observed in the bilateral renal arteries, and there is a slight decrease of the flow velocity to the renal arteries (fig . Changes of the wall pressure following implantation of a fenestrated stent graft were observed in the simulation, as is shown in figure 12 . It was observed that high pressure was seen within the aneurysm sac prior to fenestration . After implantation of the stent - graft, the maximum wall pressure was much lower inside the aneurysm sac . As shown in figure 12, the wall pressure in the proximal renal arteries was similar to that observed in the common iliac arteries, but the wall pressure in the distal renal arteries was much lower than that observed in the common iliac arteries . The areas of high wall shear stress were mainly situated in the regions of enhanced recirculation or vortices . This was apparently observed at the level of the renal arteries because of the vortices caused by the protruded renal stents, which were implanted in the renal arteries . After stent - graft implantation, the maximum shear stress was significantly increased inside the aneurysm, and this was because of the laminar blood flow through the stent graft when compared to the turbulent pattern in the dilated aorta aneurysm . Although the shear stress was reduced to some extent at the proximal aneurysm neck when compared to that calculated for the pre - stent grafting (fig . A reduction of the shear stress at the renal arteries is most likely caused by the presence of stent wires inserted into the renal arteries, as is shown in figure 13 . Our study is the first report to investigate the hemodynamic effect of fenestrated stents on the renal arteries . Although based on two sample patients, our results provide a basis for testing the effect of placing a fenestrated vessel stent into the renal artery, and our research findings provide insight into the treatment outcome of fenestrated endovascular repair . The purpose of implanting a stent - graft is to exclude the aneurysm from the systemic blood circulation so that the aneurysm gradually shrinks and becomes smaller while the blood flows through the new conduit, which is produced by the stent graft . For this purpose of treatment the unique characteristics of fenestrated stent grafting involve creating an opening in the graft material with inserting fenestrated stents into vessels, and mainly the renal arteries . In addition, a fenestrated stent normally protrudes into the aortic lumen by less than 7 mm, as was reported in our previous studies (15). Therefore, there exists a potential risk for fenestrated stents to interfere with the renal blood flow . However, this was not observed in our study as the calculated velocity to the renal arteries did not show significant changes following implantation of fenestrated stents, and this indicates the safety of placing fenestrated stents into the renal arteries, and even with the presence of a certain length of stent protrusion . Previous studies have been performed to investigate the fluid - stent graft interaction based on aaa models, yet these studies were focused on the situations of infrarenally or suprarenally fixation of stent grafts (26 - 30). There are few studies that have focused on flow analysis in the situation of fenestrated endovascular repair and this situation has not been systematically studied . In our study, realistic aaa models generated from two patients who were treated with fenestrated stent grafts were used to simulate the blood flow patterns and the velocity changes . Moreover, we simulated the actual intraluminal appearance of the fenestrated renal stents in relation to the abdominal aorta and renal arteries, which reflects the real treatment of patient . It is within our expectation that flow recirculation or a vortex was observed at the proximal renal arteries because of the intra - aortic protruded stents; however, the effect of fenestrated stents on the renal velocity was minimal, based on our analysis . Our results could be used as guidance for following up fenestrated repair although the intra - aortic stent protrusion is less than 7 mm in most of the situations, there exists the possibility that the stent protrusion could be as long as 10 mm or more in some cases, as was reported in our previous studies (15, 16). Thus, a simulation of various lengths of stent protrusion could provide an in - depth study of the hemodynamic effect of fenestrated stents . Moreover, the thickness of stent wires could increase since it is possible for the blood material to adhere to the wires and so this may affect the flow of blood into the renal artery . This was confirmed by a previous experimental study showing that small bits of materials were deposited onto the wire, leading to the increase of the cross - sectional area of the stent wire (30). A simulation of wire thickness of more than 0.4 mm deserves to be performed to reflect this situation and to analyse the subsequent flow interference . Therefore, a further flow analysis based on different wire thicknesses is needed so that a robust conclusion can be drawn . Studies have shown that low shear stress could lead to a reduction of the cross - sectional area of the renal ostium owing to the presence of stent wires (because of formation of neointimal hyperplasia on the stent surface) (31, 32). It has been reported that augmentation of the wall shear stress is accompanied by a local reduction in the neointimal hyperplasia (31). Another potential risk of low shear stress is the formation of artery plaque or atherosclerosis in the aortic branches (32). Our flow analysis observed the reduced wall shear in the renal arteries following insertion of fenestrated renal stents, and this indicates the potential risk of interference with the renal hemodynamics or the development of stenosis . From a clinical point of view, we consider that hemodynamic analysis of the interference of the renal stents is important for understanding the long - term safety of fenestrated stent grafting, although further studies are needed to confirm it . Despite the realistic models used in our study first, the aorta models were rigid rather than elastic . In the normal physiological situation, although our analysis was based on a two - way fsi that reflects the effect of pulsatile forces on the arterial wall, movement of the aortic wall during the cardiac cycles was not considered in our results . This explains to some extent that the wall pressure measured in the renal arteries was lower than that in the iliac arteries as we used rigid models in our simulation, as is demonstrated in figure 12 . Gaillard et al . (33) in their study reported that for the rigid model, the vortex created during the cycle in the distal segments does not impact on the wall (with the vortex remaining confined to the proximal part). However, in the soft model, the vortex migrates to the distal part during the cardiac cycle and impacts the wall, and so it can weaken it . Our analysis based on rigid models also resulted in the low flow rate to the renal arteries when compared to the high flow rate noticed at the common iliac arteries, as is shown in figure 7 . In the normal physiological condition, the renal arteries have low peripheral resistance, and so high flow volume with persistent diastolic flow reaches the renal arteries and this leads to a high flow velocity profile . In contrast, a low flow volume with a low flow velocity profile is seen in the common iliac arteries due to the high peripheral resistance and the low diastolic flow . The low flow rate to the renal arteries is particularly obvious in the systolic phase, and it is mainly because of the simulated blood flow running through the rigid tube (model) with side branches (simulated renal arteries) rather than the blood flow passing through the elastic arteries with movement during the cardiac cycles . Despite this limitation, our analysis of the flow velocity to the renal arteries in the presence of fenestrated renal stents is valid as the simulated stents protrude into the aortic lumen; thus, their effect on the flow analysis is not determined by movement of the arterial wall (like in aorta models). Second, only two cases were tested in our study, which is another limitation . Further studies composed of more patients with different aortic geometries should be performed to enable drawing a robust conclusion . Last, although we included a case with type i endoleak in the simulated models, we did not perform measurements of the sac pressure . The fsi simulations reported by li and kleinstreuer (34) indicated that the stent - graft migration force is greatly dependent on the difference in the pressure levels between the stent - graft and the aneurysm cavity . Traditional imaging - based follow - up of aaa after evar has been restricted to detecting endoleaks and the changes in the aaa morphology and it has proved to be unreliable in preventing aneurysm rupture (35). Pressure measurements of the aneurysm sac are increasingly being recognized as the most accurate indication of aaa exclusion . Further studies based on soft aorta models with a focus on the pressure level differences between the aneurysm sac and the stent - graft could be valuable for detecting endoleaks, which cannot be detected by routine imaging techniques, and for predicting stent - graft migration . In conclusion, our preliminary study using the fsi method shows that the interference of fenestrated stents with the renal blood flow is minimal and our study demonstrates an insignificant hemodynamic effect, indicating the safety of placing fenestrated stents into the renal arteries . The wall shear stress was reduced to some extent following implantation of the renal stents, indicating the potential risk of thrombus formation or stenosis at the renal arteries . Further studies that will include various size stent protrusions and different wire thicknesses, as well as measurements of the aneurysm sac pressure, are necessary for improving our understanding of the long - term safety of fenestrated stent graft repair of aaa.
From november 2008 through december 2011, mrsa of companion animal origin was routinely isolated from specimens submitted for diagnostic purposes to vet med labor gmbh in ludwigsburg, germany, or to the institute of microbiology and epizootics, freie universitt berlin, in berlin, germany . S. aureus was confirmed as described (7) and stored in glycerol stocks at 80c . Pcr routinely used to confirm methicillin resistance and species identity had failed to produce a positive signal for meca in 10 mrsa isolates from companion animals (2 isolates from dogs, 7 from cats, and 1 from a guinea pig) (8). We screened these 10 isolates for the meca homologue by using the pcr method published by cuny et al . (5) and sent the amplicons obtained to lgc genomics gmbh (berlin, germany) for sequencing . Automated antimicrobial drug susceptibility testing was performed by using the biomrieux vitek 2 system (nrtingen, germany) according to the manufacturer s instructions . The following drugs were tested according to clinical and laboratory standards institute guidelines m31a3: penicillin, ampicillin sulbactam, oxacillin, gentamicin, kanamycin, enrofloxacin, marbofloxacin, erythromycin, clindamycin, tetracycline, nitrofurantoin, chloramphenicol, and trimethoprim sulfamethoxazole, (9). All isolates were further characterized by spa typing, multilocus sequence typing, and microarray hybridization by using the alere identibac s. aureus genotyping chip (alere technologies gmbh, jena, germany) as described (1012). All pcr amplicons demonstrated 100% identity with the dna sequence of mecalga251 (national center for biotechnology information no . The strains originated from geographically diverse areas (5 federal states of germany) and were isolated from different infection sites (table). All strains were identified as mrsa by the vitek 2 system (growth in the presence of 6 g / ml cefoxitin according to the vitek 2 advanced expert system), although oxacillin mics were rather low (0.5 g / ml) or moderately high (4 g / ml) (table). Imt, institute of microbiology and epizootics, freie universitt berlin, berlin, germany; oxa, oxacillin; st, sequence type; vb, vet med labor gmbh, ludwigsburg, germany . All isolates were positive for nuc and negative for meca according to pcr to detect mrsa (8), and all were positive for mecalga251 according to pcr to detect the novel meca homologue (5). As has been described for atypical mrsa, 4 strains belonged to st130 and 1 strain belonged to st1945 (differs from st130 by 1 allele) (35). The remaining 5 isolates were assigned to st599 (differs from st121 by 2 alleles) (table). St599 has been reported for methicillin - susceptible isolates from humans in europe, asia, and africa (www.mlst.net). The figure shows a minimum spanning tree based on 4,197 entries of the s. aureus multilocus sequence type database (http://saureus.mlst.net/) as of january 19, 2012 (figure, panel a) and a detailed view of the branches and sts harboring strains with the novel meca homologue published (figure, panel b) (35). Minimum spanning tree based on multilocus sequence typing data from 4,197 staphylococcus aureus strains (a) and an enlarged view of 1 phylogenetic group (b). Each circle represents a distinct sequence type (st), and circle size is proportional to st frequency . Green indicates mecalga251-positive s. aureus strains of companion animal origin reported in this study and sequence data from published multilocus sequence typing results (35); red indicates st599 methicillin - resistant s. aureus; and blue represents st2024 methicillin - sensitive s. aureus isolated from a wild rat . Microarray hybridization data revealed that the agr type i and capsule type 5 seem to be associated with st599 and agriii and that clonal complex (cc) 130 isolates harbor the capsule type 8 encoding gene . Cc130 and st599 isolates were positive for the surface - associated proteins clfa, clfb, fnba, and bbp . All st599 strains produced a positive hybridization result for 1 of the gene variants encoding the toxin responsible for toxic shock syndrome (tst1 or tst - bov), and all but 1 of them harbored the enterotoxins c (sec) and l (sel), indicating the presence of an s. aureus pathogenicity island that encodes superantigens (13). Positive or ambiguous hybridization signals for ccrb1, ccra3, and ccrb3 were obtained for 5 isolates, suggesting the presence of the sccmecxi in those strains, according to the results of shore et al . Our findings of cc130 and st599 mrsa harboring mecalga251 in several companion animal species suggest that in germany, the presence of the meca homologue in mrsa is not exclusively associated with cc130 . This finding supports the hypothesis that some, if not all, mrsa strains that harbor the novel meca variant can cause infections among a broad variety of hosts, as has been shown for mrsa of human, equine, canine, and other companion animal origins (1,7). All currently known mecalga251carrying mrsa were observed in a distinct section of the s. aureus population (figure, panel b). Whether this phylogenetic group possesses the ability to integrate the novel meca variant needs to be further investigated . In the past however, all isolates were correctly identified as mrsa by the vitek 2 system, as reported (4). Of the 10 isolates, a recent study identified cats as a potential natural reservoir for s. aureus of cc133, a genetic lineage that has also been reported for s. aureus of ruminant origin (14). Moreover, we have identified a cc130 strain (mssa_st2024, t8403) from a wild rat (imt21250; id4035) (www.mlst.net). In addition, cc130 mrsa containing the meca homologue has only recently been reported for isolates from humans in germany (5). Although many investigators focus on livestock - associated mrsa, and because particular companion animal lineages of mrsa seem to be lacking, transmission of mrsa between companion animals and human family members in close proximity might be underestimated, especially in cases of recurrent infection (15). The emergence of mrsa harboring the novel meca homologue has consequences for the verification methods for mrsa used in veterinary medicine; implementation of new methods will be inevitable . Their supposed restriction to only a few genetic lineages and the potential risk for interspecies transmission of atypical mrsa between companion animals and their owners in household environments needs further elucidation.
A 67-year - old male (height, 165.4 cm; weight, 69.1 kg) with a history of hypertension and hyperthyroidism was scheduled to excise a laryngeal mass because of right vocal fold leukoplakia . The patient's preoperative laboratory test, chest x - ray, pulmonary function test results were unremarkable . Transnasal fiberoptic laryngoscopy taken 2 months prior to the surgery showed right vocal cord palsy (fig . The initial vital signs of patient in the operating room were systolic / diastolic blood pressure of 150/70 mmhg and oxygen saturation of 98% on room air . General anesthesia was induced with 120 mg propofol and 40 mg rocuronium, followed by preoxygenation with 5 l oxygen . Two min after injecting the rocuronium, the patient was intubated with a 6.5 mm endotracheal laser tube (medtronic, laser - shield ii 6.5 mm i.d . Anesthesia was maintained with 67 vol% desflurane in an oxygen - air mixture under a 0.3 fraction of inspired oxygen (fio2). Total gas flow was 4 l / min . At the end of the surgical procedure, 15 mg pyridostigmine and 0.4 mg glycopyrrolate were administered after irregular spontaneous breathing of the patient . Five min after administering the anticholineasterase, we removed the tube after confirming clear consciousness of the patient and inspiratory pressure less than 40 mmhg . The patient responded well to commands and 5 sec grip strength was good on both hands . The patient was placed on a face mask for manual ventilation and inhaled racemic salbutamol sulfate (100 g as salbutamol) twice through the face mask to facilitate bronchodilation . Laryngeal edema, right vocal cord palsy, and paradoxical adduction of the left vocal cord during inspiration were detected by laryngoscopy . Hydrocortisone sodium succinate (100 mg) was administered intravenously to alleviate the laryngeal edema and 1 mg midazolam was administered intravenously twice for anxiolysis . The patient still complained of dyspnea and inspiratory stridor after 5 min so we applied 5 cmh2o manual ventilation by face mask to support the patient's self - ventilation and the dyspnea and inspiratory stridor improved immediately . 2). Immediately after applying cpap, the dyspnea and inspiratory stridor were relieved . We detected left vocal fold abduction from the midline on inspiration and widening of the glottic gap on the exam . We decided to maintain 5 cmh2o cpap applied via a face mask during transport to the intensive care unit (icu). An arterial blood gas analysis (abga) was done immediately in the icu and showed ph 7.4, paco2 39.8 mmhg, pao2 104.2 mmhg and sao2 97.7% . He had maintained oxygen saturation> 95% on a high - flow nasal oxygen cannula system (optiflow, fisher & paykel healthcare limited, auckland, new zealand) under settings of fio2 0.35 and flow 35 l / min (fig . Another transnasal fiberoptic laryngoscopy was performed 9 hours after extubation and no left vocal fold adduction movement was detected on inspiration . The results of an abga at that time were ph 7.411, paco2 43.0 mmhg, pao2 65.6 mmhg, and sao2 93.6% . No left vocal fold adduction movement was detected on inspiration during transnasal fiberoptic laryngoscopy (fig . Pvfm is a disorder that presents paradoxical adduction of the vocal fold during inspiration and abduction during expiration . It was reported first by patterson et al . In 1974 . At that time, pvfm was considered as neurologic or psychiatric disorder, therefore it was described as " munchausen's stridor " . The symptoms of pvfm are inspiratory stridor, shortness of breath, choking sensation, voice changes, and cough due to the limited airway opening associated with paradoxical vocal fold adduction . The major differential diagnoses are asthma, bilateral vocal cord paralysis, unilateral vocal cord paralysis, vocal cord granuloma, subglottic or glottis stenosis, laryngomalacia, tracheomalacia, benign or malignant neoplasm of the upper airway, and palatine or lingual tonsil hypertrophy . Pvfm is often misdiagnosed as asthma because its clinical symptoms are similar with those of asthma . Patients with pvfm are resistant to asthmatic medical therapy, therefore, they tend to be exposed to unnecessary long - term steroid therapy and escalated drug use . Features that help distinguish pvfm from asthma are inspiratory stridor heard loudly over the larynx, rare sputum production, and associated voice changes . The organic causes of pvfm are brain stem compression, upper or lower motor neuron injury, and a movement disorder similar to parkinson's disease . The functional causes of pvfm include conversion disorder, anxiety disorder, depression, personality disorder, and stress disorder . According to a 2002 study by forrest et al . Patients with psychologically based pvfm present with conversion disorder alone or accompanying diseases, such as asthma, gastroesophageal reflux disease, laryngeal sicca, chronic laryngitis, and laryngeal sensory neuropathy . Neurologic factors consist of focal respiratory dystonia with or without spasmodic dysphonia, multiple sclerosis, and autonomic dysfunction . The gold standard for diagnosing pvfm is direct visualization of paradoxical inspiratory adduction of the vocal fold by laryngoscopy while the patient is complaining of symptoms, such as inspiratory stridor and dyspnea . . A pulmonary function test (pft) can be helpful when there is a cut - off or flattening of the inspiratory limb on spirometry that suggests an extrathoracic obstruction . Clinical symptoms and laryngoscopic findings are essential for diagnosing pvfm but a pft is not . Typical findings on transnasal fiberoptic laryngoscopy are adduction of all parts of the true vocal cord and posterior diamond - shaped glottal chinking on inspiration . If the patient is asymptomatic at the time of laryngoscopy, some specific examination, such as metacholine challenge, exercise, histamine, and pungent smell, should be performed to trigger pvfm . Treatments for pvfm include reassurance, speech therapy, psychotherapy, biofeedback, benzodiazepines, and inhaled anticholinergics . If the patient does not improve with these therapies, positive pressure ventilation, a bag - valve - mask, or cpap should be considered . In an emergency situation, such as postoperative pvfm, invasive procedures, such as endotracheal intubation or tracheostomy, are performed under a falling oxygen saturation condition due to near complete vocal cord obstruction . In this case, the patient was self - medicating with a benzodiazepine preoperatively because of concern about the right hand tremor . The spo2 of the patient was maintained> 97% after extubation, therefore, we decided to apply cpap rather than performing invasive procedures, such as re - intubation or a tracheostomy . Applying cpap in a patient with pvfm lowers expiratory flow and increases lung volume, which helps the glottis to open and relieves dyspnea . Cpap also reduces the effort needed for inspiration by establishing a favorable pressure gradient for inhalation . However, few patients with pvfm encounter an emergency situation, such as severe dyspnea . Therefore, rapid diagnosis and treatment are very important for a patient's prognosis . Pvfm is very difficult for anesthesiologists to diagnose in the post - anesthesia care unit because the gold standard for pvfm is transnasal fiberoptic laryngoscopy . However, pvfm should be considered when patients complain of dyspnea and stridor after endotracheal extubation . In conclusion, oxygen supply and positive pressure ventilation could be applied first in a case of pvfm when the patient complains of severe inspiratory dyspnea after endotracheal extubation . If the patient continues to suffer from prolonged dyspnea after supplying oxygen and positive pressure ventilation, cpap could be effective before performing invasive procedures, such as endotracheal intubation or a tracheostomy.
Signaling between nervous and immune systems is in part due to the fact that these two systems share ligands and receptors . The cellular components involved in these interactions within the central nervous system (cns) are mainly mastocytes, also called mast cells, and glia . In human brain, mastocytes are very scarce and are preferentially located in perivascular territories . By contrast, glial cells comprise about 90% of the total cell content in the cns and are classified as microglia and macroglia (astrocytes, oligodendrocytes, and ependymal cells). Representative of the immune system in the cns are mastocytes and microglia, two cell types derived from hematopoietic cells of the bone marrow that migrate to the brain before closure of the blood brain barrier (bbb) [2, 3]. The cns challenged by different aggressions frequently elicits immune and inflammatory responses [4, 5]. Mastocytes and microglia are efficient sensors of adverse endogenous or exogenous conditions of the cns [2, 6]. Moreover, stress conditions induce rapid mastocyte degranulation via the hypothalamic peptide corticotropin - releasing hormone (crh) and exogenous danger molecules like polyinosinic - polycytidylic acid (poly (i: c)), bacterial lipopolysaccharide (lps), and peptidoglycan (pgn), which are detected by mastocytes and microglia via toll - like receptors (tlrs) [8, 9]. Also, glucocorticoids (gcs) play a relevant role in stress - induced potentiation of neuroinflammatory responses by sensitizing microglia to proinflammatory stimuli . As part of these responses, glial tlrs, connexin hemichannels (cx hcs), pannexin (panx) channels might be key players in acute and chronic neurodegenerative diseases characterized by open bbb, demyelinization, and neuronal degeneration . The causes of various chronic diseases that affect the cns, such as alzheimer's disease (ad), parkinson's disease (pd), and multiple sclerosis (ms), are complex and can be related to multiple factors . Notably, the innate host defense has been demonstrated to play an active role in promoting neurodegeneration [12, 13]. However, the possible role of these cellular and molecular elements during brain ontogenesis and the consequences in the adult cns remain unknown . This review presents possible implications of glial toll - like receptors (tlrs) and cx hc and panx channels activation after potentiation by stress in cns dysfunctions . During pregnancy, viral infections are common and emerge to predispose the offspring to develop psychiatric diseases [14, 15]. Viral mimic polyinosinic: polycytidylic acid [poli (i: c)] resembles the structure of double - stranded rna (dsrna) generated in host cells during viral replication, and it is recognized by tlr3 that activates the innate immune response . The administration of poly (i: c) is a way to trigger the innate immune response, which mimics the early phase of viral infections, and avoids the use of infectious agents, and treatments can be standardized and experiments may be easily compared . All together, they represent an interesting area because perinatal infections, particularly those of viral etiology, are frequent and have been associated with diverse alterations of adult cns, including schizophrenia and autism [19, 20]. Tlrs are highly conserved germ line - encoded pattern - recognition receptors that initiate innate immune responses via recognition of pathogen - associated molecular patterns (pamps) as well by recognition of danger - associated molecular patterns (damps) that correspond to endogenous ligands released after tissue injury or cellular stress, such atp, histones, heat - shock proteins, mrna, high - mobility group box-1 protein (hmgb1), surfactant proteins a and d, and mitochondrial proteins . Activation of tlrs triggers a cascade of intracellular events leading to activation of several transcription factors, including nf-b, activator protein-1 (ap-1), and ifn - regulatory factor-3 (irf-3) and -7 that regulate the expression of various cytokines and chemokines, responses that are performed in the cns mainly by mastocytes and microglia . In addition, activation of innate immune responses via tlrs is a prerequisite for the generation of adaptive immune responses that become relevant in autoimmune diseases such as experimental autoimmune encephalomyelitis (eae). The number of molecular members that comprise the tlr family is ten in humans (tlrs 110) and twelve in mice (tlrs 19; tlrs 1113). Some tlrs can be expressed on the cell surface (tlrs 1, 2, 4, 5, 6, and 10) or in intracellular compartments (tlrs 3, 7/8, and 9), but others can be found in both the cell membrane and intracellular compartments (tlr3 and tlr7; endosomes and endoplasmic reticulum). Each tlr detects distinct pamps derived from viruses, bacteria, mycobacteria, fungi, or parasites . For example, tlr3 and tlr7/8 detect ds and single - stranded (ss) rnas from virus, respectively; tlr4 responds to lps from gram - negative bacteria; and tlr9 senses bacterial dna that contains unmethylated cytosine - guanosine dinucleotides (cpg) [2225]. In the adult brain, mastocytes are mainly found in leptomeninges and thalamus close to the bbb [26, 27], but they are also present early in brain ontogeny [28, 29]. Mastocytes can be activated by antigens that induce crosslinking of ige bound to mast cells, cd47 recognition, calcium ionophore, atp, compound 48/80, and also by recognition of damps or pamps [26, 27]. If these activators bind to mastocytes for a short period of time (from seconds to a few minutes), they lead to rapid degranulation and release bioamines, proteoglycans, proteases, atp, tnf- and chemokines stored in preformed granules, whereas activations of longer durations lead to the release of newly formed cytokine (tnf-, il1, and granulocyte macrophage colony - stimulating factor (gm - csf)), and chemokine (c c motif) ligand 3 (ccl3), enzymes (tryptase, chymase, carboxypeptidase), lipid mediators (prostaglandins, leukotrienes, thromboxanes, and platelet - activating factor), and nitric oxide (no), mediating the recruitment of effector cells, fluid extravasation, and tissue inflammation [30, 31]. Murine mastocytes express the mrna of tlrs 14 and 69 but not tlr5 [3236]. Moreover, human mastocytes express the mrna of tlrs 110 with the exception of tlr8 [9, 3739]. In mastocytes, tlr ligands, such as poly (i: c), lps, r-848, and cpg oligodeoxynucleotide, promote il-6 and tnf- secretion as well as regulated upon activation, normal t cell expressed and secreted (rantes) and macrophage inflammatory protein (mip) without significant degranulation [35, 38, 40, 41]. More specifically, in rodent mastocytes, binding of lps to tlr4 induces the release of de novo expressed (without degranulation) and secreted tnf-, il-5, il-10, and il-13 but not gm - csf, il-1, or leukotriene c4 (ltc4), while binding of pgn to tlr2 induces degranulation that includes histamine release [9, 34, 37]. In three different mouse models, where tlr3, tlr4, and tlr7 were specifically deleted in mastocytes, the recruitment of effector cd8 t cells, neutrophils, and dendritic cells, respectively, this implies that mastocytes recognize, respond, and coordinate immune responses, features that are suppressed by trls 3, 4, and 7 . Not only ligands, but also immunological host environments are decisive for mastocyte activity . In human mastocytes, prolonged lymphotoxin - alpha (lta) and pgn exposure downregulate fcri, decreasing degranulation products after an antigen crosslinking reaction . Poly (i: c) treatment also decreases degranulation in an in vitro allergic model, affecting mastocyte adhesion to fibronectin and vitronectin through conformational inactivation of cd29, the receptor of fibronectin . Moreover, lps and pgn induce mastocytes migration in vitro after brief treatment with il-6 and ccl5/rantes, respectively . The activation and migration of mastocytes occur in several neurologic disorders including ms [46, 47], pd, amyotrophic lateral sclerosis (als) [49, 50], ad, traumatic injury, ischemic and hemorrhagic stroke [53, 54], and viral infections . Mastocytes activation and migration are critical for the increased bbb permeability and progression of neuroinflammation . Additionally, proteases released during mastocyte degranulation can also degrade myelin components, contributing to myelin damage in the cns and peripheral nervous system . Microglia can rapidly respond to pathogens through their tlrs but do not sense apoptotic cells through the same mechanism [58, 59]. Moreover, levels of tlrs expressed by microglia vary depending on the stages of development or pathological conditions . Tlr activation induces a cascade of intracellular events leading to the activation of several transcription factors, including nf-b, ap-1, irf-3, and irf-7 that regulate the expression of many molecular elements of inflammatory responses . In human microglia, activation of tlr3 by agonists such as poly (i: c) induces a strong proinflammatory response that allows microglia to mediate the development of t - helper 1 (th1) cells . Moreover, infection with the west nile virus (a retrovirus that produces dsrna) in mice lacking tlr3 shows reduced microglial activation and more resistance to lethal infection with reduced viral load and inflammatory responses in the brain compared to wild - type mice . Mastocytes release several cytokines in response to tlr2 activation including tnf-, il-4, il-5, il-6, and il-13 . Meanwhile, the activation of tlr4 causes release of tnf-, il-6, il-13, il-5, il-10, and eotaxin [34, 6365]. Also, numerous chemokines including ccl5/rantes, can also induce a proinflammatory profile in microglia [37, 38, 59, 66]. Il-33 derived from microglia modulates the activation of p2 receptors on mastocytes inducing secretion of il-6, il-13, and monocyte chemoattractant protein-1 (mip-1), which in turn can modulate the microglia activity . It induces microglia to secrete tnf-, il-6, and ros and activate in microglia proteinase - activated receptor-2 (par-2), a g protein - coupled receptors widely expressed in neurons, astrocytes, and microglia that are implicated in the pathogenesis of ischemia and neurodegeneration, because it induces widespread inflammation [7173]. The activation of microglial par-2 also upregulates p2x4 receptors and promotes release of brain - derived neurotrophic factor, tnf-, and il-6 that upregulate the expression mastocyte of par-2, which results in activation and release of tnf- . It is interesting to note that mastocytes but not microglia have been described to be the first responder in cns injuries, such as perinatal hypoxia - ischemia . Many cells produce tnf- in response to several stimuli, but mastocytes store tnf- in granules, and thus they can release it before other cells including microglia and endothelial cells . Additionally, the recruitment and activation of mastocytes occur previous to responses elicited by neurons, glia, and endothelial cells . Therefore, mastocytes initiate acute inflammations in response to a stimulus, and when inhibited, the brain damage decreases, as observed when the early mastocyte response is inhibited with cromolyn (a mastocyte stabilizer), and then significant neuroprotection is observed . A strong link between lps, the tlr4 agonist, and brain injury both in fetal and newborn animals has been demonstrated . Lps injected into developing mouse and rat brains has been shown to induce injury in white matter . Moreover, systemic lps administration to preterm fetal sheep induces cerebellar white matter injury, and in vitro assays demonstrate that tlr4 gene deletion prevents lps - induced oligodendrocyte death . In astrocytes, the expression of tlrs is limited in astrocytes, probably because of the neuroectodermal origin of astroglia . These cells express tlr2, which increases in response to proinflammatory stimuli [22, 80]. They also express tlr3 that responds to poly (i: c), hence producing among other cytokines il-6 that contributes to inflammation in humans and mice [8082]. The gene profile of astrocytes activated via tlr3 shows neuroprotective mediators and cell growth factors, that is, differentiation and migration molecules comprising a neuroprotective response rather than a proinflammatory phenotype [83, 84]. Tlr4 has been shown to participate in stroke - caused brain damage [8587] and in ad [88, 89]. Likewise, tlr4 could play a pivotal role in demyelinating diseases, such as ms . Both tlr agonists and cytokines induce the expression of chemokines ccl2, ccl3, ccl5, intercellular cell adhesion molecule-1 (icam-1), and vascular cell adhesion molecule-1 (vcam-1). Moreover, lps and poly (i: c) induce the production of il-6, tnf-, ifn-4, ifn-, and inos . Lps and dsrna in parallel induce astrocyte activation, which leads to il-1, il-1, il-6, tnf-, gm - csf, lt, and tgf-3 secretion, although macrophage migration inhibitory factor (mif) secretion is inhibited . However, no effect has been found on anti - inflammatory cytokines such as il-2, il-3, il-4, il-5, il-10, tgf-1, tgf-2, and tnf- [11, 93]. Recently, in addition to tlr2, tlr3, and tlr4, tlr1, tlr5, tlr6, and tlr7/8 have been found in astrocytes, but their functional roles remain unknown [22, 84]. Therefore, the understanding of the detailed mechanisms of tlr signaling in astrocyte activation in cns inflammatory conditions still needs further investigation . The expression and function of tlrs in oligodendrocytes, unlike other glial cells, have been poorly studied . Only tlr2, -3, and -4 have been evaluated, being these receptors related to the regulation of inflammatory processes, gliosis, and remyelination after injury [95, 96]. Knockout mice for tlr2 and tlr4 exposed to spinal cord injuries show a lower remyelination capacity, and thus it is believed that these receptors would have a key role in the formation of myelin . Astrocyte dysfunction triggers primary microglial activation, which induces demyelination [78, 97]. Furthermore, injection of lps in the bone marrow induces a rapid oligodendrocyte loss, followed by an increase in oligodendrocyte number . After acute demyelination induced by lps, a more widespread distribution of oligodendrocyte precursor cells is triggered by the activation of microglia / macrophages, which is an event that accelerates remyelination [99, 100]. Rats treated with zymosan, a tlr2 agonist, show oligodendrocyte and axonal loss without regeneration . In addition, rats treated with lps, that is, a tlr4 agonist, show oligodendrocyte death and demyelination [76, 101]. Also, lps - induced spinal cord damage shows significant demyelinization associated with an important reduction in the amount of oligodendrocytes . Other researchers have shown that tnf- and tnfr1 play a relevant role in oligodendrocyte death induced by tlr activation [103105]. However, bsibsi et al . Showed that zymosan and lps reduce survival, differentiation, and myelin - like membrane formation, while poly (i: c) triggers apoptosis in rat oligodendrocyte cultures . These findings suggest that tlrs play a pivotal role in oligodendrocyte differentiation and myelination, both in physiological and pathological conditions . Compared to other cell types, tlrs play direct roles in regulating various aspects of oligodendrocyte's behavior . However, the apparent contradiction between the effects of lps and zymosan on oligodendrocytes in different models has not been clarified . Future research could help to determine the functionality of tlr receptors in oligodendrocytes under physiological and pathological conditions . With regard to the neuroendocrine modulation of the activity of tlrs, this can take local, regional, and systemic routes . Local components include neuropeptides such as substance p, crh, calcitonin gene - related peptide (cgrp), and endogenous opioids released by peripheral nervous system . Among the regional components, the sympathetic and parasympathetic innervations release neurotransmitters (adrenaline and acetyl choline), and neuropeptides (neuropeptide y or vasoactive intestinal peptide (vip)) play a relevant role . Also at a regional level, a neuronal component regulates immunity through the innervation of immune organs and release of noradrenaline, and also a hormonal component regulates immunity systemically by means of adrenaline released from the medulla of the adrenal glands, whereas the systemic factors include the neuroendocrine system through the hypothalamic - pituitary - adrenal (hpa) axis and the anti - inflammatory effects of gcs . Furthermore, neuropeptides including cholecystokinin (cck), somatostatin, melanocyte - stimulating hormone (msh), vip, and gastrin also reduce the inflammatory response . Additionally, il-1 participates in several aspects of the immune response to infections such as regulation of inflammation and modulation of adaptive immune responses against viral infections [108, 109]. The inflammasome is a multiprotein complex that activates a platform for caspase-1 and caspase-1-dependent proteolytic maturation and secretion of interleukin-1 (il-1). The signal 1 corresponds to tlr ligands or tnf-, and the signal 2 includes atp, amyloid- (a), k efflux, pore - forming toxins, and silicic and uric acid crystals [111113]. After tlr2 and tlr4 activation, secretion and maturation of cytokines il-1 and il-18 depend on caspase-1 cleavage of their premature forms . In both cases, inflammasome complex proteins mediate caspase-1 activation in the presence of high concentrations of extracellular atp through activation of p2x7 receptors [114, 115]. Activation of p2x7 receptor leads to a large membrane pore formation identified as panx1 channels [116, 117], which recently has been found critical for caspase-1 activation [116, 118]. Not only in immune cells but also in neurons and astrocytes, panx1 recruitment mediates caspase-1 activation, suggesting that during infections, overall tlrs and panx1 channels could enhance inflammatory responses . One hc corresponds to one - half of a gap junction channel and is located at unapposed cell surfaces serving as communication pathway between the intra- and extracellular compartments . Two types of hcs are formed in most cells, and they are generally coexpressed . One of them is formed by connexins (cxs, 21 in humans) and the other by panxs 13 . Hcs provide a membrane pathway for releasing signaling molecules (e.g., atp, glutamate, pge2, and nad) and thus are recognized as paracrine / autocrine communication pathways under normal and pathological conditions [121, 122]. Inflammation is a key condition in neurodegeneration that occurs in postischemic brain, diabetes, ms, pd, ad, and possibly in various other neurodegenerative diseases [123, 124]. In neuroinflammatory conditions, the successive activation of different glial cells via hcs has been partially demonstrated [125, 126], and mastocytes are likely to be involved in early steps of different pathological conditions (figure 1). As mentioned previously, the degranulation response of mastocytes is an early and rapid response and might require precise coordination where hcs could be essential . Mastocytes express cxs 32 and 43, but to our knowledge, it remains unknown whether they form functional hcs . In addition, no clear evidence of panx1 expression in mastocytes has been published, but activation of p2x7 receptors leads to the formation of membrane pores permeable to molecules up to about 900 kda with single currents, similar to what has been described for panx1 channels, along with histamine release [117, 128]. Since the degranulation process depends on influx of extracellular ca, it is possible that panx1 channels participate in atp release, and then atp activates p2x7 receptors, which are ca permeable, allowing the influx of ca required for the mastocyte degranulation response . Then, glial cells become involved and microglial cells respond before astrocytes (within several minutes to few hours). In the normal cns, they express the macrophage marker cd11b, low levels of cd45, and practically undetectable levels of major histocompatibility complex (mhc) class ii molecules, cd40, and cd86 . In vitro, the microglia activation process is characterized by an upregulation of cd45, mhc class ii, and the costimulatory molecules cd40 and cd86 [130, 131]. The expression of mhc ii antigens is a characteristic feature of antigen - presenting cells, and their coexpression with costimulatory molecules is a hallmark of microglial cells' ability to interact with other cells, such as t cells . Activated, microglia proliferate and migrate to the injury site where they form cell aggregates and secrete pro- and anti - inflammatory cytokines and chemokines, no, and growth factors . The activation of microglia can be acute or chronic, and this would depend not only on the duration of an external stimulus but also on the quality of the stimulus (stress, infection, inflammation, and signals from damaged neurons). In fact, they show differences when activation is induced by stress or inflammation . For instance, acute stress induces morphological activation of microglia and increased c - fos expression in the periaqueductal gray matter but not in the surrounding midbrain . If activation is chronic, it can lead to microglial overactivation followed by microglial degeneration . Therefore, activated microglia secrete tnf- and il-1, which in astrocytes induce opening of cx43 hcs leading to the release of atp and glutamate by astrocytes, which can kill neurons through the activation of panx1 channels, p2x7 receptors, and nmda receptors in neurons . Another way of cell - cell interaction used by activated microglia can be found in cx- and panx - based channels . Microglia express low to undetectable levels of cx32, cx36, cx43, and cx45 [136139]. They also express panx1, and treatment with a2535 has been shown to increase its surface levels . Similarly, the expression of cx43 is upregulated in cultured rat / mouse microglia treated with lps or tnf- plus ifn-, calcium ionophore plus phorbol 12-myristate 13-acetate, or pgn derived from staphylococcus aureus . However, the possible functional role of cx - based hcs expressed by activated microglia remains to be elucidated . Under normal conditions, astrocytes are highly coupled with each other, forming intercellular networks, through which ca waves propagate . Extracellular atp acts as a paracrine messenger in these waves, since it activates purinergic receptors (p2x and p2y) in astrocytes of surrounding cells, thus resulting in an increase of [ca]i . The mechanisms for atp release from astrocytes may include vesicle - mediated exocytosis and diffusion through cx43 hcs [125, 145, 146] and/or channels formed by panx1 . Astrocytes also release several transmitters called gliotransmitters, including glutamate, gaba, atp, and adenosine . Increases in [ca]i can induce the release of gliotransmitters that promote increases in [ca]i in neighboring neurons, for example, through atp- and glutamate receptor - dependent pathways . The increased [ca]i occurs in local astroglia as well as in astrocytes located more distantly . Gliotransmitters might affect diverse neuronal functions including arborization and neuronal plasticity as well as more complex functions such as fear memory . Thus, astrocytic cx hcs and panx1 channels might be molecular targets to prevent undesired effects induced by stress . Most astrocytes also express cx30 and cx43, and at least cx43 forms hcs that are activated by proinflammatory cytokines, hypoxia - reoxygenation, and high glucose . For instance, lps does not induce cell permeabilization to fluorescent dyes in primary cultures highly enriched with astrocytes of newborn brains, but astrocytes cocultured with microglia respond to lps with a large increase in cx43 hc activity . Moreover, the effect of lps is mimicked by exogenous applied tnf- and il-, indicating that astrocytes do not respond to lps in the absence of microglia . Moreover, astrocytes previously exposed for 24 h to medium conditioned by a-treated microglia (cm - a) are permeabilized via cx43 hcs . As part of the mechanism, tnf- and il-1 have been shown to mimic the effect of cm - a, and neutralizing tnf- with soluble receptors and il-1 antagonists abrogated this effect . Recent in vivo studies have demonstrated that cx43 hcs are critical mediators of postischemic white and gray matter dysfunction and injury . Moreover, upregulation of astroglial panx1 channels and cx43 hcs has been found using an experimental model of brain abscess, suggesting that both channel types could play an orchestrated function in some inflammatory responses . Cx43 hcs of reactive astrocytes favor the release of excitotoxic compounds, atp, and glutamate, which activate neuronal p2x7 receptors, nmda receptors, and panx1 channels, hence promoting neurodegeneration . Activation of neuronal panx1 channels by atp and glutamate released through cx43 hcs from astrocytes exposed to cm - a was shown to induce neuronal death . Therefore, it has been proposed that blockade of astroglia and/or neuronal cx hcs and panx1 channels of the inflamed nervous system may represent a strategy to reduce neuronal loss in various pathological states [157159]. Additionally, the effect of the maternal environment on the developing cns in the offspring has been analyzed in fetal nonhuman primates . To this end, mothers were subject to a high - fat diet (hfd), and the cns of the fetuses showed increased levels of il-1 and il-1 type 1 receptor, as well as a rise in microglia activation markers, suggesting the activation of the local inflammatory response . Under the previous conditions, it is possible that microglia and astrocytes also present upregulation of hc activity, but this needs experimental demonstration . Oligodendrocytes might respond within the same time frame as astrocytes, since they can communicate via gap junctions as previously described herein . These cells are responsible for producing and maintaining myelin from the earliest stages of embryonic development to adulthood . Like other cells of the cns, oligodendrocytes have low renewal capacity . However, oligodendrocyte precursor cells induce remyelination, following the loss of myelin as a consequence of an injury . Many of their functions are accomplished by the expression of a variety of interactions between cx- and pannexin - based channels . Oligodendrocytes form gap junction channels with cell bodies of adjacent oligodendrocytes and between layers of myelin, called reflective gap junctions; oligodendrocytes form gap junctions with astrocytes as well . Collectively, this gap junction communicated network helps to absorb and remove extracellular k and glutamate released during neuronal activity, thus generating a spatial buffer where ions and molecules are diluted among cell communicated via gap junction channels [165167]. The study of demyelinating diseases, consisting of loss or destruction of myelin, has revealed panx1 channels, cx hcs, and gap junction channels as key factors in oligodendrocyte survival, as well as neuroprotection and myelin maintenance . Moreover, by means of the qpcr technique, the mrna of panxs 1 and 2 was detected in primary cultures of oligodendrocytes obtained from optic nerves of 12-day - old rats . Both were located in somas as well as in the layers of the myelin sheath . Extracellular atp mediates the ischemic damage to oligodendrocytes and is partially explained by the activation of panx1 channels . Both genetic and/or inflammatory diseases triggered by viral or toxic sources may affect myelin formation (hypomyelinating diseases) or its maintenance (demyelinating diseases) as it has been found in human diseases associated with hcs formed by mutated cxs . The first event in pathological manifestations of demyelinating disease of the cns is the disruption of the bbb that leads to access of demyelinating antibodies [161, 171174]. Also, activated t cells entering the cns mediate the release of inflammatory cells, which together with activated microglia release proinflammatory cytokines that promote oligodendrocyte death in vitro [175178]. Tnf- and ifn- can activate microglia and/or macrophage that destroy oligodendrocytes by oxidative stress [180, 181]. This repair is called remyelination, and its process, mediated by oligodendrocyte progenitor cells, is associated with functional recovery . It has been shown that chemokine- (cxcl-) 2 and proinflammatory cytokines, such as il-1 and il-6, promote oligodendrocyte progenitor cell proliferation, differentiation, and remyelination . Under inflammatory conditions, oligodendrocytes show upregulation of mhc i molecules, which are constitutively expressed, as well as fas, ifn-, and tnf- receptors (tnfri - ii), transforming them into targets for cd8 cells [175, 176, 182185]. Under control conditions there is no expression of mhc ii molecules in these cells [186, 187]. However, cultured oligodendrocytes treated with ifn- in the presence of the synthetic gc (dexamethasone) express mhc ii molecules, suggesting that under stress they could interact with cd4 t lymphocytes and either activate immune reactions or become the targets of t - cell - mediated cytotoxic attack . An excess of extracellular atp is an activator of both innate and acquired immunities, acting as a damp that is chemotactic factor for neutrophils, and a strong regulator of activation, death, and survival of microglial cells [189191]. Pathway for atp release is highly variable and includes connexin hcs, panx1 channels, volume - regulated anion channel (vrac), purinergic p2x7 receptor, and/or vesicular exocytosis [192195]. Moreover, mastocytes represent an abundant source of atp stored in granules that are released under activation conditions [196198] such as specific (e.g., ige + antigen) and nonspecific stimulation (e.g., stress, mechanic stimulation, and osmotic swelling). With regard to the participation of mastocytes in cns alterations, atp can be released by trauma - induced degranulation and thus stimulates adjacent neurites via p2x and p2y receptors . Additionally, the neuropeptide sp released from nerve terminals upon bradykinin stimulation participates in nerve mastocyte communication . This enables interactions between nerve and mast cells and initiates and represents the development of neuroimmunological synapses . Also, glial cells are involved in neuroimmune cross - communication, and atp induces glial cells to release il-1, tnf-, and il-33 . Therefore, atp released from mastocytes is an important autocrine / paracrine / exocrine factor that mediates cross - communication between different cell types . Moreover, human lad2 mast cells stimulated with ige, anti - ige, or substance p (sp) secrete mitochondrial particles, mitochondrial dna (mtdna), and atp in absence of cell death . Furthermore, mitochondria added to mast cells trigger degranulation and release of histamine, pgd2, il-8, tnf-, and il-1, and this response is partially inhibited by dnase and atp receptor antagonists . Only 30 min of immobilization stress can stimulate the hpa axis and cause degranulation in ~70% of rat dura mastocytes . This response could be triggered by neurotensin (nt) and crh acting on mastocytes increasing the permeability of the bbb [203206]. As mentioned previously, activated mastocytes release proinflammatory cytokines and atp among other bioactive compounds that promote microglia, and astrocyte activation and both reactive glia promote neuronal damage [123, 124]. Related to this, acute or chronic stress through gcs sensitizes microglia to a subsequent proinflammatory challenge, suggesting that stress should worsen the outcome of neuroinflammation . To our knowledge, it remains unknown if signal transduction of proinflammatory agents via tlrs and activity of hcs is enhanced by gcs or stress . Related to the issue presented previously, various neurodegenerative disorders present activation of microglia in different brain regions and restraint combined with water immersion induces massive microglial activation in the hippocampus, hypothalamus, thalamus, and periaqueductal gray matter [207, 208]. Although the precise mechanism of microglia activation induced by stress remains unknown, it is likely that bioactive molecules released by activated mastocytes (see what is mentioned previously) lead to the activation of microglia and, therefore, induce progression of neurodegenerative changes . In an ex vivo approach, rats were first pretreated in vivo with ru486 (gc receptor antagonist) and then exposed to an acute stressor (inescapable tail shock; is), and 24 h later, hippocampal microglia were isolated and stimulated with lps . Microglia obtained from rats not treated with a gc receptor antagonist showed an increase in gene expression of proinflammatory cytokines (il-1 and il-6). However, in rats pretreated with ru486, the sensitization of microglial to proinflammatory stimuli did not occur . Astrocytic signaling is potentiated by gcs (i.e., methylprednisolone and dexamethasone) via long - range calcium waves, and an increase is observed in resting cytosolic ca levels, as well as the extent and amplitude of calcium wave propagation (twofold) compared to control conditions . Furthermore, it is known that stress affects microglial function and viability during adulthood and early postnatal life . Experiments both in vitro and in vivo have shown that stress hormones can affect the function and viability of microglia . However, little is known if stress during pregnancy affects microglia of the offspring . In a recent report, prenatal stress effects on microglia of the offspring were studied . In this model, prenatal stress during embryonic days 1020 consisted of 20 min of forced swimming . In the offspring, a reduction in the number of immature microglia in the two main brain reservoirs of amoeboid microglia, corpus callosum, and internal capsule was observed . Moreover, accelerated microglial differentiation into ramified forms in the internal capsule and brain regions, such as the entorhinal cortex, parietal lobe neocortex, thalamus, and septum, was seen in the neonates in relation to an increase in plasma corticosterone in the pregnant dam . The stimulation of microglial tlr3 with its ligand leads to the release of il-6, il-12, tnf-, and ifn- among others (figure 1). In connection to this, the importance of tlr in various cns diseases (i.e., infection, trauma, stroke, neurodegeneration, and autoimmunity) has been described . This is how viral infections have been implicated in the onset of ms by stimulation of tlr3 . Additionally, in an animal model of schizophrenia, the stimulation of pregnant mothers with poly (i: c) results in reduced neuronal arborization of the offspring, which is correlated with a status of higher activation . Interestingly, cx hcs participate in neurite outgrowth and release of atp and glutamate, which also affect neuronal arborization [214, 215]. It is interesting to note that sensitivity to drug abuse behavior, as well the neuroinflammatory response to a subsequent proinflammatory challenge (as noted previously), is associated with stress and stress - induced release of gcs . Neuroinflammatory mediators derived from glia have an important role in the development of drug abuse . This is how neuroinflammatory mediators, such as proinflammatory cytokines, are induced by opioids, psychostimulants, and alcohol, all of which modulate many effects including drug reward, dependence, tolerance, and analgesic properties . An interesting aspect is that drugs of abuse may directly activate microglial and astroglial cells via tlrs, which mediate the innate immune response to pathogens . A key aspect is the timing of stress exposure relative to inflammatory challenge, and if a proinflammatory stimulus (e.g., lps) is added immediately before stress exposure, stress induces an anti - inflammatory effect, which is reflected in the inhibition of the increase in brain il-1 levels . The importance of stress associated with infections is given by the fact that the acute or chronic stress sensitizes the inflammatory responses of the cns to immunological challenges . Microglia show an increase in expression of mhc ii, tlr4, and the f4/80 antigens . Therefore, stress changes the microenvironment of the cns to a phenotype with inflammatory characteristics . One explanation to this phenomenon is that gcs sensitize microglia to infections [10, 218]. In peripheral blood monocytes from individuals under chronic stress, an increase in the expression of genes with promoter response elements for nf-b is observed as well as allows expression of genes that have promoter elements for gc receptors . Otherwise, in older stressed or chronically depressed adults, an increase in inflammatory response occurs when they are challenged with antigens, showing depressive characteristics and elevated levels of il-6 after immunization with influenza vaccines . Further evidence that supports this notion comes from observations in older caregivers of patients with dementia, who also presented an elevation of il-6 for over four weeks after vaccination with influenza vaccines, whereas this elevation was not observed in non stressed individuals . Furthermore, stress worsens immunity and brain inflammation, which is important in ms and neuropsychiatric disorders [221226]. Under stress, the neuropeptides crh and nt are secreted and thus can activate microglia and mast cells, which in turn release molecules with proinflammatory properties . This results in maturation and activation of th17 autoimmune cells and disruption of the bbb that leads to t cells entry into the cns enhancing the brain inflammation, which might support the pathogenesis of ms . Nt also stimulates secretion of vascular endothelial growth factor (vegf) and induces expression of crh receptor-1 in mast cells [20, 206, 227]. Several lines of evidence associate microglia with the pathogenesis of ms because activation of microglia is prominent and precedes t - lymphocyte infiltration and demyelination . Activated microglia release glutamate and no causing neuronal death and bbb disruption [228, 229]. With regard to the participation of mastocytes in the pathogenesis of ms, patients with this disease show elevated levels of tryptase (that activate microglia) and histamine in cerebrospinal fluid (csf) [68, 230] therefore, several lines of evidence suggest an important role of mastocytes and microglia in neuroinflammatory diseases . Therefore, both cell types represent therapeutic targets to be considered for treatment of ms and other neuroinflammatory diseases . Among the factors relevant to the development of autism spectrum disorders (asd), stress during pregnancy and the first 6 months of postnatal life has been associated with increased risk of asd . As mentioned previously, crh also activates mast cells, resulting in the release of several proinflammatory cytokines including il-6, which in turns may increase the bbb permeability [222, 234, 235]. Recently, a decrease in the mitochondrial function in approximately 60% of patients with autism has been demonstrated [236238]. The brain of these patients shows lines of evidence of neuroinflammation [239242], with high levels of mitochondrial dna . Additionally, elevated levels of nt that could activate mast cells have been detected in children with autism . The involvement of mast cells and brain inflammation is related to mitochondrial fission and translocation to the cell surface during degranulation, which leads to release of atp and mitochondrial dna . The importance of atp is that it can maintain inflammation by activating mast cells [225, 246]. This is how prenatal stress modifies the phenotype, distribution, and activation statuses of microglia in the offspring . Different stressors, together with the activation of the inflammatory immune response, enhance the effects of proinflammatory molecules or conditions, showing synergistic effects . Viral infections are the most common causes of infection during prenatal life, and maternal respiratory infection can also increase the risk of the offspring to develop certain mental disorders . The most direct evidence for this comes from a prospective study of pregnant women with medically documented respiratory infections, where the risk for schizophrenia in the offspring is increased 3-fold by infection in the second trimester . Evidence that supports this phenomenon comes from models of cocultures between astroglia and microglia treated with dexamethasone . In these experiments, functional membrane properties of astrocytes in cocultures are differentially regulated, which might reflect steroid effects in adjacent glial components in vivo . In cocultures with 30% microglia, dexamethasone - treated cocultures show significant increased gap junctional intercellular communication, which could facilitate the propagation of inflammatory signal along astrocytic networks . Therefore, if a stressor is sufficiently sustained, this may reflect neurochemical processes that can make the organism more vulnerable to pathological stimuli producing behavioral and neurochemical responses [250, 251]. This can be reflected in an increased susceptibility to diseases of the nervous system, such as the progression of depressive disorders and anxiety, and can even affect the course of neurological diseases [250, 251]. Furthermore, activated microglia affect the expression of cx hcs in astrocytes, which in turn increases the astrocytic atp and glutamate release with deleterious consequences on neurons . Therefore, these lines of evidence represent an aspect to be addressed in a model of stress in pregnant animals, in which one can analyze the effects of stress on microglia of the offspring in terms of activation and its effect on astrocytes, which could promote neuronal damage, with cx hcs and panx1 channels being possible therapeutic targets . Additionally, the synergistic effect of stress and stimulation with viral infection (for which rna viral mimics poly (i: c)) has not been studied in offspring of pregnant females subjected to stress, which is also a novel approach and can be correlated with a possible susceptibility of offspring to diseases of the nervous system . An important aspect is that when microglia are strongly activated, they remain in a preactivate state for years, which means that microglia are excessively responsive to even slight stimuli . This fact also has been linked to the activation of microglia by viral infections early in life and that can be later reactivated more rapidly compared to microglia in normal state [252, 253]. Therefore, the possibility of having microglia (using minocycline) and mastocytes activation (with grh - r antagonists) as therapeutic targets opens the possibility of their modulation as treatment for various neuropsychiatric disorders, viral infections, and other neuroinflammatory pathologies of the cns . In summary, parental stress is proposed to induce potentiation of neuroinflammatory responses by first: activating directly mast cells through crh recognition . Second: mast cells proinflammatory mediators prime microglia, astrocytes and olygodendrocytes, modifying their phenotype, distribution, and activation statuses in the offspring, but mainly promoting hc expression . Third: sensitized microglia exposed to inflammatory stimuli (i.e., tlr3 ligands) (figure 1) are activated and secrete cytokines (tnf-, il-1). They also show increased functional expression of panx1 channels and cx hcs through which atp and glutamate are released to the extracellular milieu . Astrocyte and oligodendrocyte become activated and release atp and glutamate in an hc depending way, and thus they promote neurodegeneration (figure 2).
