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pressure is a fundamental thermodynamic variable that can modulate protein structure, dynamics, and function. the effect of hydrostatic pressure on protein stability has been studied extensively, including many studies that have explored how protein denaturation occurs under high - pressure conditions. an ellipse - shaped zone of stability in the temperature pressure plane has been proposed on the basis of theoretical models, molecular dynamics (md) simulations, and experiments. in addition to studying the stability of proteins in equilibrium under high - pressure conditions, pressure perturbation can also be used to study protein folding kinetics. microsecond pressure - jump (p - jump) techniques have recently extended the range of such experiments to near the folding speed limit. here we ask whether such fast protein refolding from the pressure - denatured state can be seen from beginning to end in a full - atom explicit solvent md simulation, and whether the speed limit from the pressure - denatured state has the same time scale as the 1 s speed limit observed in temperature - jump (t - jump) experiments and single - molecule experiments., is slower than backbone torsional transitions or end - to - end contacts of short loops, and attributed to either reduced diffusion on a rough energy landscape, or equivalently to extremely short - lived high - free - energy folding intermediates. md simulation has been used for studying the effect of pressure on protein thermodynamics and protein denaturation, but kinetic refolding and the molecular time scale have proved elusive so far. recent developments in both hardware and software have pushed md simulations up to the millisecond time domain. at the same time, a new generation of microsecond kinetics p - jump experiments can access the md time scale, and would benefit greatly from interpretation based on the atomistic detail available from simulations. so far simulations have only observed the trapping process associated with non - native helix formation after a p - jump, and it remains to be seen whether the burst phase observed in experiments translates into fast and complete refolding in silico. we previously applied the ultrafast p - jump technology to study the refolding kinetics of an alanine - rich mutant of five - helix bundle -repressor, ya. in addition to a 1 ms. after 18.6 s of conformational search, the simulation revealed a 0.9 s stretch of productive structural assembly, bracketed by three - helix alignment and loop formation motions that were almost, but not quite, concerted. this 0.9 s time scale agrees with the molecular time scale of 12 s measured by t - jump experiments on near - downhill folding -repressor mutants. yg consists of the wild - type -repressor sequence residues 685, with mutations tyr22trp, gln33tyr, ala37gly, and ala49gly. it has a melting temperature of tm = 55 c and a folding time constant of 22 s near tm in aqueous buffer. for md simulations the sequence was used without c - terminal amidation or n - terminal acetylation. the initial structure of the -repressor fragment was taken from the protein data bank (pdb code 1lmb). from a crystal structure of the similar ya mutant, we know that the mutant and wild - type native structures are quite similar. for p - jump experiments, the protein was grown in e. coli and purified and lyophilized as described previously. md simulations were performed in explicit solvent using the tip3p water model and the charmm22 force field with cmap corrections for protein and ions. the force field has excess helix propensity, which may accelerate trapping (from non - native helix) and folding (from native helix). the initial protein structure was placed in a cubic box of 24 282 water molecules at 55 mm nacl salinity, neutralized with extra ions employing vmd. the simulated system, including protein, water molecules, and ions, measured 91.1 in each dimension at t = 325 k and p = 1 bar and contained 74 197 atoms. all simulations were carried out with periodic boundary conditions in a constant particle number, temperature, and pressure ensemble (npt), in five steps. step (1) : in a p - jump simulation, pressure was increased from 1 bar to 5 kbar in 0.15 s at a rate of 1 bar/30 ps while maintaining the temperature at t = 325 k. step (2) : in a high - temperature and high - pressure unfolding simulation, temperature was increased to 525 k while maintaining pressure at p = 5 kbar, running the simulation for 0.15 s. step (3) : after the protein unfolded in step (2), the temperature was dropped back to 325 k and pressure was kept at p = 5 kbar while the denatured protein was equilibrated at high pressure for 1 s in a high - pressure equilibrium simulation. step (4) : in a pressure - drop simulation, pressure was jumped downward from 5 kbar to 1 bar in 0.15 s at a rate of 1 bar/30 ps while maintaining the temperature at t = 325 k. steps (1)(4) were carried out on general purpose supercomputers using namd 2.9. step (5) : the resulting pressure - denatured state under refolding conditions was employed as the initial state for a refolding simulation, carried out on the special purpose supercomputer anton for 32 s. constant temperature (t = 325 k) and constant pressure (p = 1 bar) were maintained during the refolding simulation. the simulation algorithm and features of the namd program are described in ref (41). the system to be simulated was first subjected to 6000 steps of conjugate gradient minimization and equilibrated for 300 ps with harmonic restraints applied to all the heavy atoms of the protein. the simulation was then continued for 3 ns without restraints at a constant pressure of 1 bar using nos hoover langevin piston barostat and at a constant temperature of 325 k with langevin damping constant of 5.0 ps. in the subsequent simulations of steps (1)(4), constant temperature was maintained using langevin dynamics with a damping constant of 1.0 ps and multiple time stepping employed with an integration time step of 2.0 fs, short - range forces being evaluated every time step and long - range electrostatics evaluated every three time steps. cutoff for short - range nonbonded interactions was 8.0 ; long - range electrostatics was calculated using the particle - mesh ewald method. all bonds involving hydrogen in the protein were constrained using rattle, while the geometries of water molecules were maintained using settle. the refolding simulation in step (5) was carried out on the anton platform. multiple time stepping was employed, with an integration time step of 2.0 fs. short - range forces were evaluated every time step and long - range electrostatics every three time steps. cutoff for the short - range nonbonded interactions was 9.28 ; long - range electrostatics was calculated using the k - gaussian split ewald method with a 64 64 64 grid. refolding kinetics experiments were performed on a home - built p - jump apparatus as described previously. briefly, an 810 l dimple was machined into a sapphire cube with a side length of 3/8-in. the sample consisting of 300 m protein in 50 mm phosphate buffer at ph 7 with either 0 or 1 m guanidine hydrochloride (guhcl) was then pipetted into the dimple and sealed with a double - layer of mylar - coated aluminum foil to prevent mixing between the sample and the pressurization fluid (water). stainless steel burst membrane, to which it was connected by a pressurization channel. the sample and burst membrane were pressurized hydrostatically to 1.2 kbar using a pressure pump (high pressure equipment company, erie, pa). the burst membrane was ruptured by passing 10 ka of current (95 v) through it, releasing the sample pressure back to 1 bar within 23 s. the sample was optically excited with a frequency - tripled ti : sapphire laser (kmlabs, boulder, co), which generated femtosecond pulses of 285 3 nm light separated by 12.5 ns. fluorescence was collected and the photons were delivered to a photomultiplier (r7400u-03, hamamatsu corp., bridgewater, nj) using an optical waveguide (oriel instruments, stratford, ct). we used a band - pass filter (b370, hoya, santa clara, ca) to avoid interference from the excitation light. the signal was recorded and digitized at 100 ps time resolution using an oscilloscope with a 2.5 ghz bandwidth (dpo7254, tektronix, beaverton, or). we chose -repressor mutant yg (y22w / q33y / a37g / a49 g) as a model system to study complete fast protein refolding after a pressure drop. it is the largest fast - folding protein folded in silico to date by all - atom md simulations. fast folding of various -repressor mutants has been studied previously using temperature - jump, pressure - jump, and rapid microfluidic mixing techniques. in order to later compare with the denatured simulation and refolding simulation, we first performed a 0.3 s md simulation of yg at t = 325 k and p = 1 bar, starting from the crystal structure. this 0.3 s simulation will be referred to hereafter as the native simulation. the average values of several structural characteristics, such as radius of gyration (rgyr), were determined from the native simulation and defined as the protein s native values, shown as red solid lines in figure 1. structural characterization of the yg unfolding trajectory. -content is the fraction of residues that are in the -helical conformation, and rgyr is the radius of gyration. the native values, calculated from a 0.3 s equilibrium simulation of the native structure at t = 325 k and p = 1 bar, are shown as red solid lines. the pressure applied through the simulation, shown as the color background, varies from 1 bar (white) to 5 kbar (blue). the temperature is kept at 325 k, except for the time window between 0.15 and 0.3 s, where 525 k is used to unfold the protein. protein coloring runs blue to red from the n - terminus to the c - terminus. the denaturation simulation followed a procedure described in a previous p - jump md simulation study that did not observe refolding of the ya mutant. briefly, we started with the native state of yg, shown in the t = 0 s conformation in figure 1. the pressure was gradually increased from 1 bar to 5 kbar over 0.15 s, while temperature was held constant at t = 325 k. in figure 1, the value of the pressure is depicted by the background color changes from white (1 bar) to blue (5 kbar). the protein remains in its native conformation in the first 0.15 s of upward p - jump simulation. high pressure can unfold a protein, but such high - pressure denaturation is a slow process that takes place on a time scale of seconds or even longer. therefore, 0.15 s of pressurizing is too short for observing any discernible conformational change. to accelerate the protein unfolding process, we heated the system to t = 525 k and simulated the system for another 0.15 s, while keeping pressure at p = 5 kbar. as shown in figure 1, the protein rapidly unfolds as evidenced by the increase of c-root - mean - squared deviation (rmsd) relative to the crystal structure (> 20). the content of secondary structure, -helix in particular, drops from the native value of 65.5% to a value in the 1030% range. during the unfolding, the protein also assumes some extended conformations with rgyr of more than 30. the high - t - p denatured state, obtained after the high - temperature and high - pressure unfolding simulation, is shown in figure 1 as the conformation at t = 0.3 s. the high temperature used in the simulation unfolds the protein, but also likely disrupts the protein more than when only high pressure is used for denaturation. to obtain a state more representative of the pressure - denatured ensemble, the high - t - p denatured state was equilibrated for 1 s at p = 5 kbar and t = 325 k. the most striking observation in the equilibration, shown in figure 1, is that the -helix content recovers from 30.0% to 60.0%, which is already close to the native value of 65.5%. the existence of high -helix content at high pressure indicates that pressure denaturation is mainly breaking the tertiary contacts, but does not perturb the secondary structure considerably, as proposed previously. the result is consistent with the finding that pressure does not affect the helix coil equilibrium significantly, based on replica exchange md simulations of -helical peptide using a different force field. recent experiments by neumaier. have shown that high pressure can slightly stabilize a helix, which explains the frequently observed helical structures in pressure - denatured proteins. notably, the pressure - denatured state after the high - pressure equilibration, shown in figure 1 as the conformation at t = 1.3 s, already contains helices 1 and 4. we performed a 0.15 s downward p - jump simulation that initiated the refolding process. significant amount of helical structure, resulting from the high - pressure equilibration, did not change substantially during the downward jump. the entire 1.45 s denaturation simulation and downward p - jump simulation rendered as one trajectory are shown in movie s1 in the supporting information (si). the time evolution of the c-displacement per residue relative to the crystal structure and the secondary structure per residue are shown in figure s1. following the downward p - jump simulation, we carried out a 32 s md simulation at p = 1 bar and t = 325 k to investigate fast refolding. the protein, except for the last helix (helix 5), folded into the native state after 19 s. the c-rmsd of the protein relative to the crystal structure (pdb code 3kz3) is shown in figure 2b, and the secondary structure is shown in figure 2c ; see figure s2 for additional quantities of interest). a particular mechanism by which yg mutant folds in our trajectory is punctuated by two fast events separated by a longer conformational search, as shown in figure 2d. protein refolding trajectory from the simulation at t = 325 k and p = 1 bar after pressure jump. (a) distinct molecular rearrangements observed at the bottleneck (transition - state passage). at each time point, the folded residues (c displacement relative to the crystal structure 2) are colored blue. (b) c-rmsd values for the protein core (residues 780), calculated relative to the crystal structure 3kz3. the native range is defined by the mean value (red solid line) standard deviation (green dashed line) from a 0.3 s equilibrium simulation of the native structure at t = 325 k and p = 1 bar. (c) time evolution of the secondary structure throughout the trajectory. the secondary structure of the crystal structure is shown on the left side of the panel. (d) time evolution of per - residue c displacements from the crystal structure throughout the refolding trajectory. (e) the time window between 18 and 20 s is enlarged to reveal the sequence of rearrangements at the bottleneck (see movie s3 in si). the color bar runs from blue (close to crystal structure) to red (far from crystal structure). in a first fast step within 2 s of the p - jump, helices 1 (residues 727) and 4 (residues 5870) adjusted their orientation and registration to reach a near - native conformation. subsequently, a number of factors prolonged the conformational search : helix 3 did not form individually until 7 s (see figure 2c), and was originally shifted toward the c - terminus by one full helical turn ; neither helix 2 nor helix 3 acquired a native orientation ; helical overshoots were observed both in helices 1 and 2, where they eroded loop 1 between the two helices as shown in figure 2c, preventing the correct helix orientation from locking in. the key bottleneck was crossed in the second fast step between 18.6 and 19.5 s (figure 2e). in 0.9 s, three key rearrangements brought the protein from a compact denatured state through the transition - state region into the native basin (figure 2a). first, between 18.6 and 18.8 s, helices 2 and 3, along with loop 2 that connects them, reoriented themselves and assumed the native packing conformation relative to helices 1 and 4. the one - helical - turn shift in helix 3 and the helical overshoot in loop 1 disappeared during this structural transition. within the next 0.4 s, loop 1 assumed its native conformation, finally, loop 3 between helices 3 and 4 rearranged into its native structure at t = 19.5 s. the whole refolding trajectory is visualized in movie s2 in the si. the trajectory for the time window between 18 and 20 s is provided in movie s3 in the si. the formation of helix 5 is presumably the last step of the folding process ; this step is not observed in the simulation. it is possible that the yg in solution adopts a native state that is different from the crystal structure. indeed, an unstable helix 5 in the native state is consistent with the high b factors in the crystal structure and observed in implicit solvent md simulations. an unstructured helix 5 has also been observed before for another -repressor mutant (d14a) and attributed to the absence of c - terminus residues from the wild - type -repressor. previous fast p - jump experiments up to 0.5 ms on yg revealed a microsecond burst phase. we recently extended the capabilities of our p - jump apparatus to collect refolding kinetic data for up to 5 ms. we used this new home - built instrument to jump the pressure of the sample from 1.2 kbar to 1 bar (see materials and methods). the tryptophan in position 22 (trp22) was used as a fluorescence probe to study folding of yg after a microsecond pressure drop. tyrosine in position 33 (tyr33) was introduced to quench trp22 fluorescence in the folded state, enhancing the change in fluorescence lifetime upon unfolding. titration of yg with guhcl shows an unfolding midpoint concentration of 1.3 m (figure s3). equilibrium pressure denaturation of yg was monitored by fluorescence spectroscopy (figure s4). based on these results, we expect no pressure denaturation of yg in 0 m guhcl up to 1.2 kbar, whereas in 1 m guhcl, the protein is poised for unfolding when the pressure is increased above 1 bar. for the p - jump experiments, trp fluorescence excited at 285 nm was sampled every 12.5 ns and digitized with a time resolution of 100 ps. the fluorescence lifetimes for nata were then normalized from = 0 (before the p - jump) to = 1 (4.7 ms after the p - jump) through a linear fitting procedure, and the 0 and 1 m protein samples were analyzed on the same scale for comparison. the dead time of the instrument with a starting pressure of 1.2 kbar was determined from the nata sample, fitting its step - function - like trace to a single exponential rise of 3 s. in general, variants with a q33y mutation exhibit an increase in fluorescence lifetime upon unfolding whether the denaturation is accomplished using temperature, pressure, or a chemical denaturant. this response can be rationalized in terms of nonradiative quenching of the tryptophan fluorescence by tyrosine in the folded state. figure 3 shows the fluorescence - detected kinetics of nata, yg without guhcl, and yg in 1 m guhcl from 1.2 kbar to 1 bar. yg without guhcl data is a folded control because yg without guhcl does not undergo pressure denaturation at 1.2 kbar. the initial lifetime increase for yg in 1 m guhcl at t = 0 is a factor of 1.4 larger than that observed for yg without guhcl. this observation indicates that there is a fast burst phase, 1 ms slow phase. one of our long simulations (ya) got trapped for the duration of the simulation, whereas the other (yg) folded rapidly. we thus propose that prompt folding is roughly as likely as kinetic trapping in both the p - jump experiments and simulations. if so, this raises the same question raised by lapidus and co - workers in their microfluidics experiment and by pande from simulations : are there very slowly interconverting denatured states ? for example, a kinetic scheme such aswith u and t initially populated, can explain how some proteins (initially in u) reach the native state rapidly whereas others (initially in t) reach it slowly. if u and t bracket the fluorescence lifetime of n, it is possible to observe microsecond and millisecond phases of opposite sign (figure 3). it is well documented that pressure unfolding can be very slow due to the positive activation volumes, and that pressure denaturation forms more compact denatured states than temperature denaturation. compact states that fold more slowly than highly extended denatured states have been observed. for example, trpzip2 has a heterogeneous denatured population, whose subpopulation with blue - shifted tryptophan fluorescence (less solvent exposed tryptophan) folds more slowly to the native state than the subpopulation with red - shifted tryptophan fluorescence (more solvent exposed tryptophan). thus it remains unproven, but fully consistent with our data and simulations, that the pressure - denatured state of -repressor contains slowly interconverting compact traps. finally, we investigated what hinders the formation of helix 5 in our simulation. in the native state, helix 5 is stabilized by interacting with helix 4 through a small hydrophobic patch. formation of this patch is prevented in our simulation by several non - native salt bridges (figure 4). since helix 5 has weak helix propensity and needs to form in concert with its tertiary contact with helix 4, the non - native salt bridges that keep the c - terminus away from helix 4 hinder the formation in helix 5. one should not conclude that the non - native salt bridges hinder overall folding. they form within the first 2 s after the p - jump and remain stable for most of the trajectory (figure 4). they may play a crucial role in bringing the n- and c - terminus together, facilitating the formation of the main hydrophobic core that involves helices 1 to 4. particularly the bridges between helix 1 and the c - terminus (glu13-arg85 and arg17-glu83), may accelerate the folding reaction in its early stage, but slow it down toward the end. in that regard it is noteworthy that blue1, a two - helix bundle containing only helices 1 and 4, folds rapidly (the conformational search of helices 2 and 3 is eliminated), but truncating helix 5 in the full - length protein (results) produces a highly unstable protein with low expression level and missing secondary structure. non - native salt bridges in the refolding trajectory, monitored by the distance between oxygen bridges. the pressure jump simulation reveals key steps in moving the protein across the transition state during folding. first of all, limited computational resources only allow the production of a single or few trajectories, preventing one from drawing a statistical conclusion, as seen in the case of the molecular rate, folding rate, or the structural heterogeneity of the transition - state ensemble. second, a bias toward a certain kind of secondary structure has been observed in the current generation of force fields. although it has been shown that this force field has a helical bias, we used it successfully to fold a fast folding mutant of -repressor (hg), and it seems to explain qualitatively both trapping and prompt refolding after a p - jump (this work). the high temperature (325 k) used in the current study has a destabilizing effect on the helical structure, which can help in balancing the helical bias to some extent. however, the development of force fields that reproduce melting temperatures and hence denatured ensembles of proteins better remains an important goal. third, a chemical denaturant (guhcl) was used in the experiment to help unfold the protein at just 1.2 kbar. the simulations, on the other hand, utilized high temperature to accelerate the unfolding process. such differences between experiment and simulation can not yet be avoided altogether, but should be minimized as progress is made in both areas.. however, unfolding simulations are widely performed at very high temperature (and pressure in our case) to generate the denatured state for refolding simulation. since the interatomic interaction strengths are much less sensitive to pressure changes than temperature changes in the ranges chosen for our simulations, we expect that high - pressure simulation at less severe temperatures could yield useful initial states for refolding. | density is an easily adjusted variable in molecular dynamics (md) simulations. thus, pressure - jump (p - jump)-induced protein refolding, if it could be made fast enough, would be ideally suited for comparison with md. although pressure denaturation perturbs secondary structure less than temperature denaturation, protein refolding after a fast p - jump is not necessarily faster than that after a temperature jump. recent p - jump refolding experiments on the helix bundle -repressor have shown evidence of a <3 s burst phase, but also of a 1.5 ms slow phase of refolding, attributed to non - native helical structure frustrating microsecond refolding. here we show that a -repressor mutant is nonetheless capable of refolding in a single explicit solvent md trajectory in about 19 s, indicating that the burst phase observed in experiments on the same mutant could produce native protein. the simulation reveals that after about 18.5 s of conformational sampling, the productive structural rearrangement to the native state does not occur in a single swift step but is spread out over a brief series of helix and loop rearrangements that take about 0.9 s. our results support the molecular time scale inferred for -repressor from near - downhill folding experiments, where transition - state population can be seen experimentally, and also agrees with the transition - state transit time observed in slower folding proteins by single - molecule spectroscopy. |
it is used extensively in the manufacture of epoxy resins, polycarbonate plastics, and food and beverage containers and also in cigarette filters and carbonless copy paper used in credit card receipts and thermal imaging papers in modern cash registers. detectable levels of bpa in urine have been shown to be present in nearly all sampled adults in recent national surveys conducted in the usa [2, 3 ]. bpa is considered to be an endocrine - disrupting chemical and has been shown to have thyroid hormone disrupting effects [1, 4 ]. recent evidence, especially from experimental animal studies, suggests that bpa exposure may be related to insulin resistance and may have a role in weight gain and obesity [5, 6 ]. furthermore, recent epidemiological studies have shown that higher urinary bpa levels are related to diabetes mellitus as well as to self - reported cardiovascular disease [8, 9 ]. recent studies have also shown that bpa exposure is related to hyperlipidemia, as well as to increased levels of serum markers of oxidative stress, inflammation. hypertension is a major public health problem, and it is also known to be a strong, independent risk factor for cvd. several of the biological mechanisms that are known to be associated with increased risk of hypertension, including thyroid dysfunction, weight gain, insulin resistance, hyperlipidemia, oxidative stress, and higher systemic inflammation, are also related to increased risk of developing hypertension. however, no previous study to our knowledge has examined the association between bpa levels and hypertension in humans. therefore, we examined the independent association between urinary bpa levels and hypertension in the 2003 - 2004 national health and nutritional examination survey (nhanes), a contemporary, multiethnic sample of us adults. detailed description of nhanes study design and methods are available elsewhere. in brief, the nhanes survey included a stratified multistage probability sample representative of the civilian noninstitutionalized us population. selection was based on counties, blocks, households, and individuals within households and included the oversampling of non - hispanic blacks and mexican americans in order to provide stable estimates of these groups. subjects were required to sign a consent form before their participation, and approval was obtained from the human subjects committee in the us department of health and human service. the current study sample consisted of participants aged greater than 20 years among whom urinary bpa was available (n = 1, 488). we further excluded participants with missing data (n = 108) on covariates included in the multivariable model, including level of education, smoking status, serum or fasting glucose levels, body mass index (bmi), and cholesterol levels. this resulted in 1380 participants (49.6% women), 580 of whom had hypertension. age, gender, race / ethnicity, smoking status, alcohol intake (g / day), level of education, history of diabetes and oral hypoglycemic intake or insulin administration were assessed using a questionnaire. individuals who had not smoked > 100 cigarettes in their lifetimes were considered never smokers ; those who had smoked > 100 cigarettes in their lifetimes were considered former smokers if they answered negatively to the question do you smoke now ? and current smokers if they answered affirmatively. body mass index (bmi) was calculated as weight in kilograms divided by height in meters squared. rigorous procedures with quality control checks were used in blood collection, and details about these procedures are provided in the nhanes laboratory / medical technologists procedures manual. serum glucose was measured using the modified hexokinase method at the university of missouri diabetes diagnostic laboratory. diabetes mellitus was defined based on the recent guidelines of the american diabetes association as a serum glucose > 126 mg / dl after fasting for a minimum of 8 hours, a serum glucose > 200 mg / dl for those who fasted 6.5%, or a self - reported current use of oral hypoglycemic medication or insulin. previous measures of bpa in biological matrixes involved techniques such as gas chromatography (gc) or high - performance liquid chromatography. to achieve enhanced sensitivity and selectivity over previous methods, in the current nhanes, measures of environmental phenols were derivatized to alkyl or acyl derivatives before gc / mass spectrometry (gc / ms) analysis. using solid phase extraction coupled to high - performance liquid chromatography and tandem mass spectrometry, detection levels of 0.12 nanograms per milliliter (ng / ml) in 100 l of urine were achieved, sufficient for measuring urinary bpa levels in nonoccupationally exposed participants. seated systolic and diastolic blood pressures were measured using a mercury sphygmomanometer according to the american heart association and the seventh joint national committee (jnc7) recommendations. up to 3 measurements patients were considered hypertensive if they reported current blood - pressure - reducing medication use and/or had systolic blood pressures > 140 mm of hg and/or diastolic blood pressures > 90 mm of hg. urinary bpa was categorized into tertiles (4.0 ng / ml). the odds ratio ((or) (95% confidence interval (ci)) of hypertension associated with higher bpa levels was calculated using multivariable logistic regression models by taking the lowest tertile as the referent. we used two models : the age, sex - adjusted model and the multivariable model, additionally adjusting for race / ethnicity (non - hispanic whites, non - hispanic blacks, mexican americans, others), education categories (below high school, high school, above high school), smoking (never smoker, former smoker, current smoker), alcohol intake (nondrinker, moderate drinker, heavy drinker), bmi (normal, overweight, obese), hypertension (present, absent), diabetes (present, absent), and total serum cholesterol (mg / dl). trends in the or of hypertension across increasing urinary bpa categories were determined by modeling bpa as an ordinal variable. sample weights that account for the unequal probabilities of selection, oversampling, and nonresponse were applied for all analyses using sas (version 9.2 ; sas institute, cary, nc) and sudaan software ; ses were estimated using the taylor series linearization method. overall, this was a middle - aged multiethnic sample with approximately 32% subjects obese, 10% subjects having diabetes mellitus, and 36.3% % having hypertension. table 2 shows the association between increasing levels of bpa and hypertension. overall, we observed a positive association between increasing bpa levels and hypertension in both the age, sex - adjusted model and the multivariable - adjusted model.. table 3 shows the association between increasing bpa levels and hypertension within subgroups of race / ethnicity, bmi, and diabetes status. we found that the positive magnitude of association between increasing bpa levels and hypertension was consistently present within all these various stratified subgroups with the multivariable or ranging from 1.4 to 1.9. also, there was no statistically significant interaction between bpa levels and these variables (p - interaction > 0.10 in all stratified analyses). first, we examined the association between urinary bpa and hypertension using urinary bpa as a continuous variable (with natural logarithmic transformation). consistent with the categorical bpa analysis, there was a significant positive association between urinary bpa as a continuous variable and hypertension with an odds ratio (95% ci) of 1.18 (1.071.31) ; p value = 0.001 in the age - sex - adjusted model and 1.11 (1.011.22) ; p - value = 0.04 in the multivariable model for every 1 unit increase in the log - transformed urinary bpa level. second, to examine the nonlinearity of the association between urinary bpa and hypertension, we added a quadratic term for bpa in the multivariable logistic regression models. we found that the quadratic term was not statistically significant (p - value = 0.459 in the age-, sex - adjusted model and 0.784 in the multivariable - adjusted model). in a large multiethnic, representative sample of us adults, we found that increasing urinary bpa levels were associated with hypertension. the observed association was found to be independent of confounding factors such as smoking, bmi, alcohol intake, diabetes mellitus, and serum cholesterol level. our study adds to the emerging evidence of the role of environmental exposure to bpa on health outcomes in humans [8, 9 ] by being the first study reporting an association between bpa exposure and hypertension, which is a major public health condition and a strong risk factor for cvd. bpa is an environmental chemical used as a constituent monomer in polycarbonate plastics, which are used extensively in drinks containers and food packaging, and in the production of oxidants used in the lining of canned goods. exposure to bpa is believed to be mainly through dietary intake with additional exposure through water, dental sealants, inhalation of household dusts, and exposure through skin. recent studies have documented that over 93 to 95% of the general population has measurable concentrations of bpa metabolites in urine [2, 3 ]. several lines of recent evidence suggest that an association between urinary bpa levels and hypertension may be biologically plausible. animal studies have suggested that bpa exposure may have a role in the development of hypertension thorough several mechanisms, including bpa 's role in weight gain and obesity development potentially through its actions on preadipocytes [23, 24 ], role as an estrogen, potential interactions with estrogen - related receptor gamma, actions as a thyroid hormone antagonist, role as a peroxisome proliferator - activated receptor gamma antagonist, and its role in influencing pancreatic endocrine function. in a recent experiment showed that mice exposed to long - term exposure to bpa developed hyperinsulinemia, insulin resistance, and glucose intolerance. furthermore, bpa has been shown to induce endothelial cell injury mediated through oxidative stress [2931 ] and elevations in lipids in animal models. in epidemiological studies in humans, bpa levels were found to be associated with abnormal liver function enzymes, higher levels of fasting glucose, insulin, and homa - ir insulin resistance [8, 9 ], all are factors known to be associated with hypertension development [15, 33 ]. however, there are no previous studies in humans for comparison. it is in this context that our results regarding objectively measured systolic and diastolic blood pressures to define hypertension in a large sample of us adults are important. in the current study, we have demonstrated a consistent, positive association between higher bpa levels and hypertension that was independent of major confounders and also consistent in stratified analyses by race / ethnicity, bmi, and diabetes mellitus. the main strengths of our study include its nationally representative sample, use of rigorous study methods to collect the data, and the availability of extensive data on confounders [19, 22 ]. the main study limitation is that the current study is cross - sectional in nature, making it impossible to draw cause - effects in the observed associations., we found that in a nationally representative sample of us adults, higher bpa levels were positively associated with hypertension independent of confounding factors such as age, gender, smoking, bmi, alcohol intake, diabetes mellitus, and cholesterol levels. if confirmed in future prospective studies, reducing environmental exposure to bpa may have a role in the prevention of hypertension. | background. bisphenol a (bpa) is a common chemical used in the manufacture of polycarbonate plastics and epoxy resins, with > 93% of us adults having detectable bpa levels in urine. recent animal studies have suggested that bpa exposure may have a role in several mechanisms involved in the development of hypertension, including weight gain, insulin resistance, thyroid dysfunction, endothelial dysfunction, and oxidative stress. however, no previous human study has examined the association between markers of bpa exposure and hypertension. methods. we examined urinary bpa levels in 1380 subjects from the national health and nutritional examination survey 2003 - 2004. main outcome - of - interest was hypertension, defined as blood pressure - reducing medication use and/or blood pressures > 140/90 mm of hg (n = 580). results. we observed a positive association between increasing levels of urinary bpa and hypertension independent of confounding factors such as age, gender, race / ethnicity, smoking, body mass index (bmi), diabetes mellitus and total serum cholesterol levels. compared to tertile 1 (referent), the multivariate - adjusted odds ratio (95% confidence interval) of hypertension associated with tertile 3 was 1.50 (1.122.00) ; p - trend = 0.007. the association was consistently present in subgroup analyses by race / ethnicity, smoking status, bmi, and diabetes mellitus. conclusions. urinary bpa levels are associated with hypertension, independent of traditional risk factors. |