Serious complications, such as renal scarring, hypertension, and chronic renal failure can result following a delay in diagnosis and treatment . The prevalence of renal scarring following febrile uti has been reported as 10% to 65% . Risk factors, including sex, not being circumcised, constipation, and vesicoureteral reflux (vur), increase the incidence of uti . Vur is the backflow of urine from the bladder to the ureter and, in some cases, to the pelvis and calyces . Previous studies suggest that the prevalence of vur in children ranges from 25% to 40% . Although voiding cystourethrography (vcug) is currently used as a reliable imaging method for diagnosing vur, it is painful and expensive and exposes the patient to radiation . Considering the side effects of vcug, the lack of vur in more than 50% of children with uti, and the spontaneous recovery from low - grade vur, researchers have sought cost - effective noninvasive markers for predicting vur . Soylu et al . Reported that fever higher than 38 and c - reactive protein (crp) of more than 50 mg / dl were suitable predictive markers for the presence of vur and high - grade vur, respectively . In this regard, the present study was conducted to determine the predictive value of clinical, laboratory, and imaging findings in the diagnosis of vur in children with their first febrile uti . This prospective cross - sectional study examined 153 children aged 1 month to 12 years with their first diagnosed febrile uti in qazvin's children's hospital, qazvin, iran, in 2012 through 2013 . This hospital is the only referral hospital for children in qazvin province that is affiliated with the qazvin university of medical sciences . The sample size was calculated on the basis of p=58% (sensitivity for feve r>38.5 to discriminate patients to correct groups), d=0.08, =0.05, 1-=0.95, =0.2, and 1- (statistical power of study)=0.8 and by using the following equation: consecutive sampling continued until the desired sample size was reached . The inclusion criteria for children with febrile uti were as follows: (1) first febrile uti; (2) having symptoms of febrile uti, such as fever, chills, vomiting, diarrhea, and irritability in infants, and fever, vomiting, abdominal and flank pain, dysuria, and frequency in children; (3) abnormal urinalysis (the presence of leukocyturia, a positive urinary nitrite test, etc . ); (4) positive urine culture (urine culture more than 110 colonies of a single pathogen in a midstream urine sample or clean catch method or 110 colonies of a single pathogen via urinary catheterization, or presence of any number of colonies of organism in urine culture taken by suprapubic method); (5) performance of renal ultrasound, dimercaptosuccinic acid (dmsa) renal scanning, and vcug . Children meeting the following criteria were excluded from the study: (1) using antibiotics; (2) failing to undergo vcug; (3) having accompanying and underlying disease, such as septicemia and immune disorders; and (4) having structural abnormalities of the urinary system (such as ureteropelvic junction obstruction, neurogenic bladder, etc .) Except vur . First, the symptoms of the disease were recorded, and, before the start of antibiotic therapy, serum samples were delivered to the laboratory to test white blood cell count, neutrophil count, platelet count, erythrocyte sedimentation rate (esr), and crp quantitative level . All laboratory examinations were performed by use of standard methods in the laboratory department of qazvin children hospital . The renal ultrasound was performed within the first 48 hours of admission, the renal vcug was performed at the end of treatment when the patients were discharged from the hospital, and the dmsa renal scan was done in the first week of admission . The ultrasound and vcug were carried out by a radiologist, and the dmsa renal scan was performed and interpreted by a nuclear medicine specialist . Any report of hydronephrosis or hydroureteronephrosis without evidence of mechanical obstructions, such as ureteropelvic junction obstruction, ureterovesical junction obstruction, and posterior urethral valves, in the renal ultrasound, and any report of reduced uptake on the dmsa renal scan for pyelonephritic changes in the kidneys were considered as suspicious for vur . According to the results of the vcug, the patients were divided into two groups: a group with vur and a group without vur . The severity of vur was graded according to the international study of reflux in children . Grades 1 and 2 were regarded as low - grade vur, and grades 3, 4, and 5 were regarded as high - grade vur . The sensitivity, specificity, positive (ppv) and negative predictive value (npv), positive (lrp) and negative likelihood ratio (lrn), and accuracy of the clinical, laboratory, and imaging variables for diagnosis of vur were determined . Chi - square test, exact test, t - test, and nonparametric tests (mann - whitney test) were applied to analyze the obtained data . The children were included in the study after their parents agreed and signed the informed consent form . Of the 153 studied patients, the male - to - female ratio was 18:135 . The most frequent symptoms in decreasing frequency were fever (100%), chills (62.7%), and dysuria (42.5%). The most frequently grown microorganism in the urine culture was escherichia coli (80.3%). Of the 153 studied patients, vur was observed in 60 (39.2%). Comparisons of the different variables between children with and without vur and also between the low - grade and high - grade vur groups are shown in tables 1, 2, 3, 4 . By use of receiver operating characteristic curve analysis, it was shown that for predicting vur in children with febrile uti, crp20 mg / dl had a sensitivity of 61% (95% confidence limit [cl], 49 - 74), specificity of 57% (95% cl, 46 - 67), lrp of 1.43, and accuracy of 58% . In addition, fever38.2 had a sensitivity of 60% (95% cl, 47 - 72), specificity of 53% (95% cl, 42 - 62), lrp of 1.26, and accuracy of 55.5% (table 5). The sensitivity, specificity, lrp, and accuracy of the dmsa renal scan for predicting vur were 63% (95% cl, 51 - 75), 96% (95% cl, 91 - 99), 14.7, and 79.5%, respectively . Also, those of the renal ultrasound were 30% (95% cl, 18 - 41), 96% (95% cl, 85 - 97), 3.4, and 60.5%, respectively . The multivariate logistic regression analysis revealed significant positive correlations between fever>38.2 and dmsa renal scan and vur, and also between esr, positive urinary nitrite test, hyaline cast, and ultrasound and high - grade vur (table 6). This study showed that the best predictive markers for the presence of vur in children with their first febrile uti are fever>38.2 and dmsa renal scan . In addition, for high - grade vur, esr, positive urinary nitrite test, hyaline cast, and ultrasound were the best predictive markers . Given than only 25% to 40% of children with uti have vur and that vcug is invasive and expensive and exposes the gonads to radiation, researchers have looked for noninvasive markers for predicting vur to avoid unnecessary vcug . 's study on 88 children with febrile uti revealed a significant difference between two groups with and without vur in terms of fever38.5, pyuria25/high power field, and crp23.5 mg / l . However, logistic regression analyses showed that only fever38.5 was an appropriate predictor of the presence of vur . Moreover, the above researchers revealed that crp50 mg / dl was a suitable predictor for the presence of high - grade vur . Performed a study on 140 children less than 5 years old with their first febrile uti and assessed variables including age, sex, and family history of uti, crp, and renal ultrasound . Their clinical approach yielded a sensitivity of 100% and specificity of 17% for predicting all vur grades and a sensitivity of 100% and specificity of 38% for predicting vur gradeiii . A similar study conducted by leroy et al . On 149 children aged 1 month to 4 years with their first febrile uti the results of the present study were somewhat similar to those of the studies by soylu et al . And oostenbrink et al . . Although the quantitative crp level, rbc count in urine, results of renal ultrasound, and dmsa renal scan showed a significant difference between the groups with and without vur in the present study, the multivariate logistic regression analysis revealed a significant positive correlation between fever>38.2 and renal dmsa scan and vur . Also, although there was a significant difference between the low - grade vur and high - grade vur groups regarding the neutrophil count, esr, leukocyturia, positive urinary nitrite, and ultrasound results, the multivariate logistic regression showed a significant positive correlation of high - grade vur with esr, positive urinary nitrite test, hyaline cast, and renal ultrasound . In a study by tseng et al on 142 children less than 2 years old with their first febrile uti, the authors reported that the sensitivity, specificity, positive predictive value, and negative predictive value of dmsa renal scan in predicting vur were 88%, 37%, 36%, and 88%, respectively . The above authors concluded that children with a normal dmsa renal scan rarely had vur and never had high - grade vur . A study conducted by camacho et al . On 152 children with their first febrile uti showed that vur was more frequent in children with an abnormal dmsa renal scan than in children with a normal dmsa renal scan (48% vs. 12%). The above researchers concluded that the risk of renal damage was very low in children with their first febrile uti and a normal dmsa renal scan . Another study pointed out the predictive value of dmsa renal scan and renal ultrasound in the diagnosis of high - grade vur . That study reported the detection rate of ultrasound for low- and high - grade vur to be 86% and 41.7%, respectively, and that of the dmsa renal scan for low- and high - grade vur to be 88.4% and 37.5%, respectively . On the contrary, sorkhi et al . Reported that the dmsa renal scan alone or along with renal ultrasound could not predict vur . Therefore, they argued that vcug must be done to diagnose vur . In the present study, the highest lrp was respectively related to the dmsa renal scan, renal ultrasound, crp20 mg / dl, and fever higher than 38.2. the dmsa scan had high sensitivity and specificity for the diagnosis of vur . In this respect,, it can be concluded that in the case of a normal dmsa scan, the risk of vur is very low, and performing vcug is unnecessary . Although some studies mentioned procalcitonin as a predictor of vur, the relevant test is more costly than other routine tests, such as crp and esr, and is not available everywhere . We hope that the results of the present study will help to avoid unnecessary vcug in children affected by their first febrile uti . Given that the present study was conducted in one educational hospital, further multicenter studies are recommended . This study revealed that the best predictive markers for the presence of vur in children with their first febrile uti are the fever>38.2 and dmsa renal scan . Esr, positive urinary nitrite test, hyaline cast, and renal ultrasound are best predictive markers for the presence of high - grade vur.
Today, the treatment of choice is surgical excision of the tongue, but before 1900, surgery was often temida1 . Only in 1673 niels the size and shape of the teeth are directly influenced by the size of tongue3 . The shape of the teeth is determined by forces employed on the teeth, especially the muscles of the tongue, lips and cheek . Due to the effects caused by the aesthetic and functional macroglossia the macroglossia is classified as true when there is excessive enlargement of the language and a relative when there is an imbalance between the size of the tongue and oral cavity, resulting in insufficient space for organ4 . The goal is to establish technical bases associated with partial glossectomy orthodontic treatment of dentofacial deformity in patients with macroglossia . Three patients underwent orthognathic surgery associated with partial glossectomy from 1995 to 1999, a multidisciplinary team - doctor and dentist . All patients had macroglossia relative and underwent clinical assessment taking into account the respiratory function, swallowing and speech deficit, as well as changes in dental occlusion and was also performed radiological evaluation . The main problem was manifested by the presence of steep lower dent alveolar, destabilizing the use of dentures causing joint dysfunction with severe pain . She underwent a single surgical intervention in the partial glossectomy and orthognathic surgery for targeting sub apical segment dent alveolar anterior inferior . The main problem of the second patient was the anterior open bite accompanied by difficulty in breathing and articulation of words . After treatment for orthodontic tooth alignment leveling, underwent surgery for a single suspension by corticotomy posterior maxillary le fort i type, reduction of mandibullary prognathism by sagittal technical branches, targeting sub apical posterior - inferior right and partial glossectomy . The third patient had mandibullary prognathism and, during orthodontic treatment in preparation for orthognathic surgery, the surgery was anticipated, since the interposition of the tongue between the back teeth did not allow the evolution of orthodontic treatment . He underwent a partial glossectomy to reduce the transverse diameter of the tongue and mandibullary prognathism by sagittal technical branches . We used rigid skeletal fixation with titanium plates and screws so that patients could stay without intermaxillary block in the immediate postoperative period . To control tongue edema, the technique used consist of segmental resection along the median raphe of the tongue and suture by planes - figure 1 . As a routine the symptoms regressed completely and all skeletal segments remained stable . Demarcation and resection of the lingual and final appearance . The classification of macroglossia is not yet consensus in the literature . According to shafer (1968)5, or primary congenital macroglossia is due to the excessive development of the musculature, which may or may not be associated with generalized muscular hypertrophy or unilateral hypertrophy . Since the secondary macroglossia may occur as a result of a tumor in the tongue, as a diffuse hemangioma or lymphangioma, neurofibromatosis, and occasionally blocking efferent vessels in cases of malignant neoplasm of the tongue . Wolford et al . (1996)6 refer to as macroglossia on pseudomacroglossia, separating it from the true macroglossia . (1980)7 macroglossia consider the functional as a third classification, occurring when the language does not fit into the oral cavity after a surgical procedure . The true when there are histological abnormalities associated with the increase of the tongue, such as vascular malformation, stretching and tumors . Relative macroglossia includes cases of apparent increase in volume without an explanation of the language exam, as in down syndrome . The decision to refer the patient to partial glossectomy should be based on the volume of the language, mobility, position, function, symptoms, speech intelligibility, skeletal anterior open bite, interference in orthodontic treatment, drooling, swallowing and tongue recurrent trauma9 . The language has increased in volume expansive effect on the lower dental arch, being blamed as the cause and maintenance of open bite, bimaxillary protrusion or diastemas10 . Being interposed between the arches, is an important etiologic factor for malocclusion listed (figures 2 and 3). A partial glossectomy performed simultaneously with mandibullary osteotomy for treatment of patients with mandibullary prognathism and anterior open bite is advantageous to prevent recidivas11 . The tongue can cause deformity increased dental - muscle - skeletal, instability in orthodontic treatment and orthognathic surgery, masticatory disability, communication problems and respiratory6 . There are several clinical and radiographic findings, but not all features are always present and their existence is not necessarily path gnomonic for the diagnosis of macroglossia . Should be included the clinical, radiographic and functional for the interference with speech, mastication, airway and stability of orthodontic treatment and orthognathic surgery . There are basically three choices in the surgical sequence: (i) stage 1: partial glossectomy, stage 2: orthognathic surgery (ii) stage 1: orthognathic surgery, stage 2: partial glossectomy and (iii) partial glossectomy and orthognathic surgery in a single stage surgery.
However, at times, conventional laryngoscopy and intubation may not be possible and alternative techniques of airway management such as supraglottic airway devices (sads) may be required . Both classic laryngeal mask airway (clma) and proseal laryngeal mask airway (plma) have been successfully used for securing a patent airway; however, due to high - cuff pressure - related complications, they can cause mucosal damage, sore throat, hoarseness and nerve palsies . The i - gel has thermoplastic elastomer gel with non - inflatable cuff and a channel for gastric suction catheter placement that precisely mirrors perilaryngeal anatomy, thereby no cuff inflation is required . Studies are now focusing on sads, which can provide ventilation with low peak airway pressure, higher compliance, low resistance and high leak pressure so that they can provide wider margin of safety for ventilation . The primary aim of the present study was to compare oropharyngeal leak pressure of size 2 i - gel and plma for airway management in paediatric patients . The secondary outcomes measured were number of insertion attempts, insertion time, ease of insertion of size sgds (2 i - gel or plma) and orogastric catheter, quality of initial airway, fibre - optic grading and pulmonary mechanics . This prospective, randomised controlled study was conducted during april 2013april 2014 after approval of the institutional ethics committee (gmc / ta i [19d]/2013/05428/22) and registration in the clinical trial registry india (ctri/2013/08/003898). After parental written informed consent, 100 children of american society of anesthesiologists physical status i and ii of either sex, aged between 2 and 6 years, weighing between 10 and 30 kg, scheduled for elective surgeries of <1 h requiring general anaesthesia (ga) with controlled ventilation were enrolled . The exclusion criteria were patients with upper respiratory tract infection, anticipated difficult airway, non - fasting status and cardiorespiratory disease . All patients were kept fasting for 46 h and premedicated with 0.3 mg / kg midazolam syrup 30 min before surgery . Ga was induced with sevoflurane (inspired concentration 46%) with 50% nitrous oxide in oxygen, and then an intravenous (i.v .) Access was established which was followed by fentanyl 2 g / kg, i.v . After checking for mask ventilation, neuromuscular blockade atracurium besylate 0.5 mg / kg . The patient's lungs were ventilated with a facemask for 3 min to allow for full relaxation of the jaw before placing the device . Patients were allocated just before device insertion to either size 2 plma or i - gel (50 patients each) based on sequentially computer - generated numbers concealed in opaque sealed envelopes . The anaesthesiologist inserting the device could not be blinded, but the assessor anaesthesiologist and patient were blinded to the group allocation . The anaesthesiologist who inserted either of the two airway devices had performed at least 50 plma and 20 i - gel insertions . Size 2 i - gel was inserted by firmly holding the device such that the cuff outlet was facing the chin of the patient, and it was then guided along the hard palate until definitive resistance was felt . The insertion of plma was performed as per the manufacturer's recommendation, using the introducer technique . A lubricated orogastric tube (ogt) was inserted through the drain tube after insertion of sgds . Correct ogt placement was determined by suction of fluid or detection of injected air by listening with a stethoscope over the epigastrium . The plma or i - gel was then connected to the circle system of anaesthesia machine (aestiva 5 7900, ge healthcare, datex - ohmeda division, helsinki, finland) using paediatric circle system . Effective ventilation of the device was judged as a square wave capnograph trace and bilateral chest movements on gentle manual ventilation . In the event of partial or complete airway obstruction or a significant air leak, a maximum of three insertion attempts were allowed before the device was considered a failure . The time interval between picking up the i - gel or plma and obtaining an effective airway was recorded as insertion time . The ease of insertion was graded as very easy if the device could be inserted without any manipulation, easy if there was only one manipulation required and difficult if any difficulty more than that . The quality of the initial airway was assessed during manual ventilation, with the pop - off valve set to limit peak airway pressure (pip) to 20 cm h2 o. the initial airway was judged as follows: excellent no audible leak; good an audible leak with relevant loss of air but sufficient ventilation, as indicated by an etco2 <40 mm hg and poor clinically relevant loss of air and insufficient ventilation, requiring repositioning or replacement of the device . Air entry in the stomach and abnormal airway sounds over the larynx on auscultation were noted . After obtaining an effective airway, the oropharyngeal leak pressure was determined by closing the expiratory valve of the circle system at a fixed gas flow of 3 l / min and the airway pressure (maximum allowed 40 cm h2 o) at which equilibrium reached was observed . Detection of an audible gas leakage at mouth and auscultation of gas leakage in the anterior neck was also performed in both the groups . The patient's lungs were ventilated with a tidal volume of 57 ml / kg, and the respiratory rate was adjusted to maintain etco2 of 3540 mm hg with inspiratory: expiratory ratio of 1:2 . After successful insertion of the device, fibre - optic assessment of airway tube (pentax medical singapore pvt . Ltd ., 438a alexandra road, singapore) was obtained by passing fibrescope through the airway tube . The view was graded as 1 = vocal cords not seen; 2 = vocal cords and anterior epiglottis visible; 3 = vocal cords and posterior epiglottis visible; 4 = only vocal cords visible . Pulmonary mechanics including compliance, resistance, mean airway pressure, peak airway pressures as shown on anaesthesia workstation were recorded in both the groups at 2 min and 5 min after device insertion . We recorded any device failure, intraoperative displacement, gastric insufflation, regurgitation, laryngospasm, bronchospasm and airway obstruction . Data about fibre - optic position of the airway tube, ease of insertion of sgd, ease of ogt insertion, quality of initial airway, failed passage into the pharynx, malposition and the cause of failure were evaluated by the anaesthesiologist performing airway device insertion . The assessor anaesthesiologist collected data regarding oropharyngeal leak pressure, insertion time, ventilation and pulmonary mechanics . Secondary outcomes included number of attempts, ease of device and ogt insertion, fibre - optic view and pulmonary mechanics . After the completion of surgery, anaesthesia was discontinued and residual neuromuscular blockade was antagonised with neostigmine methyl sulphate 0.05 mg / kg and glycopyrrolate 0.01 mg / kg . The sample size was calculated considering a projected difference of 30% between the two groups for airway leak pressures to be significant, at 95% confidence limits, a type 1 error of 0.05 and a power of 80% . The data were analysed using international business machines corporation spss statistics for windows (version 22.0, armonk, ny). Kolmogorov non - parametric data were compared with fisher's exact test or chi - square test . Data were presented as mean standard deviation with p <0.05 as statistically significant . A total of 122 children were screened, 10 children were excluded due to upper respiratory tract infection and in 12 children the parents refused to participate in the study . There were no differences in demographic characteristics of patients between the groups as shown in table 1 . The oropharyngeal leak pressure in i - gel group was 29.5 2.5 (95% confidence interval [ci], 2830) cmh2 o as compared to 26.1 3.8 (95% ci, 2427) cmh2 o in plma group (p = 0.002) [table 2, figure 2]. Insertion time was shorter for i - gel as compared to plma and none of the patients had failures in the insertion of sads . The number of attempts, ease of insertion of sgds and fibre - optic grading of airway tube were comparable as shown in table 2 . The quality of initial airway was better in i - gel as compared to plma (p = 0.018). Pulmonary mechanics including peak airway pressure, mean airway pressure, resistance and compliance were similar in both the groups as shown in table 3 . Success rates of ogt placement and fibre - optic grading were similar in both the groups . None of the patients experienced any adverse event including intraoperative displacement of sads, gastric insufflation, regurgitation, laryngospasm, bronchospasm and airway obstruction in both the groups . Patient baseline demographic characteristics airway characteristics of supraglottic devices oropharyngeal leak pressure of i - gel versus proseal laryngeal mask airway in paediatric patients undergoing general anaesthesia with controlled ventilation airway parameters after placement of supraglottic airway device the study exclusively compared oropharyngeal leak pressure of size 2 i - gel with size 2 plma and pulmonary mechanics in paediatric patients under ga with controlled ventilation . We chose size 2 to bring uniformity in the use of two devices in children aged 26 years weighing 1030 kg as the recommended weight range for the size 2 plma is 1020 kg, whereas it is 1025 kg for the i - gel. The results of our study demonstrated higher leak pressure in size 2 i - gel as compared to size 2 plma. This could be attributed first to the unique non - inflatable cuff of i - gel, which mirrors the perilaryngeal anatomy appropriately . Second, the leak pressure of i - gel appears to improve with time due to thermoplastic material, which forms a more efficient laryngeal seal after warming to body temperature . To obviate this effect, we measured the leak pressure after 5 min of correct placement of i - gel . Currently, some studies show higher leak pressure in i - gel as compared to plma while others show similar leak pressure with both the devices . In a meta - analysis, i - gel was compared with several types of laryngeal mask airways (lmas) in children and authors reported no evidence for differences in rate of insertion at first attempt, insertion time, ease of insertion or gastric tube insertion . The mean oropharyngeal leak pressure was 3.29 (2.254.34) cm h2 o higher with i - gel as compared to other lmas . In an another meta - analysis, i - gel was found to be equally effective and provided a significantly higher leak pressure as compared with plma and clma . However, in these studies, authors used different sizes of sads and none of these studies compared size 2 i - gel versus plma in paediatric patients undergoing surgery under ga with controlled ventilation . In the present study, time taken for successful insertion of i - gel was less as compared to plma group . This difference was first due to the less flexible stem in i - gel as compared to plma and second due to the presence of prefilled elastomeric cuff in i - gel allowed to omit the step of inflating the cuff . This is in contrast with saran et al ., who found comparable insertion times with both the devices . This might be attributable to different techniques of insertion of plma as saran et al . The insertion time for i - gel group in the present study was shorter as compared to previous studies . Success rate, number of insertion attempts, ease of sgd and ogt insertion was found to be comparable in both the groups . The quality of initial airway in the present study was superior in i - gel as compared to plma, which are similar to other studies . Our study showed similar fibre - optic grading in both the groups, which was consistent with other studies . The present study found similar pulmonary mechanics including resistance, compliance, peak airway pressure and mean airway pressure in both the groups . Saran et al . Measured only peak airway pressure and so far no other study has measured pulmonary mechanics in detail . In the present study, there were no significant haemodynamic changes on comparing i - gel and plma and were accordance with published literature . The present study found similar margin of safety for ventilation in i - gel as compared to plma in patients weighing between 10 and 30 kg receiving ga with positive pressure ventilation . The novelty of the study is firstly, higher oropharyngeal leak pressure in i - gel as compared to plma which ensures wider safety margin for ventilation in children 26 years weighing 1030 kg and secondly, comparison of pulmonary mechanics in these children on controlled ventilation with size 2 i - gel or plma, which has not been reported earlier in literature . First, the study involved patients with a normal airway and whether the same outcome can be extrapolated to patients with difficult airway is subject to performance of similar large - scale studies in patients with difficult airway . Second, the blinding was not possible for the anaesthesiologist inserting the device and fibre scope . Third, the airway device insertion was done under muscle relaxant effect, so the results are not necessarily the same for spontaneously breathing and less deeply anaesthetised patients . Fourth, our findings only apply to use of the size 2 devices in children aged 26 years weighing 1030 kg . Size 2 i - gel exhibited superior oropharyngeal leak pressure and quality of airway in paediatric patients with controlled ventilation as compared to same sized plma although the pulmonary mechanics were similar.
Aging is the most prominent risk factor for the occurrence of neurodegenerative diseases among others, including oxidative stress (keller et al ., 2005; jain et al ., 2011), telomere length (harris et al ., 2006), genetic mutations (anderton et al ., 2002) and head injury (maiese et al ., 2008). In the united states there are over 35 million of people with a mean age of 65 years and even older, that mainly die from age - related diseases (drago et al ., aging increases susceptibility of people to environmental stressors, thereby increasing the chance to develop neurodegenerative conditions, most likely because the self - repair ability is compromised and tissues and/or organs undergo a progressive decline (musumeci et al ., 2014a). The aging process is associated with a number of structural, biochemical, functional and neurocognitive changes in the brain . The structural changes include expansion of cerebral ventricles, regional decreases in cerebral volume (raz et al ., 2005), loss of neural circuits and reduced brain plasticity (burke and barnes, 2006; kolb and gibb, 2011), thinning of the cortex (shahani et al ., 2006), decrease in both of the grey and the white matter volume (bartzokis, 2011), changes in neuronal morphology (sowell et al ., 2003) and formation of neurofibrillary tangles (hedden and gabrieli, 2004; neill, 2012). Among the age - related biochemical changes significant decreases in dopamine receptors d1, d2, and d3 (wang et al ., 1998; kaasinen et al ., 2000) and decreasing levels of different serotonin receptors and their transporters such as 5-hydroxytryptamine transporters (5-htts) (chang and martin, 2009; chang et al ., 2009) have been repeatedly reported . Among the neuropsychological changes, alterations in orientation (benton et al ., 1981) and memory (hof and morrison, 2004) are the most common ones . Moreover, many age - related neurodegenerative diseases are characterized by accumulation of disease - specific misfolded proteins in the central nervous system (cns) (van ham et al ., 2009). These include -amyloid peptides and tau / phosphorylated tau proteins in alzheimer's disease (ad), -synuclein in parkinson's disease (pd), superoxide dismutase (sod) in amyotrophic lateral sclerosis (als) (durham et al ., 1997), and mutant huntingtin in huntington's disease (hd) (scherzinger et al ., 1997). The association between age and protein misfolding is not clear yet, but it is probably related to alterations of molecular mechanisms triggered by aging cells, such as telomere shortening, cells shrinkage and decline of quality control over protein synthesis mechanisms (hung et al ., 2010; thanan et al ., 2014) schematic representation illustrating some of the most common risk factors that contribute to the onset and/or progression of neurodegenerative diseases and the related mechanisms driving the neurodegenerative process . Telomeres are an evolutionarily conserved repetitive nucleotide sequences (ttaggg) localized at the end of each chromosome, that are folded into a t loop structure by a protein complex called shelterin (stewart et al ., 2012). Telomeres play four fundamental roles: protecting genetic information from erosion during dna replication; protecting dna from damage; serving as a binding site for dna repair proteins; and providing information about the cell proliferation history (stewart et al ., 2012; musumeci et al ., 2015; giunta et al ., the telomere length is the sand glass of the cell since it specifies the number of divisions a cell can undergo before it finally dies; thus, it indicates the cell proliferative potential . Telomere shortening leads to the attainment of the so - called hayflick limit, which indicates the transition of cells to the state of senescence . Following this step, cells progressively enter a state of crisis, which is accompanied metabolic disturbances that culminate in massive cell death . Telomerase plays a pivotal role in the pathology of aging and cancer by maintaining genome integrity, controlling cell proliferation, and regulating tissue homeostasis . Telomerase is essentially composed of an rna component, the telomerase rna or terc, which serves as a template for telomeric dna synthesis, and a catalytic subunit, telomerase reverse transcriptase (tert). The canonical function of tert is the synthesis of telomeric dna repeats, and the maintenance of telomere length . However, accumulating evidence indicates that tert may also exert some fundamental functions that are independent of its enzymatic activity (verdun and karlseder, 2007) (please refer to figure 2). A reduction in telomerase expression contributes to telomere shortening in mitotic cells, while high levels of the enzyme in mesenchymal stem cells (mscs) contribute to their the phenomenon of telomere shortening is closely associated with aging itself, but it has been widely demonstrated that cells can also undergo premature aging due to several factors such as oxidative stress, inflammation and infections, which are able to speed up this process and determine age - related dysfunctions (hung et al ., 2010; jenny, 2012; kong et al ., 2013; kota et al ., therefore, given the involvement of these factors (and in particular of oxidative stress) in the development of neurodegenerative / age - associated diseases, it becomes of primary importance to also gain more insights on the underlying mechanisms triggered by these stressors, as this could serve to improve current therapeutic strategies based on the use of mscs to treat neurodegenerative conditions . The telomerase is composed of an rna component, telomerase rna or telomerase rna component (terc), which serves as a template for telomeric dna synthesis, and a catalytic subunit, tert . Tert besides its canonical function in telomere elongation has also a role as a transcriptional modula - tor of the wnt--catenin (-cat) signalling pathway . Tert acts as a cofactor in the -cat transcription complex; in this complex, tert interacts with brg1, a chromatin remodeling factor, to regulate the wnt/-cat signalling pathway . Tert is not only acti - vated by the wnt/-cat pathway, but -cat could also be directly regulated by tert induction, which results in maintenance of telomere length . In the mitochondria, tert also plays a role in regulating apoptosis in - duced by oxidative damage of mitochondrial dna (mtdna). Cen: centromere . A reason why telomeres are the preferred targets of oxidative insult seems to be primarily related to their dna composition, which tends to be rich in guanine residues (coluzzi et al ., indeed, the high incidence of guanine bases promotes the generation of alterations to dna bases to species called 8-oxoguanine (8-oxog), which, if not repaired, may lead to single or double strand breaks, mutations or even genomic instability (grollman et al ., 1993). Of interest, genomic instability, oxidative stress and ageing are not to be considered as independent causative factors in telomere shortening, but need to be considered as interconnected phenomena . Consistent with this theory, convergent data has identified an accelerated wnt/-catenin cascade activation as a common denominator triggered by these insults . Activation of this pathway reduces mscs proliferation potential, hampers telomerase activity and drives a cellular shift of mscs towards a differentiated / senescent phenotype (as elegantly reviewed by fukada et al .,, it is auspicable that strategies aimed at dampening the occurrence of these detrimental events in neurons or to block the wnt/-catenin intracellular pathways could have the potential to significantly impact the senescent process, including premature telomere shortening . Early neuronal cell death is a feature of neurodegenerative disorders and reduced telomere length has been associated with premature cellular senescence . Studies have shown that reduced telomere length in peripheral blood is associated with the incidence of illnesses associated to the aging phenotypes, such as dementia (thomas et al ., 2008), neurodegenerative disorders such as hd and genetic neurovascular diseases such as ataxia telangiectasia (at) (metcalfe et al ., 1996; kota et al ., since ltl is reflective of global cellular morbidity and mortality, it has been proven that it could be used as a useful tool to screen neurodegenerative disorders (sahin and depinho, 2012). It is worth emphasizing, however, that leukocytes include diverse cell populations that play complementary roles in tissue homeostasis and responses to infections and diseases, and the possibility exists that simply monitoring ltl may lead to misleading results . Indeed, the three major classifications of leukocytes are granulocytes, lymphocytes, and monocytes . These populations have different telomere lengths and erosion rates as a result of differences in telomerase activity, proliferation history, and telomere trimming (stewart et al ., 2012). These differences have to be carefully taken into account when considering a possible study of age - related processes in neurodegenerative disorders . However, a recent study has consistently showed that the ltl was reduced in individuals suffering from neurodegenerative disorders as described above, suggesting that the phenomenon of telomere shortening could at least be partly implicated or could contribute to the triggering of pathological pathways activated in these diseases (kota et al ., the reduced telomere length has been attributed to oxidative stress, aberrations in mitochondrial homeostasis, deficient dna repair mechanisms, and decreased dna methylation status (von zglinicki, 2002; blasco, 2007; gackowski et al ., 2008; copped and migliore, 2009; van groen, 2010; sahin and depinho, 2012). Interestingly, a review of data from literature concerning the potential use of ltl as a biomarker in ad and pd showed to be inconsistent in both cases, since the number of studies reporting no association between ltl and disease states almost overlapped the ones indicating a correlation between ltl shorthening and neurodegeneration (eitan et al ., 2014). Interestingly enough, a recent study reported an even longer ltl in pd patients, associating short telomeres with reduced risk of pd (schrks et al ., 2014). The reason of these inconsistencies could be dependent on the population number used in these studies, as the low number of patients together with the inter - individual variability may often result in significantly reduced statistical power, and purportedly to unreliable results . It has been shown that variability in ltl in individuals can be induced by different factors such as chronic stress, diet, lifestyle, chronic inflammation state and hormone levels (liu et al ., 2010; broer et al ., the interaction between these factors and genotype can also play a role in ltl variability (takata et al ., 2012). Certainly, further investigation in this field are needed to clarify the precise role and diagnostic or therapetic potential of ltl in neurodegeneration . The limited regeneration power of the cns represents a major challenge for the development of new therapeutic strategies efficacious to promote its functional repair . Mscs have been proposed as a viable therapeutic tool for degenerative disorders as they possess high proliferative ability and they are able to differentiate into multiple lineages (mobasheri et al ., 2014; musumeci et al ., 2014c; tanna and sachan, 2014). Mscs can differentiate into neuron - like cells and determine a paracrine effect by modulating the plasticity of damaged host tissues; by secreting neurotrophic and survival - promoting growth factors that inhibit apoptosis and promote neurogenesis, glial scar formation, immunomodulation, angiogenesis and neuronal and glial cell survival; by restoring synaptic transmitter release; by integrating into existing neural and synaptic networks; and by re - establishing functional afferent and efferent connections (siniscalco et al . In addition, low immunostimulating and high immunosuppressive properties make mscs a suitable source for cellular therapy (abumaree et al ., 2012; kwon et al ., 2014). Another point in favor to mscs employment in therapy is that cells can be transplanted directly without any prior genetic modification or reprogramming, and are able to migrate to the tissue injury sites (amado et al ., 2005). Mscs have also been proven to be useful for the treatment of pathologies in which tissue damage is caused by oxidative stress and thus in those pathologies linked to stress - induced telomere shortening and premature aging, where mscs are likely to be more resistant to oxidative insult than normal somatic cells (benameur et al ., 2015). This feature is particularly important since it makes mscs an interesting and testable model for the treatment of age - related neurodegenerative disorders . Currently, there is a great interest towards the use of mscs in pioneering therapies aimed at treating chronic and progressive neurodegenerative diseases, which are currently incurable and whose attempts to find disease - modifying therapies have failed, such as ad, pd, als and hd . It has been shown that after transplantation into the brain, mscs promote neuronal growth, decrease apoptosis, reduce the levels of free radicals, stimulate the formation of new synaptic networks from damaged neurons by supporting axonal outgrowth, modulate neuroinflammatory activities and promote proteosomal degradation of ubiquitinated misfolded proteins (caplan and dennis, 2006; mezey, 2007; uccelli et al ., 2011). Through paracrine mechanisms, mscs are also able to interact with neighbouring damaged host cells and influence their microenvironment, by sharing proteins, rnas and even mitochondria (spees et al ., 2006; olson et al ., 2012). As a proof - of - concept, mazzini et al . Demonstrated that mscs can decrease motor neuron cell death through paracrine actions when implanted into the cns of als patients (mazzini et al . Recently, the paracrine properties of bone marrow - derived mscs (bm - mscs) have been also shown in rat model of ad, suggesting their potential therapeutic role in this disease (salem et al ., 2014). The potential efficacy of human mscs (hmscs) has been also confirmed recently, as treatment succeeded to ameliorate some behavioral defects observed in a rodent model of hd, hence demonstrating that xenologous transplantation of hmscs could be considered a potentially successful approach to counteract neurodegeneration caused by hd, and perhaps other cns disorders (hosseini et al ., 2014). Mscs can be readily isolated from various tissues, show high plasticity and are capable to differentiate into many functional cell types (woodbury et al ., 2000; numerous studies have shown that bm - mscs can differentiate into cells that display neuronal or even dopaminergic characteristics both in vitro and in vivo (ni et al ., 2010; zeng et al ., 2011) a recent study reported that mouse bm - mscs provided neuroprotection by secreting a key factor, prosaposin, a molecule capable of rescuing mature neurons from apoptotic death . The secretome of bm - mscs showed to reduce toxin - induced cell death in cultures of rat pheochromocytoma cells, human rencell cortical neurons, and rat cortical primary neurons (li et al ., 2010). Unfortunately, the medical procedure to obtain bm - mscs from the bone marrow is invasive and definitely painful to patients . Therefore, efforts have been made to find more practical alternatives . Indeed, recently other mscs sources have gained clinical interest for use in regenerative medicine; and adipose tissue represents one of these sources with a broad spectrum of benefits . Human adipose tissue represents a readily available autologous source of mscs (ghasemi and razavi, 2014). Human adipose tissue - derived mscs (hat - mscs) retain morphological, phenotypic and functional characteristics resembling those of bm - mscs (zuk et al ., 2002), are stable over long term culture, expand efficiently in vitro and possess multi - lineage differentiation potential (zuk et al ., 2001; musumeci et al ., 2011; choudhery et al ., 2013; latest observations suggest that transplantation of hat - mscs into the brains of elderly mice improved both locomotor activity and cognitive functions . Transplanted cells rapidly differentiated into neurons and in part, into astrocytes, and produced choline acetyltransferase proteins, restoring acetylcholine levels in thebrain . Moreover, transplantation of hat - mscs restored neuronal integrity by stimulating the release of neurotrophic factorsby neighbouring cells (park et al ., 2013). In this regard, an aspect to be considered in mscs therapies is that it is now well - recognized that many pleiotrophic molecules endowed with neuroprotective potential, including some neuropeptides produced locally by resident glial cells or neurons (i.e., pituitary adenylyl cyclase activating polypeptide and/or vasoactive intestinal peptide), when stimulated by neighbouring cells (i.e., implanted mscs) may prevent cognitive decline caused by aging (pirger et al ., 2014), facilitate nerve recovery after injury both in the cns (reviewed by waschek, 2013) and the periphery (tamas et al ., 2012), stimulate remielination processes and glial regenerative support to neurons (castorina et al ., 2014, 2015) and are even capable to prevent retinal damage and mantain retinal barrier properties (giunta et al ., 2012; scuderi et al ., 2013) or impede oxidative insults (castorina et al ., 2012), a broad spectrum of physiopathological events that, at different degrees, are negatively impacted by senescence . Even more interesting, combinatorial administration of these molecules with mscs has been suggested to support spinal cord recovery after damage (fang et al ., 2010), inferring on the mutual reciprocity between the two, especially desirable to complement the existing gaps determined by the single therapeutic employment of mscs in aged patients affected by neurodegenerative disorders . Another source of mscs that has captured minor scientific interest is represented by dental pulp stem cells (dpscs). Dpscs have also been recognized as capable to differentiate into a variety of cell lineages (zhang et al ., 2006; huang et al ., 2009), but more studies are required to better define their potential . Other sources of stem cells are that obtained from human - exfoliated deciduous teeth (shed), which have been shown to contain multipotent stem cells (miura et al ., 2003). The importance of shed is that they are derived from a tissue similar to the umbilical cord . Notably, both kinds of dscs can be induced to differentiate into neuron - like cells and be transplanted in brain injury and/or neurodegenerative disease animal models to conduct neuroregeneration studies (sakai et al ., 2012; based on these findings, it is plausible to believe that the extracted teeth, considered a common waste product from dental extraction procedures, could be employed in the future to exploit in tissue engineering strategies as a promising substitute of bm - mscs . A major issue that has significantly limited the use of mscs - based therapy is the low yielding of viable mscs from donor tissue . In fact, in order to harvest sufficient mscs to procure some clinical benefits cells need to replicate several times in vitro . Unfortunately, a number of studies have demonstrated that mscs from various animals undergo spontaneous transformation when cultured for long terms, posing a limit to this approach . Indeed, transformed mscs show some of the features of senescent cells, with a progressive shortening of telomers and consequently, cell death (ahmadbeigi et al ., 2011; ren et al ., 2011; he et al ., 2014). Such an aging process occurring in mscs appears to be tissue - specific and has been shown to be regulated by evolutionarily conserved signaling pathways . More recently, a signaling pathway that has shown to be tightly associated to age - related cellular changes is the wnt/-catenin signaling cascade (decarolis et al ., 2008; hiyama et al ., 2010; stevens et al ., wnt/-catenin signaling plays a functional role as a key regulator of self - renewal and differentiation properties in mscs . (2014) found that activation of the wnt/-catenin pathway delays the progression of cellular senescence as shown by the decrease in senescence effectors p53 and phospho - retinoblastoma (prb), lowered senescence - associated -galactosidase (sa--gal) activity, and increased telomerase activity . In contrast, suppression of the wnt pathway promoted senescence in mscs (jeoung et al ., 2014). 2012) also showed that wnt/-catenin pathway is connected and regulates tert expression through the interaction with kruppel - like factor 4 (klf4), a core component of the pluripotency transcriptional network (a schematic representation is depicted in figure 2). Unfortunately, to date, the mechanism through which the wnt/-catenin signaling pathway regulates age - related neurogenic differentiation in mscs still remains unclear and needs further investigations . It has been assumed that aging is presumably linked to diminished organ repair capacity due to reduced functionality of mscs . For this reason, it should be taken into account that the effectiveness of mscs - based therapies are highly influenced by donor age . It was observed that progressively aging murine bm - mscs exhibit a decline in mscs number, proliferation, differentiation, angiogenic and wound healing properties, along with enhanced apoptotic and senescent features (kretlow et al ., 2008; choudhery et al ., 2012a, b). In a study using adipose tissue - derived mesenchymal stem cells (at - mscs) from both young and old donors, it was observed that both were able to form colonies, but at - msc from younger donors produce more colonies containing larger numbers of cells and increased proliferative rate than those obtained from older donors (alt et al ., 2012). Moreover, at - mscs obtained from aged donors displayed increased senescent features, as indicated by the greater expression levels of p16 and p21 genes, which have been indicated as markers of senescence (stolzing et al ., 2008). In the latter study, the expression of sa--gal was measured and it was also found at higher levels in aged at - mscs cultures, while sod activity was decreased (stolzing et al ., 2008). It was further identified that mscs from elderly donors became more granular and developed a more flat and larger morphology at passages 56, indicating the appearance of typical morphological signs of replicative senescence (khan et al ., recently, in a study conducted on hbm - mscs from young and old donors used to differentiate and promote neurite outgrowth from dorsal root ganglia neurons (drgn), brohlin and coworkers observed that treatment of hbm - mscs with growth factors induced protein expression of the glial cell marker s100 in cultures from young but not old donors . However, exogenous administration of growth factors enhanced the levels of brain - derived neurotrophic factor (bdnf) and of vascular endothelial growth factor (vegf) transcripts in both donor cell groups and partly recovered stemness properties of mscs from elderly, supporting the hypothesis stated above . Finally, in the same study it was demonstrated that mscs co - cultured with drgn significantly enhanced total neurite length only when obtained from young but not old donors . Moreover, mscs from young donors maintained their proliferation rate while those from the old ones showed increased population doubling times (brohlin et al ., 2012). These observations suggest that mscs isolated from either young or old donors may benefit of a combinatorial approach to retain, at least in part, their regenerating properties on neurons . Nevertheless, to date mscs from young donors are still to be considered the first choice mscs source to use for cns repair (figure 3). The fact that mscs can be conveniently obtained from different accessible tissues (such as bone marrow, blood, adipose and dental tissue) and demonstrate neuroprotective effects, immunomodulatory properties and self - migratory activity, makes them an attractive therapeutic tool for potential application in neurodegenerative disorders . However, there are some critical points that still need to be clarified before msc - based therapy can be adopted in clinical practice . These include the reduced stemness properties of mscs isolated from elderly or caused by long - term expansion in vitro, which could result in reduced efficacy for regenerative cellular therapy . The complex pathways involved in neurodegenerative disorders, should be evaluated with care, in the attempt to extend the current understanding of the pathogenesis of these diseases and identifying targets for intervention . To be suitable for use inneuroregenerative therapy, the mechanisms that govern the self renewal capacity of mscs, it is proposed that scientific effort should focus more on finding the appropriate microenvironment (culture conditions) that more likely will allow to yield sufficient number of functional mscs . As previously discussed, amolecular mechanism worthy of attention could be represented by the wnt/-catenin signaling pathway, whose involvement in triggering the shift of mscs towards a senescent phenotype appears to be clear . These findings, together with the evidences obtained with combinatorial approaches using neuroprotective agents, support the idea that trophic molecules, including some neuropeptides, may elicit a regulatory function on the wnt signaling cascade, which in turn, could be the key element in controlling mscs senescence (jeoung et al ., 2014). Alternatively, another critical mechanism to target could be telomere regulation, but this strategy has already reach general consensus, since studies on the mechanisms controlling telomere status and regulation in these cells have progressively gained importance in the last years . In fact, strategies to prevent telomere loss or to increase telomere length of mscs may prevent or delay degeneration and hence the onset of symptoms in neurodegenerative disorders, improving the results of mscs - based therapetic approaches . Finally, a further and reasonable method to expand mscs validity in therapy could be represented by banking younger adipose tissue for later use . Preservation of mscs at a younger age, when their biological utility is maximal, could provide a usable source of functional mscs with full regenerative potential for future applications in regenerative medicine.