of the many porous materials being developed for sustained release drug delivery systems, electrochemically etched porous silicon (psi) is an attractive candidate because of its highly tunable nanostructure (micro- to macroporous), chemically modifiable surface, biocompatibility, and biodegradability. the biodegradation product of psi is orthosilicic acid [si(oh)4 ], which is easily absorbed from the gastrointestinal tract and excreted in the urine. it has been shown that psi nanoparticles loaded with drugs can be injected intravenously and are degraded in vivo into benign products excreted by the kidneys. however, interaction between the porous matrix and the drug must be carefully considered to design an effective delivery system that provides controlled release of the drug in its active form. loaded molecules will quickly diffuse out of porous materials because of local concentration gradients unless additional interactions between the porous matrix and drug are generated. freshly prepared psi contains highly reactive surface si h species with underlying elemental silicon, both of which are good reducing agents. for example, the reduction potential of psi is sufficient to reduce, and thereby inactivate, many organic and inorganic drugs such as doxorubicin, daunorubicin, and cisplatin. thus modification of psi is needed to both limit redox degradation of drugs and increase the affinity of the drug for the psi surface. various electrostatic or hydrophobic interactions, covalent chemistries, or pore capping reactions have been used to create more effective drug delivery systems. here we describe a new oxidative psi trapping method that is versatile and sufficiently mild to be useful with a range of drugs possessing a wide variety of characteristics (organic or inorganic, hydrophilic or hydrophobic, neutral or positively charged, and/or redox - active). the method relies on the dynamic structure of psi and physical changes that occur during its oxidation. electrochemically etched psi readily oxidizes in aqueous solutions, which generally results in reduction of the pore volume because of swelling of the pore walls as oxygen is incorporated into the silicon skeleton (scheme 1). we hypothesized that pore wall swelling could be used to trap a drug within the psi framework, provided the dimensions of the initial pores, the oxidized pores, and the drug are in a suitable size range. the redox activity of the psi matrix with the drug candidate is an additional constraint : to prevent the drug from being reduced, the silicon skeleton must be oxidized by something other than the drug being trapped. we reasoned that an oxidant of sufficient kinetic and thermodynamic competency and concentration could be preferentially reduced, leaving the trapped drug unaffected. because water is a relatively weak oxidant, stronger silicon oxidants such as nitrite, peroxide, and dimethyl sulfoxide (dmso) are candidates, depending on the characteristics of the drug being loaded. although it has a good ability to oxidize silicon or si h surface bonds, when used as a food preservative it actually suppresses oxidation ; in that case, it limits the oxidation of fatty acids by acting as a trap for free radicals derived from oxygen and other reactive oxygen species. this ability of nitrite ion to act as a chemical oxidant for silicon while simultaneously suppressing more deleterious oxidation reactions is a distinctive characteristic of this oxidant in this work. this leads to a decrease in pore diameter as the porous silicon film (bottom left) becomes partially oxidized (bottom middle) and then fully oxidized (bottom left). while we have long suspected that silicon oxidation could be used to load drugs within the psi framework, we have never specifically tested or validated that this loading technique actually leads to drug trapping and further that the drug is not degraded by the loading process. thus, in this study, we tested the redox trapping method using cobinamide, a vitamin b12 analogue that is being developed as a cyanide antidote. we chose cobinamide because its hydrophilicity, small size, and redox - active cobalt center make it difficult to load into porous matrices in its active oxidized state ; thus, it presented a greater challenge than most drugs, and if the method worked for cobinamide, it would likely work for other drugs. we compared cobinamide to rhodamine b, a cationic organic dye with limited redox activity, to prove that cobinamide s redox activity was not involved in the oxidation trapping mechanism. the distinctive uv visible absorbance spectra of cobinamide and rhodamine b provided for quick and easy detection and redox state assessment. boron - doped p - type si wafers (110, 0.011, or 0.030.05 cm resistivity, 100 orientation) were obtained from international wafer service, inc. aqueous hydrofluoric acid (48% aqueous), dimethyl sulfoxide, hydrogen peroxide (30% aqueous), hydrochloric acid (1 m), and sodium hydroxide (all acs grade) were purchased from fisher scientific. phosphate - buffered saline (1, ph 7.4) was purchased from mediatech, inc. pure aquohydroxocobinamide was produced by base hydrolysis of hydroxocobalamin (purchased from sigma - aldrich). porous silicon (psi) films were prepared by anodic electrochemical etching of polished si wafers. a teflon etch cell was used that exposed 8 cm of the si wafer to a 1:1 (v / v) 48% aqueous hf / ethanol mixture. samples were etched at a constant current density of 32 ma / cm for 1200, 600, and 430 s for samples 13, respectively. the psi films were removed from the crystalline silicon substrate by a current pulse of 5 ma / cm for 50 s in a solution of 3.3% aqueous hf in ethanol. the free - standing psi films were rinsed with ethanol and dried in vacuo. dried psi films (10 mg) were immersed in 1 ml of 25 mm sodium nitrite (ph adjusted to 5 with hcl) within a glass vial and fractured into microparticles by ultrasonication for 30 min. the mixture was maintained at room temperature for 24 h to allow further oxidation of the particles, and the particles were washed three times each with water and ethanol by centrifugation and dried in vacuo at 60 c for 2 h. the resulting oxidized particles were added to 1 ml of 5 mm cobinamide or rhodamine b in deionized water (ph adjusted to 5 with hcl) and agitated for 16 h at room temperature. the cobinamide- or rhodamine b - loaded particles were then washed three times each with water and ethanol by centrifugation and dried in vacuo. a 1 ml solution of 5 mm cobinamide or rhodamine b and 25 mm sodium nitrite in water (ph adjusted to 5 with hcl) was added to 10 mg of dried psi films in a glass vial. the mixture was exposed to ultrasonication for 30 min to fracture the si films into microparticles and maintained at room temperature for 24 h to allow further oxidation of the particles in the presence of cobinamide or rhodamine b. the particles were washed three times each with water and ethanol by centrifugation and dried in vacuo at 60 c for 2 h. a scanning electron microscope (fei xl30) was used to obtain cross - sectional images of the psi samples. attenuated total reflectance fourier transform infrared (atr - ftir) spectra were recorded using a thermo scientific nicolet 6700 ftir instrument with a smart itr diamond atr fixture. raman spectra were recorded using a renishaw invia raman microscope with a 100 mw, 532 nm laser excitation source. desorption isotherms were acquired on the freshly etched materials at 77 k on a micromeritics asap 2020 instrument. thin film optical interference spectra of the porous si samples were recorded in a 180 reflection configuration using an unpolarized tungsten light and an ocean optics 4000 ccd spectrometer fitted with a bifurcated fiber - optic cable. porous si microparticles loaded with cobinamide or rhodamine b were immersed in 1 ml of either deionized water or aqueous phosphate - buffered saline (ph 7.4) at a particle concentration of 100 g / ml and agitated at room temperature. the supernatant containing released cobinamide or rhodamine b was collected at set times (2, 8, 24, 48, 72, 96, and 120 h) and replaced with fresh water or buffer. concentrations of released cobinamide were determined by adding excess potassium cyanide to the solution to convert cobinamide to the dicyano form and then measuring the absorbance value at 370 nm (= 30000 m cm). concentrations of released rhodamine b were determined by the absorbance at 544 nm (= 106000 m cm). in this study, we were interested in determining how the different porous silicon morphologies generated from silicon wafers with different resistivities (under similar reaction conditions) would affect oxidation of the different silicon structures and how those differences would affect the loading and subsequent release of cobinamide from the silicon matrix. porous silicon films were prepared from single - crystal silicon 100 wafers with three different resistivities : 1.20, 0.65, and 0.05 cm (samples 13, respectively). the silicon wafers were subjected to anodic electrochemical etching in a 1:1 (v / v) aqueous 48% hydrofluoric acid / ethanol electrolyte solution. a constant current density of 32 ma / cm was used to prepare all three samples, but the etch time was varied so that the thickness of each psi sample was 10 m. we used the spectroscopic liquid infiltration method (slim) to determine the thickness and total open porosity of the etched films and found average open porosities of 60% for samples 1 and 2 and 40% for sample 3. these measurements were confirmed by cross - sectional scanning electron microscopy (figure s1 of the supporting information) and gravimetric analysis. the nanostructure of the pores was characterized using nitrogen adsorption desorption analysis, with the resulting isotherms exhibiting type iv hysteresis loops, typical of mesoporous materials (figure 1). porous silicon films were prepared with different pore morphologies to systematically study the influence of pore size on loading and subsequent release of cobinamide from the silicon matrix. the measured brunauer teller (bet) surface areas of the samples were 548, 482, and 299 m / g for samples 13, respectively. as expected, the pore size increased as the concentration of boron dopant increased for samples 13 as determined by the mean barrett halenda (bjh) pore diameters of 3.6, 4.7, and 5.6 nm, respectively. nitrogen adsorption desorption isotherms for porous silicon particles prepared from p - type silicon wafers with resistivities of 1.20, 0.65, and 0.05 cm for 13, respectively. the adsorption and desorption branches of the isotherms are shown as filled and empty circles, respectively. the surface chemistry of psi plays a large role in the adsorption of drugs within the porous matrix. therefore, we first studied the interaction between cobinamide and the psi surface of samples 13 using fourier transform infrared (ftir) spectroscopy (figure 2 and figures s2 and s3 of the supporting information). the presence of silicon hydride species on the surface of freshly prepared psi was confirmed by ftir signals assigned to si h vibrations, at 2108 and 2084 cm (sih2 and sih stretching modes, respectively). to study the initial reaction of cobinamide with the surface of the silicon matrix, ftir spectra of the psi samples were monitored after exposure to aqueous solutions (ph adjusted to 5 with hcl) with and without cobinamide for 2 h. the ftir spectrum of the cobinamide - exposed sample (figure 2c) has an amide stretching frequency at 1660 cm, which is characteristic of free cobinamide [ftir of free cobinamide (figure s4 of the supporting information) ] and absent in a control sample exposed to deionized water (figure 2b). because the samples were thoroughly washed and dried before ftir analysis, the presence of the cobinamide peaks in the spectrum confirms loading of cobinamide in all three psi samples (13). a broad silicon oxide band (1100 cm), along with peaks assigned to oxsi - hy species (22002250 cm), was also present in both the water- and cobinamide - treated samples. the height of the silicon oxide band compared to those of the oxsi - hy bands was larger in the cobinamide - exposed sample than in the water - exposed sample. in addition, the magnitudes of the si h vibrational bands decreased only slightly for the sample exposed to water alone, while the cobinamide - exposed sample displayed a complete loss of the si these findings suggest that the rate of silicon oxidation was increased in the presence of cobinamide. fourier transform infrared (ftir) spectra of a freshly etched porous silicon sample of 2 (bottom), 2 exposed to water (middle), or an aqueous cobinamide solution (top) for 2 h. the baselines of the middle and top spectra are offset from the x - axis for comparison. the redox interaction between cobinamide and the silicon matrix was investigated further using visible reflectance and absorbance spectroscopy. the optical reflectance spectrum of psi was used to monitor the rate of silicon oxidation in the presence of cobinamide, while the absorbance spectrum of cobinamide was used to determine whether any redox degradation of cobinamide had occurred. first, silicon oxidation was analyzed by monitoring changes in the optical reflectance spectrum over time of psi sample 2 exposed to cobinamide. the fourier transform of these reflectance spectra gave values of 2nl, or an effective optical thickness of the psi film. a value of 2nl can be used to assess how the refractive index (n) of the material changes over time [assuming any changes in sample thickness (l) are negligible ]. exposing psi to aqueous cobinamide caused a steady decrease in the value of 2nl over time, which can be attributed to a decrease in the refractive index of the sample due to the conversion of si (nd = 3.8) to sio2 (nd = 1.54) (figure 3). the absorbance spectrum of the cobinamide solution after it had been exposed to psi for 2 h revealed significant changes compared to that of the original cobinamide solution (figure 4a). the cobalt center of cobinamide was in the co oxidation state initially ; however, the appearance of new absorption peaks at 315 and 470 nm suggested the co center was reduced to co by the psi. the absorbance of the cobinamide supernatant obtained after the sample had been exposed to psi for 24 h revealed a complete loss of the lowest - energy absorption band leaving only a weak, ill - defined absorption spectrum at wavelengths of < 400 nm. such changes suggest substantial alterations, if not complete loss of the cobalt corrin ring coordination. to protect the loaded cobinamide from redox degradation, the effect of added oxidants peroxide, nitrite, and dmso was evaluated. in these experiments, the oxidant was introduced into the cobinamide solution prior to the addition of the psi sample, so both cobinamide and the oxidant were present in the solution during drug loading. we found that cobinamide was unstable in the peroxide and dmso solutions but was stable in nitrous acid generated from an acidic sodium nitrite solution [25 mm nano2 (ph 5) ] (eq 1).1the shift in the 2nl value measured for psi samples exposed to cobinamide dissolved in the acidic nitrite solution decreased much more rapidly compared to the shift in 2nl observed upon exposure to cobinamide without nitrite present. experimental optical response vs time showing the oxidation of psi sample 2 upon exposure to aqueous cobinamide solutions with () or without () nitrite present. the effective optical thickness (2nl, where n is the average refractive index and l is the total thickness of the sample) was obtained from the fast fourier transform of the optical reflectance spectra of both samples obtained at each time point. the quantity 2nl (%) is defined as (2nl 2nl0)/2nl0 100%, where 2nl0 is the value of 2nl measured immediately after introduction of the solutions. in addition, when cobinamide was loaded into the acidic nitrite solution, no change occurred in the relative height or shape of the peaks observed in the solution absorbance spectrum of cobinamide after exposure to psi for 2 or 24 h (figure 4b). instead, only the overall intensity of the spectrum decreased because of the decreased concentration of free cobinamide in solution, as it became trapped within the psi matrix. the reflectance and absorbance data both suggest that psi preferentially reduced the nitrite, thus protecting cobinamide from redox degradation. to confirm that cobinamide trapped within the psi matrix was indeed protected by the acidic nitrite solution, loaded samples were exposed to sodium hydroxide (ph 9) to dissolve the porous matrix and release the trapped cobinamide. the absorbance spectra of the released cobinamide loaded in samples 1 and 2 were unchanged compared to the spectrum of preloaded cobinamide, whereas the absorbance spectrum of sample 3 showed signs of redox degradation of the released cobinamide. visible absorbance spectra of cobinamide solutions without (a) or with (b) added nitrite. absorbance spectra of solutions were recorded before they were exposed to psi particles (), and absorbance spectra of the supernatants were recorded after periods of psi particle exposure of 2 (---) or 24 h (). therefore, raman spectroscopy was used to elucidate the difference in cobinamide protection between silicon sample 3 (derived from the more highly doped p - type si) and samples 1 and 2. the raman spectra of samples 1 and 2 contained peaks identical to those in the spectrum of unloaded cobinamide, with no evidence of elemental (i.e., unoxidized) silicon, whereas the spectrum of sample 3 had an additional peak at 520 cm assigned to the si thus, while the silicon matrices of samples 1 and 2 became fully oxidized, some unoxidized silicon remained after the oxidation trapping of cobinamide in sample 3. once a certain thickness of silicon oxide had formed on the pore surface, the room - temperature oxidation process was expected to slow considerably. thus, the pore walls of samples 1 and 2 were sufficiently thin to allow complete oxidation of the si skeleton. the unoxidized silicon remaining in sample 3 after oxidative loading most likely became exposed during the dissolution of the psi matrix. this exposed elemental silicon was then able to interact with loaded cobinamide, resulting in the reductive degradation of the drug. raman spectra of preloaded cobinamide (free cbi) and cobinamide - loaded psi particles prepared from p - type silicon wafers with resistivities of 1.20 (1-cbi), 0.65 (2-cbi), and 0.05 (3-cbi) cm via oxidative trapping of cobinamide with aqueous nitrite solutions (oxtrap). two different loading methods were compared to determine which provided a higher loading efficiency and more sustained release of cobinamide. for these studies, acidic nitrite solutions were used with psi samples 1 and 2 to avoid the redox degradation of cobinamide associated with sample 3. for the oxidative trapping method (oxtrap), cobinamide was dissolved in an acidic nitrite solution, and the solution was added to freshly etched psi films ; the mixture was ultrasonicated for 30 min to form microparticles and maintained at room temperature for 24 h to complete the oxidation process. for the postoxidation loading method (postox), the psi films were also maintained in the presence of the nitrite oxidizing agent during the 30 min ultrasonication and 24 h oxidation periods, but cobinamide was added to the particles after the 24 h oxidation step. optical microscope images of the loaded microparticles (figure 6) revealed that the average size of the microparticles was very similar for the two types of postox samples studied (21 5 m for 1 and 22 6 m for 2). bright field optical microscope images of microparticles prepared via the oxidation trapping (oxtrap, top) and postoxidation (postox, bottom) methods of loading cobinamide. the psi microparticles were prepared via a 30 min ultrasonication of psi films of sample type 1 (left) and 2 (right). likewise, ultrasonication of either sample 1 or 2 subjected to the oxtrap method yielded particles with similar sizes (46 14 m for 1 and 48 14 m for 2). however, the particles prepared using oxtrap were significantly larger than those prepared using postox for both 1 and 2. presumably, the simultaneous cobinamide loading and matrix oxidation that occur with the oxtrap method generate a stronger, composite particle that is not as easily fractured by ultrasound energy. the cobinamide loading efficiency was measured by extracting the loaded cobinamide from the porous si microparticle matrix via treatment with a sodium hydroxide solution (ph 9). the basic solution dissolved the si and sio2 components of the microparticle, releasing the drug payload into solution. we quantified the cobinamide by converting it into the dicyano form (by adding excess potassium cyanide) and measuring the optical absorbance at 370 nm (= 30000 m cm). the average cobinamide loadings were 20 8 and 86 10 g of cobinamide / mg of si for sample 1 and 74 6 and 112 6 g of cobinamide / mg of si for sample 2 for the postox and oxtrap particles, respectively. thus, for both samples 1 and 2, the oxtrap method yielded greater cobinamide loading than the postox method, with sample type 2 showing the largest relative increase (1.54-fold increased mass loading of drug, relative to that of the postox method). the increased cobinamide loading seen with the microparticles of sample 2 is attributed to the pore size (4.7 nm) being larger than that of sample 1 (3.6 nm). the reduction in the average pore size upon oxidation of the silicon matrix is expected to show a more pronounced effect on the efficiency of drug loading as the pore size approaches the diameter of the drug payload, and this is observed in the data. when introduced before oxidation in the oxtrap method, cobinamide can more freely penetrate the pores, and the silicon oxide can then grow around the cobinamide molecules. we next tested the retention of cobinamide within the oxidized silicon matrix for the different microparticle formulations. in pure water, the rate of silicon dissolution is relatively slow, which is expected to delay the release of cobinamide trapped within the silicon oxide matrix. if cobinamide is not physically trapped, it will be free to diffuse out of the pores even if the silicon matrix has not degraded. consistent with these arguments, the release of cobinamide from the postox particles was significantly more rapid than from the oxtrap particles for both sample types 1 and 2 (figure 7a), with 50% of the loaded drug released within 3 h for postox versus 3 days for oxtrap formulations. this confirms that oxidation of the psi skeleton in the presence of cobinamide will effectively trap the drug in the pores and that this process increases the level of retention of cobinamide in the resulting microparticles. we observed no significant difference in the cobinamide release rate between samples 1 and 2 for the postox - loaded samples and only a slight difference for the oxtrap particles. the slight reduction in the release rate for sample 2 could be due to the slightly larger amount of cobinamide loaded in the particles, which may act to shield the silicon matrix from water dissolution. release of cobinamide into water (a) or aqueous phosphate - buffered saline (b) from oxidized psi particles 1 (circles) and 2 (squares) loaded with cobinamide via the postoxidation (postox, empty symbols) or oxidation trapping (oxtrap, filled symbols) method. the data were averaged from three samples, and the error bars indicate the standard deviation. the release of cobinamide from the microparticles was also tested in phosphate - buffered saline (pbs), a more physiologically relevant solution (figure 7b). the psi matrix degraded more quickly in pbs than in water, although as with pure water, the release profile showed a smaller rate of cobinamide release for the oxtrap than for the postox microparticles for both samples 1 and 2. however, the difference in release rates was less pronounced for the pbs experiments than for the water experiments because of the increased rate of dissolution of silicon in pbs. in addition, for a given preparation (oxtrap or postox), we observed no significant difference between the release profiles of sample types 1 and 2. to determine if the oxidation trapping method could be used for molecules other than cobinamide, we repeated the loading and release studies of sample type 2 using rhodamine b. we loaded rhodamine b like cobinamide using both the postox and oxtrap methods. as for cobinamide, a larger mass loading was achieved with the oxtrap protocol (87 5 g of rhodamine b / mg of si) than with the postox method (7 1 g of rhodamine b / mg of si). the release profiles (in deionized water) for the delivery of rhodamine b from postox particles showed more sustained release than from postox particles (figure 8). the data confirmed that the oxtrap method is not specific to cobinamide, providing larger mass loading and more effective trapping for both types of molecules. release of rhodamine b into water from type 2 oxidized porous silicon particles loaded with rhodamine b via the postoxidation [postox () ] or oxidation trapping [oxtrap () ] method. the data are the means of three samples, and the error bars indicate the standard deviation. this study focused on the ability of porous si to shrink its pore dimensions upon mild oxidation of the porous skeleton, to enhance the loading capacity and extend the duration of release of small hydrophilic drugs. via oxidation of the silicon matrix in the presence of the molecular payload (oxtrap), the molecule was more effectively trapped than it was after simple diffusional loading into a preformed oxide matrix. it was found that the pore and molecule size is a critical parameter in this process ; the pore and molecule must be appropriately matched to allow the molecule to diffuse into the unoxidized pore and not diffuse out of the oxidized pore. for cobinamide, the optimal unoxidized pore diameter was between 3.6 and 4.7 nm. for redox - active drugs, the pore wall thickness is an additional important factor. we found that material with smaller pore diameters (and thus thinner pore walls) could be completely oxidized to sio2 under mild (room temperature, aqueous nitrite) conditions, whereas material with thicker pore walls retained elemental silicon within the porous skeleton. this residual silicon then reacted with and degraded the cobinamide model drug under long - term aqueous release conditions. in addition, we showed that aqueous acidic nitrite (no) acts as a mild oxidant that does not degrade the model drugs but is sufficiently reactive to oxidize the si skeleton, outcompeting the reduction of cobinamide by si and thus preserving the activity of the molecule. we found that porous si samples prepared in a 1:1 (v / v) hf / ethanol solution from p - type silicon wafers with a resistivity of 0.65 cm possess pore walls sufficiently thin to become fully oxidized, whereas the pore walls in porous si made from silicon with a resistivity of < 0.5 cm are too thick and do not become fully oxidized under the oxtrap reaction conditions. this approach may be generally applicable to a wider range of drugs, including more sensitive protein and oligonucleotide therapeutics. | an approach for the preparation of an oxidized porous silicon microparticle drug delivery system that can provide efficient trapping and sustained release of various drugs is reported. the method uses the contraction of porous silicon s mesopores, which occurs during oxidation of the silicon matrix, to increase the loading and retention of drugs within the particles. first, a porous si (psi) film is prepared by electrochemical etching of p - type silicon with a resistivity of > 0.65 cm in a 1:1 (v / v) hf / ethanol electrolyte solution. under these conditions, the pore walls are sufficiently thin to allow for complete oxidation of the silicon skeleton under mild conditions. the psi film is then soaked in an aqueous solution containing the drug (cobinamide or rhodamine b test molecules were used in this study) and sodium nitrite. oxidation of the porous host by nitrite results in a shrinking of the pore openings, which physically traps the drug in the porous matrix. the film is subsequently fractured by ultrasonication into microparticles. upon comparison with commonly used oxidizing agents for psi such as water, peroxide, and dimethyl sulfoxide, nitrite is kinetically and thermodynamically sufficient to oxidize the pore walls of the psi matrix, precluding reductive (by si) or oxidative (by nitrite) degradation of the drug payload. the drug loading efficiency is significantly increased (by up to 10-fold), and the release rate is significantly prolonged (by 20-fold) relative to control samples in which the drug is loaded by infiltration of psi particles postoxidation. we find that it is important that the silicon skeleton be completely oxidized to ensure the drug is not reduced or degraded by contact with elemental silicon during the particle dissolution drug release phase. |
eosinophilic gastroenteritis (eg) is a rare inflammatory disorder, characterized by eosinophilic infiltration in one or multiple areas of the gastrointestinal (gi) tract from the esophagus to the rectum. clinical presentations depend on the region of the gi tract involved and the depth of bowel wall involvement. the three main patterns include predominant mucosal disease, predominant muscle layer disease and predominant subserosal disease. reports from different parts of the world provide important information regarding epidemiology, disease characteristics and management. there have been several case series of patients with eg from the western world, australia and east asia. to our knowledge, there has not been report of a series of patients with eg from the middle east region. the aim of this study was to describe clinical characteristics and treatment response in a series of eg patients with long - term follow up from iran. this study was approved by the institutional review board of digestive disease research center, tehran university of medical sciences. using prospectively updated databases at shariati hospital and private outpatient clinics of the authors from january 1997 to december 2010, we identified and reviewed medical records of all patients with the diagnosis of eg. eg was defined by the presence of all the following criteria : 1) gi symptoms attributable to eg, 2) biopsies showing marked eosinophilic infiltration of one or more regions of the gi tract from the esophagus to the colon (> 20 eosinophils per high power field) and 3) exclusion of parasitic infection or extraintestinal infiltration of eosinophils. we collected the following data : demographic characteristics, presenting symptoms, date of symptoms onset, date of diagnosis, duration of follow up, absolute eosinophil count, treatment regimen and number of possible relapses. relapse was defined by the presence of one of the following criteria : 1) reappearance of gi symptoms with re - elevation of peripheral blood eosinophilia, 2) reappearance of gi symptoms and restarting immunosuppressive drugs by an experienced gastroenterologist and 3) reappearance of gi symptoms and re - demonstration of eg on biopsy specimen after previous normalization of biopsy specimens. patients were divided into the following groups : 1) those with predominantly mucosal disease as defined by infiltration of mucosa with eosinophils, no evidence of intestinal obstruction or eosinophilic ascites ; 2) those with predominant disease in muscular layer as defined by the presence of intestinal obstruction and eosinophilic infiltration of muscular layer, and absence of eosinophilic ascites ; and 3) those with predominant subserosal disease manifested by eosinophilc infiltration of the gi tract and eosinophilic ascites. mean age of the patients was 45.115.5 (range : 27 - 75) years. of the 22 patients, 18 (82%) had small bowel, 11 (50%) had gastric, 5 (23%) had colonic, and 2 (9%) had esophageal involvement (table 1, figures 1a-1c). twenty (90%) patients had mucosal involvement, one (5%) had muscular involvement and one (5%) had subserosal involvement. a. small bowel series in a patient with small intestinal eg showing mucosal thickening, nodularity and ulceration in the jejunum (straight arrow) and terminal ileum (curved arrow). b. abdominal ct scan from the same patient showing dilated loops of small bowel (straight arrow). a loop of jejunum has marked mucosal thickening and nodularity (curved arrow). c. photomicrograph of the jejunal biopsy from the same patient showing mild shortening of intestinal villi associated with moderate to severe expansion of the lamina propria by an increased number of plasma cells, lymphocytes and numerous eosinophils. the eosinophils tended to form small aggregates and collections around the crypts with occasional exocytosis to the crypt epithelium. three (14%) patients had anemia and 4 (18%) had positive fecal occult blood tests. the mean eosinophilc count in patients with eosinophilia was 4720 4213 (range : 500 - 10720) per microliter. the presenting symptoms included abdominal pain in 15 (68%), diarrhea in 6 (27%), pruritus in 6 (27%), weight loss in 6 (27%), nausea / vomiting in 4 (18%), fever in 3 (14%) and dysphagia in 1 (5%) patients. the median duration between symptom onset and diagnosis was 12 (range 1- 48) months. associated autoimmune disorders included celiac disease (1), sarcoidosis (1) and ulcerative colitis (1). patients