These viruses infect a variety of species, including aquatic birds, poultry, pigs, horses, dogs, and humans, causing significant morbidity and mortality . In the case of human influenza viruses, in addition to the burden of yearly epidemics, there is the ever - present threat of an influenza pandemic . Pandemics occur when a novel strain of influenza virus of animal origin evolves the ability to infect and efficiently transmit among humans . While some influenza pandemics, like the 1918 h1n1 pandemic that killed more than 40 million people worldwide, have had devastating consequences, others, including the h1n1 pandemic in 2009, which resulted in an estimated 18,000 deaths, have been considerably milder . Unfortunately, the emergence of new human pandemic viruses, as well as the subtype and virulence of the causative viruses, is still unpredictable . Although there are vaccines available for certain influenza viruses, they are strain specific, and the generation and general distribution of a new influenza virus vaccine take more time than the spread of a new virus, severely limiting the impact of vaccines in the first wave of a pandemic . Influenza antiviral drugs are of broader specificity, but resistance to those drugs and problems with availability limit their use . In order to mitigate the possible impact of an influenza pandemic, we need more research and development in the generation of improved vaccines and antivirals with broad cross - reactivity against multiple influenza viruses . In addition, by investigating the factors responsible for the generation of human pandemic viruses, we can better recognize the risks that animal influenza virus strains pose to humans and build eradication campaigns to target specific viral strains circulating in animals . Critical to this issue is the question of what makes an influenza virus transmissible in humans and animals . A better understanding of influenza transmission will lead to the development of countermeasures for viral transmission that can enhance our pandemic preparedness plans . Several mutations and changes associated with increased airborne transmission of avian influenza viruses in ferrets have been identified in recent years (13). However, previous attempts to identify adaptive changes in highly pathogenic avian h5n1 viruses associated with transmission have failed, supporting the possibility that h5n1 viruses might be structurally unfit for mammalian transmission (4). More recently, two independent laboratories lead by ron fouchier and yoshihiro kawaoka have ruled out this hypothesis and identified specific mutations that allow the h5n1 virus to accomplish airborne transmission in ferrets (unpublished observations). While this provides important information on the adaptability of h5n1 viruses, more research needs to be conducted to understand the possibility that avian h5n1 could evolve to become transmissible in humans, to predict its pathogenesis in humans, and to find the molecular mechanisms responsible for host specificity in influenza virus transmission . While research on influenza virus transmission is critical for finding ways to better tackle this pathogen, it is important to conduct such research using appropriate biocontainment and biosafety conditions to minimize possible risks of virus release to the environment . Risk assessment is a crucial tool in selecting biocontainment levels for research on potentially dangerous pathogens . According to the biosafety in microbiological and biomedical laboratories (bmbl) manual (5), the definitive reference book for biosafety issues, biological risk associated with pathogen research is determined by three elements: the activities that can result in human exposure to the pathogen, the probability that such exposure would cause an infection, and the consequences of such an infection . Although ferret - adapted h5n1 viruses probably have attenuated infectivity and pathogenesis for humans, to minimize all possible risks one should use biocontainment facilities and practices that prevent human exposure . Preventing the escape of viruses from the laboratory can be achieved by housing research activities in a facility equipped with interlocked rooms with negative pressure and high - efficiency particulate air (hepa) filtered air circulation and using the appropriate decontamination and/or sterilization practices for material leaving the facility . Since human infection with influenza viruses occurs via the respiratory route, infection of laboratory personnel can be prevented by the use of powered air - purifying respirators . These practices correspond to enhanced biosafety level 3 (bsl3), as described in the bmbl manual . The effects of accidental exposure to the virus in an enhanced bsl3 facility can be minimized by vaccinating personnel with an h5n1 vaccine and through the use of antiviral drugs . Increased biocontainment, or bsl4, h5n1 viruses, on the other hand, are sensitive to the antivirals zanamivir and oseltamivir, and infection with the virus is preventable by vaccination with the h5n1 inactivated vaccine . Influenza virus research is important for the development of novel intervention strategies for preventing and mitigating influenza epidemics and pandemics . As scientists, we have the responsibility to avoid the undue restrictions of the highest level of biocontainment if enhanced bsl3 facilities can provide the appropriate biosafety . The use of bsl4 containment would not decrease the risk of virus release any more than enhanced bsl3 containment, but it would result in an unnecessary burden that would restrict research on h5n1 influenza transmission to a few facilities and considerably decrease the speed of research on this important pathogen.
, it was found that the thyroid gland had the maximum amount of selenium per gram of tissue . Autoimmune thyroiditis (ait), the prototype of autoimmune diseases, is characterized by t - cell - mediated autoimmune destruction of thyroid cells . Environmental factors, such as iodide intake, immunotherapeutic agents, or viral infections that may initiate the disease . In areas, where selenium deficiency is prevalent, higher incidence of thyroiditis has been reported due to a decreased activity of selenium - dependent glutathione peroxidase enzyme within thyroid cells . Severe nutritional selenium deficiency leads to an increased rate of thyroid cell necrosis and invasion of macrophages . Whether this it may be assumed, however, that thyroid cell damage may initiate or maintain autoimmune thyroiditis, especially in patients susceptible to the development of autoimmune diseases . To study the effect of selenium supplementation in patients with autoimmune thyroid disease . Patients of all age groups and both sexes with autoimmune thyroid disease (as defined by an anti- thyroid peroxidase antibody [tpoab] level more than 150 iu / ml) irrespective of the baseline thyroid status . Patients with overt hyperthyroidism who are on antithyroid drugs, patients on any other medication, which may alter the immunity status of the patients, and pregnant patients were excluded from the study . Patients were randomized into two age and tpoab - matched groups; 30 patients received 200 g sodium selenite / day, orally, for 3 months, and 30 patients received placebo . The differences in antibody concentrations at the beginning and end of the study were determined by t - test for paired samples . Patients of all age groups and both sexes with autoimmune thyroid disease (as defined by an anti- thyroid peroxidase antibody [tpoab] level more than 150 iu / ml) irrespective of the baseline thyroid status . Patients with overt hyperthyroidism who are on antithyroid drugs, patients on any other medication, which may alter the immunity status of the patients, and pregnant patients were excluded from the study . Patients were randomized into two age and tpoab - matched groups; 30 patients received 200 g sodium selenite / day, orally, for 3 months, and 30 patients received placebo . The differences in antibody concentrations at the beginning and end of the study were determined by t - test for paired samples . In the selenium treated group 27 patients were female, and three patients were male (m: f = 1:9), which was comparable with the ratio in the placebo - treated group (1:7.3) the mean ages at presentation in both the groups were 34 2.5 and 31 3.4 years, respectively . At study entry, the mean tpoab concentrations were identical for both groups (selenium treated group, 669 205 iu / ml; placebo, 729 277 iu / ml). Out of the total 30 patients in the selenium treated group, 6 patients were overtly hypothyroid, 15 were subclinical hypothyroid, 6 were euthyroid, and 3 were subclinical hyperthyroid . There were comparable numbers of patients in each subgroup in the placebo - treated group also . The mean tpoab concentration decreased significantly by 49.5% (p <0.013) in the selenium treated group versus 10.1% (p <0.95) in the placebo - treated group . In subgroup analysis, the decrease in the mean tpoab titre was highest in the subclinical hyperthyroid group (up to 64.42%), and comparable in the other three groups (41.13%, 47.18%, and 42.64% in the euthyroid, hypothyroid, and subclinical hypothyroid groups respectively). One patient with hypothyroidism in the selenium treated group with a tpoab concentration of> 1000 iu / ml, had completely normalized antibody concentrations after 3 months . It was also found that those patients with tpoab greater than 1000 iu / ml revealed a mean 31.38% reduction in the selenium - treated patients, compared with no significant change in tpoab in the placebo group . . Glutathione peroxidase can reduce hydrogen peroxides and phospholipid hydroperoxides, and hence can reduce the production of free radicals and reactive oxygen species . These mechanisms may contribute to reduced inflammatory activity in the organ - specific autoimmune response, and may explain the improvement of autoimmune thyroiditis in our study . Based on the link described above between selenium and the thyroid, several studies applying organic and inorganic selenium compounds were undertaken in patients, with ait in areas with low to borderline - low - selenium content . A prospective placebo - controlled clinical study with selenium in ait conducted in the selenium deficient area of bavaria in southern germany, by grtner et al . In 2002 showed a 36% reduction in anti - tpo titers in the selenium - treated group, whereas a further reduction of up to 60% was seen in a subgroup of patients with basal anti - tpo levels above 1200 iu / ml . Supplementation of selenium has a significant impact on inflammatory activity in thyroid - specific autoimmune disease . It would be of interest to determine whether early treatment with selenium in patients with newly developed autoimmune thyroiditis may delay, or even prevent the natural course of these diseases.
Aorto - enteric fistula is a direct communication between the abdominal aorta and the gastrointestinal tract . Primary aorto - enteric fistula is defined as a spontaneous erosion of the aorta into the gastrointestinal tract and must be distinguished from secondary aorto - enteric fistula that may complicate aortic surgery, including endovascular procedures, and is ten times more frequent [2, 3]. About 250 cases of primary aorto - enteric fistula are reported in the medical literature while its prevalence in large autopsy series has been reported to vary between 0.04 and 0.07% [4, 5]. Secondary aorto - enteric fistulas are more common with an incidence varying from 0.4 to 2.4% of abdominal aortic vascular procedures [2, 3, 6, 7, 8]. Due to its fixed position and close relation to the abdominal aorta, the duodenum is the commonest location of aorto - enteric fistula, usually in its third or fourth part . Common causes of primary aorto - duodenal fistula (padf) include atherosclerotic aneurysms [9, 10] and aortic infections . Salmonella, klebsiella, mycobacteria, spirochetes and fungi are the microbial species most commonly involved [10, 11, 12, 13]. Uncommon causes of padf include carcinoma of the pancreas, biliary stones, trauma, ingested foreign bodies, peptic ulcer disease, benign cysts and duodenal diverticular disease . Four cases of idiopathic padf are reported [21, 22, 23, 24], while seven cases of padf following abdominal radiotherapy for malignancy are described in the medical literature [25, 26, 27, 28, 29, 30, 31]. We report a case of padf developfing 25 years after surgery and radiotherapy and successfully treated at our institution . We discuss the details of the procedure and review the literature pertaining to this uncommon condition . A 61-year - old man with a recent history of erosive gastritis and duodenal ulcer presented to the emergency department of our hospital with hematemesis, melena and circulatory collapse . His past medical history was remarkable for a left radical orchiectomy and para - aortic lymph node dissection followed by radiotherapy (total dose 45 gy) given 25 years previously for seminoma . An episode of intestinal obstruction due to adhesions was treated surgically a few months after completion of the radiotherapy . One month before admission he had an episode of acute gastroenteritis due to salmonella enteritidis confirmed by stool cultures . His medications included oral amoxicillin, 1 g twice daily, and esomeprazole, 40 mg at bedtime . On examination laboratory values included a white blood cell count of 20.96 10/mm, a hematocrit of 33%, a hemoglobin concentration of 11.5 g / dl and a platelet count of 244 10/mm . Liver function tests, electrolytes, amylase, lipase, blood urea nitrogen and creatinine were normal, as were prothrombin and partial thromboplastin coagulation tests . The patient was resuscitated with a combination of colloids and crystalloids, a nasogastric tube was inserted, revealing copious bright red blood . Upper gastrointestinal endoscopy showed the presence of active bleeding from the second part of the duodenum . After the endoscopy an intravenous infusion of omeprazole at high doses was initiated . In spite of these measures, one hour later massive hematemesis recurred with marked hypotension . The patient was therefore transferred to the operating room for emergency laparotomy . Through a midline laparotomy the gastric antrum and first part of duodenum were opened longitudinally . As no ulcer was found and a large amount of blood was coming from the distal lumen, the incision was extended . The duodenum was mobilized with great difficulty because of extensive and dense adhesions due to the previous surgery and radiotherapy resulting in conglomeration of duodenum, aorta and inferior vena cava . Eventually the source of the bleeding was identified as a small - diameter, direct communication between the aorta and the third part of the duodenum . There were no signs of aneurysmatic dilatation, aortitis, or peri - aortic sepsis . The bleeding was initially controlled by manual compression and the defect in the aortic wall directly repaired with interrupted 2/0 prolene sutures (fig . As the duodenum was widely lacerated and devascularized, we decided to remove it together with the gastric antrum 2) and a feeding jejunostomy tube inserted . During the procedure the patient received a total of 11 units of packed red blood cells, 4 units of fresh frozen plasma and 4 units of platelet concentrate . The postoperative course was complicated by the development of a bilio - pancreatic fistula on day seven with an output of about 400 ml / day which was treated with octreotide and total parenteral nutrition . Emergency relaparotomy showed that the bleeding was originating from a large omental vessel and a partial dehiscence of the duodeno - jejunal anastomosis . Hemostasis was carried out and fibrin glue (tissucol) was applied to the duodenal remnant in an attempt to control the fistula . A further 5 units of packed red blood cells and 2 units of fresh frozen plasma were transfused . The subsequent clinical course was complicated by persistent fistula and low - grade sepsis with fever and leukocytosis . A ct scan of the abdomen showed multiple fluid collections which were managed with percutaneous ultrasound - guided drainage and intravenous antibiotics . The patient received a further 5 units of packed red blood cells and 1 unit of fresh frozen plasma . Eventually the fistula output decreased to less than 50 ml / day and oral intake could be resumed . A follow - up ct scan 3 months after surgery showed a few small remaining fluid collections and the fistula had closed completely . The patient is alive and well 19 months after discharge and ct scan shows no residual fluid collections . Histhological examination of the duodenal wall and surrounding tissue at the fistula site showed signs of chronic radiation damage . Padf occurs when a degenerative process in the aortic wall erodes directly into the adjacent duodenum . Its incidence is considerably lower than that of secondary aorto - duodenal fistula, which occurs after reconstructive aortic surgery, and less than 200 cases have been reported in the medical literature . Astley cooper in 1822 first reported a case of padf caused by the rupture of an aortic aneurysm, and heberer in 1957 successfully treated a patient with padf by resection of a saccular aneurysm and direct suture and repair of the aorta and duodenum . Padf is associated with an atherosclerotic aortic aneurysm in over 80% of cases [1, 8, 9, 10, 11] and aortic wall infection in most of the remaining 20% [10, 11, 12, 13]. Rare causes of padf include pancreatic cancer, duodenal ulcers, gallstones, duodenal diverticulitis, duodenal trauma, foreign body ingestion and benign cysts . Only 7 cases have been reported in the medical literature: 6 cases following external radiotherapy and one case after intraoperative radiotherapy (table 1). In addition, 3 cases of arterio - enteric fistula following surgery for gastric cancer and a combination of intra- and postoperative radiotherapy have been reported . The fistulas originated from the left gastric, the celiac trunk and the superior mesenteric artery, respectively, and are therefore not included in the present review . Bleeding originated from the third part of the duodenum in 4 cases and from the fourth part in 3 cases . In 3 cases, as in ours, the condition for which the radiotherapy had been given was metastatic seminoma of the testis . The time interval between the treatment and the bleeding episode was extremely wide, ranging from 2 weeks to 25 years in our case . In 5 cases extensive surgical dissection in the paraaortic region had been carried out before the radiotherapy was given but, as no aortic reconstruction was made, the fistulas are rightly to be considered primary . The pathogenesis of postirradiation padf is unclear, particularly in cases where many years, 25 in our case, have elapsed since the treatment was given . Acute radiation damage to the aortic wall may result in aortic wall necrosis and rupture during or shortly after the treatment [36, 37], but the reasons why a communication between aorta and duodenum should develop years or decades after irradiation are far from clear . Radiation damage to the duodenum following high - dose radiation is well described and is explained by its fixed location in the retroperitoneum . It may result in bleeding, ulceration, perforation or fistula formation, often requiring emergency surgical exploration . The histological changes seen in chronic radiation include mucosal and submucosal damage due to obliteration of small vessels, telangiectasis and new vessel formation . The role of chronic infection is not clear but is likely to be a contributing factor in the development of a fistula, as well as the pulsatile stress of the underlying aorta onto a weakened duodenal wall . Although the small bowel is certainly most sensitive to radiation damage, large vessels, including the aorta, may also suffer from radiation injury . Apart from acute rupture during or shortly after radiotherapy, chronic changes are well described, including thrombosis of mural vessels (vasa vasorum) resulting in fibrosis, stenosis and occlusion 10 or more years after treatment and accelerated arteriosclerosis and periarterial fibrosis 20 or more years later . Despite the fact the damage induced by radiotherapy to both the duodenal and aortic wall is well known, the series of events leading to the formation of a fistula between the two structures are still elusive . Most authors seem to favor the hypothesis that a chronic ulcer slowly erodes through the duodenal wall and eventually perforates a weakened and fibrosed aortic wall . In fact an ulcer appeared to be at the origin of the fistula in cases no . 3, 5 and 6 [15, 25, 30]. In the first case the aorta showed a large band of necrosis involving the media and the adventitia while stains for microorganisms were negative . In the second case, occurring 6 years after radiotherapy, a radiation ulcer was found at laparotomy in the third part of the duodenum that had penetrated into the aorta . Emergency endoscopy appeared to show a forrest type 1a bleeding ulcer of the duodenal bulb while at laparotomy no ulcer was seen in that location . As blood appeared to be coming from the distal lumen, the incision was extended along the anterior wall of the duodenum until the fistula was found in the posterior wall of the third portion . We therefore believe that emergency endoscopy resulted in a misdiagnosis that can be well explained if we consider the dramatic setting in which it was carried out . As no evidence of a chronic ulcer was seen in the surgical specimen, the cause of the fistula remains elusive in our case . Clinical presentation of padf is usually dramatic with massive hematemesis, although the bleeding may be occasionally less severe and cause melena only . In about half the cases a minor herald bleed may precede a major episode hours or even days before a catastrophic hemorrhage . Unfortunately the true cause of the bleeding is often misdiagnosed as originating from an ulcer or other condition and therefore adequate measures are not taken to prevent a second, often fatal bleed . The reasons for this repeat bleed are not entirely clear but it is likely due to spasm of the duodenal muscle layer in response to the bleed and hypotension secondary to hypovolemia . In the majority of cases a free interval of 4 - 24 h is observed but episodes occurring weeks later are described . In our case a free interval of just one hour occurred, which allowed us to carry out an upper gastrointestinal endoscopy, although this led to a wrong diagnosis . On the other hand, endoscopy is rarely diagnostic in this condition because the orifice of the fistula is generally small and hidden among the folds of the duodenal mucosa . The main purpose of endoscopy is therefore to rule out other causes of bleeding, but it is rarely diagnostic and direct demonstration of an aorto - enteric fistula is a rare event and in most cases the examination is negative or nondiagnostic . On the other hand the misdiagnosis of a bleeding duodenal ulcer may lead to a delay of emergency, life - saving surgical intervention . In a hemodynamically stable patient ct can provide very useful information . A gas - containing fluid collection in the paraaortic tissues is an indirect sing of infection surrounding the retro - duodenal aorta . Also, the presence of an aortic aneurysm following an episode of massive upper gastrointestinal bleeding should alert to the possibility of an aorto - enteric fistula . Angiography is diagnostic in only a minority of cases, as the fistula can be demostrated only in cases of active bleeding who are rarely stable enough to be subjected to this procedure . Further diagnostic evaluation should be pursued only if the clinical condition of the patient permits it . In cases of severe, repeated bleeding the only reasonable measure to be adopted to save the patient's life is surgical intervention . In the largest published series of 118 cases of aorto - duodenal fistula, only 25% of patients underwent surgical repair and only 60% of these survived the operation . Emergency surgery by an experienced vascular surgeon provides the only chance of identifying the origin of the fistula and repair it in order to prevent exsanguination and rapid death . Over 75% of padfs originate from an aneurysm of the infrarenal descending aorta and in this case the treatment consist in aortic reconstruction using prosthetic material, excision of the fistulous tract and repair of the remaining defect in the duodenal wall . The notion that local contamination is demonstrated in cultures in about 15% of cases does not justify the choice of an extraanatomic bypass with its higher morbidity and mortality . This approach should be reserved only to cases with signs of gross local contamination or abscess formation within the periaortic tissues . In the rare cases of radiation - induced padf a primary surgical repair without use of foreign material appears to be justified . The radiation - damaged portion of the duodenal wall should be excised as a primary repair would certainly result in dehiscence, enteric fistula and abscess formation . A protective sleeve of prosthetic material inserted between the aorta and reconstructed bowel has been used in only one case of the published series and should be considered as a potentially useful adjunct . A pedicled omental graft, as in our case, should also be considered as a protection of the primary repair of the aortic wall . Clinical presentation of padf is usually dramatic with massive hematemesis, although the bleeding may be occasionally less severe and cause melena only . In about half the cases a minor herald bleed may precede a major episode hours or even days before a catastrophic hemorrhage . Unfortunately the true cause of the bleeding is often misdiagnosed as originating from an ulcer or other condition and therefore adequate measures are not taken to prevent a second, often fatal bleed . The reasons for this repeat bleed are not entirely clear but it is likely due to spasm of the duodenal muscle layer in response to the bleed and hypotension secondary to hypovolemia . In the majority of cases a free interval of 4 - 24 h is observed but episodes occurring weeks later are described . In our case a free interval of just one hour occurred, which allowed us to carry out an upper gastrointestinal endoscopy, although this led to a wrong diagnosis . On the other hand, endoscopy is rarely diagnostic in this condition because the orifice of the fistula is generally small and hidden among the folds of the duodenal mucosa . The main purpose of endoscopy is therefore to rule out other causes of bleeding, but it is rarely diagnostic and direct demonstration of an aorto - enteric fistula is a rare event and in most cases the examination is negative or nondiagnostic . On the other hand the misdiagnosis of a bleeding duodenal ulcer may lead to a delay of emergency, life - saving surgical intervention . In a hemodynamically stable patient ct can provide very useful information . A gas - containing fluid collection in the paraaortic tissues is an indirect sing of infection surrounding the retro - duodenal aorta . Also, the presence of an aortic aneurysm following an episode of massive upper gastrointestinal bleeding should alert to the possibility of an aorto - enteric fistula . Angiography is diagnostic in only a minority of cases, as the fistula can be demostrated only in cases of active bleeding who are rarely stable enough to be subjected to this procedure . Further diagnostic evaluation should be pursued only if the clinical condition of the patient permits it . In cases of severe, repeated bleeding the only reasonable measure to be adopted to save the patient's life is surgical intervention . In the largest published series of 118 cases of aorto - duodenal fistula, only 25% of patients underwent surgical repair and only 60% of these survived the operation emergency surgery by an experienced vascular surgeon provides the only chance of identifying the origin of the fistula and repair it in order to prevent exsanguination and rapid death . Over 75% of padfs originate from an aneurysm of the infrarenal descending aorta and in this case the treatment consist in aortic reconstruction using prosthetic material, excision of the fistulous tract and repair of the remaining defect in the duodenal wall . The notion that local contamination is demonstrated in cultures in about 15% of cases does not justify the choice of an extraanatomic bypass with its higher morbidity and mortality . This approach should be reserved only to cases with signs of gross local contamination or abscess formation within the periaortic tissues . In the rare cases of radiation - induced padf a primary surgical repair without use of foreign material appears to be justified . The radiation - damaged portion of the duodenal wall should be excised as a primary repair would certainly result in dehiscence, enteric fistula and abscess formation . A protective sleeve of prosthetic material inserted between the aorta and reconstructed bowel has been used in only one case of the published series and should be considered as a potentially useful adjunct . A pedicled omental graft, as in our case, should also be considered as a protection of the primary repair of the aortic wall . Radiation - induced padf is a rare and life - threatening cause of massive gastrointestinal hemorrhage . Its etiology has not been fully elucidated but it likely involves chronic inflammatory changes, fibrosis and accelerated atherosclerosis, resulting in weakening of the aortic and duodenal walls . A history of abdominal radiotherapy, no matter how many years before, should be carefully sought in all patients presenting with massive gastrointestinal bleeding . Endoscopy, ct scan and angiography can be useful in the evaluation of these patients, but are diagnostic in only a minority of cases and a high index of suspicion remains the key to prompt diagnosis and effective treatment . The diagnosis of padf is generally only confirmed at exploratory laparotomy and all too often is not considered in the differential diagnosis . Once the diagnosis is established or suspected, an urgent laparotomy is mandatory . Direct reconstruction and pedicled omentum plasty, associated with duodenal resection, our report confirms previous observations that high - energy radiation is a possible etiologic factor in spontaneous padf, and calls attention to the fact that a fistula may develop even decades after the completion of radiotherapy.
In last two decades following advances in neuroimaging studies, there has been growing interest in studying the neurological soft signs (nss) and cognitive function in obsessive - compulsive disorder (ocd), a disorder which was earlier explained only on basis of psychoanalytical theories . Dysfunction in brain functioning implies that ocd should be characterized by neurological abnormalities which can be either hard signs refer to impairment in basic motor, sensory, and reflex behaviors . In contrast, nss are described as nonlocalizing neurological abnormalities that cannot be related to impairment of a specific brain region or are not believed to be part of a well - defined neurological syndrome . However, even if they are nonlocalizing, some nss can suggest dysfunction in particular neural networks and, thus, can give additional information concerning abnormalities in the functional organization that characterize some psychiatric diseases . Cognition denotes a relatively high level of processing of specific information including thinking, memory, perception, motivation, skilled movements, and language . A constellation of these core cognitive deficits in various combinations and severity also have a role in the explaining the neuroanatomy and psychopathology of the disease . Efforts for assessment of nss in ocd have met with variable success with some studies reporting significantly higher nss in the domains of complex motor coordination, involuntary movements, mirror movements, and primitive reflexes in patients with ocd . However, there has been lack of consistency and specificity in the findings of the studies . Studies have reported impairment in visuospatial ability, executive function, attention, concentration, and working memory in ocd subjects . These deficits further lead to impairment in social and occupational functioning leading to increased distress and disability in ocd patients . Although western studies have looked at these issues, there are few studies from india investigating the role of nss and cognitive deficits in adult ocd . The study was conducted in the outpatient department of psychiatry in a tertiary care general public hospital after obtaining permission of the institutional review board . The study group comprised fifty subjects with the international classification of diseases-10 diagnosis of ocd, with age more than 18 years . Subjects with intellectual subnormality, organic mental disorders, and spectrum of psychotic disorders (schizophrenia, schizoaffective disorder, and bipolar disorder) as the major axis i disorder were excluded from the study . The control group comprised fifty subjects matched with study group for age and education with no past, present, or family history of major psychiatric disorder in first - degree relatives . All participants gave written informed consent to a protocol approved by the institutional review board . Nss were assessed using heidelberg soft neurological signs scale, a 16-item scale . A sufficient internal reliability and it assess five factors: motor coordination (ozeretski test, diadochokinesis, pronation - supination, finger - to - thumb opposition, and speech articulation), sensory integration (gait, tandem walking, and 2-point discrimination), complex motor tests (finger - to - nose test, fist - edge - palm test), right / left and spatial orientation (right / left orientation, graphesthesia, face - hand test, and stereognosis), and hard signs (includes arm - holding test, mirror movements). It assesses six cognitive domains: short - term memory recall task (two learning trials of five nouns and delayed recall after approximately 5 min), visuospatial abilities (clock - drawing task and a three - dimensional cube copy), executive functions (alternation, phonemic fluency, and verbal abstraction task), attention, concentration and working memory (sustained attention task, a serial subtraction, digits forward and backward), language (naming, repetition, aforementioned fluency task), and orientation to time and place . All analyses were performed with the statistical package for social sciences software (spss inc ., whitney u - test for comparison of mean between the study and control group was used for the analyses . For correlational analyses, pearson correlation (p) the mean age of ocd subjects was 30.62 years with range between 18 and 59 years . Among them, 64% of the subjects were males, 56% of the subjects were college educated, 34% were up to secondary level, 6% were primary educated, and 4% were illiterate . As revealed in table 1, scores were significantly higher in ocd subjects in all the tests for motor coordination (ozeretski's test, p = 0.001; diadochokinesis, p <0.001; pronation - supination, p = 0.004; finger - to - thumb opposition, p <0.001; and speech articulation, p <0.001), sensory integration (gait, p = 0.002; tandem walking, p <0.001; and 2-point discrimination, p <0.001), complex motor tasks (finger - to - nose test, p <0.001 and fist - edge - palm test, p <0.001), and hard signs (arm - holding test, p <0.001 and mirror movements, p <0.001). Ocd subjects scored significant higher in all the tests of right / left and spatial orientation (right / left orientation, p <0.001; graphesthesia, p = 0.007; and stereognosis, p <0.001) except for test of sensory extinction (face - hand test, p = 1.000). Comparison of neurological soft signs as shown in table 2, ocd patients fared worse on all subsets of montreal cognitive assessment compared to controls . Ocd patients showed significant deficits in short - term memory task (p <0.001), visuospatial ability (p <0.001), attention, concentration and working memory (p <0.001), and language (p <0.001). There was no significant difference for orientation (p = 0.53) between ocd patients and controls . The mean age of ocd subjects was 30.62 years with range between 18 and 59 years . Among them, 64% of the subjects were males, 56% of the subjects were college educated, 34% were up to secondary level, 6% were primary educated, and 4% were illiterate . As revealed in table 1, scores were significantly higher in ocd subjects in all the tests for motor coordination (ozeretski's test, p = 0.001; diadochokinesis, p <0.001; pronation - supination, p = 0.004; finger - to - thumb opposition, p <0.001; and speech articulation, p <0.001), sensory integration (gait, p = 0.002; tandem walking, p <0.001; and 2-point discrimination, p <0.001), complex motor tasks (finger - to - nose test, p <0.001 and fist - edge - palm test, p <0.001), and hard signs (arm - holding test, p <0.001 and mirror movements, p <0.001). Ocd subjects scored significant higher in all the tests of right / left and spatial orientation (right / left orientation, p <0.001; graphesthesia, p = 0.007; and stereognosis, p <0.001) except for test of sensory extinction (face - hand test, p = 1.000). As shown in table 2, ocd patients fared worse on all subsets of montreal cognitive assessment compared to controls . Ocd patients showed significant deficits in short - term memory task (p <0.001), visuospatial ability (p <0.001), attention, concentration and working memory (p <0.001), and language (p <0.001). There was no significant difference for orientation (p = 0.53) between ocd patients and controls . Nss were present to a significantly greater extent in ocd patients than controls in our study . These findings are in accordance with numerous prior studies reporting significantly higher total nss scores in ocd subjects . Nss in our ocd patients were significantly higher in all the subscales of motor coordination, sensory integration, complex motor tests, right / left and spatial orientation, and hard signs . Higher scores for motor coordination, complex motor tasks, and hard signs seen here are also reported by other studies . Although the initial study by hollander 1990 did not report higher scores in sensory integration and right / left and spatial orientation, subsequent studies by bolton 1998 and guz 2004 have reported higher scores also on these subtests as well . Bolton 1998 also demonstrated that ocd subjects had neurological signs in certain categories such as motor coordination, sensory integration, hard signs, tardive dyskinesia, catatonic, and extrapyramidal signs similar to patients with schizophrenia . The significant impairment in cognition observed in our study in the domains of visuospatial ability, executive function, attention, and working memory is in accordance to other studies . Visuospatial and visuoconstructional deficits in tests using ability to draw complex figures are the most consistent findings in ocd . Ocd patients have shown impairment on both, verbal memory on measures of new learning such as the california verbal learning and nonverbal memory such as visual reproduction and delayed recognition of figures . These findings may suggest impairment in encoding and retrieval of new information in ocd patients . Similarly, the finding of short - term memory task deficit in this study may be explained on the above basis as well as the impaired attention seen in our patients . Although recent research has reported deficit in verbal fluency in ocd, there is scanty literature studying other language deficits such as repetition and naming . The orbitofrontal cortex and basal ganglia are the most consistently associated with ocd in imaging studies, ocd symptoms attributed to be caused by the hyperactive orbito - fronto - thalamic circuit . These imaging findings are corroborated by the finding that disrupting connections between ofc, acc, thalamus, and basal ganglia by means of anterior capsulotomy or cingulotomy result in a symptomatic improvement in most ocd patients . Further ocd symptoms are reported damage to the basal ganglia, especially the caudate and orbitofrontal cortex . Also, dysfunction of the basal ganglia secondary to a streptococcal infection or encephalitis lethargic has also been associated with the development of ocd . Although nss are described as nonlocalizing neurological abnormalities, neuroimaging studies have suggested associations of nss with activation changes in the sensorimotor cortex, supplementary motor area, cerebellum, basal ganglia, and thalamus . Similarly, the cognitive deficits, namely, visuospatial, executive, attention, concentration, and memory observed in the study are indicative of the underlying neuroanatomical and neurophysiological changes, mainly in the frontal lobe . The presence of confounding factors such as comorbid depressive or psychotic symptoms is known to influence nss and cognition . However, study by trivedi et al . Comparing ocd patients without any other comorbid axis i disorder with healthy controls reported significant impairment in executive function, sustained attention abilities, and spatial working memory in ocd patients . Similarly, rao et al . Reported significant neuropsychological deficits in thirty recovered ocd patients in comparison with thirty matched healthy controls . These findings are indicative that neuropsychological deficits are possibly stated independent and are indicative of underlying neuroanatomical deficit . As this was a cross - sectional study, the effect of therapeutic interventions on the scores of nss and cognitive functions could not be assessed . The presence of nss and cognitive deficits in ocd is indicative of underlying neuroanatomical and neurophysiological dysfunction and that ocd is a brain disease . Ocd affects the younger population and is known to cause significant impairment in individual social and occupational productivity . This disability can now be linked to the defective higher mental functions secondary to neurological dysfunction and not just the obsessive symptomatology as thought previously.
Successful outcomes of endodontic treatment depend on the identification of all roots and root canals which in turn guarantees complete extirpation of pulp tissue, proper chemomechanical cleaning and shaping and three - dimensional obturation of the root canal system with an inert filling material . Failure of at least one of these stages entails high risk of unsuccessful root canal treatment of the tooth with a subsequent development or persistence of a periapical lesion . Normally, mandibular first molars have one mesial root and one distally . The mesial root has two canals (mesiobuccal and mesiolingual), ending mainly in two distinct apical foramina . The distal root typically has one root canal, although if the orifice is particularly narrow and round, a second distal canal may be present . Anatomical variations in the number of roots as well as canal configuration in mandibular molars are not uncommon [3, 4]. One of the major anatomical variations is the presence of an additional third root, also called the radix entomolaris (re) which is located distolingually in mandibular molars . In very rare cases, when this additional root is located mesiobuccally, it is called radix paramolaris [5, 6]. Anatomical studies have reported an association between the presence of a separate re in the first mandibular molar and certain ethnic groups . In populations with mongoloid traits, such as chinese, eskimos, and american - indians, it occurs with a frequency of 5 to more than 30% [7, 8]. In african population, a maximum frequency of 3% was found [9, 10], whereas in europeans the incidence was even less . Using full - mouth periapical radiographs, investigated the incidence of radix entomolaris in german population . Seven patients were found to have a three - rooted mandibular first molar with an overall incidence of 1.35% . In indian population, garg et al . Examined 1054 periapical radiographs and reported 5.97% of occurrence of re in mandibular first molars . The same method was used by karale et al . Who reported a higher incidence (6.67%) of re . Knowledge of occurrence, location, and incidence of any tooth anatomical variation is important as it has a significant role in clinical dentistry . Many epidemiological studies have highlighted the importance of watching re while performing root canal treatment on mandibular first molars . Dental schools have been very recently established in palestine and there is no single research dealing with teeth anatomy and the incidence of anatomical variation in our country . With a huge number of dentists and very few specialists in endodontics, this research has been conducted so as to provide information about various anatomical variations that could be encountered during endodontic therapy . The purpose of this study was to evaluate the percentage of permanent mandibular first molar teeth with three roots in a palestinian population using conventional digital x - rays in two different angles . Three hundred and twenty two mandibular first molars from 185 females and 137 males of different ages scheduled for root canal treatment at the dental center of the arab american university were included in this clinical investigation . The study sample represents all patients who needed primary root canal treatment or referred for retreatment over a 2-year period . The age of the 322 participants ranged from 11 to 62 (mean = 37) years . This study was approved by the ethics committee of the school of dentistry research centre . The criteria used to indicate the presence of re were clear distinction of an extra root, indicated by the crossing of translucent lines defining the pulp space and periodontal ligaments, originating in the upper half of the distal root . One endodontist and one paediatric dentist (both instructors of undergraduate dental students) served as examiners in this study . Disagreement in the interpretation of the radiographs was discussed between the two investigators until a consensus was reached . At least two preoperative radiographs were taken for each tooth undergoing root canal treatment using a digital x - ray sensor (dr . Suni, san jose, california, usa). One radiograph was taken from orthoradial position and the other taken either 30 mesially or distally . When the radiographs revealed a case of re, another radiograph for the opposing side was taken . After obtaining adequate anaesthesia, the tooth was isolated with rubber dam and root canal treatment was initiated . The pulp chamber was irrigated with 3% sodium hypochlorite and carefully examined with an endodontic probe (dg-16, dentsply, gloucester, uk). All canals were scouted using k - file number 10 (dentsply, maillefer, ballaigues, switzerland). Working length was estimated using an apex locator (novapex, forum technologies, rishon le - zion, israel) and confirmed with a working length radiograph with k - files introduced into the canals . Ah plus (dentsply detrey, konstanz, germany) was used as a sealer . A postoperative radiograph was taken to assess the technical quality of root canal filling . When satisfactory, a permanent filling was placed . Figures 1, 2, and 3 show an example of a mandibular first molar with three roots . The incidence of re and comparison of the occurrence between males and females and between the right and left sides of the mandible were recorded . Comparison of the incidence and the correlations between males and females and left- and right - side occurrences were analyzed by using the pearson chi - square test with spss (15.0; spss inc ., three hundred and twenty two patients comprising 185 females and 137 males formed the study sample . There was no significant difference in the incidence of three - rooted mandibular first molars between females (7/185) and males (5/137) (table 1). However, there was a significant difference between the right side (8/12) and the left side (4/12) (p <0.05). Knowledge of both normal and abnormal anatomy of teeth dictates the parameters for execution of root canal therapy and can directly affect the probability of success . Therefore, practitioners should be familiar with the existence as well as the prevalence of teeth abnormalities . Mandibular first molars seem to be the most frequent teeth in need of root canal treatment as they are the first permanent teeth to erupt . Nonetheless, anatomical variations of the root canal system in molars are not appreciated by a great number of general practitioners . The presence of a third root (re) may complicate the endodontic treatment and lead to failure as a result of canal missing . While conducting root canal treatment in mandibular first molars, clinicians should be aware of this morphological abnormality . De moor et al . Type ii refers to an initially curved entrance which continues as a straight root / root canal . Type iii refers to an initial curve in the coronal third of the root canal and a second curve beginning in the middle and continuing to the apical third . The infrequent occurrence of re requires that the clinician be cautious in diagnosis and management of the lower molar teeth . Although it is not necessary, an additional root is often associated with an increased number of cusps and an increased number of root canals with a more prominent occlusodistal or distolingual lobe . Radiographs taken at different angulations reveal the basic information regarding the anatomy of a tooth and can thus help to detect any aberrant anatomy such as extra canals and/or roots . When the outline of the distal root or the root canal seems unclear on the preoperative radiograph, the presence of a hidden third root should be suspected . Studies have shown that a second radiograph should be taken from a more mesial or distal angle (30 degrees) which could probably reveal the presence of re . Periapical radiographs were used in this study because they are routinely used in the dental school of the arab american university throughout endodontic steps . This technique is noninvasive and inexpensive and allows for interstudy comparisons relating to gender and bilateral occurrence difference for three - rooted mandibular first molars . On the other hand, this method has some disadvantages . As radix entomolaris is mostly situated in the same buccolingual plane as the distobuccal root, a superimposition of both roots can appear on the radiograph resulting in an inaccurate diagnosis . The digital system offers many advantages over the conventional radiography such as ease and speed of use, reduction in time between exposure and image interpretation, less radiation dosage to the patient, elimination of chemical waste hazard, and the ability to digitally manipulate the captured image . Unfortunately, a more advanced technology represented by the cone beam computed tomography (cbct) is not available . Cone beam computed tomography scans were recently shown to be a valuable tool in several stages of endodontic treatment as they provide an immediate and accurate three - dimensional radiographic image . Preoperatively, these images give information about the internal and external tooth anatomy which include number and location of roots and canals, root and canal curvatures, size of the pulp chamber, and the degree of calcification . Cbct images allow a complete elimination of the superimposition of structural images outside the area of interest and provide a high - contrast resolution and data from a single computed tomography imaging process . They also provide three dimensional images in the axial, coronal, or sagittal planes . In cases of re, cbct shows the exact position of distolingual root and hence it helps in tracking the curvature and prevents iatrogenic event that might occur in relation to canal curvature like instrument separation, perforation, ledge formation, and so forth . When the occurrence of re is confirmed or suspected on the radiograph, the access cavity preparation should be modified from the classic triangular access to a more rectangular or trapezoidal outline . The orifice of re is mainly located disto- to mesiolingually from the main distal canal . If the entrance of re canal is not clearly visible after removal of the pulp chamber roof, a more thorough inspection of the pulp chamber floor and wall, especially in the distolingual region, is necessary . The introduction of dental operating microscope (dom) has changed the face of endodontics . Although the operating microscope is not used in our clinics, its use is recommended in routine endodontic practice as it offers an excellent illumination and magnification of the operating field and provides a tremendous advantage in locating and treating extra canals . Coelho de carvalho and zuolo reported that the dom had enabled them to locate 8% more canals in mandibular molars . Al - nazhan has examined 251 mandibular first molars of saudi patients (clinically and radiographically). He reported an incidence of 6% of re amongst saudi population . In their clinical investigation conducted on chinese population, yu et al . Screened 378 cases of mandibular first molars with root canal therapy and reported an incidence of 27% of teeth with re . In our study, the overall incidence of patients with three - rooted mandibular first molars was 3.73% . This finding was in a range of previous reports on middle easterners [24, 26]. However, it was low when compared with data reported for asian races: 24.5% in koreans, 32% in chinese, and 25.6% in taiwanese . In the present study, the same result was reported by other studies [11, 12, 28, 30]. When considering the right and left sides of the mandible, three - rooted mandibular first molars occurred more frequently on the right side than on the left side . These findings were in accordance with some previous studies [12, 28] and different from some others [11, 31] which reported that re can occur more on the left side . Loh report a bilateral occurrence of three rooted mandibular permanent first molars from 50 to 67%; however, in our study the bilateral occurrence was only 33.3% . This percentage was higher than the study on german population and much lower than the studies [2729] involving asian subjects (koreans, chinese, and taiwanese, resp . ). General practitioners as well as specialists in endodontics should always think about a possible third root (re) when planning a root canal treatment for a mandibular first molar . Careful clinical and radiographic examination is indispensable in the diagnosis of any anatomic variation in the root canal system of any tooth prior to initiating treatment . As this study revealed that the incidence of a third root in palestinian population was within the range of previous reports from the middle east but considerably lower than the percentage from the far east, the use of conventional two - dimensional radiographs for the assessment of re would be probably considered as a limitation in the clinical approach and methodology of this study . Hopefully, future research in palestine would be able to study a larger and more varied population utilizing cone beam computed tomography.