were followed for a median duration of 36.5 (range : 4 - 123) months. two patients with mucosal disease had spontaneous remission with fasting and supportive care, however neither developed relapse after a mean follow up of 44 months. twenty patients were treated with prednisolone at initial doses of 15 to 50 mg per day, which was tapered over 6 to 8 weeks. one patient with eosinophilic esophagitis and recurrent dysphagia required several courses of endoscopic esophageal dilation in addition to systemic corticosteroids. five out of 15 (33%) developed relapse (range : 1 to 4 relapses) after their initial course of corticosteroid therapy. a repeat course of prednisolone resulted in resolution of symptoms in those who experienced relapse. however, 2 patients with several relapses required maintenance treatment with azathioprine to prevent further episodes of relapse. one of the azathioprine treated patients did not relapse after starting azathioprine ; but the second patient who had serosal involvement required low dose prednisolone in addition to azathioprine to maintain remission. first reported in 1937, eg is a rare but increasingly recognized inflammatory disorder of the gi tract characterized by the presence of severe eosinophilic infiltration in one or multiple segments of the gi tract. the pathogenesis of eg is unknown, but it may be related to the destruction of intestinal epithelium by the release of eosinophilic major basic protein from the resident eosinophils. patients with predominantly mucosal disease present with diarrhea, abdominal pain, vomiting and nutritional deficiency (i.e., iron deficiency anemia). those with predominantly muscular involvement present with symptoms of gastric outlet obstruction or intestinal obstruction. patients with involvement of serosal and subserosal layers present with eosinophilic ascites. although peripheral eosinophilia is an important guide to diagnosis, it was absent in 19% of our patients. the 81% rate of peripheral eosinophilia in our study was similar to previous case series of eg in the western countries or east asia. in our patients, in this study, the median duration between symptom onset and diagnosis was 12 (range 1- 48) months. on the other hand, therefore, gastroenterologists need to be vigilant, including eg in the differential diagnosis of patients with gi symptoms who have associated peripheral eosinophilia, a remarkable personal history of allergy, or those whose gi symptoms do not respond to regular therapeutic measures. our patients responded well to a 6 to 8 week course of corticosteroid therapy. in this study, the relapse rate after initial corticosteroid therapy was 36%. therefore, patients need to be followed in terms of clinical symptoms and peripheral eosinophil counts to detect early signs of relapse. in conclusion, we report a series of patients with eg with long term follow up from iran. the clinical symptoms of eg patients are mostly non - specific and may be confused with functional bowel disorders. therefore, gastroenterologists need to be aware of this treatable condition, and take mucosal biopsies during endoscopies in patients with suspected eg. | background eosinophilic gastroenteritis (eg) is a rare inflammatory disorder of the gastrointestinal (gi) tract. there have been several case series of patients with eg from the western world and east asia. however, there has not been a report of patients with eg from the middle east region. the aim of this study is to describe clinical characteristics and treatment response in a series of eg patients from iran. methods we retrospectively reviewed charts with a diagnosis of eg from 1997 to 2010 at shariati hospital and the private clinics of the authors. clinical characteristics of the patients were evaluated, and the treatment response and relapse rate were assessed. results twenty - two patients (9 male) with eg were identified. mean age of the patients was 45.115.5 (range : 27 - 75) years. median duration between symptom onset and diagnosis was 12 (range 1- 48) months. twenty (90%) patients had mucosal involvement, one (5%) had muscular involvement and one (5%) had subserosal involvement. patients were followed for a median duration of 36.5 (range 4 - 123) months. two patients had spontaneous remission with supportive care. the remaining 20 patients responded well to oral corticosteroid treatments. the relapse rate was 33%. episodes of relapse were successfully controlled with a repeat course of corticosteroids. two patients with several relapses required maintenance treatment with azathioprine. conclusion the clinical characteristics and treatment responses of eg patients from iran are similar to reports from other parts of the world. patients need to undergo close follow up after treatment to detect early signs of relapse. |
maximal oxygen consumption (vo2 max) is the amount of oxygen the human body can utilize in muscles and/or tissues. vo2 low aerobic fitness is associated with congestive heart failure, anemia, and obstructive pulmonary disease. vo2 max is determined by both the capacity of oxygen delivery to the blood stream and the capacity of oxygen extraction from blood. botanical supplements have been studied for decades, some of which have been associated with health or performance benefits. echinacea is an herbal supplement that is derived from the north american purple coneflower plant and has shown some potential mechanisms that, with supplementation, could augment oxygen transport. echinacea supplementation was found to increase vo2 max likely through an increase in the number and size of red blood cells, hemoglobin, and hematocrit associated with increases in serum erythropoietin. recent research suggests echinacea induced erythrocythemia and vo2 max results in an increase in serum erythropoietin (epo) [1, 5 ]. this glycoprotein hormone, primarily produced in the kidneys, regulates red blood cell production. epo improves exercise performance by increasing oxygen blood transport, which results in a greater vo2 max. the increase in red blood cell mass is proceeded by a decrease in plasma volume to control hemoconcentration. the regulation of epo production is based upon oxygen concentration in the blood and is increased by any condition that results in a decrease in the quantity of oxygen that is transported in the blood to the tissues [1, 8 ]. running economy (re) is the energy cost reduction of submaximal running evaluated through the measurement of oxygen consumption during steady state exercise [8, 9 ]. potential mechanisms for improved submaximal re ensuing from erythrocythemia include an increase in the amount of adenosine triphosphate (atp) produced per mole of oxygen consumed, a decrease in the amount of atp necessary for running at a given speed, or a combination of both mechanisms [1, 8, 9 ]. another adaptation resulting from erythrocythemia is a reduction in the heart rate [8, 9 ]. based upon the literature, it is reasonable to assume that echinacea will increase epo and, in turn, affect erythrocyte production. however, oxygen availability is generally not a limiting factor for exercise in normobaric environments. therefore, an increase in epo alone may not be sufficient to increase exercise performance or maximum aerobic capacity, where both delivery and extraction of oxygen by tissue are necessary to increase vo2 max. recent studies have shown an increase in supplementation usage, which may be related to the growing competitiveness in sports on both the collegiate and professional level [1416 ]. in a study of male and female elite figure skaters, supplement use was reported to prevent illness and disease, to increase energy, and to enhance performance. given the available information regarding echinacea, it is important to evaluate whether or not supplementation is effective at increasing maximum aerobic capacity in the absence of an increased volume of physical activity and exercise. the purpose of this study was to determine the effects of oral echinacea supplementation on vo2 max and economy during submaximal treadmill walking and running in human participants. it was hypothesized that echinacea supplementation might augment running performance in humans based upon previous evidence of changes in epo hormones levels demonstrated in previous research [1, 5 ]. the local institutional review board approved the study for the use of human subjects. all subjects provided informed written consent prior to beginning the study and were aware of their right to withdraw at any time. participants were tested for maximum aerobic capacity, provided with a 30-day supply of supplements, and were asked to stringently adhere to their typical routine and levels of physical activity and exercise. following the completion of the 30-day supplementation period, each participant was again tested for maximum aerobic capacity. all subjects reported consumption of all doses of the required supplement. participant information can be seen in table 1. the botanical supplement (vo2- advantage, second wind llc, highland park, il, usa) contained only substances that are generally regarded as safe by the united states food and drug administration. the product contains a concentrated dose of echinacea purpurea (8 grams day), as well as rhodiola rosea, cordyceps sinesis, quercetin, beta alanine, catechins, vitamin c, vitamin b-3, vitamin b-12, iron, folic acid, inositol, and alpha - lipoic acid. the subjects were provided with a 30-day supply of the supplement, and all subjects were compliant in consuming the required doses. the graded exercise test format included the use of the same equipment during both pre- and posttesting sessions. the subjects ran on a track master tmx 425 treadmill (full vision inc., newton, ks.) during the assessment. participant 's expired air was sampled and analyzed with a parvomedic trueone 2400 metabolic measurement system (parvomedics, sandy, ut). the system utilized a mixing chamber and was set to report data every 10 seconds. listed accuracy for the gas sensors in the unit are paramagnetic o2 analyzer 0.1%, infrared co2 analyzer 0.1%, and pneumotach 2%. the test was concluded when the oxygen consumption was determined to have reached a plateau, or the participant volitionally ceased exercise. participants were determined to have reached vo2 max if they demonstrated a plateau in oxygen consumption (.45). paired samples t - test analysis did not reveal a significant difference in maximum aerobic capacity, t(12) = 0.67, p =.516. presupplementation maximum aerobic capacity (m = 51.0, sd = 6.8) was similar to postsupplementation values (m = 51.8, sd = 6.5). the 3 minutes of absolute oxygen consumption (l o2/min) was averaged for the comparison. the repeated measures anova did not reveal a main effect for changes in economy pre to post, f(1,11) = 0.067, p =.800, nor an interaction effect for time (pre, post) by stage (14), f(3,33) = 0.669, p =.577. based upon the present investigation, a botanical supplement with a concentrated dose of echinacea is not sufficient to increase maximum aerobic capacity. this finding is in contrast to previous reports in the literature that reported increases in vo2 max with echinacea supplementation. in that study, a similar dose of 8 grams per day of echinacea was administered for 28 days (the present study used 30 days), and significant increases in vo2 max in the supplement group were noted. however, the increase in maximum aerobic capacity in the supplement group was very small, indicated by an increase of only 1.5%. this result is likely to be statistically significant but the practical significance is questionable given the very small effect of the supplement. a more recent study is in agreement with the present study which found that an 8 gram per day dose of echinacea was not found to increase the maximum aerobic capacity of trained distance runners. it may be important to note that all three studies utilized a similar does of echinacea purpurea, which allows for comparison of the effects. in the present investigation and the baumann. study, there was almost no change in the mean aerobic capacity of the participants who received the supplement. in the whitehead. however, the longest use of the supplementations resulted in no difference in vo2 max. in regard to economy, the present investigation did not reveal any changes in walking or running economy during the first 4 stages of the graded exercise test. this is, again, in contrast to the changes reported by whitehead. this study reported significant, but very minimal, changes in walking / running economy during the first 2 stages of a graded exercise test (stage 1 : 1.5% increase in economy ; stage 2 : 1.67% increase in economy). based upon the available evidence, it seems unlikely that echinacea supplementation has a meaningful impact on exercise economy during walking and running. the limitations of the present study involved the use of self - reported compliance for adherence to the supplementation regime, the use of a self - reported instrument (lpa survey) to examine potential changes in physical activity and exercise behavior, and the use of supplement that had components other than echinacea. though these limitations are necessary to disclose, the nature of the study was likely similar to the use of the supplement in a real - world setting. therefore, the results are applicable to those who have an interest in exercise or sport nutrition. based upon the present study, the use of a botanical supplement with a concentrated dose of echinacea purpurea can not be recommended to increase aerobic capacity or economy during exercise in healthy, recreationally active adults. given the information regarding the ability of echinacea to augment epo levels, these supplements should be evaluated for use in populations where oxygen delivery limits functional capacity, such as those individuals with disease states or those persons who will transition to living at altitude. | the present investigation evaluated the efficacy of a botanical supplement that delivered a concentrated dose of echinacea purpurea (8 grams day1). the participants were 13 apparently healthy, recreationally active college students (vo2 max : 51 ml o2/kgmin). the participants were provided with a 30-day supplementation regime. data regarding maximum aerobic capacity was collected through pre- and posttesting surrounding the 30-day supplementation regime. the participants were instructed to maintain normal levels of physical activity and exercise during the experimental period. the levels of physical activity and exercise were monitored via the leisure and physical activity survey. the participants did not report any significant increases in aerobic physical activity or exercise during the supplementation period. paired samples t - test analysis did not reveal a significant difference in maximum aerobic capacity, t(12) = 0.67, p =.516. presupplementation maximum aerobic capacity (m = 51.0, sd = 6.8) was similar to postsupplementation values (m = 51.8, sd = 6.5). this study suggests that botanical supplements containing a concentrated dose of echinacea purpurea is not an effective intervention to increase aerobic capacity of recreationally active individuals. |
alzheimer 's disease (ad) is characterized as a progressive neurodegenerative disorder and considered as prominent cause of dementia in the elderly. the main characteristics of this disease are difficulties in household handling routine and cognitive and emotional disturbance in the elderly. the treatment of ad is a clinical challenge. with the development of cholinesterase inhibitors and a n - methyl - d - aspartate antagonist (memantine), therapeutic decisions have to be guided by clinical studies and should consider the physiopathogenesis and epidemiology of the disease. the main objective of these clinical trials is to reduce the behavioral and psychological symptoms of dementia (bpsd) and to improve cognition and the functional activity status, thus reducing the impairment of instrumental activities of the daily living (iadls) and to lower the institutionalization rates (nursing home placement). unfortunately, only a limited number of trials have dealt with this topic and with follow - up periods shorter than two years. in spite of the absence of sufficient therapeutic effectiveness in mild and moderate ad, these drugs are still considered as the first line of treatment for ad (1). studies of cost - effectiveness suggest that memantine (2,3) and donepezil (4) are useful in the reduction of institutionalized care and/or cognitive impairment in patients with ad. recently, two clinical trials showed no improvement of the cognitive deficit (5,6) or reduction in the institutionalization rate (6). searching for alternatives, many herbal products have been tested and employed in the treatment of ad, but with different clinical responses (7). the assessment of these drugs through randomized controlled trials should be useful to identify effective products in the treatment of ad. searching at medline (during april 2006, pubmed), lilacs (latin american and caribbean health science literature : 40th edition, may 2001, the last research was performed in april 2006) ; cochrane library (issue 1, 2006) ; dissertation abstract (usa, during april 2006) ; adear (alzheimer 's disease clinical trials database, until april 2006) ; national research register (1/2006) ; current controlled trials (the last research was performed in october 2005) ; psychinfo journal articles (during the year of 2006) ; relevant web sites ; and scanning of reference list of relevant articles. search for keywords in mesh (medical subject heading (mesh) with the words alzheimer disease, dementia, cognition disorders was performed first. in the second part, the keywords were herbal and phytotherapy. the crossover results of the two searches were evaluated by the jadad 's measurement scale (8). the articles were selected using the criteria listed below : the studies should be randomized ; double - blind and controlled (with a control group and a treatment group). studies