Tramp mice spontaneously develop autochthonous prostate tumors as a result of a transgene (sv40) controlled by the probasin promoter . While tramp tumors can be harvested at any age, it should be noted that tramp prostatic tumors generally remain small until the mice reach ages greater than 20 weeks . Remove the urogenital tract (ugt), by cutting open the peritoneal cavity and pulling back the adipose tissue . Locate the bladder; it lies directly between the two large, white seminal vesicles . Hold onto the bladder with forceps . Keeping the scissors closed, smooth away adipose tissue and cut down on the urethra as close to the pelvic girdle as possible and sever the vas deferens . While cutting down, simultaneously pull up on the bladder . This will release the entire ugt that can now be micro - dissected to obtain each of the lobes of the prostate . Use a dissecting microscope to visualize the ugt and clear away any excess adipose tissue . Using forceps, hold onto the urethra and collect the ventral, lateral and anterior lobes of the prostate . Using forceps, tease apart the prostate tissue and place it into the digestion (dissociation) buffer . Place tumor tissue in 1 ml dissociation buffer (100 u / ml collagenase type iv and 100 g / ml dnase in rpmi + 10% fbs). Each tumor may require unique dissociation conditions; for tramp prostatic tumors, use 1 ml and for b16 tumors, use 5 ml (see below). Note: the grade of collagenase (i - iv) will depend on the cell population to be isolated; myeloid cell isolation is best with type iv, whereas lymphocyte yield is higher using type i. place tube in 37 c incubator for 30 min . Pipette up and down with a 1 ml pipette to get an easily flowing single cell suspension . Filter suspension through 70 micron filter and wash 3x with macs separation buffer supplemented with 10% fbs for myeloid cell isolation . Then rinse the pellet with 10 ml macs buffer and centrifuge again with the same settings . Next, add 1 g anti - cd16/32 antibody (ab) per 10 cells (200 l 2.4.g2 hybridoma culture supernatant) and incubate at room temperature for 10 min . Note: the amount of fcr- blocking antibody (ab) to be added may vary depending upon the frequency / number of fcr- cells in the tissue and the volume of the cell suspension; therefore, this step may require optimization . Add 100 l of " pan - dc " or 10 l of anti - cd11b, anti - cd11c or anti - pdca-1 microbeads per 10 total cells, depending on the desired cell population . Note: the amount of microbeads required may vary depending upon the frequency / number of cells of the desired population in the tissue; therefore, this step may require optimization . Mix well by gently flicking tube (do not vortex) and incubate for 15 min at 4 - 8 c, shielded from light . Do not incubate on ice . Wash cells by adding 10 ml of macs buffer . Prime the column by rinsing with an appropriate amount of macs buffer for the column selected: for lc and ms columns use 500 l . For ls or ld columns use 1 ml . After priming the column, use one column for every 1 x 10 cells . Collect unlabeled cells (pass - through) and wash the column a minimum of three (up to five) times with 500 l volume for a total of 1.5 - 2.5 ml of fluid wash . Perform wash step 1 by adding macs buffer to the column; it is important to keep the column flowing . Do not let column stand without flow or allow it to run dry (this cannot be understated, as drying of the column can ruin purity and lead to significant loss of cell viability . ). For wash step 2, rinse the column with dissociation buffer mix (containing collagenase + dnase as described in step 1.7); this serves as a " harsher " wash step to flush out sticky debris from dead or dying tumor cells . Perform wash step 3 with macs buffer; if the flow through does not look completely clear after wash step 3, continue to wash for 2 additional wash steps with macs buffer (for a total of 5 washes). For large subcutaneous tumors (e.g., b16 melanoma), during the last wash step, apply gentle pressure with a gloved fingertip to the top of the column; this releases extra debris for a cleaner, purified cell population . This step is not required for solid tissue tumors such as prostate; instead, use extra wash steps, washing a total of at least 5 - 6 times . Collect the magnetically labeled cells by firmly applying the plunger supplied with the column . Count cell number and confirm purity for the selected cell population by flow cytometric analysis . For the identification of dc populations, suggested markers include cd45, cd11c, pdca-1, b220 and cd11b . Suggested macrophage markers include cd45, cd11b, f4 - 80, and ly6c . This protocol works best with tumors that are 250 mm or less (estimated by measuring bisecting diameters of the tumor). B16 tumor cells were injected subcutaneously into c57bl/6 mice and tumor measurements were recorded every 2 days 8 . Mice with tumors measuring 250 mm are euthanized by co2 asphyxiation . Using autoclaved surgical instruments rinsed with 70% etoh, cut tumor into small (<3 mm) pieces and incubate in 5 ml dissociation solution (rpmi medium supplemented with 5% fbs, collagenase type i (200 u / ml) and dnase i (100 g / ml)) for 30 min at 37 c, pipetting (using a 1,000 l pipet tip) and vortexing every 10 minutes during the incubation . If myeloid cells will be subsequently isolated, substitute 5% fbs and collagenase type i with 10% fbs and collagenase type iv (200 u / ml), respectively . After incubation, pass cell suspension through a 70 m cell strainer and wash twice with 10 ml macs buffer (prepared per manufacturer's instructions, miltenyi biotech). Optional - for very large tumors (> 300 mm), inflammatory cells can be pre - enriched using density gradient centrifugation (percoll or ficoll). Aspirate the wash supernatant and add 1 g anti - cd16/32 antibody/10 cells (200 l of clone 2.4.g2 culture supernatant) and incubate at room temperature for 10 min . Without washing, add 1 g anti - thy1.1 pe antibody per 10 cells, mix well by gently flicking the tube and incubate for 30 minutes on ice, shielded from light . Wash the cells to remove unbound primary antibody by adding 10 ml macs buffer and centrifuge at 400 xg for 5 minutes . Aspirate the supernatant and add 20 l anti - pe microbeads (miltenyi biotech) per 10 total cells, according to manufacturer instructions . Mix well by gently flicking tube (do not vortex) and incubate at 4 c (do not use ice) for 15 minutes, shielded from light . Caution: vortexing can diminish the integrity of the beads . Wash cells by adding 10 ml of macs buffer and centrifuge at 400 xg for 5 minutes . Aspirate supernatant, and resuspend cells in 500 l macs buffer . For tumors of a larger size (> 300 mm), use 1 ml buffer . Most tumors will also flow through ls and ld columns, but require further digestion and mechanical disruption to achieve the appropriate level of single cell suspension . Apply cell suspension onto the column and allow to completely flow through (without letting the column run dry). Next, wash with 500 l macs buffer, and repeat washing 3x . Only add new buffer when the column reservoir is empty, but do not let the column stand without flow . This is a critical step for large subcutaneous tumors: after the last wash step, with a gloved finger tip, apply gentle pressure to the top of the column while still on the magnetic separator . Next, wash the column 1 more time with 500 l macs buffer . Remove column from the separator and place it in a clean 15 ml conical tube, add 2 ml macs buffer onto the column . Flush out the magnetically - labeled cells by firmly pushing the plunger into the column . Purity at this step is typically between 75 - 80% . To achieve> 90% purity, repeat the magnetic separation procedure as described in step 1.10 - 1.12 using a new ms column . The ms column is smaller and more compact so the suspension will run more slowly through the column, but will yield higher numbers and purity of the cell of interest . Count cell number for further experiments and check purity by flow cytometric analysis . For isolation of adoptively transferred t cells such as those described in this protocol, allelic markers for identification include cd45 and thy1.1 (transferred cells) and thy1.2 (host t cells). The yield of a particular cell population (i.e. Macrophages, dc, t cell, etc .) Will vary depending on the size of the tumor and treatments that were administered during tumor growth . A prostate from an untreated 14 - 16 week old tramp mouse should yield between 8x10 - 1x10 cd11c / pdca-1 (dc) cells at 90 - 95% purity or 1x10 - 1.5x10 f4/80/cd11b (macrophages) at 80 - 90% purity from 300 mg of tissue following the isolation protocol above as shown in figure 1 . The number of each of these cells slightly increases upon adoptive transfer of tumor - antigen specific t cells . Poor purity is usually a result of insufficient washing, allowing the column to dry (which can result in tumor debris retention in the column), or insufficient fc receptor blocking . Similarly, the total cells isolated from b16 tumors will also vary depending on tumor size at the time of tissue harvest and adoptive transfer of t cells (transfer of 5x10) with or without dc vaccine (transfer of 1x10). Very few antigen - specific t cells infiltrate the tumor unless an antigen - pulsed dc vaccine is also given one day after t cell transfer . If a dc vaccine is administered to a mouse bearing a small, palpable (<50 mm) tumor, a yield of approximately 3x10 thy1.1 t cells, with a purity of 80 - 85%, would be considered a " good " harvest as shown in figure 2a . However, if larger tumors are harvested, total yield and purity will be reduced . Macrophages (f4/80/cd11b) are usually a smaller percentage of the total cells in b16 tumors . Figure 2b shows that utilizing cd11b, from a tumor that is 250 mm, yields approximately 1x10 macrophages at 90 - 95% purity . Additionally, figure 2b shows that the dc population in b16 tumors are heterogeneous . Unlike prostate tumors, two subpopulations: cd11c / pdca-1 (plasmacytoid dc) and cd11c / pdca-1 (conventional dc) can be obtained from b16 melanoma tumors . An estimated 2x10 pdc and 4x10 cdc reduced purity is usually a result of not clearing the tumors cells from the column . Step 2.12 is a critical step to remove the small clump of melanoma cells that persists at the base of the column . Following this step improves the effectiveness of the wash steps and results in better purity . Dcs (cd11c / pdca-1) and macrophages (f4/80/cd11b) were isolated from a tramp prostate tumor . Dot plot values represent percentage of cells of interest pre- or post - purification . (a) adoptively transferred thy1.1/cd8 t cells and (b) myeloid cells including cd11c / pdca-1 plasmacytoid dcs, cd11c / pdca-1 conventional dcs and f4/80/cd11b macrophages were isolated from subcutaneous b16 melanoma tumor from thy1.2 mice . Tramp mice spontaneously develop autochthonous prostate tumors as a result of a transgene (sv40) controlled by the probasin promoter . While tramp tumors can be harvested at any age, it should be noted that tramp prostatic tumors generally remain small until the mice reach ages greater than 20 weeks . Remove the urogenital tract (ugt), by cutting open the peritoneal cavity and pulling back the adipose tissue . Locate the bladder; it lies directly between the two large, white seminal vesicles . Hold onto the bladder with forceps . Keeping the scissors closed, smooth away adipose tissue and cut down on the urethra as close to the pelvic girdle as possible and sever the vas deferens . While cutting down, simultaneously pull up on the bladder . This will release the entire ugt that can now be micro - dissected to obtain each of the lobes of the prostate . Use a dissecting microscope to visualize the ugt and clear away any excess adipose tissue . Using forceps, hold onto the urethra and collect the ventral, lateral and anterior lobes of the prostate . Using forceps, tease apart the prostate tissue and place it into the digestion (dissociation) buffer . Place tumor tissue in 1 ml dissociation buffer (100 u / ml collagenase type iv and 100 g / ml dnase in rpmi + 10% fbs). Each tumor may require unique dissociation conditions; for tramp prostatic tumors, use 1 ml and for b16 tumors, use 5 ml (see below). Note: the grade of collagenase (i - iv) will depend on the cell population to be isolated; myeloid cell isolation is best with type iv, whereas lymphocyte yield is higher using type i. place tube in 37 c incubator for 30 min . Pipette up and down with a 1 ml pipette to get an easily flowing single cell suspension . Filter suspension through 70 micron filter and wash 3x with macs separation buffer supplemented with 10% fbs for myeloid cell isolation . Then rinse the pellet with 10 ml macs buffer and centrifuge again with the same settings . Next, add 1 g anti - cd16/32 antibody (ab) per 10 cells (200 l 2.4.g2 hybridoma culture supernatant) and incubate at room temperature for 10 min . Note: the amount of fcr- blocking antibody (ab) to be added may vary depending upon the frequency / number of fcr- cells in the tissue and the volume of the cell suspension; therefore, this step may require optimization . Add 100 l of " pan - dc " or 10 l of anti - cd11b, anti - cd11c or anti - pdca-1 microbeads per 10 total cells, depending on the desired cell population . Note: the amount of microbeads required may vary depending upon the frequency / number of cells of the desired population in the tissue; therefore, this step may require optimization . Mix well by gently flicking tube (do not vortex) and incubate for 15 min at 4 - 8 c, shielded from light . Do not incubate on ice . Wash cells by adding 10 ml of macs buffer . Prime the column by rinsing with an appropriate amount of macs buffer for the column selected: for lc and ms columns use 500 l . For ls or ld columns use 1 ml . After priming the column, use one column for every 1 x 10 cells . Collect unlabeled cells (pass - through) and wash the column a minimum of three (up to five) times with 500 l volume for a total of 1.5 - 2.5 ml of fluid wash . Perform wash step 1 by adding macs buffer to the column; it is important to keep the column flowing . Do not let column stand without flow or allow it to run dry (this cannot be understated, as drying of the column can ruin purity and lead to significant loss of cell viability . ). For wash step 2, rinse the column with dissociation buffer mix (containing collagenase + dnase as described in step 1.7); this serves as a " harsher " wash step to flush out sticky debris from dead or dying tumor cells . Perform wash step 3 with macs buffer; if the flow through does not look completely clear after wash step 3, continue to wash for 2 additional wash steps with macs buffer (for a total of 5 washes). For large subcutaneous tumors (e.g., b16 melanoma), during the last wash step, apply gentle pressure with a gloved fingertip to the top of the column; this releases extra debris for a cleaner, purified cell population . This step is not required for solid tissue tumors such as prostate; instead, use extra wash steps, washing a total of at least 5 - 6 times . Collect the magnetically labeled cells by firmly applying the plunger supplied with the column . Count cell number and confirm purity for the selected cell population by flow cytometric analysis . For the identification of dc populations, suggested markers include cd45, cd11c, pdca-1, b220 and cd11b . This protocol works best with tumors that are 250 mm or less (estimated by measuring bisecting diameters of the tumor). B16 tumor cells were injected subcutaneously into c57bl/6 mice and tumor measurements were recorded every 2 days 8 . Mice with tumors measuring 250 mm are euthanized by co2 asphyxiation . Using autoclaved surgical instruments rinsed with 70% etoh, cut tumor into small (<3 mm) pieces and incubate in 5 ml dissociation solution (rpmi medium supplemented with 5% fbs, collagenase type i (200 u / ml) and dnase i (100 g / ml)) for 30 min at 37 c, pipetting (using a 1,000 l pipet tip) and vortexing every 10 minutes during the incubation . If myeloid cells will be subsequently isolated, substitute 5% fbs and collagenase type i with 10% fbs and collagenase type iv (200 u / ml), respectively . After incubation, pass cell suspension through a 70 m cell strainer and wash twice with 10 ml macs buffer (prepared per manufacturer's instructions, miltenyi biotech). Optional - for very large tumors (> 300 mm), inflammatory cells can be pre - enriched using density gradient centrifugation (percoll or ficoll). Aspirate the wash supernatant and add 1 g anti - cd16/32 antibody/10 cells (200 l of clone 2.4.g2 culture supernatant) and incubate at room temperature for 10 min . Without washing, add 1 g anti - thy1.1 pe antibody per 10 cells, mix well by gently flicking the tube and incubate for 30 minutes on ice, shielded from light . Wash the cells to remove unbound primary antibody by adding 10 ml macs buffer and centrifuge at 400 xg for 5 minutes . Aspirate the supernatant and add 20 l anti - pe microbeads (miltenyi biotech) per 10 total cells, according to manufacturer instructions . Mix well by gently flicking tube (do not vortex) and incubate at 4 c (do not use ice) for 15 minutes, shielded from light . Caution: vortexing can diminish the integrity of the beads . Wash cells by adding 10 ml of macs buffer and centrifuge at 400 xg for 5 minutes . Aspirate supernatant, and resuspend cells in 500 l macs buffer . For tumors of a larger size (> 300 mm), use 1 ml buffer . Most tumors will also flow through ls and ld columns, but require further digestion and mechanical disruption to achieve the appropriate level of single cell suspension . Wash the column with 1 ml macs buffer to prime the column . Apply cell suspension onto the column and allow to completely flow through (without letting the column run dry). Next, wash with 500 l macs buffer, and repeat washing 3x . Only add new buffer when the column reservoir is empty, but do not let the column stand without flow . This is a critical step for large subcutaneous tumors: after the last wash step, with a gloved finger tip, apply gentle pressure to the top of the column while still on the magnetic separator . Next, wash the column 1 more time with 500 l macs buffer . Remove column from the separator and place it in a clean 15 ml conical tube, add 2 ml macs buffer onto the column . Flush out the magnetically - labeled cells by firmly pushing the plunger into the column . Purity at this step is typically between 75 - 80% . To achieve> 90% purity, repeat the magnetic separation procedure as described in step 1.10 - 1.12 using a new ms column . The ms column is smaller and more compact so the suspension will run more slowly through the column, but will yield higher numbers and purity of the cell of interest . Count cell number for further experiments and check purity by flow cytometric analysis . For isolation of adoptively transferred t cells such as those described in this protocol, allelic markers for identification include cd45 and thy1.1 (transferred cells) and thy1.2 (host t cells). The yield of a particular cell population (i.e. Macrophages, dc, t cell, etc .) Will vary depending on the size of the tumor and treatments that were administered during tumor growth . A prostate from an untreated 14 - 16 week old tramp mouse should yield between 8x10 - 1x10 cd11c / pdca-1 (dc) cells at 90 - 95% purity or 1x10 - 1.5x10 f4/80/cd11b (macrophages) at 80 - 90% purity from 300 mg of tissue following the isolation protocol above as shown in figure 1 . The number of each of these cells slightly increases upon adoptive transfer of tumor - antigen specific t cells . Poor purity is usually a result of insufficient washing, allowing the column to dry (which can result in tumor debris retention in the column), or insufficient fc receptor blocking . Similarly, the total cells isolated from b16 tumors will also vary depending on tumor size at the time of tissue harvest and adoptive transfer of t cells (transfer of 5x10) with or without dc vaccine (transfer of 1x10). Very few antigen - specific t cells infiltrate the tumor unless an antigen - pulsed dc vaccine is also given one day after t cell transfer . If a dc vaccine is administered to a mouse bearing a small, palpable (<50 mm) tumor, a yield of approximately 3x10 thy1.1 t cells, with a purity of 80 - 85%, would be considered a " good " harvest as shown in figure 2a . However, if larger tumors are harvested, total yield and purity will be reduced . Macrophages (f4/80/cd11b) are usually a smaller percentage of the total cells in b16 tumors . Figure 2b shows that utilizing cd11b, from a tumor that is 250 mm, yields approximately 1x10 macrophages at 90 - 95% purity . Additionally, figure 2b shows that the dc population in b16 tumors are heterogeneous . Unlike prostate tumors, two subpopulations: cd11c / pdca-1 (plasmacytoid dc) and cd11c / pdca-1 (conventional dc) can be obtained from b16 melanoma tumors . An estimated 2x10 pdc and 4x10 cdc are expected from 250 mm b16 tumors at 80 - 90% purity . Reduced purity is usually a result of not clearing the tumors cells from the column . Step 2.12 is a critical step to remove the small clump of melanoma cells that persists at the base of the column . Following this step improves the effectiveness of the wash steps and results in better purity . Dcs (cd11c / pdca-1) and macrophages (f4/80/cd11b) were isolated from a tramp prostate tumor . Dot plot values represent percentage of cells of interest pre- or post - purification . (a) adoptively transferred thy1.1/cd8 t cells and (b) myeloid cells including cd11c / pdca-1 plasmacytoid dcs, cd11c / pdca-1 conventional dcs and f4/80/cd11b macrophages were isolated from subcutaneous b16 melanoma tumor from thy1.2 mice . This protocol can be modified, based on the size and source of the tumor (subcutaneous, spontaneous tumor, or orthotopic tumors). For larger tumors, it is recommended to increase the amount of dissociation buffer, macs buffer, and number of washes . The heartiness of the cells isolated can depend on the tme from which they are being enriched . For example, in our experience, cells isolated from spontaneous prostate tumors require gentler dissociation than cells isolated from subcutaneous b16 melanoma tumors . During dissection of the tumor, eliminate as much adipose, skin, or other debris that can prevent effective enzymatic dissociation . It is critical to completely digest tumor masses and obtain a single cell suspension to ensure proper ab labeling and to prevent columns from clogging . For myeloid cell isolation, the amount of fetal bovine serum recommended in the macs isolation buffer was increased from 2% to 10% to improve cell viability . It is also essential to keep all buffers and cells cold throughout the protocol to prevent non - specific ab binding and clogging of the column . In conclusion, to obtain highly enriched populations of antigen presenting cells and tumor antigen - specific cytotoxic t lymphocytes, we have modified and optimized an antibody - based isolation procedure utilizing the miltenyi biotech technology . Utilizing this protocol, both adoptively transferred and endogenous leukocyte populations may be enriched using abs directed against cell type - specific surface markers . One advantage of the described procedures is the reduction of tumor debris carry - over following extensive and rigorous washing . This includes the use of collagenase in the wash buffer as well as identification of a point at which added pressure to the column can eliminate column clogs by tumor cells . Obtaining a sufficient number of immune cells from tissues, especially at a purity that is suitable for functional analysis, however, in our experience, the protocol herein yields the highest number of cells, at the greatest purity, with the most consistency.
Open surgery for fracture stabilization is often inappropriate in this population due to a poor risk - benefit profile, particularly if life expectancy is short . Percutaneous vertebroplasty and kyphoplasty are appealing adjunctive procedures in patients with malignancy for alleviation of intractable pain . Described in this report is a case of a painful osteolytic vertebral metastasis that was successfully treated by a novel percutaneous vertebral augmentation system . A 42-year - old caucasian female presented with a history of metastatic lung cancer unresponsive to radiation and chemotherapy with symptoms inadequately controlled by opiates over the previous 6 months . Magnetic resonance imaging and spiral computed tomography with two - dimensional reconstruction showed an osteolytic vertebral metastasis with complete involvement of the t10 vertebral body, extending to the cortical vertebral wall anteriorly and posteriorly . The patient was treated with percutaneous vertebral augmentation (kiva vcf treatment system, benvenue medical, inc, santa clara, ca) utilizing a novel coil - shaped polyetheretherketone implant designed to minimize the risk of cement extravasation . After the minimally invasive procedure, bone cement distribution within the vertebral body was ideal, with no observed cement extravasation . No complications were reported, pain completely resolved within 24 hours, and use of intravenous narcotics was progressively diminished within 1 week . The kiva system represents a novel and effective minimally invasive treatment option for patients suffering from severe pain due to osteolytic vertebral metastasis . If left untreated, progression of these lesions results in painful microfractures with potential for vertebral level collapse and spinal cord compromise . Open surgery for fracture stabilization is often inappropriate in this patient population due to a poor risk - benefit profile . Given that the average life expectancy in patients with vertebral metastasis is 1 year,1 surgery is undesirable since the postoperative recovery consumes much of the remainder of life . Therefore, nonoperative and minimally invasive techniques are the most appropriate treatment options for vertebral metastases . Percutaneous vertebral augmentation is a minimally invasive procedure involving the injection of polymethylmethacrylate (pmma) into a vertebral body that is either partially collapsed or at high risk for collapse due to osteolysis . Vertebral augmentation is appealing as an adjunct to radiotherapy or chemotherapy in patients with malignancy for alleviation of intractable pain, despite comprehensive nonoperative management . Several studies have reported dramatic improvements in pain severity associated with osteolytic vertebral metastases following percutaneous vertebral augmentation.24 however, percutaneous vertebral augmentation is associated with higher risk of complications from pmma extravasation in cancer patients compared with osteoporotic patients due to loss of cortical integrity.5 as such, malignancy is widely considered a relative contraindication for these procedures . Described in this report is the case of a painful osteolytic vertebral metastasis that was successfully treated by percutaneous vertebral augmentation using a novel coil - shaped polyetheretherketone (peek - optima, victrex plc, lancashire, uk) implant designed to minimize the risk of pmma extravasation . A 42-year - old caucasian female was referred to our institution in june 2011 for evaluation of pain palliation using percutaneous vertebral augmentation . The patient presented with a 4-year history of metastatic lung cancer unresponsive to radiation and chemotherapy, with symptoms inadequately controlled by opiates over the previous 6 months . The patient reported pain severity of 10 on an 11-point visual analogue scale and back disability of 89% on the oswestry disability index . Magnetic resonance imaging and spiral computed tomography (ct) with two - dimensional reconstruction revealed an osteolytic vertebral metastasis with complete involvement of the t10 vertebral body, extending to the cortical vertebral wall anteriorly and posteriorly (figure 1). Immediately prior to the procedure, the patient was premedicated with intravenous antibiotics (vancomycin hydrochloride 1 g and gentamycin 100 mg). The patient was placed in the prone position on the table of the angiographic suite, and the procedure was carried out with digital hybrid fluoroscopic and ct guidance . The entire procedure was performed under local anesthesia (3 ml of 2% lidocaine hydrochloride at the skin level and deep to include the periosteum) along with continuous monitoring of vital parameters . The peek implant is part of the kiva vcf treatment system (benvenue medical, inc, santa clara, ca) and is offered in a kit containing an access needle, kirschner guide - wire, dilator with working cannula, delivery system with nitinol coil guide - wire, and the implant bone cement and its manual screw injection system . After a percutaneous approach was accomplished with an 11-gauge needle manually inserted through the costotransversal (extra - pedicular) route to reach the midline and close to the upper endplate of the t10 vertebral body (figure 2a), a kirschner guide - wire was inserted to allow the placement of a 6-gauge working cannula (figure 2b). Through the 6-gauge introducer, the delivery system was inserted to allow the deployment of a nitinol coil - shaped guide - wire into the vertebral body (figure 2c). The kiva implant was then delivered over the nitinol guide - wire (figure 2d) inside the osteolytic lesion of the vertebra (figure 2e) to form a nesting, cylindrical column . Up to four loops of the implant may be inserted into the vertebral body for a maximum coil stack of 12 mm, which re - elevates the endplate . After the coil was retracted, a total of 6 ml of radiopaque bone cement was gradually injected using a manual screw injector through the kiva implant and its inner holes under fluoroscopic monitoring (figure 2f). Post - procedural ct scan with multi - planar reformatting demonstrated a satisfactory bone cement distribution within the vertebral body and the complete absence of extravasation either anteriorly or posteriorly (figure 3). Pain severity decreased from 10 to 0 within 48 hours, and analgesic opiate use was suspended . Back the patient was followed at 15 days, 1 month, 6 weeks, and 4 months post - treatment . Complete pain relief was maintained throughout 4 months of follow - up with no further need of analgesia . Further, previously radiated vertebral levels are at elevated risk for collapse, with potentially devastating neurologic consequences . This case report shows that percutaneous vertebral augmentation using the novel coil - shaped peek kiva implant is feasible, effective, and may reduce the risk of pmma extravasation in the treatment of painful osteolytic vertebral metastases with diffuse cortical bone involvement . The results of this report are novel in that the kiva implant represents a new therapeutic option for the treatment of painful vertebral metastasis where radiation therapy, traditional surgical stabilization, or balloon - based vertebral augmentation procedures may be contraindicated . Chew et al3 conducted a systematic review on the safety and efficacy of percutaneous vertebral augmentation for spinal metastases and myeloma . Although vertebral augmentation reduced pain severity by 47%87%, serious complications were reported in up to 12% of patients . The risk of cement extravasation is also notably higher in malignancies due to high vascularization and osseous destruction.5 resulting complications may include intercostal neuralgia, radiculopathy, myelopathy, or spinal infections.6 the kiva system was designed to reduce and stabilize vertebral fractures by deploying a coiled peek implant, which is then augmented with cement . Pmma bone cement is injected through the lumen of the kiva implant, which helps to contain and control the distribution of the cement . Once cured, the cement interlocks the implant to the cancellous bone . With traditional kyphoplasty and vertebroplasty procedures, pmma is injected into cancellous bone, but there is no mechanism for cement containment . Consequently, pmma cement may leak laterally to the soft tissues, superiorly or inferiorly into the adjacent disc space, or posteriorly, where it may involve the exiting nerve root or the spinal canal.6 a recent clinical study was conducted with the kiva system in 26 patients (42 fractures) for treatment of vertebral compression fractures.7 anterior cement extravasation was identified at 4.8% of levels with no reported intracanal extravasation or adverse clinical sequelae . These results compare favorably to cement extravasation rates of 7%72% reported in previous studies.812 the mechanism of pain amelioration with percutaneous vertebral augmentation is currently unknown, although two main hypotheses prevail . Polymethyl methacrylate cement stabilizes vertebral microfractures, which eliminates painful vertebral body and periosteal micromovements.13 in addition, the thermal polymerization of pmma following injection ablates pain receptors in trabecular bone, in vertebral periosteum, and in vascular structures.14 the combination of these two proposed factors leads to immediate postoperative anterior column stability and pain relief.4 the kiva system represents a novel and effective minimally invasive treatment option for patients suffering from severe pain due to osteolytic vertebral metastasis . The novel design of the kiva implant may reduce the risk for pmma extravasation versus traditional vertebral augmentation procedures . Prospective studies are needed to validate the safety and effectiveness of this device in patients with vertebral metastasis . Written informed consent was obtained from the patient for publication of this case report and accompanying images.
The causes of malnutrition in hd and pd patients include low energy intake induced by anorexia, loss of nutrients due to dialysis, dietary restriction before dialysis, increased protein catabolism and acidosis . The need for protein increases in pd patients due to severe peritoneal protein loss which is aggravated due to peritoneal infections . In addition to the foregoing, inflammation and inflammatory factors also play an important role in the development of malnutrition in these patients . Some complications of prolonged inflammatory process include protein loss, adipose and muscle atrophy, increased catabolism, oxidative stress, and atherosclerosis . Following the deterioration of the nutritional status, esrd results in inflammatory responses with various mechanisms such as decreased clearance of proinflammatory cytokines, release of oxidative substances such as free oxygen radicals and reduced intake of antioxidants such as vitamin e. recent studies have shown that patients suffering from malnutrition have a higher rate of mortality compared to well - nutritioned patients . The risk of cardiovascular diseases among hd patients with protein - energy malnutrition (pem) is higher compared to well - nourished hd patients . Traditional risk factors for cardiovascular diseases such as a high body mass index (bmi) and serum total cholesterol cannot explain the high prevalence of cardiovascular diseases in hd patients with pem . Nevertheless, heart failure is an important cause of mortality and morbidity in esrd patients . The prevalence of hypertension, left ventricular hypertrophy, ischemic heart disease, and heart failure is higher in patients with chronic renal failure on dialysis . Their cardiovascular mortality is about 1030 times more than normal populations . In one study, the prevalence of some cardiovascular disease in esrd patients includes 14% coronary artery disease, 19% angina pectoris, 31% cardiac failure, 7% dysrhythmia, and 8% peripheral vascular disease . On echocardiography 15% had systolic dysfunction, 32% left ventricular dilatation, and 74% left ventricular hypertrophy . According to above studies, however, no study has evaluated the relationship of echocardiographic findings and malnutrition in dialysis patients . Therefore, we compared the frequency distribution of malnutrition between hemodialysis and peritoneal dialysis patients and its relationship with echocardiographic findings . This case control study was done in a dialysis center, both hd and continuous ambulatory pd, in 20112012 in isfahan university of medical sciences, iran . The exclusion criteria included: patients with severe infection during 1 month before our study, change of dialysis method, history of rheumatic heart disease, cardiomyopathy, and primary pulmonary hypertension were not included . Malnutrition - inflammation score (mis) index was used for the evaluation of malnutrition . Mis consists of ten components, each with four levels of intensity, from 0 (normal) to 3 (severe). The ten components include dry weight change, dietary intake, gastrointestinal symptoms, work capacity, associated diseases, reduced fat stores or loss of subcutaneous fat, reduced muscle mass symptoms, bmi, serum albumin, and total iron - binding capacity levels . The sum of scores ranged from 0 (normal) to 30 (severe malnutrition). According to studies, the grading of severity of malnutrition according to mis score is as follows: lack of malnutrition to mild malnutrition: mis <9moderate malnutrition: mis = 918severe malnutrition: mis> 18 . Lack of malnutrition to mild malnutrition: mis <9 moderate malnutrition: mis = 918 severe malnutrition: mis> 18 . The patient's demographic information including age and sex along with their echocardiographic information such as ejection fraction, regurgitation of aortic, mitral, tricuspid, pulmonary valves, and mis were recorded . All the data were analyzed by spss version 18.0, inc, chicago, ill ., usa using descriptive statistics (mean and standard deviation of variables) and chi - square tests to compare mis based on the type of dialysis and echocardiographic findings and mann whitney u - test . Thirty - nine patients (72.2%) were men and 15 (27.8%) were women with a mean age of 59.77 1.33 . Thirty - five patients (63.60%) were men and 20 (36.4%) were women with the mean age of 54.21 1.30 . Table 1 shows the frequency distribution of mis in pd and hd groups and compares them . The frequency distribution of the malnutrition - inflammation score index there was no significant difference between the two groups in terms of the frequency distribution of malnutrition indicators (mis) (p> 0.05). The relationship between the type of dialysis and echocardiographic findings were analyzed using chi - squared test . In the hd group, there was no significant relationship between mis and echocardiographic findings (p> 0.05), except for aortic and mitral valve insufficiencies (p <0.05). Table 2 indicates that there was no significant relationship between mis and echocardiographic findings in pd patients (p> 0.05). The relationship between malnutrition - inflammation score and echocardiographic findings in hemodialysis and peritoneal dialysis groups many factors that appear to lead to these two conditions overlap, as do assessment tools and such criteria for detecting them as hypoalbuminemia . Both these conditions are related to poor dialysis outcome . Low appetite and a hypercatabolic state are among common features . Pem in dialysis patients has been suggested to be secondary to inflammation; however, the evidence is not conclusive, and an equicausal status or even opposite causal direction is possible . Possible causes of mis include comorbid illnesses, oxidative and carbonyl stress, nutrient loss through dialysis, anorexia and low nutrient intake, uremic toxins, decreased clearance of inflammatory cytokines, volume overload, and dialysis - related factors . Mis is believed to be the main cause of erythropoietin hyporesponsiveness, high rate of cardiovascular atherosclerotic disease, decreased the quality of life and increased mortality and hospitalization in dialysis patients . Because mis leads to a low bmi, hypocholesterolemia, hypocreatininemia, and hypohomocysteinemia, a therefore, obesity, hypercholesterolemia, and increased blood levels of creatinine and homocysteine appear to be protective and paradoxically associated with a better outcome . There is no consensus about how to determine the degree of severity of mis or how to manage it . Successful management of mis may ameliorate the cardiovascular epidemic and poor outcome in dialysis patients . In the comparison between the frequency distribution of mis in the participants of pd and hd groups, despite the 1.8% participants with severe malnutrition in the hd group, no statistically significant differences was observed . Although it was calculated in this study that there is no significant relationship between mis and echocardiographic findings (ejection fraction, aortic, mitral, pulmonary, and tricuspid valve insufficiencies) in pd participants, there was a significant relationship between mis and echocardiographic findings and aortic and mitral valve insufficiencies, and other echocardiographic findings had no significant relationship . The study by liu et al . Who evaluated peripheral vascular disease in hd patients, found that the rate of cardiac death, death due to infection, peripheral vascular disease, and cardiovascular hospitalization was higher in them compared to healthy participants . In another study, by pecoits - filho, it was found that the risk of diastolic heart failure was higher in patients with chronic renal failure compared to the general population . It can be concluded that if severe malnutrition aggravates in hd patients, the chance of a significant relationship between mis and echocardiographic findings increases . Finally, this project is recommended to be conducted with a lager sample size to repeat these results so that they are proven with greater certainty . Aen contributed in the conception of the work, conducting the study, revising the draft, approval of the final version of the manuscript, and agreed for all aspects of the work.ak contributed in the conception of the work, collecting data, conducting the study, revising the draft, approval of the final version of the manuscript, and agreed for all aspects of the work.ma contributed in the conception of the work, conducting the study, revising the draft, approval of the final version of the manuscript, and agreed for all aspects of the workga contributed in the conception of the work, revising the draft, approval of the final version of the manuscript, and agreed for all aspects of the work.fm contributed in the conception of the work, analyzing data, revising the draft, approval of the final version of the manuscript, and agreed for all aspects of the work . Aen contributed in the conception of the work, conducting the study, revising the draft, approval of the final version of the manuscript, and agreed for all aspects of the work . Ak contributed in the conception of the work, collecting data, conducting the study, revising the draft, approval of the final version of the manuscript, and agreed for all aspects of the work . Ma contributed in the conception of the work, conducting the study, revising the draft, approval of the final version of the manuscript, and agreed for all aspects of the work ga contributed in the conception of the work, revising the draft, approval of the final version of the manuscript, and agreed for all aspects of the work . Fm contributed in the conception of the work, analyzing data, revising the draft, approval of the final version of the manuscript, and agreed for all aspects of the work.
Functional imaging studies investigating therapy - induced recovery from aphasia after left - hemisphere stroke are rare (for review see). This holds true even more for research with patients, who suffer from chronic nonfluent aphasia and concomitant apraxia of speech (aos), a dysfunction of higher - order aspects of speech motor control characterized by deficits in programming or planning of articulatory gestures [2, 3]. Research results point out so far that neural correlates of functional recovery seem to involve both hemispheres . While in patients with small left - hemisphere lesions activation occurs to a greater extent in perilesional regions, in patients with large lesions involving the perisylvian language zone, there tends to be more activation of regions homologous to left - hemisphere language areas [4, 5]. Successful recovery seems to be correlated with perilesional activation; persistent right - hemisphere activation, however, seems to indicate slow and incomplete recovery [1, 611]. So far, only few studies demonstrated a direct impact of speech therapy on language recovery in chronic aphasia [12, 13]. The observation that even severely impaired aphasia patients are sometimes able to produce sung words more effectively than spoken words prompted many researchers and therapists to implement singing in the treatment of patients suffering from both motor speech disorders as well as aphasia [1423]. Melodic intonation therapy (mit), a form of speech therapy that was developed already in the 1970s, combines melodic intonation and rhythmic hand tapping with the objective to activate homologous language - capable regions in the right hemisphere [14, 22]. The pet - study of belin et al . Included seven nonfluent aphasia patients, who received melodic intonation therapy (mit) by comparing repetition of untrained mit - loaded words to repetition of the same words spoken with a natural intonation . Repetition of words using mit strategies resulted in a significant increase of activation in left broca's area as well as decrease in right - hemisphere regions . This result contradicts the essential objective of mit to engage homologous right - hemisphere language regions . One criticism of this study is that activation changes were not measured by means of pre- and posttreatment image acquisition . However, schlaug et al . Demonstrated treatment - associated fmri changes in the right - hemisphere encompassing premotor, inferior frontal, and temporal lobes in a patient suffering from broca's aphasia treated with mit compared to a control patient . Based on their post- and pretreatment results with diffusion tensor imaging, schlaug et al . Conclude that the right arcuate fasciculus can be remodeled by intense, long - term mit . As pointed out in detail in our previous studies, the greater bihemispheric organization for singing compared to speech might offer a chance for patients suffering from neurological speech and language disorders [2527]. Singing combines pitch and intonation processing but also temporal processing and we are particularly interested in the latter . Temporal organization is an essential characteristic of language and speech processing and seems to be extremely vulnerable to left - hemisphere brain damage . Lesion studies from the field of language as well as music demonstrate that patients with left - hemisphere lesions have problems with rhythm and time perception [2831]. Many studies confirm deficits in aphasia patients with regard to temporal structuring of speech but also in aos [3, 32, 6971]. Stahl et al . Investigated the importance of melody and rhythm for speech production in an experimental study with 17 nonfluent aphasia patients . While language production in aphasia patients may be nonfluent because of language - systematic reasons, for example, word retrieval deficits or agrammatic speech, temporal structuring in patients suffering from aos is affected because of deficits in temporal coordination or sequencing of speech movements [34, 35]. Extended segment and intersegment durations caused by disturbed anticipatory coarticulation result in slowed speech with visible and audible groping [3739]. A number of treatment approaches from the field of speech therapy implemented strategies to control for rhythm or rate of speech production with patients suffering from aos . Examples of such approaches are finger tapping, prolonged speaking, vibrotactile stimulation, metronomic pacing [3, 43, 44], or metrical pacing . Taking the temporal organization of speech into account, we developed rhythmic - melodic voice training (sipari), a music therapy technique that is based on specific use of the voice [17, 25]. Over the past years, we performed several behavioral studies with patients suffering from chronic aphasia and aos, which demonstrated that especially nonfluent patients significantly improved by this training [25, 26, 46, 47]. Since 2010, this treatment method is included in a cochrane review . In a prestudy with 30 healthy control subjects, we investigated the neural substrates of chanted vowel changes in rhythm sequences by functional imaging in order to find explanations for the efficacy of this treatment . Chanting is a rudimentary or simple form of singing, for example, on one pitch only . According to our findings, rhythm structure is a decisive factor concerning lateralization as well as activation of specific, language - related areas during simple singing . With increasing demands on motor and cognitive capabilities additional activation of inferior frontal areas of the left hemisphere occurred, particularly in those areas, which are described in connection with temporal processing and sequencing [4951]. These activations do not only comprise brain regions, whose lesions are causally connected with language disorders, but also regions of the left hemisphere (broca's area, insular cortex, and inferior parietal cortex), whose lesions are reported to cause aos [52, 53]. Our current study aims at investigating how the above - mentioned rhythmic - melodic voice training influences brain activation in patients with chronic nonfluent aphasia and concomitant aos . If it was possible to activate left - hemisphere language - related areas, as our imaging data with healthy subjects suggest, this might point to specific processes of reorganization, for example, improved temporal sequencing of sublexical speech components . Maybe, this explains at least in parts the efficacy of this treatment, which we already demonstrated in several behavioral studies mentioned above . Therefore, only three patients with severe chronic nonfluent aphasia and concomitant aos could be recruited from the aphasia center north rhine westphalia (aphasiker - zentrum nrw e.v .) For participation in this pilot study . Independently from the confirmation of the patients' therapists, three experienced speech therapists diagnosed the patients with aos on the basis of direct observations involving inconsistently occurring phonemic and phonetic errors, initiation problems, prolonged segment durations, prolonged intersegment durations (sound / syllable / word segregation), disturbed prosody, visible groping, and effortful speech (see [36, 45]). All patients were right - handed as determined by means of the edinburgh handedness scale, german speaking, and were included in the study 18 months after the incident . They had no perceptual hearing impairments and their auditory comprehension was sufficient to understand the instructions . Their capacity regarding concentration and attention was good and their general health condition was stable enough for continuous participation during the 6-month treatment period of this research study . Apart from general school education, none of the patients had any special musical training . All patients gave written informed consent in line with the declaration of helsinki and the institutional review board of the rwth aachen . This study was undertaken in compliance with national legislation . At the age of 53, u. suffered a left - hemisphere ischemic stroke involving in parts the area of the middle cerebral artery with secondary hemorrhage . Clinical symptoms were spastic hemiparesis on the right and severe broca's aphasia and moderate aos . Up to the accident, mr . U. was an industrial consultant in a leading position . At the age of 44, a. suffered an extensive left - hemisphere cerebral hemorrhage encompassing frontotemporal regions and the area of caudate nucleus with extension to the basal ganglia as well as left internal capsule . Clinical symptoms were spastic hemiparesis on the right and severe global aphasia as well as severe aos . Up to the accident, mrs . H. suffered an extensive left - hemisphere ischemic stroke in the area of the middle cerebral artery with displacement of the midline . Clinical symptoms were spastic hemiparesis on the right and both severe global aphasia and aos . Up to the accident, mr . A summary of the patients' characteristics is given in table 1 . Before and after therapy patients underwent the same fmri procedure as 30 healthy control subjectsin our prestudy in order to investigate if changes in brain activation occur due to the applied training . Tasks of our fmri paradigm comprised repetition of chanted vowel changes in rhythm sequences with differently demanding complexity levels for the following reasons: chanting is a rudimentary or simple form of singing, for example, on one pitch only and facilitates evaluating the influenceof rhythm structure because melodic components are reduced . / i/) requires exact temporal coordination and sequencing of speech movements . By focusing onsublexical processing with a single vowel change, we minimized the influence of semantic and lexical components of speech processing (for more details on the fmri tasks see). (8 vowels, alternately /a / i/) and differed as follows: (1) vowel changes with regular groupings, (2) vowel changes with regular groupings and rests, and (3) vowel changes with irregular groupings (see musical notations of figures 1(a), 1(b), and 1(c)). The length of each stimulus was electronically set to 4 sec . With a max . Patients had to listen and to immediately repeat the heard stimuli after the presentation had stopped . We used an event - related design with a total of40 trials per condition and 40 randomly included null - events . The stimuli were presented in a pseudo - randomized order with a mean interstimulus interval of 9 sec . The presentation time took 4 sec . And the duration of the repetition period varied according to the estimated jitter time . The paradigm was implemented in presentation (neurobehavioral systems) and synchronized to the scanner . Stimuli were presented binaurally through mr - compatible headphones with a sound absorption of 30 dba (resonance technology). All conditions were performed with eyes closed . Concerning movement artefacts, we point out that we compared three conditions utilizing the same response modality, that is, overt chanting . This allows generation of statistical maps that indicate activity more related to cognitive function than to movement . Since our tasks are essentially demanding with regard to cognitive abilities (e.g., attention, short - term memory), which are often impaired in patients with frontal lobe damage, a sparse temporal scanning design was not used in this study . We wanted to avoid attention loss and consequently lower functional response caused by relatively long interscan intervals, which are required in sparse temporal schemes . Moreover, stimuli were constantly sung at a frequency of 220 hz, which is beyond the main frequency peaks of the scanner spectrum . A remark in advance is that auditory stimulation was regarded as separately modeled condition in this design, which is not part of this paper . Auditory presentation and reproduction were time - shifted; patients did not sing along but after the presentation had stopped . Hence, the expected auditory activations in the auditory areas caused by the auditory stimulus presentation will not be present in the reported results (see). Recorded data of pre- and postrhythmic chanting of all patients were analyzed by 2 professional musicians (singer and percussionist) post hoc . Tone repetitions (a total of 8 tones per stimulus) had to be timed correctly with a max . Comparison of pre- and posttreatment performance of the patients for each of the three tasks was statistically assessed by mcnemar's test using exact binomial probability calculations (see table 3). Additionally, two experienced speech therapists of the aphasia center north rhine westphalia (aphasiker - zentrum nrw e.v .) Who were blinded to the experiment performed two well - established diagnostic procedures for the german language as control tests at baseline and at the end of the 6-month treatment period in order to assess potential changes in language and speech motor capabilities . One instrument used for assessment of the efficacy of the treatment was the aachener aphasie test (aat). The aat is a standardized procedure for evaluating type and severity of aphasia, developed and validated in the german language, subsequently translated into several european languages including english, and also validated and standardized in dutch and italian . The aat may also be applied repeatedly in order to assess the efficacy of speech therapy interventions . The presence and type of aphasia were established using the alloc classification procedure, a nonparametric discriminant analysis computer program using the normative data of the aat . The aat consists of six description levels for spontaneous speech (communicative verbal behavior, articulation and prosody, automatized language, semantic structure, phonemic structure, and syntactic structure) and five subtests (token test, repetition, written language, naming, and comprehension) for the assessment of specific language impairments . For an assessment of the degree of language impairment related to the entire group of aphasia patients, the aat assesses percentile scores from the score values of the five subtests, that is, token test, repetition, written language, naming, and comprehension . The percentile score found for one test value indicates the percentage of patients of the exercise sample (n = 376) who have achieved the same or a lower score . Although our primary focus was on expressive verbal behavior and motor speech performance, written language and comprehension were also assessed because reliable data regarding the speech profile can only be achieved if the aat is administered in its complete version . Apart from that, a more general transfer to other language modalities like written language and comprehension could not be excluded . Although not standardized, the hierarchical word list (hwl) is the first german diagnostic procedure, which allows systematic assessment of the symptoms caused by aos . The procedure contains a word / nonword repetition test (48 words and 48 matched nonwords) with word length varying between one and four syllables . Half of the items are phonologically simple (single consonants in syllable onsets or codas) and half are complex (consonant clusters in onsets or codas). 96 items) are assessed in a quantitative analysis as regards number of assessable items, phonetic structure, phonemic structure, and speech fluency . Qualitative analysis evaluates speech effort, groping, syllabic speech, and deviant word accent on a scale from 0 (without abnormality) to 3 (very pronounced) on an overall visual analogue scale . Functional images were obtained with a whole - body 3 t siemens trio mri - system . Participants were fixated in the head coil using velcro straps and foam paddings to stabilize head position and minimize motion artefacts . After orienting the axial slices in the anterior - posterior commissure (ac - pc) plane functional images were acquired using a t2 * -weighted echo planar imaging (epi) sequence with a repetition time (tr) of 2200 ms, an echo time (te) of 30 ms, and a flip angle (fa) of 90 degrees . 640 volumes consisting of 41 contiguous transversal slices with a thickness of 3.4 mm were measured . A 64 64 matrix with a field of view (fov) of 220 mm was used, yielding an effective voxel size of 3.44 3.44 3.74 mm . Functional images were preprocessed and analyzed using spm8 (wellcome department of cognitive neurology london, uk). During preprocessing, images were realigned and unwarped in order to correct for motion and movement - related changes in magnetic susceptibility . Translation and rotation correction did not exceed 1.8 mm and 1.9, respectively, for any of the participants . The anatomical t1 images of the patients were coregistered to the mean functional image using a rigid - body transformation implemented in spm8 so that activation maps could be displayed on the structural images . As this study included patients with extended lesions, which may cause problems with the normalizing algorithm, finally, all functional images were smoothed using a gaussian filter of 8 8 8 mm to increase signal - to - noise ratio in the images . In the first - level statistical analyses, each preprocessed functional volume was entered into a subject specific, fixed - effect analysis using the general linear model approach for time - series data suggested by friston and coworkers [60, 61] and implemented in spm8 . The data were high - pass filtered using a set of discrete cosine basis functions with a cut - off period of 128s in order to exclude low frequency confounds . For each of the 3 conditions of interest statistical parametric maps (spms) were evaluated and voxels were considered significant if their corresponding linear contrast t values were significant at a voxelwise threshold of p = 0.05 (fdr - corrected). Each patient received 50 individual therapy sessions (60 minutes, twice a week) over a period of 25 weeks . During this period, no speech therapy took place . In order to control for comparable treatment conditions patients received exactly the same treatment program . We emphasize that none of the stimuli of the fmri paradigm was trained during the treatment period of 6 months . The applied rhythmic - melodic voice training (sipari) comprises six components: singing, intonation, prosody, breathing (german: atmung), rhythm, and improvisation . The efficacy of this treatment could be demonstrated in several behavioral studies with patients suffering from chronic aphasia and aos [25, 26, 46, 47]. In 2010 a pseudorandomized controlled study with chronic nonfluent aphasia patients (mean duration of aphasia: 11,5 years), which examined the effects of the sipari method, was included in a cochrane review . Focusing initially on melodic speech components, which are mainly processed in the right hemisphere, a stepwise change to temporal - rhythmic speech components is carried out with the objective to stimulate phonological and segmental capabilities of the left hemisphere . To this end, an essential core of the treatment constitutes rhythmic singing with differently demanding complexity levels as concerns motor and cognitive capabilities . Since this treatment has been developed especially for severely impaired patients, an essential part of the verbal material comprises sublexical tasks (i.e., single vowels, vowel changes, consonant - vowel changes, etc .) In order to enable those patients to practice motor and cognitive function like planning, programming, and sequencing, that is, basics of language processing . The objective is a general transfer from the level of sublexical speech components to the level of words and phrases . In terms of linguistics, sipari intervenes at the interface of phonological and phonetic encoding where access to the mental syllabary is supposed to take place [62, 63]. By embedding segmental and syllabic speech elements in rhythmic sequences with differently demanding complexity levels, apart from the fact that grouping or chunking (i.e., bundling events together into larger units) serves to enhance maintenance of information in working memory [65, 66], temporal - rhythmic chunking promotes speech motor processes by training intersyllabic programming, which is supposed to play an important role in phonetic planning . In contrast to other treatment approaches mentioned in the introduction, which use pacing techniques or synchronous singing to an external timekeeper, sipari focuses on encouraging self - initiated planning and sequencing performance . Therefore, we give special emphasis on vocal training in connection with cognitive function, for example, executive control and working memory . This implies training of auditory short - term maintenance of melodic and rhythmic information in order to enable patients in a second step to coordinate the maintained information with verbal material . Treatment objectives are to improve motor, linguistic, and cognitive functions and thus to support speech motor processes and also language - systematic processes, that is, to encourage planning, programming, and sequencing . We did not include a description of the method because detailed information on the method as well as selection of exercises has been already published elsewhere [17, 25, 46, 68]. To determine how neural activity differed before and after therapy, both subtractions (i.e., pre - minus post - therapy and post - minus pre - therapy) were performed for all conditions . The anatomical localizations were determined by two experienced experts (neuroanatomist and neuroradiologist) from the university hospital aachen . U. subtraction post - minus pre - therapy yielded significant activation in the left hemisphere, comprising the basal ganglia (caudate nucleus), insula, and inferior frontal regions . A. subtraction post - minus pre - therapy demonstrated significant activations in both hemispheres, including superior and middle temporal gyrus . (3) mr . H. subtraction post - minus pre - therapy yielded bilateral activation of the superior temporal gyrus, however, more pronounced in the left hemisphere . U. while subtraction pre - minuspost - therapy demonstrated significant activation in the right precentral gyrus, the reverse subtraction yielded a shift of significant activation to the left precentral gyrus and superior temporal gyrus . The reverse subtraction, however, showed significant activation of the left superior temporal gyrus and bilateral inferior frontal gyrus . H. pre - minuspost - therapy subtraction showed activation in the right middle and superior temporal gyrus . The reverse subtraction demonstrated significant bilateral activation in the superior temporal gyrus and precentral gyrus activation in the right hemisphere . H., neither subtraction pre - minuspost - therapy nor the reverse subtraction showed any results . U. while no significant activation could be found in subtraction pre - minuspost - therapy, the reverse subtraction yielded significant activation in the left hemisphere comprising the superior and middle temporal gyrus . With regard to anatomical locations, cluster sizes, and t values see table 2 . Recorded data of pre- and post- rhythmic chanting of all patients were analyzed by 2 professional musicians (singer and percussionist) post hoc . Comparison of pre- and posttreatment performance of the patients for each of the three tasks was statistically assessed by mcnemar's test using exact binomial probability calculations (see table 3). All patients improved statistically significant (p <0.001) in condition (1) vowel changes with regular groupings . Analyses of condition (2) vowel changes with regular groupings and rests yielded statistically significant improvements for mr . U. achieved a statistically significant improvement (p <0.012) in condition (3) vowel changes with irregular groupings . Clinically significant improvements could be assessed in the subtests token test, repetition, and naming . U. less speech effort and less syllabic speech (improvement of 1.5 points each out of an overall scale of 3 points) and for mrs . A. and mr . H. less speech effort and less groping (improvement of 1 point each). Difficulties with temporal coordination or sequencing of speech movements are frequently reported in patients suffering from aphasia and aos [3, 32, 6971]. Our own experiences are in accordance with these findings and prompted us to develop rhythmic - melodic voice training sipari [17, 25], which was applied in this study (see sections 1 and 3.4.1). The major objective of this pilot study was to investigate how this training influences brain activation in three patients with severe chronic nonfluent aphasia and aos (1 broca's, 2 global aphasia patients). Before and after therapy each patient underwent the same fmri procedure as 30 control subjects in our prestudy . To determine how neural activity differed before and after therapy, both subtractions (i.e., pre - minus post - therapy and post - minus pre - therapy) were performed for all three conditions (see section 4.1 and figures 1(a), 1(b), and 1(c)). In addition, pre- and posttreatment results of patients' vocal production as well as their language and speech motor performance were examined by cognitive methods . Musical analyses of the recorded data revealed that before therapy none of the patients had any strategy to manage the demands of this condition . It should be mentioned that this condition comprises either no vowel change within one beat or the same tone durations and regular changes within one beat . From beat to beat tone durations change in even - numbered ratios (see musical notation of condition (1) figure 1(a)). A conceivable strategy to keep the respective stimulus in short - term memory in order to reproduce it afterwards could be, for example, to group vowel changes on the basis of tones with equal durations [64, 72, 73]. We suggest that due to therapy patients could use an adequate strategy in the post - therapy assessments more effectively . First, musical analyses corroborate this assumption . Secondly, subtraction post - minus pre - therapy resulted in brain activation comprising areas, which are described not only in connection with temporal processing and sequencing [4951] but also with language and speech processing, for example, inferior frontal gyrus, insula, basal ganglia (caudate nucleus), and particularly superior temporal gyrus [1, 7476]. However, while significant activations of the broca's aphasia patient (mr . U.) Were found exclusively in the left hemisphere, in both global aphasia patients (mrs . A. and mr . H.) significant activations were measured in perilesional and also in homologous areas in the right hemisphere (see figure 1(a)). Moreover, comparison of all three conditions points to increases or changes of activation that differ depending on task demand; for example, for all patients, activation was most pronounced in this post - minus pre - therapy comparison . However, these findings are all the more remarkable as our study also included chronic global aphasia patients with large lesions . H. also improved in basically all measures regarding his language performance a possible explanation could be that in contrast to the other two patients at least parts of his arcuate fasciculus are still intact . However, we cannot verify this assumption because diffusion tensor imaging data are not available . Further research is needed, especially if we compare our results with other studies, which investigated the therapeutic effect of singing on language rehabilitation . For instance, schlaug et al . Demonstrated treatment - associated fmri changes in the right - hemisphere encompassing premotor, inferior frontal, and temporal lobes in a patient suffering from broca's aphasia treated with melodic intonation therapy (mit). Based on their post- and pretreatment results with diffusion tensor imaging, schlaug et al . Even conclude that the right arcuate fasciculus can be remodeled by intense, long - term mit . Musical analyses of condition (2) vowel changes with regular groupings and rests demonstrated that mr . H coped better with this task already at the beginning of the treatment (see table 3). U. a shift of activation from right precentral gyrus in the pre - minus - posttreatment subtraction to left precentral gyrus and superior temporal gyrus activation in the reverse subtraction (see figure 1(b)). The same way, data of mr . H. demonstrate that activation changed from right middle and superior temporal gyrus activation in the pre - minus - posttreatment subtraction to bilateral superior temporal gyrus activation in the reverse subtraction . Additional activation could be measured in the right precentral gyrus . What is special about this condition is that implementation of rests brings about higher demands on timing because legato and staccato vocalization changes from beat to beat (see musical notation of condition (2) figure 1(b)). This way of vocalization requires precise execution of articulatory movements because staccato and legato vocalizations change from beat to beat . Particularly, the initial phase of vocal preparation becomes the focus of attention, which is reported to be dominated by the left hemisphere . Since findings of our prestudy with healthy subjects also corroborate this assumption, this may explain the shift from right to left superior temporal and precentral gyrus (mr . U.) Representing improved auditory - motor interaction in a task, which requires exact executed vocalization . H., who additionally activated left superior temporal gyrus but had to compensate with regard to motor preparation by activating right precentral gyrus due to his lesion in the left homologue . Musical evaluations confirmed more correct entries and improved legato and staccato differentiation in the post - therapy analyses . While mrs . A. was not able to manage this task before therapy, musical evaluation as well as post - minus pre - therapy subtraction point to improved planning with significant activations in the left superior temporal gyrus . Bilateral inferior frontal gyrus activation but also activation of right cingulate gyrus suggest that this task was demanding for mrs . A. based on the results of our prestudy, we assume that these activations are related not only to sustaining attention in order to maintain temporal information in memory but also to coordination of response generation and respective action planning . The only patient, who developed a strategy to manage condition (3) vowel changes with irregular groupings post - therapy, was mr . U. his post - minus pre - therapy subtraction data yielded significant left - hemisphere activation in the superior and middle temporal gyrus (see figure 1(c)). Since irregularity of this condition caused by implementation of syncopations, dottings, and rests further increases the demands on auditory - motor interaction, activity seems to be focused on this area, which is reported to be interfacing with motor planning systems for sublexical aspects of speech [75, 76]. One may object that posterior superior temporal gyrus activation in basically all of our post - minus pre - contrasts, with an asymmetry towards the left, might only indirectly reflect any improvements but merely auditory processing . This may hold true if we had limited our analysis to the fmri data only . Post- and pretest comparisons revealed clinically significant improvements for all patients in the aachener aphasie test (aat) concerning the subtests token test (mr . U. and mr . H.), repetition (mr . H.), and naming (mrs . A. and mr . Furthermore, all patients achieved a clinically significant increase in profile, thus testifying to the fact that an improvement in the overall range of all five subtests occurred (see figure 2). These improvements are remarkable as they concern expressive language capabilities (in particular naming) of two severely impaired global aphasia patients (mrs . H.). Particularly in connection with further improvements in the token test (mr . H.), which represents a measure to evaluate the severity of the aphasic disorder, these results imply that more comprehensive activation of language - systematic processes must have been initiated . Likewise, this assumption is corroborated by substantial improvements in spontaneous speech for all patients (see table 4). Our findings are in line with our previous therapy studies [25, 26, 46, 47]. We suggest that specifically focusing on improving cognitive function (e.g., auditory short - term and working memory performance), which is one of the main objectives of the applied treatment, is an essential reason for these improvements (see section 3.4.1 and). Moreover, assessments of speech motor capabilities of the patients revealed improvements concerning number of assessable items (mr . H.), phonetic structure (mr . All patients produced the items with significantly less speech effort in the posttest, two of them, namely, the global aphasia patients with severe aos (mrs . A. and mr . The improvements concerning phonetic structure and phonemic structure are remarkable insofar as they indicate that not only retrieval of motor plans for phones but also sequential organization of movements for a series of phones improved, exactly those processes that are particularly impaired in patients with aos [34, 80]. Likewise, one frequently cited temporal characteristic of apraxic speech is a reduction in overall speech rate . Since qualitative analysis yielded that all patients improved in speech fluency, it would appear that also patients with aos benefit from a treatment, which combines motor and cognitive training . In this therapy study including three patients with chronic nonfluent aphasia and aos, we demonstrated the effects of rhythmic - melodic voice training (sipari) by functional imaging . While post - minus pretreatment imaging data of the broca's aphasia patient (mr . U.) Yielded significant left - hemisphere activation in perilesional regions, activation patterns of both global aphasia patients (mrs . A. and mr . H.) comprised perilesional regions as well as homologous areas in the right hemisphere . A neural correlate of a system, which is supposed to interface with motor planning systems for sublexical aspects of speech [75, 76], was consistently located in the left superior temporal gyrus . This auditory - motor circuit provides the essential neural mechanisms for phonological short - term memory [81, 82]. Functional reintegration of this region is mentioned in the literature in connection with language improvement [1, 10, 8385]. Although patients of our study are already in the chronic stage and have large lesions, they improved significantly with regard to language but also speech motor capabilities . They recruited parts of the neural network that we previously found in healthy subjects using the same fmri paradigm, for example, inferior frontal gyrus, insula, and basal ganglia . In addition, our findings indicate that also in severely impaired patients activations vary with task demand . Therefore, further research will elucidate potential influences in greater detail, for example, the relationship between rhythm structure, grouping strategy, and phonological working memory . Based on our results, we assume that, for example, an improvement of short - term storage of sublexical phonological material and, as a result of this, improved temporal sequencing possibly represent one essential prerequisite for improvements of speech motor but also language capabilities . Planning, programming, and sequencing include motor as well as cognitive capabilities . In this context, the singing voice may serve as a gateway be it that linguistic as well as musical components are applied systematically.