should establish methodological procedures in the crossover or be conducted at the same time. in the case of being crossover, a washout period of at least 7 days was required. patients included in the researches had their diagnosis rated into three degrees as follows : mild, moderate and severe forms of ad, according to the criteria from the national institute of neurological and communicative disorders and stroke ad and related disorders association (nincds - adrda) (9). the models used were as follows : mini - mental beginning values between 10 and 26 (initial and mild group) or < 10 (initial group). clinical trials should last for at least 1 month (4 weeks). neuropsychiatry symptoms progression should be measured with numerical score using the assessment scale (adas - noncog, range of score, 070), npi (neuropsychiatric inventory, range of score 0120), clinical global impression of change or behavioral rating scale for geriatric patients. the final score should be quantified using a combination of adl and iadl methodological procedures. exclusion criteria : the herbal product has already been target of a quantified systematic review study. in this case, only the results of the studies will be considered. jadad 's measurement scale : methodological quality was assessed using a scale developed and validated by jadad. this scale assesses the completeness of reporting using three items with a five points maximum score. a bonus point is given if an adequate method to generate the random sequence is described. if there is an explicit statement that the study is double - blind item 2 is scored. item 3 is scored if there is either an explicit statement that all patients included were also analyzed or if the number and reasons for dropouts in all groups are given separately. for being classified as adequately reported a trial should score at least three of five points, a cut - off point is recommended by the author of the scale (10). all extraction and quality assessments were performed by at least two independent reviewers using standard forms developed for each review. two herbs and two herbal formulations were identified to have effectiveness in the treatment of cognitive disturbance of ad in the systematic review : salvia officinalis (11), melissa officinalis (12), and yi - gan san (13) and ba wei di huang wan (bdw) (14). gingko biloba was previously identified in one meta - analysis (15), and only the conclusions of the study will be considered. another study will be conducted with huperzine a, a product derived from a chinese herb huperzia serrata, to evaluate the safety and efficacy in the treatment of ad in a multicenter randomized controlled trial of its effect on cognitive function (16). the studies of salvia (11), melissa (12), yi - gan san (13) and bdw (14) have reached jadad 's measurement scale of 3. the researches had a follow up of 1 month (yi - gan san) (13), 2 months (bdw) (14) and 4 months (salvia and melissa) (11,12). two studies compared herbal medicines and control samples, using intention to treat [salvia (11) and melissa (12) ]. none of the studies evaluated the institutionalization rate or compared the active principle with the current therapies with acetyl cholinesterase inhibitor or memantine. the results of this systematic review identified four studies with methodological quality assessing s. officinalis (11), m. officinalis (12), yi - gan san (13) and bdw (14). these herbs and formulations presented efficiency in reducing the mild and moderate symptoms of ad. gingko biloba presented statistically significant mild effectiveness in the treatment of cognitive deficit in ad. the meta - analysis study of cochrane (13) concluded that additional controlled studies would be necessary in order to recognize cognitive improvement with the use of gingko. there is still need of a prospective study with an appropriate duration and representative sample to identify if g. biloba reduces the development of ad (17). another plant with a large application perspective is h. serrata, after multicenter trial confirmation underway (16). melissa and yi - gan san showed reduction in the cognitive deficits and a good sedative effect in patients with ad (12,13). previous clinical studies showed that the extract of m. officinalis reduces laboratory - induced stress (18) and might have benefits in mood improvement (1921). the use of these herbs and formulations should be well tolerated, (22) and adverse effects have not yet been reported (23). further studies should be conducted to compare the current therapies for ad and the use of these herbal remedies in controlling the symptoms of ad. it has been suggested that the chemical composition of the essential oil of the melissa and salvia leaf extracts are monoterpene aldehydes, polyphenol flavonoids (including rosmarinic acid) (24) and monoterpene glycosides (25). all of these components have many observable effects in vitro, which include powerful anti - oxidative activity (26,27) and an affinity to nicotinic and muscarinic receptor in the human cerebral cortex (28). this last mechanism is of special interest, as modulation of cholinergic systems should play a role in improving the cognitive function, especially in ad. yi - gan san is a mixture of 7 different dried plants, many of them (unticariae sinensis and angelicae root) with possible actions in the serotoninergic and gaba system. var purpurea makino, cornus officinalis sieb et zucc (cornaceae), dioscorea batatas decne root (dioscoreaceae), alisma orientale juzep rhizome (alimataccae), poria cocos wolf, paeonia suffruticosa andr. studies have suggested that bdw enhances the choline acetyltransferase activity and increases the acetylcholine content of the frontal cortex in a murine model (29,30). major methodological limitations of the four studies are small size of samples and short - term duration. there is no description of the chemical composition and/or possible active principles of the different products employed in studies involving yi - gan san (13) and bdw (14) formulation (table 1). in the study with bdw (13), sepia sp. and face powder were used as placebo ; however, the way that the shellfish formulation was performed was not described. in the control group of the study using the yi - gan - san (14) formulation, 25 mg per day of tiapride hydrochloride were introduced. this intervention (contamination) occurred in 44% of the individuals and was responsible for the symptoms, among them dizziness. generally, crude herbal drugs are natural products and their chemical composition depends on several factors such as geographic source of the plant material, climate in which it was grown, and time of harvest. commercially available herbal medicinal products also vary in their content and concentration of chemical constituents from batch to batch and when products containing the same herbal ingredient are compared between manufacturers. even when herbal products are standardized for content of known active or marker compounds to achieve more consistent pharmaceutical quality, variations in the concentrations of other constituents can be observed. the use of a protocol such as the consolidated standards of reporting trials (consort), composed of 22 items, will probably minimize the limitations of rtc with phytotherapic agents (31). the use of herbal medicines in the treatment of ad should be compared with the pharmacological treatment currently in use. such studies should include the identification of the active principle in order to improve the validation of the clinical trial. further large - scale, multicenter studies are necessary to determine the effectiveness of these substances in the cognitive deterioration of ad. until then, this review provides some evidence of the benefit of melissa, salvia, yi - gan san and bdw in the treatment of ad. phytotherapic interventions in the alzheiemer 's disease selected rct adas - cog, alzheimer 's disease assessment scale ; cdr - sb, clinical dementia rating - sum of the boxes ; che - i, acetyl cholinestarase inhibitor ; mmse, mini - mental state examination ; npi, neuropsychiatric inventory ; hplc, high - performance liquid chromatography. formula containing 4 g de atractylodis lanceaea rootstock ; 4.0 g of poria cocos wolf ; 3.0 g of cnidium monnieri rootstock ; 3.0 g of urticaria and angelica sinensis root ; 2.0 g of bupleuri radix ; 1.5 g of glycyrrhizae uralensis rhizoma ; and 3.0 g of uncariae ramulus et uncus. purpurea makino (scrophulariaceae) ; 4 g of cornus officinalis sieb et zucc (cornaceae) ; 4 g of dioscorea batatas decne root (dioscoreaceae) ; 3 g od alisma orientale juzep rhizome (alimataccae) ; 3 g of poria cocos wolf (poriacea) ; 3 g of paeonia suffruticosa andr. (paeoniaceae) ; 1 g of cinnamomum cassia blume (lauraceae) ; and 1 g of aconitum carmichaeli debx. | the treatments of choice in alzheimer 's disease (ad) are cholinesterase inhibitors and nmda - receptor antagonists, although doubts remain about the therapeutic effectiveness of these drugs. herbal medicine products have been used in the treatment of behavioral and psychological symptoms of dementia (bpsd) but with various responses. the objective of this article was to review evidences from controlled studies in order to determine whether herbs can be useful in the treatment of cognitive disorders in the elderly. randomized controlled studies assessing ad in individuals older than 65 years were identified through searches of medline, lilacs, cochrane library, dissertation abstract (usa), adear (alzheimer 's disease clinical trials database), national research register, current controlled trials, centerwatch trials database and psychinfo journal articles. the search combined the terms alzheimer disease, dementia, cognition disorders, herbal, phytotherapy. the crossover results were evaluated by the jadad 's measurement scale. the systematic review identified two herbs and herbal formulations with therapeutic effects for the treatment of ad : melissa officinalis, salvia officinalis and yi - gan san and bdw (ba wei di huang wan). ginkgo biloba was identified in a meta - analysis study. all five herbs are useful for cognitive impairment of ad. m. officinalis and yi - gan san are also useful in agitation, for they have sedative effects. these herbs and formulations have demonstrated good therapeutic effectiveness but these results need to be compared with those of traditional drugs. further large multicenter studies should be conducted in order to test the cost - effectiveness of these herbs for ad and the impact in the control of cognitive deterioration. |
myocardial bridging (mb) is a congenital coronary anomaly that is defined when a segment of a major epicardial coronary artery is tunneled in the myocardium. the incidence of myocardial bridging is elaborated as 1.516% in angiographic studies and may rise up to 80% at autopsy. mb is most commonly located in the middle left anterior descending artery (lad) and the prominent angiographic finding is systolic compression of the involved epicardial coronary artery. although mb is a phenomenon with a benign course, it has also been reported to cause myocardial ischemia, acute coronary syndrome, coronary vasospasm, atrioventricular block, transient ventricular dysfunction, ventricular septal rupture, ventricular tachycardia, and sudden cardiac death. these reports suggest that at least some of the patients with mb may have a predisposition for major cardiac events. it has been postulated that the adverse events in the reported cases are related to the effect of mb on the coronary blood flow. on the other hand, the electrophysiological effects and arrhythmogenic potential of these effects on the coronary flow are not well - described. previous research has shown the importance of detecting myocardial repolarization abnormalities in the prediction of arrhythmogenic potential. qt dispersion (qtd) is defined as the minimum qt interval subtracted from the maximum and cqtd is defined as the qtd corrected for the rate. the recently defined parameters of tp - e interval (defined as the difference between the qt interval and the qt - peak time period) and tp - e / qt index were reported to indicate myocardial repolarization abnormalities better than qtd and cqtd parameters. increased tp - e interval has been associated with cardiovascular mortality and ventricular tachyarrhythmias. in this study, we analyzed the effects of exercise on myocardial repolarization parameters in patients with and without myocardial bridging. patients enrolled in this prospective study were consecutive patients who underwent diagnostic coronary angiography for suspected coronary artery disease (cad) at ondokuz mays university hospital between january 2011 and april 2014. fifty patients who were diagnosed as having isolated mb at coronary angiography (group i) and 48 patients with normal coronary angiograms (group ii) were included in this study. patients with coronary atherosclerosis, those with acute coronary syndromes, left ventricular systolic dysfunction (lvef 0.05) (table 2). no significant differences were observed between the result of exercise testing of the groups (p>0.05). no rhythm abnormalities or hemodynamic deteriorations were detected in the two groups during exercise testing. additionally, the exercise testing yielded a positive result in 9 patients (18%) in the mb group, while it was negative in 41 patients (82%). the changes in the ventricular repolarization parameters of the patients during exercise are shown in table 4. qt max and qt min intervals during peak exercise showed a significant decrease in comparison with the baseline values in the two groups (372.312.1 vs. 327.810.7 ms, p<0.001 ; 335.814.1 vs. 285.110.5 ms, p<0.001 ; 368.311.5 vs. 304.515.1 ms, p<0.001 ; 344.913.2 vs. 275.414.9 ms, p<0.001, respectively). however, qtd and cqtd at peak exercise increased significantly in comparison to baseline values in the mb patient group (36.410.3 vs. 42.614.1 ms, p=0.003 and 39.310.1 vs. 65.416.7 ms, p<0.001 respectively). additionally, significant increases were detected in tp - e, ctp - e intervals and tp - e / qt ratio during peak exercise in comparison to baseline values in patients with mb (695.7 vs. 81.18.4 ms, p<0.001 ; 75.26.6 vs. 94.57.4 ms, p<0.001 ; 0.180.01 vs. 0.200.02, p<0.001, respectively) (figure 2). on the other hand, significant differences in qtd, cqtd, tp - e, ctp - e intervals and tp - e / qt ratio during peak exercise in comparison with baseline values were not detected in the control group (p=0.178, p=0.071, p=0.065, p=0.182, p=0.07, respectively) (table 4). multivariate analysis demonstrated that the length of mb (standardized coefficient=0.446, p<0.001) and percentage of diameter reduction (standardized coefficient=0.510, p<0.001) were independent predictors of a prolonged ctp - e interval in the multivariate stepwise logistic regression model (table 5). standardized coefficient and p values were 0.125 and 0.110 for age, 0.109 and 0.128 for lvmi, respectively. although myocardial bridge phenomenon is a condition which runs a benign course, cases associated with myocardial ischemia, acute coronary syndrome, coronary vasospasm, cardiac arrhythmia, and sudden cardiac death were reported. thus, some patients with mb may be considered at risk for major cardiac events. we detected a significant increase in ventricular repolarization parameters (qtd, cqtd, tp - e, tp - e / qt) in patients with mb in the present study. qt dispersion is a simple and effective marker of electrical heterogeneity in myocardial cells. an increase in this heterogeneity is associated with an increase in the potential for cardiac arrhythmias. several novel repolarization parameters (tp - e and tp - e / qt) were defined in recent studies. these parameters were reported to be more sensitive in detecting cardiac repolarization dispersion than conventional parameters. indeed, an increase in tp - e interval was shown to be associated with long qt syndrome, brugada syndrome, hypertrophic cardiomyopathy, and development of malignant ventricular arrhythmias during the course of myocardial infarctions. the characteristic angiographic finding of myocardial bridging is a narrowing during systole (milking effect). on the other hand, intracoronary ultrasound and doppler studies have shown that coronary obstruction may also involve the diastolic period. exercise and stress may increase the myocardial oxygen demand while shortening the diastolic duration by increasing the heart rate and contractility. for this reason, higher heart rates may be expected to cause a predisposition for ischemia in patients with mb. huang. have detected a reversible perfusion defect in patients with myocardial bridging in 201t1 single photon emission computed tomography after exercise stress testing. in another study, malignant ventricular arrhythmias induced by exercise were reported in patients with mb with evidence for concomitant ischemia. the segment proximal to the region of the myocardial bridge has been associated with atherosclerosis rather than the mb segment itself. previously, the disturbance of blood flow and high wall stress proximal to myocardial bridge have been identified as main contributors for the development of atherosclerosis in the segment proximal to the bridge. in addition, vasoactive agents (endothelin-1, endothelial nitric oxide synthase, angiotensin - converting enzyme) have been shown to be present in higher concentrations in the proximal portion of the mb artery in comparison to the mb segment, further shedding light into the mechanisms of atherosclerosis found in the proximal segment. the mechanical stress caused by systolic narrowing at the mb segment may result in endothelial damage, which in turn may lead to platelet aggregation, coronary vasospasm and eventually acs. indeed, coronary vasospasm has been described in the myocardial bridge artery segment before. as a conclusion, endothelial damage, vasospasm, and atherosclerotic processes developing in the proximal portion of the mb artery segment are among other causes of ischemia that develops in patients with myocardial bridging. myocardial fibrosis and interstitial edema development were shown in the myocardial bridge artery area in a recent histopathological study. similarly, hostiuc. have reported significant myocardial fibrosis and interstitial edema in the mb area in patients who experienced sudden death due to mb. also, death in these patients was attributed to electrical myocardial instability, which had developed in a background of myocardial fibrosis. the close association between prolongation of qt dispersion and development of ventricular arrhythmias was shown, but it may also be a predictor in the diagnosis of coronary artery disease (cad). have shown that a qtd longer than 60 ms during exercise has 70% sensitivity and 95% specificity in diagnosing cad, with a predictive value superior than 1 mm st segment depression. similarly, the association between tp - e interval and tp - e/ qt ratio with myocardial ischemia was also investigated. have shown that tp - e interval measured after a primary percutaneous coronary intervention (ppci) in patients with st elevation myocardial infarction (stemi) is a predictor of death and target vessel revascularization. have detected a significant decrease in qtc, ctp - e intervals and tp - e / qt ratio after successful thrombolysis for stemi. also, a decreased tp - e interval was shown to be a predictor of successful reperfusion in patients undergoing primary pci. in our study, positive ischemia findings were found during peak exercise in 9 patients (18%) in the mb patient group, while no ischemia finding was found in 41 (82%) patients. however, we think that absence of st segment depression in most of our patients during peak exercise did not mean absence of ischemia in these patients in the light of the information mentioned above and considering the inadequate sensitivity and specificity of exercise test in terms of detecting ischemia. although positive ischemia findings were not observed during peak exercise, prolongation in qt dispersion, tp - e interval and tp - e / qt ratio in mb patients might develop as a result of ischemia in the area supplied by the mb artery during peak exercise. hence, in the multivariate analysis we performed, we found a close relation between the length of mb and percentage of diameter reduction which might be markers of the severity of ischemia observed in the area of mb artery and prolonged ctp - e interval and we concluded that the length of mb and percentage of diameter reduction were an independent predictor of prolonged ctp - e interval. based on this information, the increased repolarization dispersion indexes in our patients may both be associated with chronic ischemia and myocardial changes developing in this background. we were not able to interpret the potential prognostic role of the exercise - induced changes of the repolarization indexes in correlation to future adverse events. to determine the predictive value of prolonged tp - e interval and increased tp - e / qt ratio, longer follow - up and large - scale prospective studies in patients with myocardial bridging are needed. we were not able to interpret the potential prognostic role of the exercise - induced changes of the repolarization indexes in correlation to future adverse events. to determine the predictive value of prolonged tp - e interval and increased tp - e / qt ratio, longer follow - up and large - scale prospective studies in patients with myocardial bridging are needed. a significant increase in qtd, cqtd, tp - e, and ctp - e intervals and tp - e / qt ratio during exercise testing was detected in patients with myocardial bridges. the observed increase in ventricular repolarization dispersion indexes may possibly develop on an ischemic background induced by exercise in the mb artery area. | backgroundalthough myocardial bridging (mb) is defined as an angiographic phenomenon with a benign course, it has also been associated with adverse cardiovascular events. the effects of exercise on myocardial repolarization in patients with mb were tested in this study, with tp - e and tp - e / qt repolarization indexes.material/methodsa total of 50 patients in whom isolated mb was diagnosed at coronary angiography (cag) (group i) and 48 patients with normal cag results (group ii) were included in this study. the participants underwent treadmill exercise stress testing according to the bruce protocol. qt dispersion (qtd) was defined as the minimum qt interval subtracted from the maximum. the tp - e interval was defined as the difference between the qt and the qt peak time period. qtd and tp - e intervals were calculated for all patients before and after exercise testing and differences between groups were compared.resultsat peak exercise, qtd and cqtd showed a significant increase in comparison to baseline values in the group of patients with myocardial bridges. significant increases were also found with exercise in the tp - e, ctp - e durations and tp - e / qt ratio of the mb patient group in comparison to the baseline values. on the other hand, significant differences in qtd, cqtd, tp - e, ctp - e intervals, and tp - e / qt ratio during peak exercise in comparison with baseline values were not detected in the control group (p>0.05).conclusionssignificant increases in qtd, cqtd, tp - e and ctp - e intervals and tp - e / qt ratio were detected in the mb patients during exercise testing. |
a methicillin - susceptible staphylococcus aureus with panton valentine leukocidin (pvl) genes was isolated from refractory breast abscesses of 12-year - old girl in japan, and classified into st88, spa - t1245 and coa - iiia. this strain harboured pvl phage sa2usa, which is usually found in st8 community - acquired methicillin - resistant s. aureus clone usa300. |
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although surgeon has a preference for patient positioning, the sitting position has several advantages including the ease of setup, excellent intra - articular visualisation for all types of arthroscopic shoulder procedures, less intra - operative blood loss, a lower incidence of traction neuropathy, and ease of conversion to the open approach if needed. surgeons who prefer the sitting position cite the ability to use general or interscalene brachial plexus block (isbpb) as an advantage. isbpb is possible for patients in the sitting position ; however, it is poorly tolerated in patients in the lateral decubitus position. interscalene brachial plexus block provides effective anaesthesia for most types of shoulder surgeries, including arthroplasty and fracture fixation. when administered by an anaesthesiologist committed to and skilled in the technique, the block has an excellent rate of success and is associated with a relatively low complication rate. interscalene brachial plexus block has several advantages for patients undergoing shoulder surgery : excellent anaesthesia, reduction in both intra - operative and post - operative doses of opiates, delay in the onset of post - operative pain, a shortened post - anaesthesia stay, and increased patient satisfaction. one of the major disadvantages of the sitting position is cardiovascular instability during the shoulder procedure. sudden, profound hypotension and bradycardia events (hbes) have been reported in more than 20% of patients undergoing shoulder arthroscopy in the sitting position. these hbes may be a form of vasovagal syncope mediated by the bezold jarisch reflex (bjr), which happens when venous pooling and increased sympathetic tone cause a low volume, hyper contractile ventricle. this leads to sudden activation of the parasympathetic nervous system and sympathetic withdrawal, causing bradycardia and hypotension. animal studies reported that serotonin (5-hydroxy tryptamine 3 [5-ht3 ]), may be an important contributing factor to the occurrence of bjr in the settings of decreased blood volume, which can be blocked by antagonising the serotonin at the level of the receptors. one previous study also reported that intravenous (iv) ondansetron attenuated the spinal induced hypotension that might be caused by bjr. the present work is a prospective, randomised study, which attempts to assess if blocking of the serotonin receptors by ondansetron (a selective 5-ht3 receptor antagonist) can help in reducing the hbes associated with shoulder arthroscopy done in the sitting position under isbpb. after obtaining institutional review board approval and informed, written consent, 100 american society of anesthesiologists (asa) physical status i or ii, age of 2050 years patients who were undergoing shoulder arthroscopy in the sitting position under isbpb were studied. sample size calculation was done based on a previous study in which the incidence of hbes in control group and study group were 24% and 6%, respectively. it was estimated that a minimum of 50 patients in each group would be required to have a 90% power of detecting a 50% difference in the hbes with 95% confidence interval. patients with a history of coronary artery disease, cardiac conduction defects, uncontrolled diabetes and hypertension, asthma, and those receiving clonidine, beta - blocker or calcium channel - blocker therapy were excluded from the study. patients underwent surgery for a variety of indications including rotator cuff repair, acromioplasty, and labral repair. one hundred patients were randomly assigned to one of the two equal groups to receive either of the following : group c : 10 ml normal saline. all patients received test drug iv over a period of 5 min before starting isbpb. a total volume of 35 ml of local anaesthetic was selected to provide the highest rate of success of interscalene block administered using the nerve stimulation technique. after establishing iv access, non - invasive monitoring of blood pressure (bp), oxygen saturation, and electrocardiogram (ecg) sedation was provided by iv administration of midazolam 1 mg and fentanyl 30 g before the block. after the proper location of the nerve, the local anaesthetic solution was injected in incremental 5 ml boluses with intermittent aspiration. all isbpb were performed by one anaesthesiologist, and all surgeries were performed by the same group of surgeons. after the isbpb, non - invasive bp was measured every 5 min with more frequent measurements if needed, and continuous ecg monitoring. beach chair position, which was achieved by elevating the back of the operating table to 6080 and flexing both the knees and hips to 90 with the patients feet resting properly on a footboard. sensory blockade was evaluated every 5 min by pinprick test and the motor block was tested by asking the patient to raise his arm, to abduct / adduct the thumb, and to flex the forearm on the arm. intra - operatively, midazolam boluses of 0.02 mg / kg and fentanyl 1 g / kg boluses were given if the patient complained of pain. the amount of midazolam and fentanyl administered and the degree of sedation were recorded (sedation score : 1 = awake, 2 = awake but sedated, 3 = asleep but responsive to verbal stimuli, and 4 = asleep but responsive to tactile stimuli). a hbe was defined as ; heart rate decrease of more than 30 bpm in 0.05). side of surgery in both the groups was comparable and non - significant for statistical analysis. to summate, all the demographic characteristics such as age, weight, asa grade, and side of the shoulder operated were comparable in both the groups and were found to be statistically non - significant (p > 0.05) [table 1 ]. there was no difference in sedation score, nil per oral status and iv fluids in both the groups ; p > 0.05, and statistically not significant [table 2 ]. characteristics of groups there were no signs and symptoms suggestive of spinal, epidural or stellate ganglion blockade or intra - vascular injection in two groups. hypotension and bradycardia events occurred in three patients in group t (6.1%), and 11 patients in group c (22.44%) which was significantly higher than in group c ; p - 0.030 [table 3 ]. the onset of these hbes was comparable in the two groups [table 3 ]. there was no significant difference in the block onset time in the two groups ; (21.82 3.6 min), and (22.1 2.5 min) in groups c and t, respectively ; p - 0.186 [table 4 ]. the duration of analgesia was significantly longer in group c (8.1 3.3 h) in comparison with group t (6.3 4.2 h) ; p - 0.035 [table 4 ]. details of hypotension bradycardia events nine patients in group c received ephedrine to treat hypotension, which was significantly higher compared to the other group (three patients in group t) [table 5 ]. one patient in group t and five patients in group c needed atropine to treat bradycardia [table 5 ]. there was no significant difference in the use of intra - operative midazolam or fentanyl [table 5 ]. the study was conducted with the aim to test the hypothesis that iv ondansetron would reduce the incidence of hbes in arthroscopic shoulder surgery in the sitting position after isbpb. activation of the bjr is considered one of the proposed mechanisms for the occurrence of hbes. the bjr is an inhibitory reflex that originates in the cardiac sensory receptors with vagal afferents, which are affected by chemical and mechanical stimuli. this reaction may be related to venous pooling (caused by the sitting position and epinephrine - induced beta 2-adrenergic effect) and increased inotropy (alfa - adrenergic effect of epinephrine). increase in epinephrine levels may result from decreased venous return and carotid baroreceptor stimulation, as well as exogenously from epinephrine administered with the local anaesthetic or in the irrigating solution. a low - volume hypercontractile ventricle causes stimulation of intra - myocardial mechanoreceptors (c fibres), which is followed, in susceptible patients, by an abrupt withdrawal of sympathetic outflow and an increase in vagal tone with resultant bradycardia and hypotension were observed in this setting. hypovolaemia not only causes stimulation of cardiac mechanoreceptors in the left ventricle that triggers the bjr and causes reflex bradycardia, vasodilatation and hypotension, but also results in the activation of thrombocytes to release serotonin which triggers chemoreceptors in the wall of the heart. furthermore, stimulation of 5-ht3 receptors, which are g protein - coupled, ligand - gated, fast - ion channels, increases the activity of the vagal nerve. one previous study in anaesthetised rats reported that bjr induced by serotonin participates in systemic hypotension and bradycardia. this means that bjr can also be directly triggered by the activation of the serotonin 5-ht3 receptors. based on these studies, we assumed that preventing the effect of serotonin by blocking 5-ht3 receptors by iv ondansetron can help in reducing the incidence of the hbes associated with shoulder arthroscopy done in the sitting position under isbpb. our results demonstrated that iv injection of ondansetron 4 mg significantly reduced the incidence of hbes in our patients from 22.44% in the saline group (group c) to 6.1% after injection of 4 mg ondansetron (group t). this 22.44% incidence of hbes in the saline group is consistent with the previous reports of such events. the reduction in hbes in our study after iv ondansetron is similar to a previous study done in rabbits, in which iv administration of granisetron 50 g / kg was efficacious in reducing bradycardia and hypotension associated with bjr. in one more study, it was found that granisetron injection reduced the heart rate fluctuation and hypotension during head - up tilt table tests that are likely related to bjr. and this is consistent with matrinek who concluded that injection of 4 mg ondansetron iv with atropine 0.6 mg could revert asystole during spinal anaesthesia and also with owczuk. who found that 8 mg ondansetron reduced the incidence of bradycardia and hypotension after spinal anaesthesia. the combination of the sitting position, regional anaesthetic technique, an awake patient, and surgical procedure may precipitate vasovagal episodes, which if anticipated are of minor significance, but which in the extreme may lead to cardiac arrest. iv uptake of local anaesthetic can be considered, but time from block placement to the onset of symptoms was 42.5 5.5 min and 44.1 2.7 min in group c and in group t, respectively. this is significantly longer than that which would be expected for peak local anaesthetic uptake from an interscalene brachial (isb) which is approximately 30 min for bupivacaine and < 20 min for lidocaine with or without epinephrine. roth., using mepivacaine 10 mg / kg and tetzlaff., using the same dose of alkalinised mepivacaine for isb reported no episodes of toxicity. the central nervous system symptoms of lightheadedness and nausea were unlike those of local anaesthetic toxicity and promptly resolved after the administration of atropine, glycopyrrolate or ephedrine. extensive spread of local anaesthetic to the epidural or subarachnoid space may cause cardiovascular and respiratory compromise but is rare. previous studies reported interval between positioning and haemodynamic changes ranging between 12 and 24 min but may be delayed to 1 h. our patients had similar sequence of events to those undergoing tilt - table testing and the onset of symptoms was (13.7 2.1 min), (13.3 3.6 min), in groups c and t, respectively. although the onset of analgesia was similar in all groups, the duration of analgesia was shorter in group t in comparison with group c. this can be explained by antagonising the effect of serotonin in controlling pain. although potential mechanisms of this observation were not clear, but animal studies have clarified the anti - nociceptive mechanisms of the descending serotoninergic system at the level of the spinal cord. it hyperpolarises the membrane of substantia gelatinosa neurons, inhibits the excitatory transmitter glutamate release of a - delta and c afferent fibres pre - synaptically and increases the inhibitory transmitters release including aminobutyric acid and glycine from the interneurons. the shortened duration of analgesia observed in our study is consistent with fassoulaki. who reported that iv ondansetron leads to a faster regression of the sensory block after spinal anaesthesia with lidocaine. furthermore, continuous iv administration of ondansetron decreased the analgesic potency of tramadol infusion for post - operative pain. the combination of sitting position, isbpb, and surgical procedure may result in hbes, usually of minor significance if anticipated. in extreme cases we conclude that iv ondansetron 4 mg can reduce the hbes during shoulder arthroscopy in the sitting position under isbpb. | background and aims : sudden, profound hypotension and bradycardia events (hbes) have been reported in more than 20% of patients undergoing shoulder arthroscopy in the sitting position. the present study was designed to know whether intravenous (iv) ondansetron (selective 5-hydroxy tryptamine 3-antagonist) can help in reducing the hbes associated with shoulder arthroscopy performed in sitting position under interscalene brachial plexus block (isbpb).methods : a total of 100 patients (age 2050 years) undergoing shoulder arthroscopy performed in the sitting position under isbpb were assigned randomly to one of the two groups : group c received 10 ml of normal saline and group t received 4 mg of ondansetron diluted in 10 ml of normal saline ` iv. all patients received isbpb using levobupivacaine 0.5%. assessment of motor and sensory blockade, pulse rate, systolic blood pressure, respiration, and side effects were noted every 5 min for first 30 min and every 10 min till the end of surgery. hbes were recorded in both groups.results:iv injection of ondansetron significantly reduces the incidence of hbes from 11 (22.44% in group c) to 3 (6.1% in group t). the duration of analgesia was significantly longer in group c (8.1 3.3) in comparison with group t (6.3 4.2 h).conclusion : we conclude that 4 ml of iv ondansetron can significantly reduce the hbes during shoulder arthroscopy done in the sitting position under isbpb. |