Heart failure (hf) is the final consequence of different heart diseases and it represents a prominent cause of morbidity and mortality worldwide . At the cellular level, the occurrence of hypertrophy of cardiac muscle cells represents a common feature of failing myocardium [2, 3] and is considered as an adaptive response to increased external load in the presence of pathological situations such as hypertension or myocardial infarction . However, sustained overload eventually leads to contractile dysfunction and hf through incompletely understood mechanisms [46]. Dysfunctional vascular regulation is an important component of the pathophysiology of hf, and reduced levels of vascular endothelial growth factor (vegf) have been observed in myocardium models of advanced hf . Angiogenesis was enhanced during the acute phase of adaptive cardiac growth but reduced as hearts underwent pathological remodelling and it has been demonstrated that inhibition of vegf signalling at the myocardium level leads to the transition from compensatory hypertrophy to cardiac failure, since both heart size and cardiac function are angiogenesis - dependent . A large number of preclinical studies have raised hope that increasing the expression of vegf and/or other proangiogenic cytokines at the myocardial level show beneficial effects especially in animal models of postmyocardial infarction [7, 8]. Nevertheless, the clinical trials based on the proangiogenesis hypothesis have failed to provide conclusive results on the therapeutic benefits of clinical approaches aimed at improving myocardial angiogenesis especially in the early phases after myocardial infarction . It should also be noticed that although most experimental studies point to a positive role of angiogenic cytokines at the cardiac level, few studies have addressed the potential role of circulating proinflammatory and proangiogenic cytokines in patients with hf . Since human serum contains a myriad of cytokines, a major limitation to the study of the proangiogenic capability of human serum is also due by the fact that most in vitro and in vivo angiogenic tests are complex and not easy to be reproduced [9, 10]. On these bases, in the present study, we have adopted a simple and reproducible in vitro endothelial cell proliferation assay in order to investigate the proangiogenic effects of human sera obtained from both healthy individuals and from a limited group of hf patients . The differential capability to promote in vitro endothelial cell proliferation was correlated with the presence and level of a variety of cytokines, analysed with the multiplex technology, and, for the hf patients, with relevant clinical parameters, such as ntpro - bnp levels and occurrence of cardiovascular events in the follow - up . The healthy group was represented by 66 subjects (age range: 2560 years). The study approval was granted by the ethics review board of the azienda ospedaliero - universitaria, arcispedale sant'anna, ferrara, and informed consents were obtained in accordance with the declaration of helsinki of 1975 . The main demographic and clinical parameters of patients were abstracted from clinical records and are reported in supplementary table 1 (see supplementary material available online at http://dx.doi.org/10.1155/2014/257081). Hf diagnosis was based on history of hf of at least six months duration, reduced exercise tolerance, objective left ventricular functional impairment (lvef), and raised level of n - terminal pro - brain natriuretic peptide (ntpro - bnp) above the normal range at hospital entry . Hf staging was performed by the new york heart association (nyha) classification and on the basis of ntpro - bnp value . Serum of healthy individuals and of hf patients was obtained from blood samples collected from an antecubital vein . After clot formation, samples were centrifuged at 3000 rpm for 15 min and serum was immediately stored frozen at 80c in single - use aliquots . Human umbilical vein endothelial cells (huvec) were isolated from umbilical cords as previously described, with some modification [1214]. Briefly, after cannulation and rinsing with cord buffer (pbs supplemented with 0.011 m glucose), the umbilical vein was infused with type 1 collagenase solution (0.4 mg / ml; worthington, lakewood, nj) and the umbilical cord was placed for 20 min at 37c for enzymatic digestion . Vein was flushed with warm egm-2 medium (lonza, walkersville, md) and the resulting endothelial cell suspension was centrifuged for 10 min at 150 g . Primary cultures of huvecs were seeded into 25 cm flasks precoated with fibronectin (bd bioscience, becton dickinson, san jos, ca) at 5 g / cm and cultured in egm-2 medium at 37c in a humidified atmosphere with 5% co2 . Primary cultures of huvec were dissociated with 0.025% trypsin/0.025% edta (gibco brl, grand island, ny) and collected by centrifugation for cell banking and/or to perform in vitro experiments . Cell viability was monitored by light microscopic analysis of the cell monolayers after hematoxylin - eosin staining or by quantitative examination after detachment of the monolayers by means of trypan blue dye exclusion, as described in [15, 16]. For this study, 80% of confluent huvec monolayers (passages 25) the purity of primary huvec cultures was evaluated by flow cytometry analysis performed on a bd facsaria ii (bd), as previously described [14, 17]. Briefly, detached cells were resuspended in 200 l of pbs containing 1% bsa (sigma - aldrich, saint luis, mo) and incubated 30 min at 4c with the following antibodies (ab): fitc - conjugated anti - cd146 (miltenyi biotec, gladbach, germany, clone 541 - 10b2), horizon v450-a - conjugated anti - cd144 (bd, clone 55 - 7h1), pe - a - conjugated anti - cd31 (miltenyi biotec, clone ac128), alexa fluor 647-a - conjugated anti - cd105 (bd, clone 266), pe - cy7-a - conjugated anti - cd34 (bd, clone 581), apc - h7-a - conjugated anti - cd45 (bd, clone 2d1), and horizon v500-a - conjugated anti - cd14 (bd, clone m5e2). Data files were collected and analysed using the facsdiva software program (version 6.1.3; bd). Only cell suspensions characterized by endothelial cell purity equal to or greater than 98% and low levels of apoptosis, evaluated by annexin - v / pi double staining [18, 19], were used for the in vitro assays . Cell proliferation experiments were performed using the xcelligence real - time cell analyzer (rtca - dp version; roche diagnostics, mannheim, germany), which monitors continuously the cellular events recording label - free changes in electrical impedance (reported as cell index). Briefly, the background impedance was performed using the standard protocol provided in the software with 100 l egm-2 complete medium (supplemented with 2% of fetal bovine serum and specific endothelial growth factors) per well, in 16-well plates . In each assay, four thousand endothelial cells (huvec) were seeded in 100 l of complete egm-2 in quadruplicate in fibronectin - precoated wells and left to equilibrate at room temperature for 30 min before data recording . Cultures were grown at 37c in a humidified atmosphere with 5% co2 over - night until preestablished cell index . Then cultures were washed two times with fresh rpmi medium before the addition of rpmi medium supplemented only with 20% human serum derived from the control subjects or from the hf patients . In selected experiments, human serum was added to the cell cultures after 30 min of preincubation with neutralizing ab anti - human vegf (peprotech inc ., rocky hill, nj) or with control ig (sigma). In parallel, as control, endothelial cell cultures were exposed to recombinant vegf165 (peprotech). Data were analysed using the xcelligence software (version 1.2.1) and expressed as mean sd of cell index normalized to the last cell index recorded before the time of cells treatment (addition of human serum). Serum samples were frozen and thawed only once before performing the milliplex map human cytokine / chemokine panel (merck millipore, billerica, ma), a bead - based multiplex immunoassay, which allows the simultaneous detection and quantification of the following 29 human cytokines / chemokines: egf, eotaxin, g - csf, gm - csf, ifn-2, ifn-, il-10, il-12(p40), il-12(p70), il-13, il-15, 1l-17, il-1 receptor antagonist (ra), il-1, il-1, il-2, il-3, il-4, il-5, il-6, il-7, il-8, ip-10 (cxcl10), mcp-1, mip-1, mip-1, tnf-, tnf-, and vegf . Samples were processed following the manufacturer's recommended protocol and analyzed by using a magpix instrument provided with the milliplex - analyst software that uses a five - parameter nonlinear regression formula to calculate cytokine / chemokine concentrations from the standard curves . For each set of experiments, values were reported as median and/or means sd . The results were evaluated by using student's t- and the mann - whitney rank - sum tests, when appropriate . One of the purposes of this study was to set up a reliable and reproducible biological assay using huvec as cellular target for monitoring the overall presence of circulating cytokines with a proangiogenic activity . For this purpose, purity of endothelial cell cultures was determined by flow cytometry analysis as cells expressing (98%) cd146, cd144, cd31, cd105, and cd34 and negative for cd45 and cd14 (figure 1(a)). In order to monitor and reproducibly measure endothelial cell proliferation, we adopted a system which measures the impedance of the cell monolayer (cell index, ci) in real - time and in label - free manner without disturbing / altering the culture (figures 1(b) and 1(c)). For this assay, endothelial cells were seeded and let to adhere and proliferate in the presence of complete medium (supplemented with 2% of fetal bovine serum and specific endothelial growth factors) until reaching a preestablished cell index value, settled in the range from 2.5 to 3.5 . Subsequently, to assess and comparatively measure the effects of human serum samples on endothelial cell proliferation, medium was removed and cultures were extensively washed before the addition of medium supplemented only with 20% human serum (figures 1(b) and 1(c)) collected from healthy subjects and hf patients . Cell index was recorded for up to 48 hours after treatment and analysis was carried out after normalization (figures 1(b)1(d)). In the first group of experiments, we analysed the ability of sera obtained from healthy subjects in promoting / affecting endothelial cell proliferation . In this analysis, we observed that, at the time points examined, sera obtained from older (mean age: 52 7.6) subjects induced a significantly (p <0.05) higher huvec proliferation compared to sera obtained from younger (mean age: 29 8.6) healthy subjects (figures 1(c) and 1(d)). This unexpected finding suggested differences between the sera of young and older healthy individuals in terms of angiogenic and/or angiostatic cytokine levels . Therefore, to check this hypothesis, we next evaluated the levels of several circulating cytokines and chemokines using the luminex technology that allows the simultaneous detection and quantification of a panel of 29 analytes (table 1). Sera obtained from older individuals showed significantly (p <0.05) higher levels of eotaxin and ifn-2 together with significantly (p <0.001) higher levels of il-7, il-8, mip-1, and vegf (table 1 and figure 2). On the other hand, sera from younger healthy individuals showed significantly (p <0.05) higher levels only of egf as compared to older individuals (table 1 and figure 2). We next analysed whether the huvec proliferation assay might also be useful to stratify sera of hf patients in relationship to relevant clinical parameters . For the purpose of this pilot study, we have enrolled 29 hf patients whose main characteristics, including cardiac functionality parameters and cardiovascular risk factors and therapy, are reported in supplementary table 1 . Twenty - three patients (79.3%) had ischaemic aetiology, whereas 6 (20.7%) satisfied the criteria for idiopathic dilated cardiomyopathy or had hf because of hypertension and valvular disorders . All patients were receiving guidelines pharmacological therapy consisting of ace inhibitors (55.2%), angiotensin ii receptors blockers (34.5%), -blockers (82.8%), antialdosterone drugs (41.4%), and diuretics (89.7%). We did not observe significant differences between nyha class groups as for hf aetiology and the most common cardiovascular risk factors: age, diabetes, hypercholesterolemia, smoking habits, history of hypertension, and coronary diseases familiarity . Based on the normalized cell index values, determined as previously described for sera from healthy subjects, we observed that sera obtained from the hf patients exhibited different effects on huvec proliferation, as exemplified in figure 3(a). The results of this assay allowed us to subdivide hf samples into two groups (figure 3(b)): a group (n = 18) with a high proliferation index (referred to as high endothelial cell index) and another group (n = 11) with a significantly lower proliferation index (referred to as low endothelial cell index). Of note, the observed differences in the ability to promote endothelial cell proliferation between the 2 hf patient subgroups were not due to differences of ages (age of the high ci hf patients: 69.8 12.2; age of the low ci hf patients: 73.1 8.7). In parallel, we have measured the levels of the same panel of cytokines / chemokines previously analysed in the sera of healthy subjects . As shown in table 2, sera from hf patients belonging to the high ci endothelial proliferation group showed higher levels of several cytokines / chemokines with respect to patients' samples of the low ci endothelial proliferation group, including the angiogenic cytokines vegf (p <0.05), il-8 and mip-1 (figure 4(a)). It is noteworthy that the levels of vegf observed in the group low of hf patients (table 2 and figure 4(a)) were comparable to those previously observed in younger healthy donors (table 1 and figure 2). The important, but not exclusive, contribution of vegf to the in vitro endothelial cell proliferation in response to hf patients' sera was underscored by experiments carried out using neutralizing ab anti - vegf (supplementary figure 1). Of interest, within the cytokines / chemokines analyzed in hf patients, the levels of il-12p70, il-8, mcp-1, mip-1, and vegf correlated (p <0.05) positively with the endothelial proliferation index assessed in two distinct time points (36 and 48 hours), with il-8 and vegf showing a higher correlation (figure 4(b)). The evaluation of potential correlation with key clinical parameters revealed no significant correlations between these cytokines and the left ventricular ejection fraction, while a significant correlation was observed between il-12p70 and ntpro - bnp levels (r = 0.25, p <0.05). Comparison of the cytokine levels between the whole hf population and healthy individuals showed significantly (p <0.05) higher levels of few cytokines, including il-12p70 and il-8, in the hf patients (supplementary table 2). Although the vegf levels in the hf patients' sera were higher than the levels measured in healthy controls (254.9 287.1 pg / ml versus 155.4 24.6 pg / ml, resp .) The difference was not statistically significant . Anyhow, it has to be underlined that the small numbers of subjects limited the overall statistical analysis . In order to understand the potential clinical relevance of our in vitro endothelial proliferation assay, pointing to a subdivision of the hf patients into two groups (high versus low endothelial cell proliferation index), we first analysed the distribution of nyha classes within the two groups . As shown in figure 5(a), although we did not observe a significant correspondence between the classification of the hf patients in high / low and the nyha classes, the patients group was represented by a higher percentage of patients belonging to classes i - ii (52% in low ci versus 29% in high ci). Moreover, the low ci patients showed lower levels of ntpro - bnp (median: 510.1 pg / ml; mean sd: 687.6 357.6 pg / ml) as compared to high ci patients (median: 1141.5 pg / ml; mean sd: 1402.5 743.1 pg / ml), although the difference did not reach statistical significance (p = 0.07). On the other hand, the two groups were very similar for the left ventricular ejection fraction (low ci patients: median 33%, mean sd 31.2% 7; high ci patients: median 34%, mean sd 32.7% 8.2, p = 0.3). Next, we analysed the subdivision of the hf patients into the two groups, in relation to the clinical events they experienced in two years of follow - up . For this purpose, we considered as outcome the occurrence of major cardiovascular events: mortality (of cardiac origin), acute myocardial infarction, percutaneous transluminal coronary angioplasty, implant of defibrillator, and surgery of aortic aneurysm . Of interest, we observed a marked different distribution of the event - free hf patients between the high ci and low ci groups . In particular, while the hf patients that did not experience any major clinical cardiac events constitute the 90.1% of the group low ci, the majority (72.2%) of the group high ci was constituted by patients that experienced major events in the follow - up (figure 5(b)). Therefore, from these observations, it emerges a trend indicating that sera from patients with severe pathology are prone to enhance endothelial cell proliferation and, in particular, serum exhibiting high endothelial proliferation activity could indicate a higher risk for the hf patient of worsening the disease . In our study, by employing a standardized in vitro endothelial proliferation assay, we have demonstrated for the first time the following: (i) endothelial cell proliferation in response to serum samples from healthy individuals increased with age and was coupled to different serum levels of proangiogenic cytokines, including vegf and il-8; (ii) the endothelial cell proliferation index determined in response to serum samples from hf patients was correlated with circulating levels of several proinflammatory / proangiogenic cytokines (il-12p70, mcp-1, mip1, il-8, and vegf), with il-12p70 showing a positive correlation also with the levels of levels of ntpro - bnp; (iii) hf patient sera exhibiting high endothelial proliferation activity could indicate a higher risk for the patient of worsening the disease . Our current findings are unprecedented and somewhat counter intuitive if one considers the important role of cardiac angiogenesis in counteracting hf . Indeed, dysfunctional blood vessel formation is a major problem in advanced hf, regardless of the aetiology . However, clinical trials of vegf gene therapy in patients with coronary artery disease or peripheral artery disease have not, to date, demonstrated clinical benefit . In this respect, only few studies have tried to evaluate the levels of proangiogenic cytokines in cardiovascular patients, and mostly were carried out in patients with acute myocardial infarction (ami) [2026]. After an initial attempt to evaluate the predictive role of serum cytokines in patients presenting at the emergency room with chest pain and patients with ami [2224], korybalska et al . Demonstrated that serum vegf measured in patients with ami (n = 106) was significantly higher in patients than in healthy controls and correlated with clinical and angiographic parameters . Moreover, iribarren et al . Demonstrated that median serum concentration of vegf was significantly higher (260 pg / ml) in a large cohort of patients with ami (n = 695) with respect to age - matched healthy controls . Thus, although in our pilot study we have analysed only 29 hf patients, our data are in line with these previous studies obtained in patients with ami, extending the notion that elevated levels of circulating proangiogenic cytokines are predictive of a poor prognosis, not only in patients with ami but also in patients with established hf . In this respect, it is of interest that other studies have clearly demonstrated that elevated levels of proangiogenic cytokines, and in particular of vegf, have a well - established pathological clinical significance in different clinical settings, such as in patients affected by different types of cancer [27, 28]. Moreover, although the role of vegf in vascular diseases, such as atherogenesis, still remains controversial [29, 30], it has been recently demonstrated that vegf - a gene transfer induced proatherogenic changes in lipoprotein profiles in a apo mouse model . Finally, the levels of circulating vegf have also been associated to adhesion and inflammation markers in normal healthy population . It is noteworthy that the in vitro endothelial proliferation assay, we have applied on sera derived from both healthy subjects and hf patients, represents a reproducible and reliable tool able to summarize the overall biological effects on the endothelium driven by the cytokines / chemokines milieu present in the peripheral blood . Although we are aware that the endothelial cell proliferation assessed in our assay could be the result of the contribution of additional cytokines beside those measured by our multiplex assay, our findings confirm the key role of the circulating vegf in the promotion of endothelial cell proliferation and also suggest the potential contribution of other circulating cytokines, including the il-12p70 . It is of interest the correlation observed between il-12p70 and ntpro - bnp that has not been previously reported in hf patients and that could deserve further investigation . Moreover, although it will be necessary to assess the in vitro endothelial proliferation assay in a higher number of clinical cases, our pilot study on the hf patients' sera suggests its potential prognostic value.
She said her headache started abruptly a week previously and that this co - occurred with impaired color perception and blurred vision . When she visited our office, she still complained of blurred vision, especially in her left eye, but her vision was 20/20 and color vision was normal in both eyes . Both of her optic discs were swollen and goldmann perimetry showed enlarged blind spots bilaterally [figs . Lumbar puncture was performed and the opening pressure was checked at 130 mm h2o; the csf composition was normal . Brain magnetic resonance imaging (mri) and magnetic resonance venography (mrv) neuroimaging results were also normal; and no empty sella, globe flattening, or venous narrowing was detected by brain imaging . She was on no medication regimen, and had no other significant past medical history . We recommended another lumbar puncture, but she refused; and thus, because of the definite bilateral disc swelling and headache, which are typical features of iih, we prescribed acetazolamide 500 mg twice daily . A week later, she told us that her headache and blurred vision had subsided, and thus, we gradually tapered the dosage during follow - up . At last visit, 4 months after the onset of her symptoms, both optic discs were normal without any swelling or temporal pallor and she no longer complained of a headache or visual symptoms . The sizes of enlarged blind spots were prominently reduced on follow - up goldmann perimetry [fig . Bilateral optic disc edema with surrounding retinal nerve fiber layer swelling in a 52-year - old woman; who presented with blurred vision, impaired color perception, and headache . (a) bilateral disc swelling with a blurred disc margin in a 15-year - old girl who presented with headache, pulsatile tinnitus, and transient visual obscuration (b) visual field tests of the patients before and after treatment with acetazolamide . Initial visual fields showing the presence of enlarged blind spots in both eyes (a and c)follow - up visual fields demonstrating diminished enlarged blind spot sizes after treatment (b and d) a 15-year - old girl presented with a headache, pulsatile tinnitus, and transient visual obscuration of 2 months duration . She had bilateral disc swelling with a blurred disc margin and goldmann perimetry showed enlarged blind spots in both eyes [figs . Brain mri / mrv was also normal with no empty sella, globe flattening, or venous narrowing . A few days after the initial lumbar puncture, the puncture was repeated, and the rechecked csf opening pressure was 205 mm h2o . Even though her icp was still within the normal range, iih was strongly suspected since she had several typical symptoms, and she told us her headache improved after the lumbar punctures . Three months after her first visit, she had minimal swelling of both optic discs, but did not have headache or tinnitus . Currently (at 7 months after her initial visit), she is on acetazolamide 250 mg twice daily . She said her headache started abruptly a week previously and that this co - occurred with impaired color perception and blurred vision . When she visited our office, she still complained of blurred vision, especially in her left eye, but her vision was 20/20 and color vision was normal in both eyes . Both of her optic discs were swollen and goldmann perimetry showed enlarged blind spots bilaterally [figs . Lumbar puncture was performed and the opening pressure was checked at 130 mm h2o; the csf composition was normal . Brain magnetic resonance imaging (mri) and magnetic resonance venography (mrv) neuroimaging results were also normal; and no empty sella, globe flattening, or venous narrowing was detected by brain imaging . She was on no medication regimen, and had no other significant past medical history . We recommended another lumbar puncture, but she refused; and thus, because of the definite bilateral disc swelling and headache, which are typical features of iih, we prescribed acetazolamide 500 mg twice daily . A week later, she told us that her headache and blurred vision had subsided, and thus, we gradually tapered the dosage during follow - up . At last visit, 4 months after the onset of her symptoms, both optic discs were normal without any swelling or temporal pallor and she no longer complained of a headache or visual symptoms . The sizes of enlarged blind spots were prominently reduced on follow - up goldmann perimetry [fig . Bilateral optic disc edema with surrounding retinal nerve fiber layer swelling in a 52-year - old woman; who presented with blurred vision, impaired color perception, and headache . (a) bilateral disc swelling with a blurred disc margin in a 15-year - old girl who presented with headache, pulsatile tinnitus, and transient visual obscuration (b) visual field tests of the patients before and after treatment with acetazolamide . Initial visual fields showing the presence of enlarged blind spots in both eyes (a and c)follow - up visual fields demonstrating diminished enlarged blind spot sizes after treatment (b and d) a 15-year - old girl presented with a headache, pulsatile tinnitus, and transient visual obscuration of 2 months duration . She had bilateral disc swelling with a blurred disc margin and goldmann perimetry showed enlarged blind spots in both eyes [figs . Brain mri / mrv was also normal with no empty sella, globe flattening, or venous narrowing . A few days after the initial lumbar puncture, the puncture was repeated, and the rechecked csf opening pressure was 205 mm h2o . Even though her icp was still within the normal range, iih was strongly suspected since she had several typical symptoms, and she told us her headache improved after the lumbar punctures . Three months after her first visit, she had minimal swelling of both optic discs, but did not have headache or tinnitus . Currently (at 7 months after her initial visit), she is on acetazolamide 250 mg twice daily . Johnston et al ., reported a series of atypical iih patients, one of which was a 13-year - old boy whose disc edema rapidly resolved after lumboperitoneal shunt insertion even though his initial csf pressure was normal . Subsequently, green et al ., reported the case of an 18-year - old woman iih patient with a normal icp, and introduced the term normal pressure iih to describe this variant of iih . The mechanism responsible for this iih variant is not known, but it has been proposed that patients may have different susceptibilities to icp levels . Repeat lumbar puncture or 24-h icp monitoring is occasionally suggested in patients who are suspected of having iih but with a normal icp by single lumbar puncture . We recommended a second lumbar puncture to both of our patients, but our adult patient refused, and repeat lumbar puncture in the 15-year - old girl confirmed a normal icp . We had considered continuous icp monitoring in both cases, but we decided to start treatment first because they complained of a headache and showed definite bilateral disc swelling with related visual field defects . Papilledema is a warning sign of an elevated icp and ophthalmologists should be alert when a patient has papilledema without any symptoms . It is known that papilledema is usually reversible with appropriate treatment in iih patients, but untreated papilledema can result in progressive and irreversible visual loss . Iih patients with persistent signs and symptoms can be treated using medical or surgical approaches, and carbonic anhydrase inhibitors, such as, acetazolamide, are perceived as the main medical treatment of iih . Surgical interventions, such as ventriculoperitoneal / lumboperitoneal shunting or optic nerve sheath fenestration, are currently the mainstay treatments when medical therapy is insufficient . As both of our patients responded well to acetazolamide, surgical intervention was not considered in our patients . Although increased icp, defined as an opening pressure of over 250 mm h2o, is one of the diagnostic criteria of iih, it should be kept in mind that some patients may have discs that are more susceptible to lower icp than others . Even when a patient's icp is within the normal range, the possibility of iih should always be considered in a patient with typical clinical features of iih, such as, papilledema, a headache, pulsatile tinnitus, and blind spot enlargement by visual field testing.
Subarachnoid haemorrhage (sah) is caused by aneurysmal rupture in 7085% of patients [1, 2]. In a systematic review, intraarterial digital subtraction angiography (ia - dsa) has been the main technique for detecting and characterizing intracranial aneurysms and remains the gold standard . However, ia - dsa is invasive and time consuming, and carries a risk of neurological complications of 0.51.8% with permanent deficit in 0.090.5% [46]. Serious non - neurological complications, which occur in 0.6% of patients, include groin hematoma, peripheral thromboembolism, transient hypotension and arteriovenous fistulas . Furthermore, ia - dsa may increase the risk of rebleeding [7, 8]. It has been demonstrated that three - dimensional ct angiography (3d - cta) can reliably detect intracranial aneurysms [914]. Only after replacement of ia - dsa by cta we report here our clinical experience with both 16- and 64-detector row cta as the first and intended only diagnostic and treatment decision - making study for intracranial aneurysms in patients with acute sah . Between april 2003 and january 2006 all patients presenting with a sah to the university medical centre groningen consecutively underwent cta as the first diagnostic study . Based on the cta findings, patients were selected for surgical clipping or endovascular coiling of a ruptured intracranial aneurysm . Sah was suspected on clinical grounds and confirmed by unenhanced ct or by blood pigments on lumbar puncture . The ct examinations were performed on a 16- or 64-multidetector row spiral ct machine (somatom sensation 16 or 64; siemens medical systems, erlangen, germany), based on a standard protocol . Parameters for 16-slice ct for diagnosis of aneurysm: via an intravenous cannula in the antecubital fossa, 80 ml of contrast agent (visipaque 320) was injected with a power injector at a rate of 4 ml / s . Injection of contrast agent was followed by a flush of 50 ml 0.9% saline (stellant; nacl neck angio) injected at the same rate . A manual fluoroscopic bolus - triggered system, with the internal carotid arteries as reference point and a delay of 4 s, determined the optimal timing . The cta protocol parameters were as follows: spiral mode, rotation time 0.5 s, reconstruction interval 0.75 mm at kernel h20, 120 kv/200 mas, acquisition time 10 s, scan range from the c1 vertebral body to the vertex parallel to the orbitomeatal line.parameters for 16-slice ct for diagnosis of sah: gantry un - angled, spiral mode, rotation time 0.75 s, 16-detector rows at 0.75-mm intervals, table speed 6 mm / rotation, reconstruction interval 3 mm at kernel h30 and acquisition parameters 120 kv/200 mas . The actual acquisition time was approximately 15 s.parameters for 64-slice cta for diagnosis of aneurysm: rotation time 1 s, table speed 15.4 mm / rotation, reconstruction interval 0.6 mm at kernel h20, 120 kv/260 mas, acquisition time 9 s and scan range extending from the c1 vertebral body to the vertex parallel to the orbitomeatal line . The protocol parameters for contrast agent injection remained unchanged.parameters for 64-slice ct for diagnosis of sah: gantry un - angled, spiral mode, rotation time 1 s, 64 detector rows at 0.6-mm intervals, table speed 9.6 mm / rotation, reconstruction interval 2 mm at kernel h30, acquisition parameters 120 kv/260 mas and acquisition time 14 s. parameters for 16-slice ct for diagnosis of aneurysm: via an intravenous cannula in the antecubital fossa, 80 ml of contrast agent (visipaque 320) was injected with a power injector at a rate of 4 ml / s . Injection of contrast agent was followed by a flush of 50 ml 0.9% saline (stellant; nacl neck angio) injected at the same rate . A manual fluoroscopic bolus - triggered system, with the internal carotid arteries as reference point and a delay of 4 s, determined the optimal timing . The cta protocol parameters were as follows: spiral mode, rotation time 0.5 s, reconstruction interval 0.75 mm at kernel h20, 120 kv/200 mas, acquisition time 10 s, scan range from the c1 vertebral body to the vertex parallel to the orbitomeatal line . Parameters for 16-slice ct for diagnosis of sah: gantry un - angled, spiral mode, rotation time 0.75 s, 16-detector rows at 0.75-mm intervals, table speed 6 mm / rotation, reconstruction interval 3 mm at kernel h30 and acquisition parameters 120 kv/200 mas . The actual acquisition time was approximately 15 s. parameters for 64-slice cta for diagnosis of aneurysm: rotation time 1 s, table speed 15.4 mm / rotation, reconstruction interval 0.6 mm at kernel h20, 120 kv/260 mas, acquisition time 9 s and scan range extending from the c1 vertebral body to the vertex parallel to the orbitomeatal line . Parameters for 64-slice ct for diagnosis of sah: gantry un - angled, spiral mode, rotation time 1 s, 64 detector rows at 0.6-mm intervals, table speed 9.6 mm / rotation, reconstruction interval 2 mm at kernel h30, acquisition parameters 120 kv/260 mas and acquisition time 14 s. source images were transferred to a remote computer workstation (odelft benelux diagnostic imaging) for viewing . Initial careful review of axial images was considered imperative . During this review any areas of concern could be noted . Two - dimensional maximum intensity projection (mip) views and three - dimensional (3-d) surface - rendered and volume - rendered reconstructions were reformatted from the raw image date on a vitrea computer workstation by one of the neuroradiologists . From april 2003 until april 2004 the ia - dsa studies were produced on a digital angiographic unit (siemens multiskop with infimed image processing) with a 512512 pixel matrix . From april 2004 onwards the studies were performed on a siemens axiom artis angiographic unit with a 10241024 pixel matrix . Selective four- or six - vessel angiography using a standard projection format was performed initially and additional views were obtained if required to identify the parent vessel and aneurysm neck more clearly . The amount of contrast medium (visipaque 270) used was 8 ml for the internal carotid artery and 6 ml for the external carotid artery, and the injection rate was 6 ml / s when the tip of the catheter was in the internal carotid artery and 34 ml / s when the tip of the catheter was in the external carotid artery . The rate of injection into the vertebrobasilar system was 68 ml / s to a total amount of 8 ml . In certain situations, the c - arm rotates in a continuous 200 arc around the patient s head during a prolonged intraarterial catheter injection of contrast medium (28 ml visipaque, injection rate 4 ml / s). The raw date images were transferred to a leonardo workstation (ax applications) from which 3-d volume - rendered reconstructions were reformatted . The presence of an aneurysm, its size and morphology, its parent and feeding vessels and the collateral circulation at the circle of willis were determined by one of the diagnostic or interventional neuroradiologists . If multiple aneurysms were detected, the usual criteria were applied to decide which aneurysm was responsible for the haemorrhage . These criteria included the unenhanced ct findings (distribution of blood) and the size and irregularity of the aneurysm . A ruptured aneurysm in association with an intraparenchymatous haemorrhage was most often selected for clipping of the aneurysm and surgical evacuation of the haematoma . Patients categorized as inconclusive or negative underwent ia - dsa . In patients with a perimesencephalic blood distribution, one ia - dsa examination was performed . In patients with a nonperimesencephalic blood distribution cta was considered false - negative when ia - dsa revealed a ruptured aneurysm or when rebleeding occurred . The positive predictive value, negative predictive value, sensitivity, specificity and accuracy of cta per patient were calculated . The chi - squared test was used to compare the performance of 16-slice cta and 64-slice cta for the identification of intracranial aneurysms . The ia - dsa findings in patients in the inconclusive category were compared with the cta findings to assess whether ia - dsa actually provided any additional information . Between april 2003 and january 2006 all patients presenting with a sah to the university medical centre groningen consecutively underwent cta as the first diagnostic study . Based on the cta findings, patients were selected for surgical clipping or endovascular coiling of a ruptured intracranial aneurysm . Sah was suspected on clinical grounds and confirmed by unenhanced ct or by blood pigments on lumbar puncture . The ct examinations were performed on a 16- or 64-multidetector row spiral ct machine (somatom sensation 16 or 64; siemens medical systems, erlangen, germany), based on a standard protocol . Parameters for 16-slice ct for diagnosis of aneurysm: via an intravenous cannula in the antecubital fossa, 80 ml of contrast agent (visipaque 320) was injected with a power injector at a rate of 4 ml / s . Injection of contrast agent was followed by a flush of 50 ml 0.9% saline (stellant; nacl neck angio) injected at the same rate . A manual fluoroscopic bolus - triggered system, with the internal carotid arteries as reference point and a delay of 4 s, determined the optimal timing . The cta protocol parameters were as follows: spiral mode, rotation time 0.5 s, reconstruction interval 0.75 mm at kernel h20, 120 kv/200 mas, acquisition time 10 s, scan range from the c1 vertebral body to the vertex parallel to the orbitomeatal line.parameters for 16-slice ct for diagnosis of sah: gantry un - angled, spiral mode, rotation time 0.75 s, 16-detector rows at 0.75-mm intervals, table speed 6 mm / rotation, reconstruction interval 3 mm at kernel h30 and acquisition parameters 120 kv/200 mas . The actual acquisition time was approximately 15 s.parameters for 64-slice cta for diagnosis of aneurysm: rotation time 1 s, table speed 15.4 mm / rotation, reconstruction interval 0.6 mm at kernel h20, 120 kv/260 mas, acquisition time 9 s and scan range extending from the c1 vertebral body to the vertex parallel to the orbitomeatal line . The protocol parameters for contrast agent injection remained unchanged.parameters for 64-slice ct for diagnosis of sah: gantry un - angled, spiral mode, rotation time 1 s, 64 detector rows at 0.6-mm intervals, table speed 9.6 mm / rotation, reconstruction interval 2 mm at kernel h30, acquisition parameters 120 kv/260 mas and acquisition time 14 s. parameters for 16-slice ct for diagnosis of aneurysm: via an intravenous cannula in the antecubital fossa, 80 ml of contrast agent (visipaque 320) was injected with a power injector at a rate of 4 ml / s . Injection of contrast agent was followed by a flush of 50 ml 0.9% saline (stellant; nacl neck angio) injected at the same rate . A manual fluoroscopic bolus - triggered system, with the internal carotid arteries as reference point and a delay of 4 s, determined the optimal timing . The cta protocol parameters were as follows: spiral mode, rotation time 0.5 s, reconstruction interval 0.75 mm at kernel h20, 120 kv/200 mas, acquisition time 10 s, scan range from the c1 vertebral body to the vertex parallel to the orbitomeatal line . Parameters for 16-slice ct for diagnosis of sah: gantry un - angled, spiral mode, rotation time 0.75 s, 16-detector rows at 0.75-mm intervals, table speed 6 mm / rotation, reconstruction interval 3 mm at kernel h30 and acquisition parameters 120 kv/200 mas . The actual acquisition time was approximately 15 s. parameters for 64-slice cta for diagnosis of aneurysm: rotation time 1 s, table speed 15.4 mm / rotation, reconstruction interval 0.6 mm at kernel h20, 120 kv/260 mas, acquisition time 9 s and scan range extending from the c1 vertebral body to the vertex parallel to the orbitomeatal line . Parameters for 64-slice ct for diagnosis of sah: gantry un - angled, spiral mode, rotation time 1 s, 64 detector rows at 0.6-mm intervals, table speed 9.6 mm / rotation, reconstruction interval 2 mm at kernel h30, acquisition parameters 120 kv/260 mas and acquisition time 14 s. source images were transferred to a remote computer workstation (odelft benelux diagnostic imaging) for viewing . Initial careful review of axial images was considered imperative . During this review any areas of concern could be noted . Two - dimensional maximum intensity projection (mip) views and three - dimensional (3-d) surface - rendered and volume - rendered reconstructions were reformatted from the raw image date on a vitrea computer workstation by one of the neuroradiologists . From april 2003 until april 2004 the ia - dsa studies were produced on a digital angiographic unit (siemens multiskop with infimed image processing) with a 512512 pixel matrix . From april 2004 onwards the studies were performed on a siemens axiom artis angiographic unit with a 10241024 pixel matrix . Selective four- or six - vessel angiography using a standard projection format was performed initially and additional views were obtained if required to identify the parent vessel and aneurysm neck more clearly . The amount of contrast medium (visipaque 270) used was 8 ml for the internal carotid artery and 6 ml for the external carotid artery, and the injection rate was 6 ml / s when the tip of the catheter was in the internal carotid artery and 34 ml / s when the tip of the catheter was in the external carotid artery . The rate of injection into the vertebrobasilar system was 68 ml / s to a total amount of 8 ml . In certain situations, rotational 3-d angiography was performed to better delineate the anatomic details of an aneurysm . The c - arm rotates in a continuous 200 arc around the patient s head during a prolonged intraarterial catheter injection of contrast medium (28 ml visipaque, injection rate 4 ml / s). The raw date images were transferred to a leonardo workstation (ax applications) from which 3-d volume - rendered reconstructions were reformatted . Source images were transferred to a remote computer workstation (odelft benelux diagnostic imaging) for viewing . Initial careful review of axial images was considered imperative . During this review any areas of concern could be noted . Two - dimensional maximum intensity projection (mip) views and three - dimensional (3-d) surface - rendered and volume - rendered reconstructions were reformatted from the raw image date on a vitrea computer workstation by one of the neuroradiologists . From april 2003 until april 2004 the ia - dsa studies were produced on a digital angiographic unit (siemens multiskop with infimed image processing) with a 512512 pixel matrix . From april 2004 onwards the studies were performed on a siemens axiom artis angiographic unit with a 10241024 pixel matrix . Selective four- or six - vessel angiography using a standard projection format was performed initially and additional views were obtained if required to identify the parent vessel and aneurysm neck more clearly . The amount of contrast medium (visipaque 270) used was 8 ml for the internal carotid artery and 6 ml for the external carotid artery, and the injection rate was 6 ml / s when the tip of the catheter was in the internal carotid artery and 34 ml / s when the tip of the catheter was in the external carotid artery . The rate of injection into the vertebrobasilar system was 68 ml / s to a total amount of 8 ml . In certain situations, the c - arm rotates in a continuous 200 arc around the patient s head during a prolonged intraarterial catheter injection of contrast medium (28 ml visipaque, injection rate 4 ml / s). The raw date images were transferred to a leonardo workstation (ax applications) from which 3-d volume - rendered reconstructions were reformatted . The presence of an aneurysm, its size and morphology, its parent and feeding vessels and the collateral circulation at the circle of willis were determined by one of the diagnostic or interventional neuroradiologists . If multiple aneurysms were detected, the usual criteria were applied to decide which aneurysm was responsible for the haemorrhage . These criteria included the unenhanced ct findings (distribution of blood) and the size and irregularity of the aneurysm . The surgical and endovascular findings were compared to the cta findings . In general, ruptured aneurysms in the anterior circulation were selected for either coiling or clipping . A ruptured aneurysm in association with an intraparenchymatous haemorrhage was most often selected for clipping of the aneurysm and surgical evacuation of the haematoma . Patients categorized as inconclusive or negative underwent ia - dsa . In patients with a perimesencephalic blood distribution, one ia - dsa examination was performed . In patients with a nonperimesencephalic blood distribution cta was considered false - negative when ia - dsa revealed a ruptured aneurysm or when rebleeding occurred . The positive predictive value, negative predictive value, sensitivity, specificity and accuracy of cta per patient were calculated . The chi - squared test was used to compare the performance of 16-slice cta and 64-slice cta for the identification of intracranial aneurysms . The ia - dsa findings in patients in the inconclusive category were compared with the cta findings to assess whether ia - dsa actually provided any additional information . Excluded from the study were 68 patients, of whom 24 were excluded because of a nonaneurysmal cause of the sah including trauma (n = 17), arteriovenous malformation (n = 6) and anticoagulant therapy (n = 1), 3 because of hypertension and intracerebral haematoma, 4 because of comorbidity or advanced age, 2 were excluded because of poor clinical condition and 1 because if poor clinical grading and advanced age, and 31 died from the initial effect of sah, rebleed or vasospasm with ischemia . The study included 224 patients, 89 men and 135 women with a mean (sd) age of 52.7 10.7 years (range 2279 years). Their clinical condition just before treatment was classified according to the original hunt and hess grading system: 99 patients were classified as grade i, 45 as grade ii, 58 as grade iii, 20 as grade iv, and 2 as grade v . Of the 224 patients, 140 underwent 16-slice cta and 84 underwent 64-slice cta . The cta results were categorized as proven ruptured intracranial aneurysm (133 patients, 59%), inconclusive (31 patients, 14%), or negative for aneurysm (60 patients, 27%). In this category 133 the distributions of the locations and sizes of the aneurysms are shown in tables 1, 2, 3 and 4 . An overview of the results in this subgroup is presented in fig . 1 . Table 1location of symptomatic intracranial aneurysms in 224 patientsaneurysm locationcta - positive (n = 133)cta - inconclusive (n = 31)cta - negative (n = 60)coiling (n = 78)clipping (n = 55)coiling (n = 8)clipping (n = 12)anterior circulationanterior communicating artery3425551pericallosal artery22middle cerebral artery318161internal carotid artery3111posterior communicating artery1781anterior choroideal artery1posterior circulationbasilar tip11vertebral junction1posterior cerebral artery1posterior inferior cerebellar artery511superior cerebellar artery111 patients had no proven aneurysm.five patients had false - negative cta, in four of whom a ruptured aneurysm was diagnosed on repeat angiography.table 2size distribution of symptomatic intracranial aneurysms in 224 patientssize (mm)cta - positive (n = 133)cta - inconclusive (n = 31)cta - negative (n = 60) <54712459704101414315241125111 patients had no proven aneurysm.five patients had false - negative cta, in four of whom a ruptured aneurysm was diagnosed on repeat angiography.table 3location of asymptomatic intracranial aneurysms in 224 patientsaneurysm locationcta - positivecta inconclusiveanterior circulationanterior communicating artery63pericallosal artery2middle cerebral artery114internal carotid artery9posterior communicating artery6posterior circulationbasilar tip1junction of vertebral artery1posterior inferior cerebellar artery1one false - negative on cta.two false - negatives on cta.table 4size distribution of asymptomatic intracranial aneurysms in 224 patients size (mm)cta - positivecta - inconclusive <5304596310141five false - negative on cta.fig . 1flow chart of cta results location of symptomatic intracranial aneurysms in 224 patients 11 patients had no proven aneurysm . Five patients had false - negative cta, in four of whom a ruptured aneurysm was diagnosed on repeat angiography . Size distribution of symptomatic intracranial aneurysms in 224 patients 11 patients had no proven aneurysm . Five patients had false - negative cta, in four of whom a ruptured aneurysm was diagnosed on repeat angiography . Size distribution of asymptomatic intracranial aneurysms in 224 patients five false - negative on cta . Flow chart of cta results the majority of patients were treated within 3 days of sah (n = 99, 75%). Of the 133 cta - positive patients, 78 (59%) were coiled and 55 (41%) were clipped . Treatment conversion was needed in three patients, in two because of difficult aneurysm morphology and in one because of rebleeding during surgery (dura not yet opened). In two patients treatment conversion was necessary because of incorrect treatment selection based on cta (fig . 2). 2cta and ia - dsa results in a 66-year - old woman with sah hunt and hess grade iii . Cta showed four aneurysms: an aneurysm of the anterior communicating artery (acoma) and two bilateral aneurysms of the middle cerebral artery and one aneurysm of the pericallosal artery . The aneurysm of the acoma was regarded as symptomatic at the time of initial sah and its cta - proven morphology showed both coiling and clipping to be a difficult challenge . The session was aborted because the neck of the aneurysm was broad and the aneurysm incorporated both a2 segments . The morphology of the aneurysm excluded complete occlusion by clipping on the 25th day . In the postoperative course again two rebleedings occurred and the patient died . At autopsy a ruptured pericallosal aneurysm was seen more distal to the clipped aneurysm . A coronal mip cta; b volume - rendered cta; c ap view dsa, selective catheterization of left internal carotid artery; d volume - rendered ia - dsa; e, f autopsy (red arrow anterior communicating artery aneurysm, yellow arrow middle cerebral aneurysm, black arrow anterior cerebral artery (a2 segment), blue arrow pericallosal artery aneurysm cta and ia - dsa results in a 66-year - old woman with sah hunt and hess grade iii . Cta showed four aneurysms: an aneurysm of the anterior communicating artery (acoma) and two bilateral aneurysms of the middle cerebral artery and one aneurysm of the pericallosal artery . The aneurysm of the acoma was regarded as symptomatic at the time of initial sah and its cta - proven morphology showed both coiling and clipping to be a difficult challenge . The session was aborted because the neck of the aneurysm was broad and the aneurysm incorporated both a2 segments . The morphology of the aneurysm excluded complete occlusion by clipping on the 25th day . In the postoperative course again two rebleedings occurred and the patient died . At autopsy a ruptured pericallosal aneurysm was seen more distal to the clipped aneurysm . A coronal mip cta; b volume - rendered cta; c ap view dsa, selective catheterization of left internal carotid artery; d volume - rendered ia - dsa; e, f autopsy (red arrow anterior communicating artery aneurysm, yellow arrow middle cerebral aneurysm, black arrow anterior cerebral artery (a2 segment), blue arrow pericallosal artery aneurysm all ruptured intracranial aneurysms were confirmed by surgery or endovascular treatment . In two patients ia - dsa was performed after surgical treatment for evaluation of coiling of asymptomatic aneurysms . In four patients (3%) a fatal rebleeding occurred during follow - up, in one patient soon after complete occlusion of the aneurysm with coiling, in one patient on the 5th day after clipping, in one patient 2 weeks after incomplete occlusion of the aneurysm with coiling and in one patient almost 3 years after clipping . In all patients the blood distribution of the rebleeding was the same as that of the primary sah . Although permission was not granted for an autopsy in two patients, it was considered most probable that the rebleeding was caused by the treated aneurysm . In two patients a ruptured pericallosal aneurysm was seen, 1.5 cm more distal from the clipped anterior communicating artery aneurysm which was regarded as symptomatic at the time of initial sah (fig . 2), and in the other a haematoma surrounding a prepontine cavernous haemangioma and an endovascular treated dissecting aneurysm of the basilar artery were seen . Five aneurysms were confirmed at surgery and subsequently clipped, 5 aneurysms were checked with ia - dsa and subsequently coiled and 19 aneurysms were confirmed with ia - dsa . Four aneurysms were diagnosed with ia - dsa during an embolization session, one of them was also embolized . However, an aneurysm of the middle cerebral artery was seen during surgery of a ruptured aneurysm of the anterior communicating artery . Clipping of the aneurysm of the middle cerebral artery was also performed . In 31 patients an overview of the results in patients in this category is presented in fig . 1 . Of patientsmore information required regarding location and orientationsymptomatic aneurysm10asymptomatic aneurysm2more information required regarding presence of intraaneurysmal thrombus in symptomatic giant aneurysm2differentiation between asymptomatic and symptomatic aneurysm1differentiation between infundibulum, vessel loop and aneurysm5fisher grade iv sah1arterial vasospasm3discrepancy between diagnosed intracranial aneurysm and distribution of blood1incomplete angiography of circle of willis3overprojection of venous structures1variance of normal intracranial vessel anatomy1amalgam artefacts1 indications for ia - dsa examination in 31 patients in 11 patients (35%) ia - dsa confirmed the results of cta . In 17 patients (55%) ia - dsa was able to give further diagnostic information required for a correct patient selection for therapy . In two patients (6%) no additional diagnostic information could was obtained from ia - dsa . In both patients vasospasm of a vertebral artery resulted in an inconclusive cta, but also excluded selective catheterization with ia - dsa . In one patient (3%) treatment selection was based on a false - positive ia - dsa . Cta was inconclusive because of amalgam artefacts in the region of the right posterior inferior cerebellar artery (pica). A second ia - dsa was performed with the intention of coiling . However, with additional views the aneurysm turned out to be a vessel loop . No ruptured aneurysms were found in ten patients (four nonperimesencephalic sah, four with perimesencephalic sah and two negative on unenhanced ct). In 20 patients only an asymptomatic aneurysm was diagnosed (tables 1, 2, 3 and 4). Of the ruptured aneurysms, 12 were clipped and 6 were coiled . An overview of the results in this category of patients is presented in fig . 1 . Of these 60 patients, 13 (22%) had negative findings on unenhanced ct, and 47 (78%) had positive findings on unenhanced ct, and of the latter 30 had a perimesencephalic blood distribution and 17 had a nonperimesencephalic sah . In 11 (85%) of those with negative findings on unenhanced ct, a second ia - dsa was performed and in one patient a second cta was performed . Cta was true - negative in all these patients . In 21 patients (70%) with perimesencephalic sah, ia - dsa was performed once, and in one of them cta was repeated once and in one cta was repeated twice . In eight patients ia - dsa was repeated once and in one patient ia - dsa was repeated twice . In this category cta was true - negative in all these patients . No rebleedings occurred . In nine patients (53%) with nonperimesencephalic sah ia - dsa was performed once, and in two of them a follow - up mra was done and in one cta was repeated . In seven patients ia - dsa was repeated twice . In five patients (29%) with nonperimesencephalic sah, cta was false - negative (tables 1 and 2). In one of these patients only ia - dsa was able to detect a 3-mm ruptured aneurysm of the anterior communicating artery, and in the other four a rebleeding occurred despite an initially negative cta and ia - dsa . Repeat angiography was performed in three patients: a ruptured aneurysm was shown by cta in two and by ia - dsa in one . The explanations for false - negative results were interpretation mistakes (two aneurysms recognized retrospectively on cta and ia - dsa), and haematoma demonstrated on cta surrounding and compressing the aneurysm leading to interpretation error on both cta and ia - dsa in one patient and on only cta in another (ia - dsa showed the aneurysm); the findings were uncertain in one patient . The diagnostic value of both 16- and 64-slice cta are outlined in tables 6 and 7 . A comparison of the results of 16-and 64-slice cta is presented in table 8 . Table 6diagnostic value of cta in ruptured aneurysmsdiagnostic valuetrue positive132 patientsfalse positive1 patienttrue negative55 patientsfalse negative6 patientspositive predictive value99%negative predictive value90%sensitivity96%specificity98%accuracy96%table 7diagnostic value of cta in additional aneurysmsdiagnostic valuetotal number detected with cta25 patients (32 aneurysms)presence checked22 patients (29 aneurysms)true positive22 patients (29 aneurysms)false positive0true negative120 patientsfalse negative4 patients (5 aneurysms)positive predictive value100%negative predictive value97%sensitivity85%specificity100%accuracy97%including 60 cta - negative patients and 60 cta - positive patients . Of the cta - positive patients, 44 were not examined with ia - dsa as standard control.table 8comparison of results of 16- and 64-slice cta for detection of intracranial aneurysms 16-slice (n = 140 patients)64-slice (n = 84 patients)cta resultpositive7459negative4515inconclusive2110ruptured aneurysmstrue positive7359false positive10true negative4213false negative42positive predictive value (%) 99100negative predictive value (%) 9187sensitivity (%) 9597specificity (%) 98100accuracy (%) 9697unruptured aneurysmstotal number on cta12 (15 aneurysms)13 (17 aneurysms)presence checked11 (14 aneurysms)11 (15 aneurysms)true positive11 (14 aneurysms)11 (15 aneurysms)false positive00true negative7545false negative1 (1 aneurysm)3 (4 aneurysms)positive predictive value (%) 100100negative predictive value (%) 9994sensitivity (%) 9279specificity (%) 100100accuracy (%) 9995patients with an inconclusive result were not included in the statistical analysis.including one cta - positive patient.including 45 cta - negative patients and 30 cta - positive patients . Of the cta - positive patients, 31 were not examined with ia - dsa as standard control.including 15 cta - negative patients and 30 cta - positive patients . Of the cta - positive patients, dsa . Diagnostic value of cta in ruptured aneurysms diagnostic value of cta in additional aneurysms including 60 cta - negative patients and 60 cta - positive patients . Of the cta - positive patients, comparison of results of 16- and 64-slice cta for detection of intracranial aneurysms patients with an inconclusive result were not included in the statistical analysis . Including one cta - positive patient . Including 45 cta - negative patients and 30 cta - positive patients . Of the cta - positive patients, 31 were not examined with ia - dsa as standard control . Including 15 cta - negative patients and 30 cta - positive patients . Of the cta - positive patients, excluded from the study were 68 patients, of whom 24 were excluded because of a nonaneurysmal cause of the sah including trauma (n = 17), arteriovenous malformation (n = 6) and anticoagulant therapy (n = 1), 3 because of hypertension and intracerebral haematoma, 4 because of comorbidity or advanced age, 2 were excluded because of poor clinical condition and 1 because if poor clinical grading and advanced age, and 31 died from the initial effect of sah, rebleed or vasospasm with ischemia . The study included 224 patients, 89 men and 135 women with a mean (sd) age of 52.7 10.7 years (range 2279 years). Their clinical condition just before treatment was classified according to the original hunt and hess grading system: 99 patients were classified as grade i, 45 as grade ii, 58 as grade iii, 20 as grade iv, and 2 as grade v . The cta results were categorized as proven ruptured intracranial aneurysm (133 patients, 59%), inconclusive (31 patients, 14%), or negative for aneurysm (60 patients, 27%). In this category 133 the distributions of the locations and sizes of the aneurysms are shown in tables 1, 2, 3 and 4 . 1 . Table 1location of symptomatic intracranial aneurysms in 224 patientsaneurysm locationcta - positive (n = 133)cta - inconclusive (n = 31)cta - negative (n = 60)coiling (n = 78)clipping (n = 55)coiling (n = 8)clipping (n = 12)anterior circulationanterior communicating artery3425551pericallosal artery22middle cerebral artery318161internal carotid artery3111posterior communicating artery1781anterior choroideal artery1posterior circulationbasilar tip11vertebral junction1posterior cerebral artery1posterior inferior cerebellar artery511superior cerebellar artery111 patients had no proven aneurysm.five patients had false - negative cta, in four of whom a ruptured aneurysm was diagnosed on repeat angiography.table 2size distribution of symptomatic intracranial aneurysms in 224 patientssize (mm)cta - positive (n = 133)cta - inconclusive (n = 31)cta - negative (n = 60) <54712459704101414315241125111 patients had no proven aneurysm.five patients had false - negative cta, in four of whom a ruptured aneurysm was diagnosed on repeat angiography.table 3location of asymptomatic intracranial aneurysms in 224 patientsaneurysm locationcta - positivecta inconclusiveanterior circulationanterior communicating artery63pericallosal artery2middle cerebral artery114internal carotid artery9posterior communicating artery6posterior circulationbasilar tip1junction of vertebral artery1posterior inferior cerebellar artery1one false - negative on cta.two false - negatives on cta.table 4size distribution of asymptomatic intracranial aneurysms in 224 patients size (mm)cta - positivecta - inconclusive <5304596310141five false - negative on cta.fig . 1flow chart of cta results location of symptomatic intracranial aneurysms in 224 patients 11 patients had no proven aneurysm . Five patients had false - negative cta, in four of whom a ruptured aneurysm was diagnosed on repeat angiography . Size distribution of symptomatic intracranial aneurysms in 224 patients 11 patients had no proven aneurysm . Five patients had false - negative cta, in four of whom a ruptured aneurysm was diagnosed on repeat angiography . Size distribution of asymptomatic intracranial aneurysms in 224 patients five false - negative on cta . Flow chart of cta results the majority of patients were treated within 3 days of sah (n = 99, 75%). Of the 133 cta - positive patients, 78 (59%) were coiled and 55 (41%) were clipped . Treatment conversion was needed in three patients, in two because of difficult aneurysm morphology and in one because of rebleeding during surgery (dura not yet opened). In two patients treatment conversion was necessary because of incorrect treatment selection based on cta (fig . 2). 2cta and ia - dsa results in a 66-year - old woman with sah hunt and hess grade iii . Cta showed four aneurysms: an aneurysm of the anterior communicating artery (acoma) and two bilateral aneurysms of the middle cerebral artery and one aneurysm of the pericallosal artery . The aneurysm of the acoma was regarded as symptomatic at the time of initial sah and its cta - proven morphology showed both coiling and clipping to be a difficult challenge . The session was aborted because the neck of the aneurysm was broad and the aneurysm incorporated both a2 segments . The morphology of the aneurysm excluded complete occlusion by clipping on the 25th day . In the postoperative course again two rebleedings occurred and the patient died . At autopsy a coronal mip cta; b volume - rendered cta; c ap view dsa, selective catheterization of left internal carotid artery; d volume - rendered ia - dsa; e, f autopsy (red arrow anterior communicating artery aneurysm, yellow arrow middle cerebral aneurysm, black arrow anterior cerebral artery (a2 segment), blue arrow pericallosal artery aneurysm cta and ia - dsa results in a 66-year - old woman with sah hunt and hess grade iii . Cta showed four aneurysms: an aneurysm of the anterior communicating artery (acoma) and two bilateral aneurysms of the middle cerebral artery and one aneurysm of the pericallosal artery . The aneurysm of the acoma was regarded as symptomatic at the time of initial sah and its cta - proven morphology showed both coiling and clipping to be a difficult challenge . The session was aborted because the neck of the aneurysm was broad and the aneurysm incorporated both a2 segments . Unfortunately, rebleeding occurred after coiling . A ruptured pericallosal aneurysm was seen more distal to the clipped aneurysm . A coronal mip cta; b volume - rendered cta; c ap view dsa, selective catheterization of left internal carotid artery; d volume - rendered ia - dsa; e, f autopsy (red arrow anterior communicating artery aneurysm, yellow arrow middle cerebral aneurysm, black arrow anterior cerebral artery (a2 segment), blue arrow pericallosal artery aneurysm all ruptured intracranial aneurysms were confirmed by surgery or endovascular treatment . In two patients ia - dsa was performed after surgical treatment for evaluation of coiling of asymptomatic aneurysms . In four patients (3%) a fatal rebleeding occurred during follow - up, in one patient soon after complete occlusion of the aneurysm with coiling, in one patient on the 5th day after clipping, in one patient 2 weeks after incomplete occlusion of the aneurysm with coiling and in one patient almost 3 years after clipping . In all patients the blood distribution of the rebleeding was the same as that of the primary sah . Although permission was not granted for an autopsy in two patients, it was considered most probable that the rebleeding was caused by the treated aneurysm . In two patients a ruptured pericallosal aneurysm was seen, 1.5 cm more distal from the clipped anterior communicating artery aneurysm which was regarded as symptomatic at the time of initial sah (fig . 2), and in the other a haematoma surrounding a prepontine cavernous haemangioma and an endovascular treated dissecting aneurysm of the basilar artery were seen . Five aneurysms were confirmed at surgery and subsequently clipped, 5 aneurysms were checked with ia - dsa and subsequently coiled and 19 aneurysms were confirmed with ia - dsa . Four aneurysms were diagnosed with ia - dsa during an embolization session, one of them was also embolized . Another aneurysm was considered a vessel loop of the middle cerebral artery on cta . However, an aneurysm of the middle cerebral artery was seen during surgery of a ruptured aneurysm of the anterior communicating artery . Clipping of the aneurysm of the middle cerebral artery was also performed . In 31 patients an overview of the results in patients in this category is presented in fig . 1 . Table 5indications for ia - dsa examination in 31 patientsindicationno . Of patientsmore information required regarding location and orientationsymptomatic aneurysm10asymptomatic aneurysm2more information required regarding presence of intraaneurysmal thrombus in symptomatic giant aneurysm2differentiation between asymptomatic and symptomatic aneurysm1differentiation between infundibulum, vessel loop and aneurysm5fisher grade iv sah1arterial vasospasm3discrepancy between diagnosed intracranial aneurysm and distribution of blood1incomplete angiography of circle of willis3overprojection of venous structures1variance of normal intracranial vessel anatomy1amalgam artefacts1 indications for ia - dsa examination in 31 patients in 11 patients (35%) ia - dsa was able to give further diagnostic information required for a correct patient selection for therapy . In two patients (6%) patients vasospasm of a vertebral artery resulted in an inconclusive cta, but also excluded selective catheterization with ia - dsa . In one patient (3%) treatment selection was based on a false - positive ia - dsa . Cta was inconclusive because of amalgam artefacts in the region of the right posterior inferior cerebellar artery (pica). A second ia - dsa was performed with the intention of coiling . However, with additional views the aneurysm turned out to be a vessel loop . No ruptured aneurysms were found in ten patients (four nonperimesencephalic sah, four with perimesencephalic sah and two negative on unenhanced ct). In 20 patients only an asymptomatic aneurysm was diagnosed (tables 1, 2, 3 and 4). An overview of the results in this category of patients is presented in fig . 1 . Of these 60 patients, 13 (22%) had negative findings on unenhanced ct, and 47 (78%) had positive findings on unenhanced ct, and of the latter 30 had a perimesencephalic blood distribution and 17 had a nonperimesencephalic sah . In 11 (85%) of those with negative findings on unenhanced ct, a second ia - dsa was performed and in one patient a second cta was performed . Cta was true - negative in all these patients . In 21 patients (70%) with perimesencephalic sah, ia - dsa was performed once, and in one of them cta was repeated once and in one cta was repeated twice . In eight patients ia - dsa was repeated once and in one patient ia - dsa was repeated twice . In this category cta was true - negative in all these patients . No rebleedings occurred . In nine patients (53%) with nonperimesencephalic sah ia - dsa was performed once, and in two of them a follow - up mra was done and in one cta was repeated . In seven patients in one patient ia - dsa was repeated twice . In five patients (29%) with nonperimesencephalic sah, cta was false - negative (tables 1 and 2). In one of these patients only ia - dsa was able to detect a 3-mm ruptured aneurysm of the anterior communicating artery, and in the other four a rebleeding occurred despite an initially negative cta and ia - dsa . Repeat angiography was performed in three patients: a ruptured aneurysm was shown by cta in two and by ia - dsa in one . The explanations for false - negative results were interpretation mistakes (two aneurysms recognized retrospectively on cta and ia - dsa), and haematoma demonstrated on cta surrounding and compressing the aneurysm leading to interpretation error on both cta and ia - dsa in one patient and on only cta in another (ia - dsa showed the aneurysm); the findings were uncertain in one patient . The diagnostic value of both 16- and 64-slice cta are outlined in tables 6 and 7 . A comparison of the results of 16-and 64-slice cta is presented in table 8 . Table 6diagnostic value of cta in ruptured aneurysmsdiagnostic valuetrue positive132 patientsfalse positive1 patienttrue negative55 patientsfalse negative6 patientspositive predictive value99%negative predictive value90%sensitivity96%specificity98%accuracy96%table 7diagnostic value of cta in additional aneurysmsdiagnostic valuetotal number detected with cta25 patients (32 aneurysms)presence checked22 patients (29 aneurysms)true positive22 patients (29 aneurysms)false positive0true negative120 patientsfalse negative4 patients (5 aneurysms)positive predictive value100%negative predictive value97%sensitivity85%specificity100%accuracy97%including 60 cta - negative patients and 60 cta - positive patients . Of the cta - positive patients, 44 were not examined with ia - dsa as standard control.table 8comparison of results of 16- and 64-slice cta for detection of intracranial aneurysms 16-slice (n = 140 patients)64-slice (n = 84 patients)cta resultpositive7459negative4515inconclusive2110ruptured aneurysmstrue positive7359false positive10true negative4213false negative42positive predictive value (%) 99100negative predictive value (%) 9187sensitivity (%) 9597specificity (%) 98100accuracy (%) 9697unruptured aneurysmstotal number on cta12 (15 aneurysms)13 (17 aneurysms)presence checked11 (14 aneurysms)11 (15 aneurysms)true positive11 (14 aneurysms)11 (15 aneurysms)false positive00true negative7545false negative1 (1 aneurysm)3 (4 aneurysms)positive predictive value (%) 100100negative predictive value (%) 9994sensitivity (%) 9279specificity (%) 100100accuracy (%) 9995patients with an inconclusive result were not included in the statistical analysis.including one cta - positive patient.including 45 cta - negative patients and 30 cta - positive patients . Of the cta - positive patients, 31 were not examined with ia - dsa as standard control.including 15 cta - negative patients and 30 cta - positive patients . Of the cta - positive patients, . Diagnostic value of cta in ruptured aneurysms diagnostic value of cta in additional aneurysms including 60 cta - negative patients and 60 cta - positive patients . Of the cta - positive patients, comparison of results of 16- and 64-slice cta for detection of intracranial aneurysms patients with an inconclusive result were not included in the statistical analysis . Including one cta - positive patient . Including 45 cta - negative patients and 30 cta - positive patients . Of the cta - positive patients, 31 were not examined with ia - dsa as standard control . Including 15 cta - negative patients and 30 cta - positive patients . Of the cta - positive patients, the distributions of the locations and sizes of the aneurysms are shown in tables 1, 2, 3 and 4 . An overview of the results in this subgroup is presented in fig . 1 . Table 1location of symptomatic intracranial aneurysms in 224 patientsaneurysm locationcta - positive (n = 133)cta - inconclusive (n = 31)cta - negative (n = 60)coiling (n = 78)clipping (n = 55)coiling (n = 8)clipping (n = 12)anterior circulationanterior communicating artery3425551pericallosal artery22middle cerebral artery318161internal carotid artery3111posterior communicating artery1781anterior choroideal artery1posterior circulationbasilar tip11vertebral junction1posterior cerebral artery1posterior inferior cerebellar artery511superior cerebellar artery111 patients had no proven aneurysm.five patients had false - negative cta, in four of whom a ruptured aneurysm was diagnosed on repeat angiography.table 2size distribution of symptomatic intracranial aneurysms in 224 patientssize (mm)cta - positive (n = 133)cta - inconclusive (n = 31)cta - negative (n = 60) <54712459704101414315241125111 patients had no proven aneurysm.five patients had false - negative cta, in four of whom a ruptured aneurysm was diagnosed on repeat angiography.table 3location of asymptomatic intracranial aneurysms in 224 patientsaneurysm locationcta - positivecta inconclusiveanterior circulationanterior communicating artery63pericallosal artery2middle cerebral artery114internal carotid artery9posterior communicating artery6posterior circulationbasilar tip1junction of vertebral artery1posterior inferior cerebellar artery1one false - negative on cta.two false - negatives on cta.table 4size distribution of asymptomatic intracranial aneurysms in 224 patients size (mm)cta - positivecta - inconclusive <5304596310141five false - negative on cta.fig . 1flow chart of cta results location of symptomatic intracranial aneurysms in 224 patients 11 patients had no proven aneurysm . Five patients had false - negative cta, in four of whom a ruptured aneurysm was diagnosed on repeat angiography . Size distribution of symptomatic intracranial aneurysms in 224 patients 11 patients had no proven aneurysm . Five patients had false - negative cta, in four of whom a ruptured aneurysm was diagnosed on repeat angiography . Size distribution of asymptomatic intracranial aneurysms in 224 patients five false - negative on cta . Flow chart of cta results the majority of patients were treated within 3 days of sah (n = 99, 75%). Of the 133 cta - positive patients, 78 (59%) were coiled and 55 (41%) were clipped . Treatment conversion was needed in three patients, in two because of difficult aneurysm morphology and in one because of rebleeding during surgery (dura not yet opened). In two patients treatment conversion was necessary because of incorrect treatment selection based on cta (fig . 2). 2cta and ia - dsa results in a 66-year - old woman with sah hunt and hess grade iii . Cta showed four aneurysms: an aneurysm of the anterior communicating artery (acoma) and two bilateral aneurysms of the middle cerebral artery and one aneurysm of the pericallosal artery . The aneurysm of the acoma was regarded as symptomatic at the time of initial sah and its cta - proven morphology showed both coiling and clipping to be a difficult challenge . The session was aborted because the neck of the aneurysm was broad and the aneurysm incorporated both a2 segments . A ruptured pericallosal aneurysm was seen more distal to the clipped aneurysm . A coronal mip cta; b volume - rendered cta; c ap view dsa, selective catheterization of left internal carotid artery; d volume - rendered ia - dsa; e, f autopsy (red arrow anterior communicating artery aneurysm, yellow arrow middle cerebral aneurysm, black arrow anterior cerebral artery (a2 segment), blue arrow pericallosal artery aneurysm cta and ia - dsa results in a 66-year - old woman with sah hunt and hess grade iii . Cta showed four aneurysms: an aneurysm of the anterior communicating artery (acoma) and two bilateral aneurysms of the middle cerebral artery and one aneurysm of the pericallosal artery . The aneurysm of the acoma was regarded as symptomatic at the time of initial sah and its cta - proven morphology showed both coiling and clipping to be a difficult challenge . The session was aborted because the neck of the aneurysm was broad and the aneurysm incorporated both a2 segments . The morphology of the aneurysm excluded complete occlusion by clipping on the 25th day . In the postoperative course again two rebleedings occurred and the patient died . At autopsy a ruptured pericallosal aneurysm was seen more distal to the clipped aneurysm . A coronal mip cta; b volume - rendered cta; c ap view dsa, selective catheterization of left internal carotid artery; d volume - rendered ia - dsa; e, f autopsy (red arrow anterior communicating artery aneurysm, yellow arrow middle cerebral aneurysm, black arrow anterior cerebral artery (a2 segment), blue arrow pericallosal artery aneurysm all ruptured intracranial aneurysms were confirmed by surgery or endovascular treatment . In two patients ia - dsa was performed after surgical treatment for evaluation of coiling of asymptomatic aneurysms . In four patients (3%) a fatal rebleeding occurred during follow - up, in one patient soon after complete occlusion of the aneurysm with coiling, in one patient on the 5th day after clipping, in one patient 2 weeks after incomplete occlusion of the aneurysm with coiling and in one patient almost 3 years after clipping . In all patients the blood distribution of the rebleeding was the same as that of the primary sah . Although permission was not granted for an autopsy in two patients, it was considered most probable that the rebleeding was caused by the treated aneurysm . In two patients a ruptured pericallosal aneurysm was seen, 1.5 cm more distal from the clipped anterior communicating artery aneurysm which was regarded as symptomatic at the time of initial sah (fig . 2), and in the other a haematoma surrounding a prepontine cavernous haemangioma and an endovascular treated dissecting aneurysm of the basilar artery were seen . Five aneurysms were confirmed at surgery and subsequently clipped, 5 aneurysms were checked with ia - dsa and subsequently coiled and 19 aneurysms were confirmed with ia - dsa . Four aneurysms were diagnosed with ia - dsa during an embolization session, one of them was also embolized . However, an aneurysm of the middle cerebral artery was seen during surgery of a ruptured aneurysm of the anterior communicating artery . An overview of the results in patients in this category is presented in fig . 1 . Table 5indications for ia - dsa examination in 31 patientsindicationno . Of patientsmore information required regarding location and orientationsymptomatic aneurysm10asymptomatic aneurysm2more information required regarding presence of intraaneurysmal thrombus in symptomatic giant aneurysm2differentiation between asymptomatic and symptomatic aneurysm1differentiation between infundibulum, vessel loop and aneurysm5fisher grade iv sah1arterial vasospasm3discrepancy between diagnosed intracranial aneurysm and distribution of blood1incomplete angiography of circle of willis3overprojection of venous structures1variance of normal intracranial vessel anatomy1amalgam artefacts1 indications for ia - dsa examination in 31 patients in 11 patients (35%) ia - dsa was able to give further diagnostic information required for a correct patient selection for therapy . In two patients (6%) patients vasospasm of a vertebral artery resulted in an inconclusive cta, but also excluded selective catheterization with ia - dsa . In one patient (3%) treatment selection was based on a false - positive ia - dsa . Cta was inconclusive because of amalgam artefacts in the region of the right posterior inferior cerebellar artery (pica). A second ia - dsa was performed with the intention of coiling . However, with additional views the aneurysm turned out to be a vessel loop . No ruptured aneurysms were found in ten patients (four nonperimesencephalic sah, four with perimesencephalic sah and two negative on unenhanced ct). In 20 patients only an asymptomatic aneurysm was diagnosed (tables 1, 2, 3 and 4). Of the ruptured aneurysms, an overview of the results in this category of patients is presented in fig . 1 . Of these 60 patients, 13 (22%) had negative findings on unenhanced ct, and 47 (78%) had positive findings on unenhanced ct, and of the latter 30 had a perimesencephalic blood distribution and 17 had a nonperimesencephalic sah . In 11 (85%) of those with negative findings on unenhanced ct, a second ia - dsa was performed and in one patient a second cta was performed . Cta was true - negative in all these patients . In 21 patients (70%) with perimesencephalic sah, ia - dsa was performed once, and in one of them cta was repeated once and in one cta was repeated twice . In eight patients ia - dsa was repeated once and in one patient ia - dsa was repeated twice . In this category cta was true - negative in all these patients . No rebleedings occurred . In nine patients (53%) with nonperimesencephalic sah ia - dsa was performed once, and in two of them a follow - up mra was done and in one cta was repeated . In seven patients ia - dsa was repeated twice . In five patients (29%) with nonperimesencephalic sah, cta was false - negative (tables 1 and 2). In one of these patients only ia - dsa was able to detect a 3-mm ruptured aneurysm of the anterior communicating artery, and in the other four a rebleeding occurred despite an initially negative cta and ia - dsa . Repeat angiography was performed in three patients: a ruptured aneurysm was shown by cta in two and by ia - dsa in one . The explanations for false - negative results were interpretation mistakes (two aneurysms recognized retrospectively on cta and ia - dsa), and haematoma demonstrated on cta surrounding and compressing the aneurysm leading to interpretation error on both cta and ia - dsa in one patient and on only cta in another (ia - dsa showed the aneurysm); the findings were uncertain in one patient . The diagnostic value of both 16- and 64-slice cta are outlined in tables 6 and 7 . A comparison of the results of 16-and 64-slice cta is presented in table 8 . Table 6diagnostic value of cta in ruptured aneurysmsdiagnostic valuetrue positive132 patientsfalse positive1 patienttrue negative55 patientsfalse negative6 patientspositive predictive value99%negative predictive value90%sensitivity96%specificity98%accuracy96%table 7diagnostic value of cta in additional aneurysmsdiagnostic valuetotal number detected with cta25 patients (32 aneurysms)presence checked22 patients (29 aneurysms)true positive22 patients (29 aneurysms)false positive0true negative120 patientsfalse negative4 patients (5 aneurysms)positive predictive value100%negative predictive value97%sensitivity85%specificity100%accuracy97%including 60 cta - negative patients and 60 cta - positive patients . Of the cta - positive patients, 44 were not examined with ia - dsa as standard control.table 8comparison of results of 16- and 64-slice cta for detection of intracranial aneurysms 16-slice (n = 140 patients)64-slice (n = 84 patients)cta resultpositive7459negative4515inconclusive2110ruptured aneurysmstrue positive7359false positive10true negative4213false negative42positive predictive value (%) 99100negative predictive value (%) 9187sensitivity (%) 9597specificity (%) 98100accuracy (%) 9697unruptured aneurysmstotal number on cta12 (15 aneurysms)13 (17 aneurysms)presence checked11 (14 aneurysms)11 (15 aneurysms)true positive11 (14 aneurysms)11 (15 aneurysms)false positive00true negative7545false negative1 (1 aneurysm)3 (4 aneurysms)positive predictive value (%) 100100negative predictive value (%) 9994sensitivity (%) 9279specificity (%) 100100accuracy (%) 9995patients with an inconclusive result were not included in the statistical analysis.including one cta - positive patient.including 45 cta - negative patients and 30 cta - positive patients . Of the cta - positive patients, 31 were not examined with ia - dsa as standard control.including 15 cta - negative patients and 30 cta - positive patients . Of the cta - positive patients, diagnostic value of cta in ruptured aneurysms diagnostic value of cta in additional aneurysms including 60 cta - negative patients and 60 cta - positive patients . Of the cta - positive patients, comparison of results of 16- and 64-slice cta for detection of intracranial aneurysms patients with an inconclusive result were not included in the statistical analysis . Including one cta - positive patient . Including 45 cta - negative patients and 30 cta - positive patients . Of the cta - positive patients, 31 were not examined with ia - dsa as standard control . Including 15 cta - negative patients and 30 cta - positive patients . Of the cta - positive patients, our primary aim was to assess whether cta is useful clinically in planning and performing clipping or coiling, especially in the acute phase in ruptured intracranial aneurysms, without recourse to ia - dsa . We demonstrated that it was possible to treat more than half of all patients with a ruptured intracranial aneurysm using only cta . By avoiding conventional angiography, it was possible to streamline the management of ruptured aneurysm during the acute phase . Further, 3d - cta was able to help in deciding whether to clip or to coil; in only two patients was treatment conversion needed due to incorrect treatment selection based on cta . We found 3d - cta to be a simple, reliable, quick and minimally invasive imaging modality that reduces the risk of complications caused by conventional angiography and reduces the delay between the patient s arrival at the hospital and treatment, leading to diminished rebleeding . Matsumoto et al . Analyzed the rate of rebleeding of ruptured aneurysms during cta and conventional angiography, and found 0% (none of 160 patients) for cta and 1.5% (5 of 317 patients) for conventional angiography . In patients with a ruptured aneurysm and intracerebral another advantage is that the radiation dosage is low compared to ia - dsa (1.0 msv at 200 mas with the cta siemens sensation 16 and 1.8 msv at 380 mas with the cta siemens sensation 64 compared with 3.56.5 msv with conventional angiography). Several other studies assessing whether cta may serve as the sole imaging method for the preoperative work - up of patients with ruptured intracranial aneurysms have been published [1726]. There is a wide variation in the percentage of patients who have had their symptomatic aneurysms treated based on cta . This may be influenced positively by the very high aneurysm prevalence and the subsequent very low negative rates of cta in some studies [22, 23, 25]. In other studies patients with a negative cta were not enrolled at all [17, 20]. In general, a mean of 1520% negative angiographies after sah is accepted . This may reflect the good awareness of the diagnosis sah in first - line and second - line health - care and the good access to cta when the diagnosis sah is considered . Furthermore, the wide variation in cta - based treatment may be partially explained by differences in hardware and software used by each group, the rate of technical failures in performing cta, scanning parameters set for screening the circle of willis and more peripheral vessels, the experience and scrutiny of the neuroradiologist evaluating each cta and the willingness of the neurosurgeon and neurointerventional radiologist to rely on cta alone in each individual case . Table 9presentation of previous studies and present studystudyno . Of patientscta - positivecta - negativecta inconclusive or no cta - based treatmenttotal patientscta - based treatmenttrue - positive ctatotal patientstrue - negative ctatotal patients218746 (55%)44 (96%)44 (100%)15 (17%)6 (60%)26 (30%)2210988 (81%)87 (99%)87 (100%)5 (5%)5 (100%)16 (15%)238462 (74%)62 (100%)62 (100%)7 (8%)0 (0%)15 (18%)199045 (100%)45 (100%)45 (100%)45 (50%)1815061 (41%)61 (100%)60 (98%)24 (16%)24 (100%)65 (43%)2512040 (27%)40 (100%)40 (100%)13 (11%)13 (100%)67 (56%)247827 (35%)27 (100%)27 (100%)20 (26%)20 (100%)31 (40%)1710093 (93%)93 (100%)93 (100%)7 (7%)209687 (91%)87 (100%)86 (99%)9 (9%)266144 (72%)44 (100%)44 (100%)15 (25%)14 (93%)2 (3%)present study224133 (59%)133 (100%)132 (99%)60 (27%)55 (92%)31 (14%)one false - negative and one false - positive ruptured aneurysm in one patient.in five patients with perimesencephalic sah, ia - dsa as the gold standard control was not performed . Presentation of previous studies and present study one false - negative and one false - positive ruptured aneurysm in one patient . In five patients with perimesencephalic sah, ia - dsa as the gold standard control was not performed . In all false - negatives were in patients with a nonperimesencephalic blood distribution, giving a false - negative rate of 29% and a risk of rebleeding of 24% . It seems unlikely that the false - negative rate of initial cta and the risk of rebleeding despite a negative initial cta in patients with a nonperimesencephalic sah might be influenced negatively by the use of cta as the first diagnostic tool . Firstly, in all patients with a rebleeding, repeat ia - dsa was also false - negative . Furthermore, the findings of other studies using ia - dsa as the first diagnostic tool were similar . In the study by urbach et al . In 67 patients with a negative initial angiogram after sah, four ruptured aneurysms were revealed by repeat angiography . Three patients presented with a nonperimesencephalic sah and one presented with a perimesencephalic sah . In the study by bradac et al . A second angiogram performed 14 weeks later revealed a ruptured aneurysm in 5 of the 40 patients . In all patients a nonperimesencephalic blood distribution was seen on ct . Because in the present study some aneurysms could be correctly identified retrospectively, we suggest that if, under strong clinical suspicion of a ruptured aneurysm, the cta is reported as normal, the study should be reviewed by a second neuroradiologist before proceeding to repeat angiography . Next, we recommend repeat cta or ia - dsa when the initial cta is negative in patients with a nonperimesencephalic sah . The substantial risk of rebleeding in patients with an aneurysmal pattern of haemorrhage in the present study indicates that some cerebral aneurysms are occult on initial cta . Pedersen et al . Reported an increase in sensitivity from 88% to 94% after 1 year s experience . Cta had a sensitivity of 50% for aneurysms <2 mm in the study of wintermark et al . . Distal pericallosal and pica aneurysms can be missed when restricting the area of coverage to the proximal circle of willis [3134]. Thrombosis of the neck of the aneurysm or of the entire sac is another possible reason . Aneurysms may be mistaken for vascular infundibula (persistent fetal nonaneurysmal dilatation of the proximal vessel) of the posterior communicating or anterior choroidal artery origins if a vessel cannot be identified arising from them . Aneurysms may masquerade as tight vascular loops if the mip thickness is wide (> 3 mm). In patients with multiple aneurysms close to bone (e.g. Carotid siphon, ophthalmic and posterior communicating artery) may be overlooked when relying on surface - rendering and volume - rendering techniques or using mip with bone editing [32, 34, 3638]. Aneurysms located within or close to the cavernous sinuses are easy to overlook unless thin - section axial and coronal mip images are reviewed on a slightly wider window width . In patients with a perimesencephalic sah the chance of finding a posterior fossa aneurysm is low: 2.55% [39, 40]. Nonaneurysmal perimesencephalic haemorrhage carries no risk of vasospasm and rebleeding and has been shown to follow a benign course with an excellent prognosis . The chance of finding an aneurysm in 5% of patients has to be weighed against the risk of complications from angiography imposed upon the remaining 95% of patients . Cta has a high accuracy for diagnosis of vertebrobasilar aneurysms and of intracranial aneurysms in general [913, 42]. In the present study, in patients with a perimesencephalic sah and a negative initial cta, no rebleedings occurred and cta was true - negative in all . Similarly, in the prospective study of huttner et al ., 69 patients with a perimesencephalic sah had a negative initial cta and ia - dsa . A repeat ia - dsa was performed in 38 patients (55%). None of the repeat ia - dsas showed any additional distinctive features with respect to the first ia - dsa . It therefore seems practical and safe to perform cta as the first diagnostic tool and to omit repeat angiography if cta is negative . A formal decision analysis based on these observations confirmed that a strategy where cta is performed and not followed by conventional angiography, if negative, results in a better utility than a strategy of cta followed by conventional angiography or of conventional angiography as primary investigation . According to the results of the present study, it seems important to distinguish the two patterns of sah on ct . Different data show that experienced radiologists can accurately discriminate between a perimesencephalic and nonperimesencephalic sah [12, 40, 45]. Early ct within 3 days is necessary for reliable assessment of the pattern of haemorrhage [12, 40, 46]. A criticism of this study might be that patients treated with endovascular coiling underwent ia - dsa as part of the endovascular procedure and thus should not be counted in the analysis of efficacy of the prospective protocol . However, a shift in management of ruptured intracranial aneurysm from surgery to endovascular treatment has appeared . The use of cta as the initial investigation for cerebral aneurysms may offset some of this increased workload whilst also improving workflow . In conclusion, in this evaluation of the use of 16-row and 64-row multislice cta in the management of ruptured intracranial aneurysms, we demonstrated that cta can be used as the first - line diagnostic modality for the management of sah patients . In cta - negative patients ia - dsa is not needed in patients with negative cta and classic perimesencephalic sah . Repeat ia - dsa or cta should still be performed in patients with a nonperimesencephalic sah, due to false - negative ctas and ia - dsas in this patient group . The remaining true indication for ia - dsa was in patients with an inconclusive cta result . In more than half of those ia - dsa provided relevant new diagnostic information.
The hepatitis b virus (hbv) is a major global public health problem affecting more than 2 billion people throughout the world (1). Despite the availability of a highly effective vaccine against hbv, 350 million people chronically infected with hbv are at increased risk of developing hbv - related liver diseases, including hepatic cirrhosis and hepatocellular carcinoma (4, 5). There are high - prevalence areas where> 8% of the population is positive for the hepatitis b surface antigen (hbsag); intermediate - prevalence areas in which 2% -8% of the population is hbsag positive and low - prevalence areas in which <2% of the population is hbsag positive (7, 8). However, because there are regions very close to low - prevalence areas and also close to high - prevalence areas, it has been suggested that splitting the intermediate category into low - intermediate - prevalence (2% - 4.99%) and high - intermediate - prevalence areas (5% - 7.99%) could better reflect regional differences and their importance (3, 9). According to a recent study on the global epidemiology of hbv infection, iran the overall prevalence of hbv infection in iran was 1.7% in the 1990s (10, 11). However, other regional studies in various provinces of iran showed hbv prevalence more than 1.7% in the last decade (12 - 14). To decrease hbv carriers in the community, using vaccination is one of the most beneficial strategies in reducing the frequency of hbv infection (15). Accurate and updated knowledge of the prevalence of hbv infection is necessary to assess the impact of prevention and control measurements, including vaccination programs, and also for bringing up to date the estimates of disease burden (9). However, only two systematic reviews have estimated the prevalence of hbv infection in the general population of iran: the study by alavian et al . (16), covering studies from seven provinces from 2001 to 2007, and the study by poorolajal et al . (therefore, there are no up - to - date national systematic reviews reporting hbv seroprevalence in the general population of iran . The present study was conducted to make an updated estimate of the prevalence of hbv infection in iran by a systematic review of peer - reviewed publications representing hbv seroprevalence in the general population of different provinces for which epidemiological information was available . The present systematic review and meta - analysis was performed according to the criteria of the prisma guidelines (18). A systematic review on literature published between january 1, 1990, and january 1, 2016, was performed to find scientific papers reporting the prevalence of hbv infection (by detection of the hbsag in samples) in the general iranian population . To identify articles, international databases (pubmed, embase, scopus, web of science, cabi, cinahl, doaj, index medicus for eastern mediterranean region - imemr, emromedex, high - wire press, and dare) and national databases (scientific information database (sid), iranmedex, and magiran) were searched for mesh terms hepatitis b, hbv, and iran and their persian equivalents in titles and/or abstracts . All potentially relevant publications were reviewed independently by two reviewers (ms - v and fs) for the eligibility criteria . Discrepancy between reviewers was resolved by consensus with an expert (sma). In this study all english or persian observational (descriptive / analytical cross - sectional studies / surveys) studies on seroprevalence of hbv with clearly described time and location of the study, proper sampling strategies (findings could be generalized to general population), reliable laboratory techniques and proper analysis methods were considered to include in the present study . The exclusion criteria were (1) studies conducted as case reports, surveillance reports, letters or correspondence, and systematic reviews or meta - analyses; (2) overlapping studies; (3) studies performed exclusively on high - risk population groups; and (4) studies with non - serum - based hbv detection assay, such as saliva testing . After review of the full texts, the following data were extracted from each study: study characteristics (first author s name, date of study, period of sample collection, and province or city of the study), participant characteristics (gender, age, population group, and sample size), hbv prevalence (hbsag seroprevalence in all cases and hbsag seroprevalence according to gender), and hbsag detection methods . The quality of the included study was assessed using a modified strobe checklist (19). Statistical analysis was done by stata software, version 13 (stata corp, college station, tx, usa). The prevalence of hbv was polled by the metan command and showed by a forest plot for the general population and also was based on both genders . The random effect model was used for meta - analysis tests . The forest plot as a graphic manner was used to show the effect size of all the studies with their confidence intervals and the results of the meta - analysis (20). Meta - regression analysis was performed for determination of responsible factors for heterogeneity using the metareg command . Publication bias was assessed by begg s and egger s tests (21, 22). Cumulative meta - analysis based on the time was performed using the metacum command . The prevalence of hbv was showed in geographic maps using esri arcmap gis version 10.1 . The present systematic review and meta - analysis was performed according to the criteria of the prisma guidelines (18). A systematic review on literature published between january 1, 1990, and january 1, 2016, was performed to find scientific papers reporting the prevalence of hbv infection (by detection of the hbsag in samples) in the general iranian population . To identify articles, international databases (pubmed, embase, scopus, web of science, cabi, cinahl, doaj, index medicus for eastern mediterranean region - imemr, emromedex, high - wire press, and dare) and national databases (scientific information database (sid), iranmedex, and magiran) were searched for mesh terms hepatitis b, hbv, and iran and their persian equivalents in titles and/or abstracts . All potentially relevant publications were reviewed independently by two reviewers (ms - v and fs) for the eligibility criteria . Discrepancy between reviewers was resolved by consensus with an expert (sma). In this study all english or persian observational (descriptive / analytical cross - sectional studies / surveys) studies on seroprevalence of hbv with clearly described time and location of the study, proper sampling strategies (findings could be generalized to general population), reliable laboratory techniques and proper analysis methods were considered to include in the present study . The exclusion criteria were (1) studies conducted as case reports, surveillance reports, letters or correspondence, and systematic reviews or meta - analyses; (2) overlapping studies; (3) studies performed exclusively on high - risk population groups; and (4) studies with non - serum - based hbv detection assay, such as saliva testing . After review of the full texts, the following data were extracted from each study: study characteristics (first author s name, date of study, period of sample collection, and province or city of the study), participant characteristics (gender, age, population group, and sample size), hbv prevalence (hbsag seroprevalence in all cases and hbsag seroprevalence according to gender), and hbsag detection methods . The quality of the included study was assessed using a modified strobe checklist (19). Statistical analysis was done by stata software, version 13 (stata corp, college station, tx, usa). The prevalence of hbv was polled by the metan command and showed by a forest plot for the general population and also was based on both genders . The random effect model was used for meta - analysis tests . The forest plot as a graphic manner was used to show the effect size of all the studies with their confidence intervals and the results of the meta - analysis (20). Meta - regression analysis was performed for determination of responsible factors for heterogeneity using the metareg command . Publication bias was assessed by begg s and egger s tests (21, 22). To correct publication bias, the trim and filled method cumulative meta - analysis based on the time was performed using the metacum command . The prevalence of hbv was showed in geographic maps using esri arcmap gis version 10.1 . Following the removal of duplicates and primary screening, 40 publications were reviewed in full, of which 20 met the eligibility criteria and were included in the meta - analysis (figure 1). Among 20 included studies, two studies (10, 11) were national, one study (year 2006) was conducted in golestan, hormozgan, and tehran provinces (23), and 17 studies were regional (table 1). Of the regional publications, two were from golestan province, covering the years 2003 - 2006 (14, 24); two were from hamadan province, covering the years 1998 - 2003 (12, 25); three were from razavi khorasan province, covering the years 1998 - 2011 (13, 26, 27); and two were from sistan and baluchestan province, covering the years 2008 - 2010 (28, 29). Other studies were from charmahal and bakhtiari (years 2012 - 2013) (30), east azarbaijan (year 2001) (31), isfahan (year 2006) (32), kermanshah (year 2010) (33), kordestan (year 2010) (34), mazandaran (years 2008 - 2011) (35), qom (year 2010) (36), and south iran (years 2008 - 2009) (37). The sample size of the studies was between 381 and 46,631 and the age of study subjects was 1 - 100 . Overall, most study subjects were females (table 1). In all included studies, the polled estimated prevalence of hbv infection in the general population of iran from 1990 to 2016 was 2.2% (95% ci: 1.9% - 2.6%) (figure 2). The highest prevalence of hbv infection (8.9%, 95% ci: 7.6% - 10.2%) was reported from golestan province (14), and the lowest prevalence (0.7%, 95% ci: 0.4% - 1.1%) was seen in kermanshah province (33). Regarding hbv infection according to gender, the prevalence of hbv infection was estimated at 3% (95% ci: 2.2% - 3.8%) and 1.7% (95% ci: 1.2% - 2.3%) for iranian males (figure 3) and females (figure 4), respectively . The results of the heterogeneity test indicated that the studies were significantly heterogeneous (p = 0.001), so the random model was used for polling the data . To determine responsible factors for heterogeneity, therefore, a subgroup analysis and a cumulative meta - analysis based on the time were performed . In the subgroup analysis, the studies were divided into two different time periods, including before and after 2010 . The polled estimated prevalence of hbv infection in the general population of iran was 2.9% (95% ci: 2.5% - 3.4%) before 2010 (1993 - 2009) and 1.3% (95% ci: 0.9% - 1.7%) after 2010 (2010 - 2014) (figure 5). In the period before 2010, there were two studies with high hbv prevalence of 8.9% and 5.1% (23, 25). Therefore, we performed sensitivity analysis and estimated hbv prevalence in studies before 2010 with the exclusion of the two mentioned studies . After sensitivity analysis, the prevalence of hbv infection in this period decreased to 2.3% (95% ci: 2% - 2.7%), and overall estimated prevalence of hbv in the general population changed to 1.8% (95% ci: 1.6% - 2.1%). After performing cumulative meta - analysis, the prevalence of hbv showed a decrease from 2.5% in 1993 to 1.7% in 2014 (figure 6). Based on the results of begg s and egger s tests for prevalence of hbv infection (p = 0.005 and p = 0.035, respectively), there was evidence of publication bias in the studies . Using this analysis, hbv prevalence in the general population of iran was estimated as 2.2% (95% ci: 1.9% - 2.6%). The results indicated a significant difference within the geographic distribution of hbv infection in the general iranian population (table 1, figure 7). The highest prevalence (6.1%, 95% ci: 3.5% - 8.7%) was observed in golestan province (in northeast iran), followed by sistan and baluchestan province (3%, 95% ci: 2.3% - 3.7%), while the lowest prevalence was reported from kermanshah (0.7%, 95% ci: 0.4% - 1.1%), kordestan (0.8%, 95% ci: 0.4% - 1.2%), and mazandaran (0.9%, 95% ci: 0.7% - 1.2%). Following the removal of duplicates and primary screening, 40 publications were reviewed in full, of which 20 met the eligibility criteria and were included in the meta - analysis (figure 1). Among 20 included studies, two studies (10, 11) were national, one study (year 2006) was conducted in golestan, hormozgan, and tehran provinces (23), and 17 studies were regional (table 1). Of the regional publications, two were from golestan province, covering the years 2003 - 2006 (14, 24); two were from hamadan province, covering the years 1998 - 2003 (12, 25); three were from razavi khorasan province, covering the years 1998 - 2011 (13, 26, 27); and two were from sistan and baluchestan province, covering the years 2008 - 2010 (28, 29). Other studies were from charmahal and bakhtiari (years 2012 - 2013) (30), east azarbaijan (year 2001) (31), isfahan (year 2006) (32), kermanshah (year 2010) (33), kordestan (year 2010) (34), mazandaran (years 2008 - 2011) (35), qom (year 2010) (36), and south iran (years 2008 - 2009) (37). The sample size of the studies was between 381 and 46,631 and the age of study subjects was 1 - 100 . Overall, most study subjects were females (table 1). In all included studies, the polled estimated prevalence of hbv infection in the general population of iran from 1990 to 2016 was 2.2% (95% ci: 1.9% - 2.6%) (figure 2). The highest prevalence of hbv infection (8.9%, 95% ci: 7.6% - 10.2%) was reported from golestan province (14), and the lowest prevalence (0.7%, 95% ci: 0.4% - 1.1%) was seen in kermanshah province (33). Regarding hbv infection according to gender, the prevalence of hbv infection was estimated at 3% (95% ci: 2.2% - 3.8%) and 1.7% (95% ci: 1.2% - 2.3%) for iranian males (figure 3) and females (figure 4), respectively . The results of the heterogeneity test indicated that the studies were significantly heterogeneous (p = 0.001), so the random model was used for polling the data . To determine responsible factors for heterogeneity, therefore, a subgroup analysis and a cumulative meta - analysis based on the time were performed . In the subgroup analysis, the studies were divided into two different time periods, including before and after 2010 . The polled estimated prevalence of hbv infection in the general population of iran was 2.9% (95% ci: 2.5% - 3.4%) before 2010 (1993 - 2009) and 1.3% (95% ci: 0.9% - 1.7%) after 2010 (2010 - 2014) (figure 5). In the period before 2010, there were two studies with high hbv prevalence of 8.9% and 5.1% (23, 25). Therefore, we performed sensitivity analysis and estimated hbv prevalence in studies before 2010 with the exclusion of the two mentioned studies . After sensitivity analysis, the prevalence of hbv infection in this period decreased to 2.3% (95% ci: 2% - 2.7%), and overall estimated prevalence of hbv in the general population changed to 1.8% (95% ci: 1.6% - 2.1%). After performing cumulative meta - analysis, the prevalence of hbv showed a decrease from 2.5% in 1993 to 1.7% in 2014 (figure 6). Based on the results of begg s and egger s tests for prevalence of hbv infection (p = 0.005 and p = 0.035, respectively), there was evidence of publication bias in the studies . Hbv prevalence in the general population of iran was estimated as 2.2% (95% ci: 1.9% - 2.6%). The results indicated a significant difference within the geographic distribution of hbv infection in the general iranian population (table 1, figure 7). The highest prevalence (6.1%, 95% ci: 3.5% - 8.7%) was observed in golestan province (in northeast iran), followed by sistan and baluchestan province (3%, 95% ci: 2.3% - 3.7%), while the lowest prevalence was reported from kermanshah (0.7%, 95% ci: 0.4% - 1.1%), kordestan (0.8%, 95% ci: 0.4% - 1.2%), and mazandaran (0.9%, 95% ci: 0.7% - 1.2%). Hbv infection is a major public health problem and considered the leading cause of chronic liver disease in iran (1, 38). In the last decade, hbv prevalence has reduced significantly in iran (38) because of the infantile mass vaccination program started in 1993, enhancement of people s awareness regarding hbv risk factors, vaccination of high - risk people, and the use of disposable syringes in vaccinations and clinical settings (39 - 43). Regardless of the present study, two previous systematic reviews have estimated the prevalence of hbv infection in the general population of iran . They estimated that hbv prevalence in iran had a rate of 2.14% (95% ci: 1.9% - 2.3%). In the second systematic review, covering six studies from 1998 to 2005 by poorolajal et al . (17), the prevalence of hbv in the general iranian population was estimated at a rate of 2.7% (95% ci: 2.2% - 3.1%). In the current study, to obtain an updated estimate of hbv prevalence in the general iranian population, a comprehensive review of the literature covering the years 1993 - 2014 was performed . According to the results, the estimated prevalence of hbv infection in the general population of iran was 2.2% (95% ci: 1.9% - 2.6%), a rate between the previous studies by alavian et al . According to an implementation of preventive measurements, including vaccination programs in last two decades, it is expected that hbv prevalence will be reduced over time . In the present study, a notable decrease of hbv infection rate was observed within the data published in the period after 2010 (1.3%) compared to the data published in the period before 2010 (2.9%). It should be noted that, in the studies before 2010, there are two studies from golestan province with the hbv prevalence of 8.9% and 5.1% (23, 25). Following sensitivity analysis and excluding the two studies from golestan province (14, 23), the prevalence of hbv infection in the studies before 2010 and overall prevalence of hbv decreased to 2.3% (95% ci: 2% - 2.7%) and 1.8% (95% ci: 1.6% - 2.1%), respectively . Despite vaccination having reduced significantly the hbv prevalence and its complications, there are some concerns in this regards, as low efficacy of hbv immunization has been reported by several studies (44 - 48). Therefore, considering some revisions in hbv vaccination may be more helpful for prevention of hbv infection (49). In this study, the prevalence of hbv infection in males was higher than females (3% vs. 1.7%). This difference might be due to the frequency of more exposure to risk factors, such as occupational risk factors in iranian males . Also, other risk factors, such as intravenous drug use, multi - partnership, and being shaved by common barbers, are more frequent in iranian men compared to women (10, 14). There was heterogeneity in the geographic distribution of hbv infection and the range of hbv prevalence was extensive, spanning from 0.7% to 8.9% . This finding suggests that various parts of iran may differ regarding possible risk factors of hbv infection, and extensive investigations should be performed to determine the geographic distribution of hbv risk factors (16). It should be noted that only two studies have examined the prevalence of hbv in the general iranian population at a national level, and both were conducted before 2,000 (10, 11). Also, at a regional level, little is known about hbv prevalence in the general population in the majority of iran s provinces . Therefore, implementation of new studies at both the national and regional levels seems to be necessary to achieve real updated information on the hbv prevalence for improving the efficiency of interventions and prevention measurements . It is worth mentioning that occult hbv infection (patients seronegative for hbsag but positive for hbv dna) is very important in public health (50 - 52), as it has been evidenced that patients with this type of hbv infection are at increased risk of cirrhosis and hepatocellular carcinoma (hcc) (51, 53) and also for the risk of hbv transfusion transmission from hbsag negative blood donors and management of bone marrow and organ transplantations (54, 55). Given that routine detection of hbv infection, which is based on the detection of hbsag, cannot detect occult hbv infections, implementation of hbv dna and screening should be considered in epidemiological studies (50). One of the strengths of the current study is that only studies that really were performed in the general population were included . (16) included the studies on the general population, blood donors, patients on surgery, and students . Additionally, in the present review, well - designed studies form peer - reviewed journals with proper sampling strategies and reliable laboratory techniques were included . Another strength of this systematic review is that it is truly updated, as it covers studies published up to jan . 1, 2016 . Iranian national databases are not functional enough because of the insufficiency of search tools, absence of user - friendly advanced search tools, and limited coverage of all national scientific journals, universities research projects, and dissertations . Another major limitation was applying standard keywords for search in iranian national databases . To solve this problem, all synonyms of standard keywords in both persian and english languages were subjected to search . According to all data on the prevalence of hbv in the general population reviewed in this study, iran was classified within the low intermediate hbv prevalence areas (2% - 4.99%), while according to recent data (after 2010), iran is classified within the low hbv prevalence areas (<2%), indicating that preventive measures conducted in iran were effective . There is a heterogeneous pattern of distribution of hbv infection between iran s provinces, indicating the necessity of continuous prevention and control measurements and the implementation of further epidemiologic studies for collecting reliable data on hbv prevalence in different parts of iran.
Vero e6 cells were infected with 100 focus - forming units of ebov expressing enhanced green fluorescent protein . After a 1-h incubation, the inoculum was removed and replaced with media (dulbecco s modified eagle's medium with 2% fetal bovine serum, penn / strep, l - glutamine) containing chloroquine (sigma, st . The supernatant was collected on days 1, 3, 5, 7, and 9 after infection and media replaced with fresh drug . Viral rna was extracted from the supernatant and quantified by real - time quantitative reverse transcription pcr as previously described (10). Concurrently, cell viability was assayed by using cell titer96 aqueous one solution (promega, madison, wi, usa) according to the manufacturer s instructions . Ec was determined by using prism6 (graphpad software, san diego, ca, usa). When added 1 h after infection, chloroquine at 5 g / ml and 25 g / ml reduced the viral loads by 0.61 and 1.07 logs, respectively (peak reduction observed on day 5), without any significant cytotoxicity (figure 1). Analysis of the data from day 5 resulted in an ec50 of 1.77 g / ml and an ec90 of 23.34 g / ml, concentrations that are comparable with previous data (8); however, reductions in viral loads at these concentrations at other time points were negligible . Although concentrations of> 50 g / ml of chloroquine reduced viral loads by 24 logs starting on day 3, this decrease was accompanied by a high level of cytotoxicity (> 50%) that was evident both in the cytotoxicity assay and microscopically resulting in poor selectivity of chloroquine . Viral loads from supernatants derived from vero cells infected with ebola virus expressing enhanced green fluorescent protein and treated with chloroquine at the indicated concentrations (0, 5, 25, 50, 100, and 200 g / ml). Six - week - old balb / c mice or syrian hamsters (both from harlan, indianapolis, in, usa) were inoculated intraperitoneally with 100 50% lethal dose of ma ebov . The mouse (11) and the hamster (12) are well - established disease models of ebov infection . Treatment groups (mice and hamsters) received 90 mg / kg of chloroquine alone (intraperitoneally). An additional group of hamsters received 50 mg / kg of chloroquine (intraperitoneally every 24 h) in combination with 2.5 mg / kg doxycycline (gavage every 12 h) and 50 mg / kg azithromycin (intraperitoneally every 24 h). After inoculation, animals were monitored at least twice daily and euthanized by using a humane endpoint scoring criteria as approved by the animal care and use committee at rocky mountain laboratories (hamilton, mt, usa). Two of 3 mock - challenged mice did not survive because of chloroquine (90 mg / kg) treatment alone (figure 2, panel a). Only 2 of 9 mice infected with ma ebov and treated with chloroquine survived, and 1 of 9 mice infected with ma ebov and treated with vehicle survived . With median survival of 7, 8, and 8 d for mock - challenged / chloroquine - treated mice, ma ebov infected / chloroquine - treated mice, and ma ebov infected / vehicle - treated mice, respectively, treatment had no significant effect on survival . This dose, although previously stated as the maximum tolerated dose in mice (8), was not well tolerated by the animals in this study and clearly did not improve survival in animals challenged with ma ebov . Survival of ma ebov - inoculated mice (a) and hamsters (b) treated with cq (90 mg / kg). C) survival of ma ebov infected hamsters treated with a combination of cq (50 mg / kg), doxycycline (2.5 mg / kg), and azithromycin (50 mg / kg). Combo, combination of chloroquine, doxycycline, and azithromycin; cq, chloroquine; ebov, ebola virus; ma, mouse - adapted . When the same dose (90 mg / kg) of chloroquine was given to hamsters challenged with ma ebov, the study had to be terminated on day 2 after treatment . Nearly all the treated animals, in both the ma ebov and the mock - challenged groups, died of acute toxicity after administration of chloroquine intraperitoneally, typically within 30 min after treatment (figure 2, panel b). In a separate study, hamsters were treated with chloroquine (50 mg / kg) in combination with doxycycline (2.5 mg / kg) and azithromycin (50 mg / kg) to additionally provide broad - spectrum antimicrobial drug coverage . Reperfusion injury of the gut after ebov disease, which would subsequently result in bacterial sepsis, has been suspected as a possible cause of death . Thus, broad - spectrum antimicrobial drugs were proposed to help in this regard . In this study, no toxicity was observed in the mock - challenged group as a result of the combination treatment . However, treatment had no effect on survival; no combination - treated or vehicle - treated groups survived, and median survival times were comparable (figure 2, panel c). Despite some activity of chloroquine against ebov in vitro, we observed no benefit to its administration in the mouse and hamster models . In the mouse model, a dose of 90 mg / kg resulted in toxicity but did not alter survival; therefore, higher concentrations of chloroquine in the mouse would not be expected to be possible . In the hamsters, this dose was already lethal on its own . In the hamster model at a lower dose (50 mg / kg) combined with doxycycline and azithromycin which together provide broad - spectrum antimicrobial coverage, in addition to doxycycline having a small antiviral effect against ebov previous anecdotal reports of the incidental use of chloroquine in patients with filovirus infections also do not support any benefit from its use (13,14). Together, these data suggest that chloroquine is unlikely to provide any protection from ebov infection in humans . Given its in vitro activity against many different viruses and its longstanding use in humans, chloroquine has been put into multiple clinical trials . During dengue virus infection, viremia did not decrease (15), and chloroquine neither prevented influenza virus infection (7) nor improved outcome of chikungunya virus infection (6) despite promising in vitro activity against these viruses . When taken together with previous findings for other less pathogenic viruses, the clinical use of chloroquine seems unlikely to provide any benefit for either prophylaxis or treatment of ebov . Moreover, chloroquine has a small therapeutic window; dosing for treatment of acute malaria is 15 mg / kg, and lethality starts at 50 mg / kg . Thus, current preclinical data do not support the continued consideration of chloroquine for use against ebov infections in humans.
The pharmacological effects of nicotine the major psychoactive ingredient in tobacco smoke are mediated by the activation of nicotinic acetylcholine receptors (nachrs), which are present in several brain regions and tied to different cellular processes (mcgehee et al ., 1995,, 1996, chiamulera, 2005, laviolette and van der kooy, 2004, metherate, 2004). As a consequence, nicotine produces a wide variety of motivational and behavioural effects, including acting on mood, emotions, cognition and motor functions (chiamulera, 2005, laviolette and van der kooy, 2004, mcgehee et al ., 1995, pontieri et al ., 1996,, the close interaction between nicotine and anxiety represents one of the main concerns, yet it is poorly understood . Nicotine modulates the physiological responses to stress and anxiety processes in both animal models and human smokers (anderson and brunzell, 2012, breslau, 1995, brioni et al ., 1993, fidler and west, 2009, file et al ., 1998, george et al .,, 2000b, george et al ., 1998, irvine et al ., 2001,, 2011, perkins and grobe, 1992, varani et al ., 2012). Stress is also a major precipitating factor for smoking relapse (shiffman et al ., 1997) and for the increase in cigarette use (skara et al ., 2001). This depiction is, however, complicated by the fact that nicotine, like many drugs of abuse, possesses both rewarding and aversive properties (grieder et al ., 2012, laviolette and van der kooy, 2003, laviolette and van der kooy, 2004; sun and laviolette, 2014). Nicotine can also induce effects in anxiety disorders such as post - traumatic stress disorder (ptsd), as evidenced by the high comorbidity between these diseases and nicotine dependence through habitual tobacco use (breslau et al ., 2004, dalack et al ., 1998, hughes et al ., 1986, lasser et al .,, 2008, ziedonis and george, 1997). In animal models, a useful protocol to study psychological stress, as well as the mechanisms involved in ptsd - like manifestations, is provided by the fear - conditioning paradigm; whereby, the subject is exposed to a conditioned stimulus (cs), such as a tone, in association with an unconditioned stimulus (us), typically a footshock . Nicotine pretreatment does not modify the acquisition or the expression of conditioned fear - responses (george et al ., 2001). Accordingly, it was reported that the acute systemic administration of nicotine or the direct infusion of nicotine into the dorsal hippocampus did not alter fear behaviours related to a cs, but did enhance the memory of the environment paired with the aversive event (davis et al . ., 2010, kenney et al ., 2012, raybuck and gould, 2010, tipps et al ., conversely, acute nicotine injection into the ventral hippocampus produced a deficit in contextual fear memories and in trace fear conditioning (kenney et al ., 2012, kutlu and gould, 2015, gould and leach, 2014, raybuck and gould, 2010). It was therefore suggested that the effects of nicotine vary according to the brain region (kutlu and gould, 2015, gould and leach, 2014, raybuck and gould, 2010). To our knowledge, no data are available on the effects that nicotine induces when administered directly in brain structures involved in the encoding of fear memories related to explicit sensory cues . Recently, we found that the higher order components of the sensory cortex, such as the secondary auditory cortex te2, are essential for the long - term storage of remote (i.e. 30 days) auditory fear memories (sacco and sacchetti, 2010). The involvement of this cortex is strictly related to emotional memory processes, and not is due to any interference with sensory or innate emotional processes (grosso et al ., 2015, thus, this cortex is particularly suitable for investigating the effects, if any, that nicotine has on explicit emotional memories, without any confounding factors from the interference with emotional or motor processes . Therefore, in this study, we address the question of whether, and if so how, acute nicotine administration into te2 can modulate the memory of cues that are predictive of threat events . Male wistar rats (age, 6580 days; weight, 250350 g) were used . The animals were housed in plastic cages with food and water available ad libitum, with a 12 h light / dark cycle at a constant temperature of 22 1 c . All the experiments were conducted in accordance with the european communities council directive 2010/63/eu and approved by the italian ministry of health (authorisation no 265/2011) and by the local bioethical committee of the university of turin . Nicotine hydrogen tartrate salt (glentham life sciences, corsham, uk) was dissolved in physiological saline (0.9% nacl) and the ph was adjusted to 7.4 . Different doses of nicotine (0.54, 27 and 54 nmol/l; 0.6 l per site, as calculated on the tartrate salt weight) were applied, based on previous studies (laviolette et al ., 2008, in particular, we applied a dose of 54 nmol/l, as this is similar to that of laviolette et al . (2008) who applied a dose of 24 nmol/0.5 l, and which has been reported to elicit rewarding effects when injected into the ventral tegmental area (vta) (laviolette and van der kooy, 2003, paxinos and watson, 1986). Muscimol (tocris bioscience, bristol, uk) was prepared at 1 mg / ml in physiological saline (letzkus et al ., 2011, the rats were infused bilaterally with either saline, nicotine or muscimol at a rate of 0.25 l / min . To allow the diffusion of the drug, the injection needle was removed after waiting for 1 min . The behavioural experiments were started at 1520 min after completion of the nicotine injection procedures or 60 min after muscimol administration . The rats were surgically implanted with bilateral, chronic, intracranial stainless steel guide - cannulae (4 mm long, 26 gauge, plastic one, roanoke, usa). First, the animals were anaesthetised with an intraperitoneal administration of ketamine (100 mg / kg; ketavet; bayer, leverkusen, germany) supplemented by xylazine (5 mg / kg; rompun; bayer) and mounted in the stereotaxic apparatus . Bilateral cannulae aimed at 2.1 mm above the te2 cortex were implanted at the following stereotaxic coordinates: 6.8 mm posterior to the bregma and 6.5 mm lateral to the midline (paxinos and watson, 1986). The cannulae were lowered below the skull surface at an angle of 19 to the vertical axis in the coronal plane (medial to lateral). To inactivate the ventral hippocampus adjacent to the te2 cortex, bilateral cannulae were implanted at 2.1 mm above this structure at the following stereotaxic coordinates: 6.8 mm posterior to the bregma 4.5 mm lateral to midline (paxinos and watson, 1986). Once secured, cannula dummies (plastic one) were used to obdurate the guide cannulae . After post - surgical recovery (810 days), injection cannulae (31 gauge) were inserted through the guide cannulae . The injector was connected through polyethylene tubing to a hamilton syringe (10 l), which was mounted on an infusion pump (harvard apparatus, holliston, usa). After completing the experiments, the cannulae placements a skinner box module was employed as a conditioning chamber, as in our previous work (sacco and sacchetti, 2010). The box floor was made of stainless steel rods (1 cm in diameter, spaced 5 cm apart) connected to a shock delivery apparatus ., the animals were left undisturbed for 2 min . After this time, a series of sensory stimuli acting as css were administered . The final 1 s of each cs was accompanied with an us consisting of a scrambled electric footshock (intensity, 0.7 ma). The rats were left in the chamber for an additional 1 min, then returned to the home cage . In the fear conditioning to acoustic stimuli, seven pure tones (8 s, 78 db, 3000 hz, 22-s inter - trial interval) were delivered as css by a loudspeaker located 20 cm above the grid floor . In olfactory fear conditioning, seven almond odours (8 s, 22-s inter - trial interval) were presented using a flow - dilution olfactometer . Clean air (1.5 l / min) was directed to a solenoid valve, which when operated, passed the air to a 15 ml bottle containing 10 ml of almond odour . Weaker olfactory fear memories were obtained by employing a footshock intensity of 0.4 ma . The animals were handled for three days (5 min per day) before the memory retention experiments . Memory was tested using a different apparatus located in a separate experimental room in order to avoid conditioned fear behaviour to contextual cues (kim and fanselow, 1992, sacchetti et al ., 1999, the apparatus consisted of a plastic cage with the floor and sides made of transparent plastic and enclosed within a sound attenuating chamber equipped with an exhaust fan, which eliminated odourised air from the enclosure and provided background noise of 60 db . Once inside a freezing response was taken as a fear index (sacco and sacchetti, 2010), where freezing was defined as the complete absence of somatic mobility, except for respiratory movements . For each animal, the amount of time (in seconds) spent freezing during the css was measured offline . The freezing behaviour was analysed by two independent observers who were blind to the animal groups (inter- and intra - rater reliabilities 90%). Freezing during the 120 s period preceding the first tone was also recorded to measure any generalisation of fear (precs period). The rats were placed on a restricted diet to maintain their body weight at approximately 90% of their free - feeding weight . A day before the behavioural protocol began, the rats were given 1 g of chocolate - flavoured food pellets (bio - serv, f07256, flemington, usa) in the home cage to familiarise them with the pellets . Animals were conditioned in the standard skinner box module described in the fear - conditioning protocol (section 2.4). The rats underwent pavlovian conditioning sessions in which the presentation of a cs was coupled with an us, consisting of the delivery of one sucrose pellet into a food cup within the chamber . All the animals were conditioned for 3 consecutive days, consisting of one conditioning session per day . Memory retention was tested 4 weeks post conditioning by presenting the css, which were not paired with any us . To minimise contextual influences, the animals were tested in an environment completely different from that employed during the learning trial (see section 2.4, the fear - conditioning paradigm). Moreover, to reduce the contribution of within - group variations in baseline responding, we analysed the differences between the cs responses and precs responses (saddoris et al ., 2009). The conditioned discriminative approach was calculated as the time the animals spent with their head in the food cup during the first 25 s of cs (8 s) and post - cs presentation (17 s) minus the 25 s preceding cs onset . The css were identical to those employed in the fear - conditioning paradigm conditioning (pure tones, 8 s, 78 db, 3000 hz). In this paradigm, the rats faced a conflict between an innate aversion to an open space and the motivation to explore it . A greater amount of time spent in the brightly lit space was linked to an index of decreased anxiety - like behaviour . The open - field apparatus consisted of a plastic opaque box (50 80 40 cm). The rats were placed in the centre of the apparatus and their behaviour was recorded for 10 min . The analyses were conducted using the smart 3.0 software (panlab, cornell, spain). The acoustic startle response was measured as an input / output function (valsamis and schmid, 2011) using a startle chamber (spsg, la jolla, california, usa). After an acclimation period of 5 min with a constant background white noise of 50 db, startle stimuli (1 s white noise) were displayed every 20 s, starting at 57 db . The startle stimulus intensity was increased between each stimulus until it reached 88 db, thus resulting in 1030 trials with startle stimuli (sacco and sacchetti, 2010). Following the experiments, rats were deeply anesthetized with intraperitoneal administration of ketamine (100 mg / kg; ketavet; bayer, leverkusen, germany) supplemented by xylazine (5 mg / kg; rompun; bayer) and intracardially perfused with saline, followed by 4% formaldehyde . Brains were cut with a freezing microtome, and injection needle tracks were identified in nissl - stained serial sections . Student's t - tests, one - way (with the total time spent freezing or the conditioned incentive responses as a dependent variable) and repeated - measure (with groups as a between - subjects variable and retention trials as a within - subjects variable) anova tests and newman keuls multiple comparisons test were employed for comparing the different behavioural groups . Initially, we addressed the question of whether and how nicotine administered locally in the te2 cortex interferes with the retrieval of remote fearful memories . Remote memory retention was assessed 1 month after fear conditioning by measuring the freezing behaviour elicited by the auditory css that had been previously paired with the us (lesburgures et al ., 2011, to minimise contextual influences, the rats were tested in a new environment (kim and fanselow, 1992, sacchetti et al ., 1999, sacchetti et al ., 2002, 1b shows the total time the animals spent freezing before the 2 min preceding and during the presentation of the seven css . Freezing responses during the 2 min preceding the cs presentation were low and similar between groups, thus indicating the absence of generalisation phenomena (kim and fanselow, 1992, sacchetti et al ., 1999). During the cs presentation, we found that nicotine decreases the fear - evoked responses at doses of 54 (n = 8) and 27 (n = 6) nmol/l, while the concentration of 0.54 nmol/l (n = 6) did not modify the freezing response with reference to the saline - injected (n = 8) rats . One - way anova showed a significant difference between groups (f3,24 = 24.70; p <0.001). The subsequent newman keuls test revealed differences between the freezing responses of the animals injected with 54 and 27 nmol/l doses of nicotine and the freezing of the saline - injected rats (p <0.05), but not between the control group and the animals injected with 0.54 nmol/l of nicotine (p> 0.05). Similar effects were observed by analysing the total freezing time of the animals during css presentation and the inter - trial time intervals (f3,24 = 9.10; p <0.001) (fig . Given that we presented seven css, the observed decreases in conditioned fear behaviour may be due to an effect on the threat memory retrieval or alternatively to an enhancement of the extinction processes that may occur during repeated presentation of css (elias et al . We therefore analysed the freezing responses during each cs presentation to test whether the freezing response changed across trials . The anova test for the repeated measures showed differences between the groups (f3,24 = 24.72; p <0.001), but not within each group across the cs presentation (f3,18 = 1.49; p> 0.05) (fig . This revealed that nicotine acts early during memory retrieval, i.e. During the first cs presentation, and similarly decreases the freezing responses to all other css . A subsequent nissl - stained inspection revealed that intra - cortical nicotine administration did not elicit permanent neuronal damage (fig . Whether the effects of nicotine were limited to the neural network specifically engaged by the auditory memory processes, we repeated the previous experiment in animals conditioned to an odour cs, a process in which the te2 is not involved (sacco and sacchetti, 2010). There was no significant difference between saline- (n = 6) and nicotine (n = 6)-injected rats during the overall cs presentation (student's t - test, t10 = 1.41, p> 0.05) (fig . 1 g), or during each cs presentation (f1,10 = 0.28; p> 0.05) (fig . However, the odour - conditioned animals displayed very strong conditioned responses, potentially masking the interference of nicotine . To rule out this possibility, nicotine administration was repeated in rats conditioned by pairing odour css with lower intensity uss . This procedure resulted in a weaker conditioning (n = 8) that was not affected by nicotine administration (n = 8), with regard to the overall css presentation (t14 = 0.53, p> 0.05) (fig . 1 g) and across each cs presentation (f1,14 = 0.07; p> 0.05) (fig . This indicates that the nicotine effects are strictly related to memory processes occurring in te2 and are not secondary to interferences from other structures, such as the amygdala or the vta, which are known to play a general role in emotional memory . Furthermore, these findings show that nicotine injection into te2 did not affect the freezing response or emotional process; rather it interferes selectively with local emotional memory processes in te2 . Several previous studies have shown that nicotine administration into the ventral hippocampus impairs contextual and trace cued fear memories (kenney et al ., 2012, kutlu and gould, 2015, gould and leach, 2014, raybuck and gould, 2010). Given that the te2 cortex is adjacent to the caudal portion of the ventral hippocampus, it may be that the nicotine effects observed in our experiment are due to nicotine spreading to this region . Despite the fact that in the aforementioned studies nicotine was administered in a region of the ventral hippocampus far from that adjacent to the te2 cortex, to clarify this point, we repeated the previous experiment by injecting a similar dose of nicotine into the region of the ventral hippocampus that is immediately adjacent to the te2 (figs . 2a b). When compared to saline - injected rats (n = 7), nicotine (n = 7) did not alter remote fearful memories to cs administration (t12 = 1.37, p> 0.05) (fig . 2c), the total freezing time during the css and the inter - trial time intervals (t12 = 0.12, p> 0.05) (fig . 2d), or freezing across each cs (f1,12 = 1.05; p> 0.05) (fig . Nicotine has also been reported to modulate sensory stimuli perception (liang et al . 2012). Nevertheless, the te2 auditory cortex is not necessary for processing simple auditory stimuli, such as those employed in the present study with the css (sacco and sacchetti, 2010). To better define whether intra - te2 nicotine administration affects auditory stimuli perception, we analysed the auditory input / output curve by assessing the startle responses as a function of the auditory stimulus intensity (db) (valsamis and schmid, 2011). On day 1, we measured the startle response in the absence of any treatment (fig . The following day, we administered the highest effective dose of nicotine (54 nmol//l; n = 8) or saline (n = 8) (fig . Anova for the repeated measures revealed no differences between groups before (f1,14 = 0.12; p> 0.05) or after (f1,14 = 0.15; p> 0.05) nicotine injection (fig . Therefore there were no significant effects of cortical nicotine administration on the perception and processing of auditory stimuli . Nicotine can enhance spontaneous motor activity and modify innate emotional behaviour when administered systemically or in specific brain regions, such as the vta and the nucleus accumbens (ferrari et al . However, the te2 cortex is neither involved in motor responses nor in innate anxiety behaviour (sacco and sacchetti, 2010). The data obtained from the animals conditioned to odour cs support this view . However, to further rule out any possible interference of nicotine administration in te2 on these processes, we tested the effects of such a manipulation on the open - field paradigm, a well - established model of anxiety . Nicotine (n = 6) or saline (n = 6) were administered in the te2 before the open - field test (fig . 3c d). The two groups did not differ in the time spent in the centre (student's t - test, t10 = 0.79, p> 0.05) or in the periphery (t10 = 0.80, p> 0.05) of the open field, two measures of the innate level of anxiety (fig . Similarly, there were no differences in the distance travelled in the centre (t10 = 1.31, p> 0.05) or in the periphery (t10 = 0.2, p> 0.05) (fig . These data support the view that the reduction in the conditioned fear - evoked response is not due to a change in the spontaneous fear state . In addition, we also measured two parameters in the two groups related to spontaneous motor activity, namely the total distance travelled (t10 = 0.71) and the averaged velocity (t10 = 0.29). No differences were detected for all instances (p> 0.05) (fig . The effects we observed could be due to a disturbance of the memory retrieval processes occurring locally in the te2 . Alternatively, it may be that nicotine induces a selective decrement in the fear - evoked responses elicited by cs presentation . In addition, are these effects strictly related to threat memories or, alternatively, does nicotine also interact with emotional memories characterised by a positive, incentive emotional content? To investigate these issues, we tested the impact of te2-nicotine administration in remote emotional memories obtained by pairing auditory css, identical to those previous employed (frequency, amplitude and duration), with delivery of a palatable food . The total time that te2-nicotine injected subjects spent with their heads in the food cup during the presentation of css minus the precs periods did not differ at all the tested dosages (54 nmol/l, n = 12; 27 nmol/l, n = 12, 0.54 nmol/l, n = 12) from the saline - injected (n = 12) rats (f3,44 = 1.27, p> 0.05) (fig . No differences were detected between groups in the baseline precs responding (f3,44 = 0.44, p> 0.05), thus suggesting that nicotine did not alter motor or motivational processes (fig . 4d). In the case of the lowest dosage (0.54 nmol/l), the low level of precs responding may be related to a potential floor effect . However, several observations argued against this possibility . Firstly, in the latter group the precs responding, even if low, is still statistically higher than zero (student's t - test, t11 = 5.14, p <0.001). More important, in this group the time that animals spent in the food cup during cs presentation (51.10 4.63) was similar either to that of saline - injected rats (59.75 4.10) and of the other groups that received nicotine (54 nmol/l, 57.79 3.81; 27 nmol/l, 50.82 5.12) (f3,44 = 1.24, p> 0.05, data not shown), thus showing the lack of nicotine's effects on appetitive memories . An alternative explanation of the present data may be that te2 is necessary for threat but not for incentive remote memories . To address this issue, we tested whether blocking te2 general activity would impair remote appetitive memories . We thus inactivated te2 through the local injection of the gabaa (-aminobutyric acid)-receptor agonist muscimol (letzkus et al . When compared with the saline - injected (n = 5) animals, the muscimol - injected (n = 6) rats showed a strongly reduced conditioned incentive response to the css (student's t - test, t9 = 5.67, p <0.001) (fig . 4f). These data indicate that the te2 cortex is necessary for the retrieval of remote appetitive memories and, therefore, that any eventual disturbance in its activity would lead to detrimental effects on such emotional memories . Overall, these results show that nicotine, at all the tested doses, did not affect incentive - memory retention, thus suggesting that intra - te2 nicotine injection did not elicit a specific memory disturbance . Thus, nicotine selectively results in a decrease in learned fear, while incentive memories remain unaffected . We therefore conclude that acutely administered nicotine in the higher order auditory cortex does not interfere with memory retrieval processes at a general level, but instead, it selectively relieves the fear aversive emotional content associated with auditory css . We then addressed the question of whether nicotine is only able to reduce fearful memories acutely, or if its effects endure for a period of time after nicotine administration . For this aim, the rats that displayed a significant reduction in conditioned freezing were re - tested 1 week after nicotine injection, in a drug - free state (fig . The freezing responses of the nicotine - treated rats were similar to those of the control animals (f3,24 = 0.70, p> 0.05) (fig . The data revealed that the effects of nicotine are not long - lasting, but are instead limited to the actual presence of nicotine in the brain . To further characterise the impact of nicotine on memory processes, we investigated whether nicotine can interfere with the memory reconsolidation processes that occur shortly after memory retrieval . It has been shown that well - established fear memories are altered when active compounds are applied immediately after memory retrieval (misanin et al ., 1968, nader et al ., 2000, sacchetti et al ., 2007, tedesco et al ., 2014), a process referred to as memory reconsolidation (nader et al ., 2000, schwabe and nader, 2014, tedesco et al ., 2014) after 1 week, memory was tested by re - presenting the css (fig . No differences were detected between the nicotine- (n = 6) and saline (n = 6)-injected animals (student's t - test, t10 = 1.09, p> 0.05) (fig . 5d), thus indicating that nicotine did not interfere with the reconsolidation mechanisms occurring after memory recall . In addition, we investigated whether nicotine interferes with the acquisition of remote memories . To this purpose, nicotine was administered before the acquisition trial, and then memory was tested 1 month later in a drug - free state (fig . 5e).when compared to saline injected animals (n = 6), rats that received nicotine before the acquisition trial displayed a statistically significant decrement in the fear - evoked responses to the css (student's t - test, t10 = 4.23, p <0.05) and in the total time spent freezing during css and the inter trial intervals (t10 = 3.96, p <0.05) (fig . 5f). Taken together with the results obtained in the experiment 1, the data indicate that nicotine affects either the formation and the retrieval of remote fear memories . So far, the data were obtained by testing memory in a contextual environment that was completely different from that in which the conditioning trials occurred . Nonetheless, as previously mentioned, the animals that systemically received nicotine did not display any significant fear - relief effects when the threat memory was tested in the same environment to which the conditioning occurred (elias et al . We therefore tested the impact of nicotine administration in the te2 on the auditory fear memories in the context of which the animals were originally conditioned (fig ., the presence of contextual cues determined an elevated fear state, as indicated by the freezing of the saline- and nicotine - injected animals during the 2 min before cs presentation (fig . 5h). During cs presentation, the freezing response was higher than during the 2 min preceding it, but similar between the saline- (n = 7) and nicotine (27 nmol/l, n = 6; 54 nmol/l, n = 7)-injected animals (f2,17 = 0.31, p> 0.05) (fig . Collectively, these data indicate that nicotine acutely affected the auditory fear memories when tested in a neutral environment . This effect was strictly attributed to the presence of nicotine and was not followed by any consequence of the original memory trace . The data suggest that nicotine relieves the fear charge embedded by auditory css but does not affect the original memory trace . This view is further supported by the evidence that, when the fear state of the animals was enhanced by the presence of contextual cues, the fear - relief effect of nicotine was abolished . In this study, we have shown that acute nicotine administration into the te2 auditory cortex before the retrieval of remote fearful memories decreases fear - evoked responses . A similar treatment did not affect remote incentive memories to auditory css previously paired with reward stimuli . We conclude that nicotine administration in the te2 selectively decreases the aversive charge of fear - predictive cues . Several observations have allowed us to rule out many confounding factors that may have interfered with the emotional memory processes . First, the effects of nicotine are not secondary to any interference with innate fear and anxiety behaviour, as indicated by the open - field test and by the high level of conditioned freezing to odour css . In addition, nicotine administration does not affect auditory sensory perception and processing, as shown by the startle reflex paradigm as well as by the high level of memory retention to similar auditory css in appetitive - conditioned rats . We can also rule out the possibility that intra - te2 nicotine administration interferes with spontaneous motor activity, thus counteracting the freezing response . In fact, nicotine injection did not affect conditioned freezing to an odour cs, and it did not change the spontaneous motor activity in the open - field paradigm results which are in line with a previous study (sacco and sacchetti, 2010). Nicotine's effects may be due to state - dependent effects . Although we cannot rule out this possibility, it should be noted that nicotine administration did not reduce auditory incentive memories or fear conditioning to odour css . Collectively, therefore, our findings indicate that nicotine injection into the te2 selectively interacts with memory information encoded at the level of the auditory cortex . Furthermore, the different effects elicited by nicotine on threat and incentive memories indicate that this drug is not involved in simply disturbing the memory retrieval process, nor does it affect the memory of the physical features of the tone acting as a cs . Therefore, we suggest that nicotine, when injected into the te2, selectively interferes with the negative aversive valence acquired by the cs . Many studies have tested the effects of nicotine on innate spontaneous behaviour . In rodent behavioural assays of anxiety - like behaviour, nicotine can either decrease (anderson and brunzell, 2012, file et al ., 1998, mcgranahan et al ., 2011, varani et al ., 2012) or promote (cheeta et al ., 2001, accordingly, nicotine elicits anxiolytic and stress - dampening, in a dose- and context - dependent manner, as well as having mood - enhancing properties in humans (evatt and kassel, 2010, laje et al ., 2001, regarding the current findings, we found that nicotine acts directly on the aversive emotional charge endowed in the perceived stimuli, independently of its action on spontaneous behaviours . The systemic administration of nicotine does not induce any effects on learned fear obtained by pairing an auditory stimulus to footshock, however it affects the extinction of contextual cues and cued fear memories (elias et al ., 2010, kutlu and gould, 2015, george et al . However, when administered systemically nicotine can cause peripheral drug - associated effects, such as a modulation of the motor and/or sensory perception processes, as well as interacting with a large variety of brain regions and neuronal mechanisms, producing a complex combination of effects . Nicotine, when administered to selected brain regions, elicits a contextual, but not cued, fear - memory strengthening, mostly due to an interaction with the memory and/or attentive processes that occur in the dorsal hippocampus (davis et al ., 2007, kenney et al ., 2012, conversely, these authors also reported that nicotine injection into the ventral hippocampus impaired rather than potentiated contextual and trace fear memories (kenney et al ., 2012, kutlu and gould, 2015, gould and leach, 2014, raybuck and gould, 2010). Therefore, it is concluded that the different behavioural effects seen with nicotine are related to regional specificity, receptor subtype specificity and/or interactions with diverse neurotransmitter systems (raybuck and gould, 2010). In this scenario, we showed that nicotine administration in the more posterior region of the ventral hippocampus did not affect auditory remote fear memories, whereas nicotine injection into the te2 auditory cortex affected learned threat tones . The odour threat stimuli and the configuration of auditory css in the conditioning environment were however unaffected, i.e. The effects of nicotine are highly cue- and site specific . It is worth mentioning that the doses employed in the present study are much higher than those employed in the aforementioned studies on the dorsal and ventral hippocampus (davis et al . . It may be therefore, that the administration of lower doses in the te2 could produce an enhancement of the cs responses, rather than a decrease . However, in most cases, nachrs are formed by a combination of different alpha and beta subunits and are highly heterogeneous across different brain structures (chiamulera, 2005, laviolette and van der kooy, 2004, mcgehee et al ., 1995, metherate et al . Thus, memory - enhancing nicotine doses (if any) can markedly differ between the hippocampus and the auditory cortex . In the latter site, in the case of auditory memory enhancement induced by intra - cortical nicotine administration, it should be also clarified as to whether these effects are due to a potentiation of the memory and/or attentive processes or rather to an improvement of the auditory stimuli perception and processing . In fact, the systemic administration of nicotine modulates tone - evoked responses in the auditory cortex (liang et al ., 2008, metherate, 2004, the doses we employed in the present study resemble those previously employed to elicit rewarding effects on spontaneous behaviour when nicotine was administered into the vta (laviolette and van der kooy, 2003, sacchetti et al . Conversely, we found that nicotine administered into the te2 did not increase the appetitive - conditioned responses to css . Given that we used cs of strong motivational value, the effects of nicotine on stimulus processing may be lost to ceiling effects . However, in our study the conditioned incentive responses of the saline - treated rats were not close to the maximum value . We therefore suggest that the observed fear - relief effects are most likely to be due to direct anxiolytic - like effects on threat memories . Nevertheless, despite unlikely, we cannot ruled out the possibility that nicotine's effects may be in part due to alteration of the motivational properties of the css . We also observed an effect of nicotine on the formation of new remote fear memories . The data therefore suggest that this substance interferes with the attribution of aversive charge to sensory stimuli, may be though a decrease in the fear - evoked content of the aversive experience, as we suggested during memory retrieval . Although a large number of studies have investigated the nachrs in several subcortical structures, such as the vta and the nucleus accumbens, much less is known about how nachrs are formed within the auditory cortex . Several genes have been identified for a large number of receptor subunits in the cortex (metherate, 2004). However, most nachrs in the sensory cortex are thought to exist as heteromers formed by 4 and 2 subunits or as 7 homomers (metherate, 2004, bieszczad et al ., 2012). High - affinity nicotine binding has been detected mostly in cortical layers 3 and 4, but also in layers 1 and 6 (metherate, 2004). In layer 4, 42-containing nachrs may be present at the presynaptic level where they can regulate thalamo - cortical transmission (metherate, 2004). In line with these observations, the te2 layer 4 is reported to be markedly activated following remote fear - memory retrieval (kwon et al ., 2012, nachrs have also been detected in interneurons present in the superficial layers (letzkus et al . Notably, interneurons in superficial layers of the auditory cortex are involved in fear memory (letzkus et al ., 2011, 2013), the activity of which is shaped by nicotine administration (letzkus et al ., 2011). Our findings have important implications for understanding the interactions between nicotine and anxiety- and fear - related disturbances, such as ptsd . Frequently, cigarette smokers have reported that they smoke to relieve anxiety (fidler and west, 2009, perkins and grobe, 1992). Stress is also a major precipitating factor in smoking relapse (shiffman et al ., 1997) and in the escalation of cigarette use (skara et al ., 2001). The present data support this view, by showing that nicotine decreases fear responses associated with perceived stimuli . Indeed, our findings have raised the intriguing idea that nicotine can relieve fear - evoked processes without impairing memory retrieval processes or decreasing the subsequent fear - memory recall in the absence of nicotine . Ptsd in particular has high comorbidity, with over 45% of ptsd sufferers reported as being smokers (breslau et al ., 2004, dalack et al ., 1998, hughes et al ., 1986, lasser et al ., 2000,, 2008, ziedonis and george, 1997). Moreover, the rates of smoking are higher in individuals with ptsd than the rates of abuse of other compounds (breslau et al ., 2004, dalack et al ., 1998, hughes et al ., 1986, lasser et al ., 2000, although it does not precisely reproduce the entire ptsd pathology, fear conditioning is widely used to study the neural mechanisms underlying threat memories and, therefore, to also gain novel insights into the pathophysiology of ptsd (taubenfeld et al ., 2009, 2014). In the present study, we focused on the effects of nicotine in the retrieval phase of memory processes, which could be similar to the mental recall / avoidance of trauma - associated stimuli observed in ptsd - suffering individuals . In this framework, our data suggest that individuals with ptsd tend to use nicotine (by smoking cigarettes) as a self - medication to relieve fear and anxiety induced by sensory cues or intrusive memories . On the other hand, our data also showed that threat memories are unaffected by nicotine after its use, i.e. Ptsd - like symptoms remain intact after nicotine use . It is worth mentioning that in our study nicotine was administered locally in the cortex and acutely in drugs - nave animals . Indeed, nicotine has very different effects depending on administration time course, route and drug history . Thus, the relationship between nicotine use and stress - related disorders requires further evaluation.
It is well established that the transition of vascular smooth muscle cells (vsmcs) from a differentiated phenotype to a dedifferentiated state plays a critical role in the pathogenesis of atherosclerosis . The phenotypic modulation in vsmcs is accompanied by accelerated migration, proliferation and production of extracellular matrix components . Eventually, these cellular events result in the formation of atherosclerotic lesions . However, the molecular mechanisms involved in phenotypic control are still unclear . Micrornas (mirnas) are a class of endogenous, small, non - coding rnas that pair with sites can in 3' untranslated regions in mrnas of protein - coding genes to downregulate their expression . More importantly, one mirna is able to regulate the expression of multiple genes because it can bind to its mrna targets as either an imperfect or a perfect complement . Thus, a mirna can be functionally as important as a transcription factor . As a group, mirnas it is therefore not surprising that mirnas are involved in the regulation of all major cellular functions . Recently, the role of mirnas in cardiac cell differentiation has been described . In this study, kwon et al . Demonstrated that mir-1 plays important roles in modulating cardiogenesis and in maintenance of muscle - gene expression by targeting transcripts encoding the notch ligand delta . The biological roles of mir-145 in diverse cancer cells have been recently identified (reviewed in). In a recent article reported by xu et al ., mir-145 was found to be a critical switch for embryonic stem cell differentiation by repressing some core pluripotency factors . Whether mirnas participate in the phenotypic control of vsmcs was unknown until recently . In this respect, three independent groups have reported exciting new discoveries regarding the critical role of the vsmc - enriched mirna, mir-145, in vsmc phenotypic modulation [8 - 10]. They identified that mir-145 plays a role not only in the differentiation of multipotent neural crest stem cells into vsmcs, but also in the differentiation of adult vsmcs . Other mirnas that may participate in the phenotypic modula tion of vsmcs are mir-143 and mir-221 [9 - 12]. This minireview summarizes the current research progress regarding the roles of mir-145 in the vsmc phenotype and the potential therapeutic opportunities of mirnas in atherosclerotic vascular disease . Ji et al . Demonstrated that mir-145 is the most abundant mirna in arteries . Its expression is significantly downregulated in rat balloon - injured arteries with neointimal lesion growth . A recent nature article by cordes et al . Showed that mir-145 expression was also decreased in mouse carotid arteries after ligation injury . More interestingly, transcripts of mir-145 were down regulated to nearly undetectable levels in atherosclerotic lesions containing neointimal hyperplasia . Our own unpublished data also revealed that mir-145 is largely downregulated in atherosclerotic mouse and human arteries, although the downregulation is less pronounced compared with that from cordes' study . If the selected atherosclerotic tissue had fewer vsmcs, the expression level of mir-145 could be lower . Recently, cheng et al . Found that mir-145 is the most abundant mirna in differentiated vsmcs . Also, its expression is quickly downregulated in subcultured dedifferentiated vsmcs and in dedifferentiated vsmcs induced by stimulation with platelet - derived growth factor (pdgf). Our unpublished data also indicate that mir-145 expression in vsmcs isolated from balloon - injured rat carotid arteries and atherosclerotic apoe - knockout mouse aortas is significantly decreased compared with that in vsmcs isolated from normal control arteries . . Found that, during the development of arteries, the expression of mir-145 is associated with the state of vsmc differentiation . Mir-145 expression is notably absent in the aorta and pulmonary arteries during later cardiogenesis, during which vsmcs and arteries are developing . In contrast, high transcript levels of mir-145 in vsmcs of the arteries are demonstrated postnatally, after vsmcs and arteries have completed their development . Cordes et al . Demonstrated that mir-145 was necessary and sufficient to induce differentiation of multipotent neural crest stem cells into vsmcs . In addition, cheng et al . Identified for the first time that mir-145 is a critical modulator for the vsmc phenotype both in vitro and in vivo . Vsmc differentiation marker genes such as sm -actin, calponin, and sm - mhc were downregulated by mir-145 downregulation, and upregulated by mir-145 upregulation . The regulatory effect of mir-145 on the vsmc phenotype was further verified by cordes and colleagues . In contrast, another co - expressed mirna, mir-143, had no significant effect on marker genes for vsmc differentiation, although it had a strong effect on vsmc proliferation . . Demonstrated that the expres sion of the mir-143/145 cluster is confined to vsmcs during development . They found that mir-143 and mir-145 are required for vsmc acquisition of the contractile phenotype because the vsmcs from mir-143/145-deficient mice are locked in the synthetic state . The regulatory effects of mirnas on the vsmc phenotype are not limited to mir-143 and mir-145 . Two recent studies revealed that mir-221 is also related to the vsmc phenotype that affects vsmc proliferation and migration . It is well known that phenotypic modulation of vsmcs is an initial cellular event in the development of atherosclerotic vascular disease . To determine the therapeutic potential of mir-145 in vascular disease, . Demonstrated that restoration of mir-145 in rat balloon - injured carotid arteries via adenovirus - mediated gene transfer significantly inhibited neointimal lesion growth . In contrast, knockout of mir-143 and mir-145 resulted in the formation of neointima in mouse arteries . The roles of mir-145 in the vsmc phenotype and atherosclerotic vascular disease are summarized in figure 1 . Expression modulation of mir-145 in the vascular walls and the potential roles of mir-145 in vascular smooth muscle cell phenotype and atherosclerosis . Its expression is significantly downregulated in developing arteries and in adult arteries containing intimal hyperplasia . The downregulated mir-145 in adult arteries will increase vsmc dedifferentiation and result in the development of atherosclerotic lesions . Mirna - based therapy may have some advantages compared with that for other molecular targets because one endogenous mirna can target its multiple target genes . Although the recent studies have demonstrated that targeting mirnas, and mir-145 in particular, may represent a new therapy for atherosclerosis, there is still a long road before mirna - based technology can be translated to clinical therapy . First, the critical mirnas responsible for the development of atherosclerosis must be further identified, especially in human atherosclerotic arteries . Second, the detailed cellular and molecular mechanisms of these critical mirnas in the prevention and treatment of atherosclerosis should be studied . Third, in addition to the biological effects of these mirnas on vsmcs, their effects on other atherosclerosis - related cellular events should be identified . Fourth, although methods are available to downregulate a mirna in vivo, technology for upregulating a mirna in the vascular walls in vivo requires development . Finally, the potential side - effects of mirna - based therapy should be studied before application in the clinic . Mirna: microrna; mir-145: microrna-145; pdgf: platelet - derived growth factor; vsmc: vascular smooth muscle cell . This work was supported by a national institutes of health grant hl080133 and a grant from the american heart association 09grnt2250567.
The app is a type i transmembrane protein with characteristics of an orphan receptor, which shares with other members of its class a particular signaling mechanism termed regulated intramembrane proteolysis (rip). The first occurs outside the transmembrane domain, usually in response to ligand binding, inducing the release of the extracellular domain . This first cleavage event elicits a conformational change that triggers the second proteolytic cleavage which takes place on the transmembrane segment . This mechanism controls several cellular processes, such as the unfolded protein response, cholesterol synthesis, and cell fate instruction . Rip of the app is mediated by three different proteases . While - and -secretases catalyze extracellular cleavage, the -secretase complex cuts at the intramembrane domain and leads to the generation of two peptides: an app active fragment, termed aicd and the a . In contrast, two independent groups indicate that aicd is produced mainly from the 695 aminoacids isoform of app through the amyloidogenic pathway (dependent on -secretase activity) [6, 7] and is therefore generated in equimolar quantities with a . The last one accumulation and the formation of various aggregates and deposits in the brain have been the main hypothesis to explain the neuropathological development of ad for almost 20 years . Initially, the study of the functions associated with the aicd was limited by the hindrance in its detection . However, recent studies showing that the levels of the aicd are increased in brains of ad patients and murine models reproducing the disease, open up the possibility that this fragment participates in the molecular mechanisms contributing to ad . The aicd is the most evolutionarily - conserved region of the app, accounting for its functional importance . Despite its relatively small size (59 aminoacids or less), it acts as a docking site for a particularly large group of intracellular proteins . Amongst this group of proteins are pin1, the x11 protein family, disabled (dab)-1, shc, jnk - interacting protein (jip)-1, and the fe65 protein family [1719], which includes fe65 itself and two closely related homologues, fe65l1 and fe65l2 . Fe65 family members contain three protein - protein interaction domains: a ww domain at the n - terminal involved in interactions with proline - rich sequences and two phosphotyrosine binding domains (ptb1 and ptb2) located at the c - terminal . The second ptb domain (ptb2) is responsible for the interaction between fe65 and the sequence 682yenpty687 of the app (following the numbering of the app695 isoform). The possibility of aicd to form multiprotein complexes through its association with fe65 and its multiple ligands (table 1) has unexpectedly expanded the potential roles of aicd . Aicd binds to fe65 in a region that is essential for a production, making fe65 a good candidate for regulating app processing . This could occur via two mutually - exclusive pathways: the amyloidogenic pathway, leading to a production mediated by the -secretase and the nonamyloidogenic pathway leading to the production of a large extracellular fragment (sapp), which is mediated by the -secretase and prevents the generation of a. fe65 acts as a potent modulator by altering the balance between the two pathways . The overexpression of fe65 in cell lines induces a dramatic increase in a secretion, whereas a secretion was decreased in fe65 knockdown cells and in hippocampal neurons of fe65/fe65l1 knockout (ko) mice . The effect on the a secretion appears to be dependent on the interaction between fe65 and app, because the knock - in mice carrying the y682 g mutation, that inhibits aicd binding to fe65, show decreased levels of a and a massive increase in sapp, as a consequence of the nonamyloidogenic pathway this is in agreement with a study showing that fe65 is a potent suppressor of the nonamyloidogenic pathway in primate cells . The mechanism by which fe65 modulates a secretion is related to its interaction with the apolipoprotein e (apoe) receptors: the low density lipoprotein receptor - related protein (lrp) and apoe receptor 2 (apoer2). Related to the participation of the aforementioned receptors, the effect of fe65 in the secretion of soluble app fragments is lost in cells lacking lrp . The functional relation with apoer2 is more complex and depends on the presence of its extracellular ligand, reelin, and its intracellular adapter, dab-1 . Reelin reduces a secretion by promoting the binding of dab1 to the app and displacing fe65, because they share the same binding region . A decrease in reelin expression in the entorhinal cortex (the first region of the brain where a deposits can be observed), displayed in pdapp transgenic mice (which carry human app with mutations swedish (swe) and indiana) and in ad patients, could seriously affect the balance of dab1 and fe65 in their binding to aicd, increasing a secretion . This has been observed in transgenic mice which lack reelin expression (reeler) and carry the mutations swedish and arctic in app . A decade ago, a possible role for the rip of app was first suggested . Since app processing seems to be similar to notch processing, it has been suggested that rip of app could be involved in transcriptional regulation . In fact, the fusion of the dna binding domain of yeast gal4 (gal4db) to the c - terminal of app induced a strong transactivation of a luciferase reporter dependent on the formation of a trimeric complex with the adapter protein fe65 and the histone acetyltransferase tip60 . A reciprocal experiment using tip60 or fe65 fused to the gal4db gave rise to some contradictory results [49, 50]. Nevertheless, a consensus model can be generated including the vast majority of observations derived from these studies (figure 1). The app acts as an anchor for fe65 and fe65-associated proteins that is,: tip60, inducing its association with membrane compartments . Membrane recruitment seems to be essential for the activation of the complex, since the overexpression of soluble aicd has no effect on transactivation . The autoinhibited conformation of fe65, produced by the association of the ww domain with a region flanked by the ptb1 and ptb2 domains . The association with the plasma membrane allows the activation of the complex, induced by the phosphorylation of tip60 by cyclin - dependent kinases (cdks). An excellent prospective candidate is cdk-5, that can be found associated with plasma membranes through its activator p35 and displays high activity in the brain . The release of the complex from the plasma membrane may be produced by the app cleavage by -secretase or additionally by the app phosphorylation at thr668 which induces a conformational change in the region recognized by fe65, decreasing the affinity for each other . Although some groups have observed aicd in the nucleus, particularly in nuclear domains such as transcriptional factories, the splicing factor compartment or directly at promoters of some genes [5961], apparently in the artificial transactivation system, the nuclear translocation of aicd is not essential to enhance luciferase expression . The n - terminal region of fe65 that includes the ww domain is necessary for nuclear translocation and therefore for its activity as a transactivating protein . Although this region lacks a nuclear localization sequence (nls), it could be directed to the nuclei by association with a protein carrying a functional nls . A good candidate to perform this function would be the nucleosome assembly protein set that binds the ww domain and is required for transactivation mediated by the fe65gal4db fusion protein . The phosphorylation of tyr547 in the fe65 ptb2 domain mediated by the abl kinase stimulates its transactivational activity, possibly preventing the association of fe65 with dexras, a ras family gtpase, that acts as an inhibitor of the complex . The search for target genes regulated by the aicd has been complex and has yielded conflicting findings . It has been reported that the aicd / fe65 complex regulates the app expression itself, glycogen synthase kinase (gsk)-3 [63, 64], tip60, the -secretase (bace1), the primate - specific caspase 4, the a degrading enzyme neprylisin [61, 65, 66], the tetraspanin kai1 [26, 63], the lipoprotein receptor - related protein (lrp1), the epidermal growth factor receptor (egfr), and the tumor suppressor p53 . Nevertheless, many of these studies have been refuted by others, which using different strategies for modulating the aicd / fe65 complex did not produce changes in the expression of the aforementioned genes [6973]. The possible origin of the reported differences is unclear, but regarding the most intensively discussed target, neprilysin, recent data may shed light on the controversy . It was shown that the aicd - binding to neprilysin gene promoter is cell type - dependent [61, 74]. Furthermore, aicd - dependent gene regulation is influenced by the passage number and cell density, providing two likely experimental explanations for this disagreement . The majority of the evidence pointing to a role of aicd in transcriptional responses derives from the use of artificial reporter systems that in fact measure the release of components from the membrane, without monitoring endogenous transcriptional activity . Besides the potential participation of fe65 in promoting the expression of several genes described above, fe65 has been also proposed to perform other nuclear functions such as the repair of dna damage . Fe65 ko mice are more sensitive to dna damage, and this can be overcome by increasing the availability of nuclear fe65 . Moreover, genotoxic damage produces a rapid translocation of fe65 to the nuclear matrix and stimulates app processing by the -secretase complex and app phosphorylation in thr668, two mechanisms that allow translocation to the nucleus of the complexes associated with aicd . Fe65 is required for efficient repair of dna double strand breaks (dsb), a function that depends on its interaction with tip60 and aicd . The fe65-dependent recruitment of tip60 to dsb sites is essential because the histone acetyltransferase activity leads to chromatin opening at the injury site, enabling the access of the complexes involved in repair . On the other hand, tip60 acetylates and activates the ataxia telangiectasia mutated (atm) kinase which in turn phosphorylates a histone h2a variant, called h2ax, which acts as a mark for the recruitment of the reparation machinery . Changes in h2ax phosphorylation could be also dependent on the stability of p53 in a mechanism that requires the accumulation of fe65 in the nuclei [81, 82]. However, the fact that phosphorylated h2ax may be also increased in fe65 ko cells under genotoxic damage suggests that complementary mechanisms may regulate this behavior . Fe65 is highly enriched in the brain where it is expressed as two isoforms produced by the alternative splicing of a 6 bp miniexon . The isoform that includes this exon (which encodes arg - glu inserted in the ptb1 domain) is expressed exclusively in neurons, whereas the isoform lacking these two aminoacids is expressed only in nonneuronal cells . Fe65 protein expression may change during development and also in pathological conditions such as ad, opening up the possibility that it participates in plastic processes in neurons, which is reflected in the phenotype of fe65 and fe65l1 double ko mouse . These mice exhibit defects in the positioning of cortical neurons characterized by the presence of ectopic neurons that break the pialmeningeal basement membrane and displace cajal - retzius neurons and also have serious defects in axonal projections . Many of these phenotypical features are shared by mice lacking some of the fe65-binding partners such as the app family and the mammalian homolog of drosophila enabled (mena). Mena belongs to a family of proteins that regulate actin dynamics and thereby modulate cell motility and morphology . Mena is located in areas of dynamic actin remodeling such as lamellipodia and growth cones and interacts with the actin - binding protein, profilin . Mena interacts with the fe65 ww domain, assembling a macromolecular complex with app that regulates axonal branching, cell motility, and possibly the dynamics of actin at the growth cone and synapsis . In a previous attempt to generate a fe65 ko, it was expressed a truncated protein lacking the n - terminal domain and translated from met261 . This 60 kda variant does not contain the ww domain and does not display the transactivation activity of the larger isoform . In spite of the expression of this smaller protein, the animal shows defects in hippocampal - dependent learning and long - term potentiation (ltp) [93, 94]. However, it is difficult to assess whether these defects are due to the 97 kda isoform loss or the appearance of this new 60 kda isoform acting as a dominant negative protein . Although the amyloid cascade hypothesis has become the mainstream in the study of ad neurobiological mechanisms, several groups have recently suggested that this should be at least reevaluated in the light of new findings [9597]. Transgenic mice that overexpress the aicd and the adapter fe65 in the forebrain (under the control of the camkii promoter) display several neuropathological features observed in various transgenic models and in the ad patients brains, with the exception that they do not show a accumulation in the brain . The expression of aicd together with fe65 seems to be essential to induce an ad - like phenotype in the transgenic model, since a single aicd transgenic mouse developed by an independent group does not present the characteristics of the double transgenic, indicating that the functional relationship between both proteins, discussed in the previous sections, is indeed essential . As in the brain of patients with ad and several other transgenic models used to study ad, the aicd / fe65 mice show an increase in gsk-3 activity . Interestingly, the double aicd / fe65 transgenic does not affect the gsk-3 mrna or protein levels, as would be expected from a previous study which suggests that the kinase should be transcriptionally regulated by the aicd / fe65 complex . Kinase activation in the double transgenic is indeed correlated with an increase in the tyr216 activating phosphorylation and a decrease in the ser9 inhibitory phosphorylation . A molecular explanation for this may be related with the fact that fe65, through its ww domain, interacts and promotes gsk-3 phosphorylation on tyr216 . Increased gsk-3 activity in the aicd / fe65 mice produces hyperphosphorylation of two direct targets: the microtubule - binding proteins, collapsin - response mediator protein (crmp)-2 and tau [11, 98]. Increased crmp-2 phosphorylation is also found in transgenic mice expressing mutated forms of app and presenilin (ps)-1 and also in the cerebral cortex of ad patients . Increased crmp-2 phosphorylation is an early event that precedes the formation of amyloid plaques and neurofibrillary tangles . Interestingly, this posttranslational modification seems to be specific for ad, since it has not been reported in other neurodegenerative conditions like the frontotemporal dementia and pick's disease [100, 101]. Hyperphosphorylation of tau is the initial event in the pathway to tau self - aggregation, forming the paired helical filaments (phfs). Phfs are found at the core of the highly insoluble intraneuronal neurofibrillary tangles, one of the two neuropathological lesions (another is the senile plaques) that characterize the ad patients brains . The aicd / fe65 mouse shares with 3xtg mice the capacity to promote the formation of tau insoluble aggregates, which are not observed in most mouse models for ad . The aicd / fe65 double transgenic mouse has nonconvulsive seizures with aging, abnormal electroencephalogram (eeg) spiking, and a greater sensitivity to seizures induced by kainic acid (ka) in young animals . It also presents several alterations in hippocampal neural circuits, characterized by abnormal sprouting of the mossy fiber terminals with increased neuropeptide y (npy) expression and loss of calbindin - positive neurons . Alterations in the eegs and seizures have been observed in ad patients and in mouse models for this pathology, such as mice r1.40 (with appswe), appps1, and pdapp [105, 106]. Aged aicd / fe65 animals (> 18 months) show neurodegeneration in the ca3 hippocampal area, although the defects in working memory (evaluated by the y maze paradigm) start at a young age (8 months). Since most of the mouse models for ad are based on the expression of mutant variants of the human app or presenilin found in cases of familiar ad, the identity of neurotoxic app fragments has not been clearly discerned yet . Several studies have shown that a deposition in senile plaques does not correlate with neuronal death and cognitive deficits present in different transgenic models [107, 108]. For example, the overexpression of wild type happ in mice produces memory deficits, tau hyperphosphorylation, synaptic loss, and neurodegeneration without inducing an increase in a levels . Surprisingly, overexpression of happ together with -secretase in mice induces a decrease in a levels and plaque deposition, but the animals suffer severe neurodegenerative disorders and learning defects . In both models, an accumulation of c - terminal fragments of app including the aicd is it therefore possible that this fragment generated along with the a may be responsible for the alterations in transgenic models of ad? Interestingly, the ad model termed pdapp, when combined with a mutated form of the aicd (d664a), shows a complete reversion of the neuropathological hallmarks of the disease, including synaptic loss, the dentate gyrus atrophy, the astrogliosis, the deficits in synaptic transmission and memory, and the behavioral abnormalities without affecting the a levels or the plaque accumulation [111114]. These results strongly suggest that the causal relationship between the a accumulation and the neuropathological defects usually associated with ad may be challenged and position the aicd as a good candidate to explain the effects observed in various transgenic models based on mutations in app and ps1 . The two hallmarks of ad, the amyloid plaques, and neurofibrillary tangles, which are elegantly related through the amyloid cascade hypothesis, are the main components in the current research on the molecular mechanisms leading to this pathology . Since its origin, the amyloid cascade hypothesis has accumulated substantial evidence in its support, which has virtually overshadowed the fact that clinical trials based on this hypothesis have been shown to be unsuccessful . One of many possibilities to explain the failure of clinical trials could be related with the fact that several mouse models express the human - mutated app found in familial ad, so it is unclear which abnormalities detected in these models are product of specific a species (like oligomers) or another toxic metabolites of app (like aicd) or simply due to effects of overexpression of happ . However, the evidence collected from the transgenic models here reviewed could help to discern whether the a species or the aicd are the key elements triggering neurodegeneration . Three independent transgenic mice lines (a single transgenic of happ, a double aicd / fe65 transgenic, and the double happ/-secretase transgenic) recapitulate the neuropathological alterations of the disease without any increase in a secretion . All of these models have an accumulation of the app c - terminal fragments . Moreover, the introduction of a point mutation in the aicd in transgenic mice expressing the happ with the swe and indiana mutations, the ad - like phenotype is reversed, in spite of increased a production . All of these evidences suggest that the aicd could be acting as the bona fide toxic intermediate in the ad progression and could become a target for future therapeutic interventions against this devastating disease.
Its onset and progression cause deterioration of sensory, motor, and autonomic nerve functions, markedly reducing patients' quality of life (qol). The polyol pathway is a side pathway metabolizing excess (or unused) glucose to sorbitol . It is thus suggested that intracellular sorbitol production (which is increased by accelerated metabolism in the hyperglycemic condition) may trigger the development and progression of dpn . Aldose reductase (ar) is a rate - limiting enzyme that controls the polyol pathway . Ar inhibitors (aris), expected to ameliorate dpn, have been extensively developed . A promising ari zenarestat not only reduced sorbitol production and improved nerve conduction velocity (ncv), but also significantly increased myelinated nerve fiber density in the sural nerve via reducing the sorbitol concentration by 80% or more . It has already been demonstrated that ranirestat orally administered for 12 weeks significantly inhibited accumulation of sorbitol within the sural nerve: a dosage of 20 mg / day reduced accumulation by 83.5% . Furthermore, the 12-week treatment improved sensory ncv (the change from baseline reached 1 m / s). Even after an additional 48-week treatment, the improved sensory ncv was long maintained and associated with ameliorated peroneal motor ncv [3, 4]. On the basis of these results, we carried out the present clinical trial to explore the effectiveness and safety of ranirestat in japanese dpn patients . This study was a multicenter (20 sites in japan), double - blind, randomized, placebo - controlled study in which patients with dpn were assigned to either ranirestat 20 mg / day or placebo administered after breakfast as a once - daily dose for 26 weeks . The 20 mg / day dose was selected because it was associated with an 83.5% inhibition of sorbitol accumulation in the 12-week biopsy study . The following procedures were performed at entry for each patient: medical history, physical examinations, nerve conduction studies (ncs), and both the toronto clinical neuropathy score (tcns) and modified tcns (mtcns) [57]. Adverse events were recorded . At weeks 12 and 26, ncs, tcns, and mtcns were repeated . The primary end point was the summed change in sensory ncv from baseline of the bilateral sural and proximal median sensory nerves . Secondary end points were the changes for individual ncvs, amplitudes, minimum f - wave latencies (mfwl), tcns, and mtcns . We enrolled patients who met the following entry criteria: age 2070 years, either sex, type 1 or 2 diabetes for at least 6 months, glycemic control stable for at least 6 weeks before entry, and hba1c (7.4% but 11.5%) [8, 9]. Dpn was diagnosed when two of the following four modified san antonio criteria were present: (1) symptoms of dpn, (2) signs of dpn, (3) abnormal results of ncs with at least two abnormal nerves (meeting this criterion was mandatory), and (4) abnormal vibration perception threshold (<10 seconds using a 128 hz tuning fork). The requirement for both sural nerves potential amplitude responses of at least 1.0 v insured the presence of viable nerve fibers to allow accurate measurements and avoided inclusion of patients with severe neuropathy who would not be expected to respond . Since a sural nerve generally shows symmetrical responses, the difference in sural nerve potential amplitude and conduction velocity between the right and left legs should be limited (amplitude <6.0 v, ncv <7.0 m / s). Patients with nondiabetic neuropathy were excluded, as well as those with any clinically significant abnormal clinical laboratory parameter or any abnormal liver function test . The study was performed in accordance with the guidelines expressed in the declaration of helsinki . Testing was standardized for measurement of temperature, side of testing, stimulation protocol, averaging of sensory potentials, and measurement of latencies and amplitudes . Standardized techniques with temperature controlled and distal distances fixed were used for ncs . The minimum temperature was maintained at 31c in the forearm and 30c in the lower calf . If limb temperature was lower than specified, the limbs tested were warmed in a heating water bath before starting the test . It was recommended to warm cold legs in hot water at approximately 40c for at least 20 min before performing the test . Unilateral ncss were performed on the nondominant median motor, dominant tibial motor, and nondominant median sensory nerves . The fixed distal surface electrode distances for motor ncs were 60 mm for the median nerve and 80 mm for the tibial nerve . Corresponding distances for sensory ncs were 20 mm proximal to the distal wrist crease for the median nerve and 140 mm for the sural nerve . Measurements of distances, response latencies, and amplitudes were performed in a standard fashion using onset latencies and baseline - to - peak amplitudes . F - waves were generated for all motor nerves with 16 supramaximal stimuli per nerve, and the minimal reproducible latency of at least three responses was measured . The examiners had access to the previous temperatures, distances, and results through this trial . Results of the screening ncs for each patient were reviewed and the eligibility of each patient was decided by the nerve conduction study assessment committee before randomization . This central supervision ensured consistency of study procedures and high quality of data under blinding . The tcns has been modified to better capture sensory test results reflecting early dysfunction in dpn, also to improve the sensitivity and specificity of the original tcns [6, 7]. The mtcns includes a symptom domain and a sensory test domain . In the symptom domain, pain, numbness, tingling, weakness, and ataxia in foot and upper limb is separated into 4 stages: 0 = absent, 1 = present but not interfering with the sense of well - being or activities of daily living, 2 = present and interfering with the sense of well - being but not with activities of daily living, and 3 = present and interfering with both the sense of well - being and activities of daily living . In the sensory test domain, pinprick, temperature, light touch, vibration, and position sense were assessed as 0 = normal, 1 = reduced at the toes only, 2 = reduced to a level above the toes, but only up to the ankles, and 3 = reduced to a level above the ankles and/or absent at the toes . The mtcns scale varies from 0 (no signs or no symptoms of dpn) to 33 (all symptoms and signs of dpn present with a maximum score of 18 symptom points and 15 sensory test points). The full analysis set was used in the efficacy analysis and included all randomized patients but excluded those receiving no investigational drug and those with no efficacy data (figure 1). Changes from baseline to the last observation in efficacy variables were compared between the treatment groups . The last observation was recorded at week 26 . When no data were available at week 26, the data were compared between groups by analysis of covariance using group as a factor and baseline values as a covariate . The changes were determined by group at each visit for which summary statistics were calculated and plotted against visit . Within - group differences were tested using the paired student t - test by group and visit . For binary data, the number and percentage were determined by group and visit and compared between the groups using the fisher exact test . For ordinal data, the number and percentage were determined by group and visit and compared between the groups using the mantel test . We screened 130 patients and excluded 57 patients for not meeting the inclusion criteria or meeting the exclusion criteria at screening (n = 54) and for withdrawing their consent prior to randomization (n = 3). Seventy - three patients were randomized to either ranirestat or placebo (40: 33), and all 73 received an investigational drug (figure 1). Some differences in hba1c between the ranirestat and placebo groups were observed at baseline . Because the magnitude of change in the individual ncv varied, the sensory ncvs were summed to comprehensively evaluate each sensory nerve's function . The summed sensory ncv (primary endpoint) was the sum of the ncv in the bilateral sural sensory nerves and proximal median sensory nerves . Distal median sensory ncv was not included in the summed sensory ncv in order to avoid the possible influence of carpal tunnel syndrome . The change from baseline to the last observation was 7.28 1.27 m / s (least squares mean [lsm] se) in the ranirestat group and 1.92 1.39 m / s in the placebo group (table 3). Analysis of covariance of the changes in the summed sensory ncv at the last observation using drug group as a factor and the summed sensory ncv at baseline as a covariate detected a significant improvement in the ranirestat group compared with the placebo group (p = 0.006). In order to investigate how the imbalance of baseline hba1c between the two groups influences the results, analysis of covariance (ancova) was conducted to assess change in summed sensory ncv from the baseline to the last observation, controlling for hba1c by adding baseline hba1c as a covariate, in reference to the ich e9 guideline . The changes in summed sensory ncv were 7.54 1.29 m / s in the ranirestat group and 1.60 1.42 m / s in the placebo group, indicating significant difference between the two groups (p = 0.003). There was no significant effect of baseline hba1c because the changes before and after adding the covariate of baseline hba1c were similar . Table 3 also shows that the improvement of ncv from baseline to the last observation in the individual nerves was consistently significant in the ranirestat group (p <0.0010.030) for all except the median motor ncv . The between - group differences in proximal median sensory ncv were significant (p = 0.019). There was a tendency of significant between - group difference in median motor ncv (p = 0.051). Analysis of covariance of the change at the last observation detected a significant difference for the proximal (p = 0.026) and distal (p = 0.019) median motor nerves between the ranirestat group and the placebo group . The improvement in the tibial motor mfwl was significant (50.12 4.69 msec versus 50.98 5.28 msec, p = 0.007). However, analysis of covariance for each nerve detected no significant improvement for the ranirestat group compared to the placebo group . The total score improved at 12 weeks in the two groups, and no between - group difference was observed in the change from baseline at the final evaluation . By domain, similar time - course changes were seen in the symptom domain in the two groups, while ranirestat change tended to increase at the final evaluation in the sensory test domain . An additional analysis in a subgroup of patients with mild to severe neuropathy according to the tcns severity classification revealed significant improvements in the sensory test domain in the ranirestat group (p = 0.037), although there was no between - group difference in total score change at the final evaluation . The prevalence of adverse events was similar in both groups: 33 of 40 patients (82.5%) in the ranirestat group and 29 of 33 (87.9%) in the placebo group . In the ranirestat group, 3 serious adverse events (appendicitis perforated, peritonitis, and spinal compression fracture) were noted, but all were judged by the investigator to be unrelated to ranirestat (table 5). This clinical trial has demonstrated that oral administration of ranirestat at 20 mg / day for 26 weeks, as compared with placebo, significantly improved the primary endpoint of summed sensory ncv: summed sensory ncvs in the ranirestat group increased after 12-week treatment and were significantly higher than that in the placebo group at the final evaluation (p = 0.006). In a proof - of - concept study conducted in north america, 12-week treatment with ranirestat 20 mg / day reduced the sorbitol concentration in the sural nerve by more than 80% and ameliorated sensory nerve ncv . These study results were reproduced in the present study carried out in japanese patients with dpn . Blood glucose control is critical for the treatment of dpn, as indicated by the results of a large - scale clinical trial . The blood glucose control status may affect the results of our study . Since an imbalance in hba1c was found at baseline in the present study, we investigated whether the blood glucose control status at baseline affected the study results . Using baseline hba1c as another covariate on the basis of the ich e9 guideline, we performed an additional analysis of summed sensory ncv and found the significance of differences between the ranirestat and placebo groups had remained unchanged (p = 0.0034): the robustness of our results was thereby confirmed . The changes from baseline to last observation in hba1c were + 0.26% in the ranirestat group versus + 0.07% in the placebo group; there were no significant changes from baseline in the two groups . Furthermore, we performed a subgroup analysis of summed sensory ncv, via dividing participants into well - controlled (improved or unchanged hba1c [hba1c 0%]) and poorly controlled (deteriorated hba1c [hba1c> 0%]). The change from baseline in summed sensory ncv was 9.4 m / s for ranirestat (n = 23) versus 4.1 m / s for placebo (n = 12) in well - controlled participants and 3.8 m / s for ranirestat (n = 14) versus 0.5 m / s for placebo (n = 19) in poorly controlled participants . In either subgroup, ranirestat produced greater changes, indicating that difference in blood glucose control during the study period had no effect on the results or conclusions in this study . Thus, we consider that baseline hba1c imbalance has no relevant effect on study conclusions . Nevertheless, as the present study is a small - scale trial with limited subgroup analysis, a further study involving a larger number of participants is desired for a valid conclusion . Dpn is a systemic neuropathy that damages both the sensory and motor nerves as well as both the upper and lower limbs . The landmark diabetes control and complication trial followed up patients receiving intensive treatment and those receiving conventional treatment for 5 years and reported that ncv at 5 years was lower by more than 1 m / s in the conventional treatment group than the intensive treatment group . In the present study, we examined and evaluated a sensory nerve and a motor nerve in both the upper and lower limbs . Compared with placebo, ranirestat significantly increased proximal - median sensory ncv (p = 0.019). These findings imply that the effect of ranirestat is not limited to a particular nerve but extends to all peripheral nerves . In this study, ncv of each nerve tested was higher by 0.83 to 2.17 m / s in the ranirestat group than in the placebo group, indicating that ranirestat and strict control of blood glucose play equally potent roles in maintaining nerve function . A clinical trial using median motor ncv (mmncv) as a parameter for long - term treatment with epalrestat (the only ari in clinical use) reported that epalrestat significantly reduced mmncv deterioration by 0.78 m / s at one year, by 1.21 m / s at 2 years, and by 1.60 m / s at 3 years as compared with the control . In this study, the difference in mmncv between the ranirestat and placebo groups was 1.06 m / s (p = 0.051). In regard to ncv, these findings indicate that ranirestat can be expected to exert an effect as potent as the existing therapeutic epalrestat . In parallel to the present trial, a phase iii clinical trial of ranirestat was carried out in north america . Reported that summed motor ncv was significantly improved by ranirestat, but summed sensory ncv was not . On the other hand, in our study, summed sensory ncv was significantly improved, whereas motor ncv was not significantly changed, although there was a tendency of significant between - group difference in median motor ncv (p = 0.051). It is difficult to clearly elucidate the reason for the different results between two trials . The number of participants in their trial was more than three times as large as ours . It may be one of reasons that summed motor ncv was significantly improved in their trial . As for summed sensory ncv, measurement of sensory ncv using surface electrodes is affected by a variety of conditions such as skin temperature and measurement site condition, because the amplitude of sensory nerve action potential (measured in microvolts) is markedly lower than that of compound muscle action potential (measured in millivolts). To overcome these difficulties, measurement in ncs was performed more precisely by standardization of measuring methods, use of common procedures, and intensive evaluation in the core laboratory to increase data reproducibility in the phase iii trial and our trial . However, a large difference in demographic characteristics of patients such as bmi might partially affect condition of measurement sites such as subcutaneous tissue; bmi (mean sd) was 25.0 3.5 in our study versus 33.1 6.8 in the north america study . Dpn is a nerve - degenerative disease that progresses slowly and is characterized by a variety of clinical manifestations including subjective symptoms (such as spontaneous pain; positive symptoms) and sensory deterioration (negative symptoms) associated with progression of nerve destruction . In this study, these various clinical symptoms were evaluated with the use of an mtcns (with symptom domain dedicated to positive symptoms and sensory test domain dedicated to negative symptoms). Both the original mtcns and japanese version are recognized as valid and reliable evaluation tools [6, 7]. In this study, the total score of mtcns improved at 12 weeks in both groups and no between - group difference in change from baseline was found at the final evaluation: no obvious effect of ranirestat was observed on clinical symptoms . Since the mtcns used in this study is based on the tcns, all patients were divided into two subgroups, based on tcns severity . One subgroup of patients with tcns total score 5 (9 in the ranirestat group and 4 in the placebo group) was excluded in order to perform an additional analysis . In the other subgroup of patients with tcns total score 6, ranirestat elicited significant improvement in the sensory test domain, as compared with placebo (p = 0.037). As sensory test results have been reported to well correlate with risk of foot ulcers, improvement in negative signs is important for preventing foot ulcers and avoiding limb amputation, which are targets of dpn treatment . Nevertheless, the efficacy of ranirestat on clinical symptoms remains to be elucidated, probably because this trial was limited by short treatment duration and presence of a placebo effect . Evaluation using mtcns in the phase iii trial of ranirestat in north america also demonstrated that mtcns scores were improved in the all groups including placebo at 12 weeks and no efficacy of ranirestat was detected at 52 weeks . Placebo effects were also noted in the recent phase ii / iii studies of ranirestat with 2-year treatment duration (in asia, europe, north america, and russia) and resulted in a failure to demonstrate significant efficacy on clinical symptoms . For evaluation of clinical symptoms of dpn, different scales have been used in different clinical trials . Placebo effects have been observed in multiple trials, perhaps not only in trials using the mtcns [16, 17]. In our and other studies, blood glucose was relatively well controlled and maintained, which might be attributable to lack of deterioration in the placebo group . Because dpn is slowly progressive, it may be necessary to design a study with longer duration of more than 2 years and with a more sensitive tool for assessing or detecting clinical symptoms . Regarding the adverse events in this study, there was no particular difference between the two groups; adverse effects on hepatic and/or renal function associated with use of other drugs were also undetectable . These findings assure the safety of the 26-week treatment with ranirestat at a daily dose of 20 mg . As compared with placebo, ranirestat administered to japanese dpn patients at a once - daily dose of 20 mg for 26 weeks significantly improved summed sensory ncv . A subgroup analysis revealed that treatment with ranirestat led to significant improvement in clinical signs (i.e., increased the sensory test domain score of the mtcns). However, this study aiming at proof - of - concept was limited by its short - term treatment duration and small number of patients . Further studies are needed to establish the efficacy of ranirestat in the treatment of dpn.
Acute acalculous cholecystitis (aac) is defined as acute inflammation of the gallbladder in the absence of gallstones and has a multifactorial pathogenesis.1 aac occurs in about 10% of all cases of acute cholecystitis . Aac has numerous causes that produce bile stasis and ischemia leading to inflammation and infection of the gallbladder . Aac tends to have a more fulminant course, is frequently associated with gangrene, perforation and empyema, and has high morbidity and mortality.2 aac has traditionally been recognized to occur in patients with serious co - morbid illnesses especially after a major operation, severe trauma, burns, systemic sepsis and prolonged intravenous hyperalimentation.3 however, there has recently been an increasing number of reports in the literature of the occurrence of aac in patients with none of the established risk factors.4 - 6 aac arising as a complication of laparoscopic appendectomy has been reported in just 1 case and it was treated conservatively.7 herein, we present the case of a 38-year - old woman who developed aac after laparoscopic appendectomy . A 38-year - old woman who was previously in good health came to our emergency room 3 days after she received laparoscopic appendectomy at a local clinic . She had acute abdominal pain which initially occurred in the epigastric and umbilical area and then migrated to the right lower quadrant after 10 hours and was associated with nausea and anorexia . Acute appendicitis had been diagnosed by abdominal computed tomography (ct) and operative findings at another hospital . Her abdominal pain and associated symptoms improved after surgery . Body temperature was 37.8. she exhibited tenderness and guarding in the epigastric region and right upper quadrant . Results of the laboratory studies were unremarkable, except for elevated c - reactive protein (crp) which was 8.04 mg / dl (normal range less than 0.5 mg / dl). Her body mass index (bmi) was 21.7 (height: 168 cm, weight: 61.2 kg). Abdominal ct showed a thickened, contrast - enhanced wall of the gallbladder (fig . Her abdominal pain subsided and crp decreased to 1.33 mg / dl after the ptgbd . The patient's postoperative progress was uneventful, and she was discharged 5 days after surgery . Aac generally occurs in patients after major surgery, in the presence of critical illnesses such as trauma, burns, and sepsis, and in the elderly.1 however there have been recent reports of acalculous cholecystitis in young healthy patients who had none of the established risk factors.6,8 the patient described in this case report was young and healthy and exhibited none of the standard risk factors other than recent surgery, although laparoscopic appendectomy is not considered to be a major operation . The commonest postulated pathogenesis of aac is bile stasis resulting in a change of bile composition and ischemia.2 factors known to contribute to bile stasis in the postoperative patient are fasting, anesthesia, dehydration, fever, and narcotics for the relief of pain.9 i believe that the most probable mechanism of aac in this woman was bile stasis that resulted from the anesthesia given to her during the appendectomy and the narcotics given for the relief of pain . Aac is difficult to diagnose because clinical signs such as abdominal pain and fever, and laboratory test results are non - specific . Delayed diagnosis of aac is associated with high morbidity and mortality due to the high prevalence of gangrene and perforation.10 early diagnosis and appropriate treatment can improve outcomes in patients with aac.11 the treatment options for aac are cholecystostomy and/or cholecystectomy.9,12 cholecystectomy generally is considered the definitive therapy, and percutaneous cholecystostomy can be performed safely and rapidly.9 standard treatment methods of aac have not yet been established, but it is usually determined by the patient's condition . I believe that early cholecystectomy in this case was an effective treatment tool for avoiding potential failure of conservative management and for preventing recurrence, although the patient was young and had none of the risk factors . As mentioned above, laparoscopic appendectomy may mask clinical signs and symptoms of aac . Therefore, when right upper quadrant pain, fever, leukocytosis, and abnormal liver function tests are observed after surgery, physicians should consider the possibility of aac, and promptly check radiological findings . Aac arising as a complication of laparoscopic appendectomy has been reported rarely, especially in young healthy patients . When abdominal pain, leukocytosis, fever and abnormal results of liver function tests are observed in patients after appendectomy, the possibility of aac should be considered . Prompt recognition and appropriate treatment of aac is necessary to minimize the associated morbidity and mortality.

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