relapse and resultant metastatic spread to distant sites of malignant neoplasms remain the leading cause of mortality associated with cancer [1, 2 ]. the classical metastatic cascade includes intravasation by cancer cells, their circulation in the lymph and blood vascular systems, extravasation, and growth into metastatic foci [3, 4 ]. however, metastasis is considered to be an inefficient process, since only very few cells among the numerous cancer cells in the circulation have the ability to invade and form distant nodules. in the past decade, two different concepts related to solid tumor progression have emerged and been intensively studied to explain these complicated phenomena. there is a growing understanding that epithelial - mesenchymal transition (emt) can contribute to invasive and metastatic tumor growth. this process is considered to ultimately promote cancer cell progression through the basement membrane and invasion into the surrounding microenvironment, such as the lymph and blood vascular systems, contributing to intra- or extravasation [68 ]. on the other hand, there is increasing data to support the hypothesis that most tumors include a minor subpopulation of cells with distinct properties similar to somatic stem cells, which are referred to as cancer stem cells (cscs) or tumor - initiating cells. cscs have been reported to have enhanced tumorigenicity, compared with the majority of tumor cells from the same tumor, and the capacity to generate heterogeneous tumor cell populations [911 ]. initially, these concepts were individually studied ; however, mani. suggested that there may be an association between emt and the gain of csc properties in breast cancer cells in 2008, and another group reported that gene expression patterns of csc - associated pathways were involved in emt. here, we review recent studies of cscs and the relationship with emt and we consider what implications this correlation may have for our ability to explain aspects of tumor progression including distant metastasis. emt was originally defined as a latent embryonic process causing epithelial cells to lose their epithelial behavior and acquire the properties of mesenchymal cells [14, 15 ]. this is a multistep process in which cells obtain molecular alterations that cause dysfunctional cell - cell adhesive interactions, loss of cell - cell junctions, and restructuring of the cytoskeleton ; all of which result in the loss of apical polarity and the acquisition of a more spindle - shaped morphology [1621 ]. for example, in vertebrates, this process facilitates the formation of a three - layered embryo by gastrulation [22, 23 ] and the mesenchymal phenotype allows migration to the proper site for organ formation. in neoplasia, a comparable process is supposed to arise on the tumor front, allowing for cellular invasion and eventual metastatic dissemination of cancer cells [17, 25, 26 ]. emt in tumor progression can be induced by several cytokines and chemokines, including transforming growth factor- (tgf). an increasing constitutive production and release of tgf by tumor cells leads to the activation of the tgf signaling pathway in an autocrine fashion, which results in an emt state with a highly invasive and metastatic phenotype [2830 ]. as significant evidence of emt in vivo, recently, giampieri and colleagues used elegant intravital imaging studies to visualize either collective or single - cell migration of cancer cells. they showed that tgf signaling could drive a switch to single - cell migration without cell - cell attachment and that the mode of migration determined the way the tumor spread. transcriptional repression of e - cadherin is mediated by members of the snail / slug family and twist, and their critical role in emt was confirmed by studies of ablation of snail in vivo. both twist and snail snail even attenuates the cell cycle, rather than promoting proliferation, suggesting that these emt regulators are also convincing metastasis genes [3335 ]. accordingly, activation of these processes in cancer cells significantly increases their metastatic potential, but some argument still exists as to whether emt is a sufficient condition for cancer metastasis. evidence for the role of emt is complicated by the fact that at the secondary site the metastatic cells likely change those cellular phenotype to show heterogeneity, permitting colonization of the distant site. several lines of experimental result have accumulated that indicate mesenchymal - epithelial transition (met) may be important for the latter stages of metastasis, when cancer cells regenerate complex growth that recapitulate the histopathological complexity of the primary tumors from which they arose [36, 37 ]. recent publications have demonstrated that met is essential for the reprogramming of fibroblasts to induced pluripotent stem cells, suggesting the correlation between the reprogramming process in some cases and repression of the emt program. another study has shown that cscs are necessary for homeostasis of metastatic cells that would require the reversion of emt. therefore, in the course of cancer progression, emt may be a transient and reversible process and not only emt but also pluripotent roles in epithelial plasticity may be essential for the establishment of cancer metastasis. the hypothesis that the majority of solid tumors contain a small subpopulation of cells with distinct properties similar to somatic stem cells is supported by both basic and clinical studies. this is a not - so - recent hypothesis that began with the discovery of a cell capable of initiating human acute myeloid leukemia. the population of cells that possessed the potential for self - renewal and differentiation was assumed to be a leukemia stem cell. in 2003, al - hajj and colleagues for the first time isolated a cd44/cd24) subpopulation of breast cancer cells and showed that the cells in this population produced tumors in a xenograft model more effectively than did the majority population of tumor cells. cd44/cd24cells were subsequently designated as cscs, and this discovery has enhanced investigation of cscs as the metastatic component of cancer, especially in breast cancer. conceptually, cscs must be engaged in the metastatic process if they are the only subset of cells capable of initiating new tumor growth. if that is the case, metastatic cells in the circulation should have the tumorigenic capacity necessary to induce tumor initiation at a distant metastatic site. this hypothesis was partly supported by reports showing that cd44/cd24 status of primary tumor was significantly correlated with distant - metastatic - free survival [41, 42 ], although the clinical relevance of the cd44/cd24 phenotype in primary breast cancer is still controversial. furthermore, cd44/cd24 breast cancer cells expressed high levels of genes related to metastasis and induced lung metastasis in vivo. in addition, analysis of genetic profiles confirmed that cd44 breast cancer cells are enriched with stem - cell markers and display activated tgf signaling and poor clinical outcomes. the expression of aldehyde dehydrogenase 1 (aldh1) has also been shown to be a csc marker, and cscs have been isolated from primary breast cancer using an aldefluor assay. a recent study demonstrated that aldh1 expression can be an independent prognostic factor for predicting metastasis in inflammatory breast cancer and that cscs have the ability to reconstitute the heterogeneity of the primary tumor at the metastatic site. aldh1 was also identified as a predictor of poor prognosis in lung and bladder cancer [47, 48 ]. in the small and large intestine, leucine - rich repeat - containing g protein - coupled receptor 5 (lgr5) was identified as a marker for stem cells and deletion of apc in lgr5-expressing cells induced their transformation within days, suggesting that lgr5 might be a limited population of cscs. from a clinical point of view, we recently showed that lgr5 was markedly overexpressed in the majority of advanced colorectal cancers (crcs) compared with normal mucosal tissue. this lgr5 expression, which was variable among crc cases, correlated significantly with lymph node metastasis, suggesting the involvement of lgr5 in the metastatic process. several studies have indicated that emt inducers can make cancer cells become more tumorigenic, giving rise to the hypothesis that tumor cells can transiently acquire stem cell - like properties as a consequence of emt. mani and colleagues performed in vitro - based experiments and reported that the induction of emt in human mammary epithelial cells resulted in the acquisition of mesenchymal morphology and the expression of mesenchymal markers. this phenotypic emt change increased the cd44/cd24 subpopulation, which exhibited the properties of stem cells, such as enhanced mammosphere - forming ability and differentiation into myoepithelial or luminal epithelial cells. they also demonstrated that transformed human mammary epithelial cells showed effective tumor - initiating ability with induction of emt. in that report, induction of emt that resulted in the enrichment of cscs was conducted by activation of the tgf pathway, as well as by ectopic expression of the transcription factors snail, slug, and twist. generally, activation of the tgf pathway inhibits tumorigenesis ; however, it is also known that the tgf pathway cooperates with other pathways to assist tumorigenesis in the malignant state [52, 53 ]. genetic signatures that predict poorer prognosis for primary breast cancer patients have been examined by comparing the gene expression profiles of cd44/cd24 cell populations with other populations [42, 44 ]. following up on the above observations on the tgf pathway, one of these studies, which performed sage profiling of cd44/cd24 and cd44/cd24 cell populations from breast cancer tissue, found expression of tgf targets, such as vimentine, ctgf, serpine1, sparc, and tgfbr2, implying tgf pathways seemed to be activated in these cells. another recent study also has shown that gene expression signature of human mammary epithelial cell line introduced emt inducers including tgf strongly correlated with the signature derived from basal b cell lines of which subtype is characterized by a stem cell - like expression profile. to induce complete emt, tgf works together with the wnt, hedgehog, notch, and ras signaling pathways, which are involved in the induction and maintenance of stem cell niches. the canonical wnt pathway, known to be a critical regulator of self - renewal in stem cell niches, is also implicated in the induction of emt in cancer and is constitutively activated in colon, skin, and hematopoietic cancers [5658 ]. in contrast, tang. reported that tgf inhibition increased the size of the csc population and promoted tumorigenesis by a mechanism that was independent of direct effects on proliferation. in that study, which used transformed human breast epithelial cells, tgf stimulation resulted in the loss of stem cell - like properties such as ability to form mammospheres. thus, further studies are needed to clarify these contradictory results on the role of tgf signaling in the regulation of tumor - initiating property and emt. identification of detectable circulating tumor cells (ctcs) in the peripheral blood of patients with solid tumors has been intensively studied for a decade, since ctcs may provide proof of principle for early primary cancer cell metastasis through the vascular network [6062 ]. several lines of evidence suggest that the finding of ctcs in the course of therapy possesses a consistent prognostic significance and is regarded as a predictive tool for response to treatments in cancer therapy [63, 64 ]. in this context, a recent study conducted in a cohort of 226 blood samples from 39 patients revealed that the majority of ctcs from metastatic breast cancer patients had emt and csc characteristics. in that study, ctcs were found in 69 of 226 (31%) blood samples taken from patients with metastatic breast cancer to investigate the expression of emt markers (twist, akt2, and pi3k) and a stem cell marker (aldh1). in the ctc - positive group, 62% were positive for the emt markers and 69% for aldh1, while in the ctc - negative group the percentages were 7% and 14%, respectively. ctcs also showed reduced expression of epithelial - specific cytokeratins. in disseminated tumor cells (dtcs), twist, which is known as an emt inducer, was overexpressed and the presence of twist - positive cells in the bone marrow prior to chemotherapy has been significantly associated with relapse. these results indicated that ctcs (or dtcs) might often exhibit characteristics of both emt and cscs, emphasizing their role in the formation of metastases. therefore, further studies focused on identifying the features of cancer cells that have escaped from the primary tumor may allow the discovery of the critical mechanisms underlying cancer metastasis and recurrence. one possible mechanism underlying chemoresistance is thought to arise from metabolic quiescence, a high level of expression of anti - apoptotic proteins [68, 69 ]. another is the high expression of atp - binding cassette (abc) family genes, such as mdr1 and bcrp1, which could catalyze the efflux of a range of structurally unrelated anticancer drugs [7072 ]. this property is shared by a subpopulation of normal stem cells known as side population (sp) cells. sp cells have been detected in several solid tumors, including breast, lung, and gastrointestinal cancers and have been shown to have the capacity for tumor initiation and self - renewal [71, 7375 ]. therefore, the identification of sp cells by flow cytometry is a promising method for the extraction of csc populations in solid tumors. induction of emt also contributes to the decreased efficacy of chemotherapy in breast, colorectal, and ovarian cancer. introduction of twist into breast cancer cells induces paclitaxel resistance and emt, as well as akt2 expression, which was amplified in breast cancer with acquired paclitaxel resistance. a detailed characterization of cell systems reveals that mesenchymal derivatives of nonsmall cell lung cancer cells display an attribute of resistance to egfr kinase inhibitor. creighton and colleagues reported in 2009 that a gene expression signature common to both cd44/cd24 and mammosphere - forming cells was found mainly in human breast cancer of the recently identified claudin - low molecular subtype, which is characterized by expression of many emt - associated genes. they also demonstrated that residual breast cancer cell populations after conventional chemotherapy may be enriched for subpopulations of cells with both cscs and mesenchymal features. consistently, cscs, isolated using cd44/cd24 from human breast cancer, demonstrated resistance against the chemotherapeutic agents and the proportion of cd44/cd24 cells increased in breast cancer patients after treatment with anticancer drugs, including docetaxel, doxorubicin, and cyclophosphamide. of the 1% of cells that survived chemotherapy using paclitaxel or 5-fluorouracil, 3035% of them were cd44/cd24, indicating selection of this population. on the basis of the above relationship between emt and cscs, using two populations of human mammary epithelial (hmle) cells one that had been induced emt by knocking down of e - cadherin and one which had not gupta and colleagues screened a collection of about 16,000 compounds to find one showing selective toxicity towards the cells that had undergone emt. as the result of the screening, salinomycin was selected for further studies and was also discovered to suppress the proportion of cscs that occur naturally as a subpopulation of breast cancer cells. pre - treatment with paclitaxel was found to increase the tumor initiation ability more than 100 folds compared with pre - treatment of salinomycin. these results point to the potential mechanisms of chemoresistance that allow csc population increased by emt to persistently survive and that may be responsible for recurrence in the primary or distant site following cancer treatment by chemotherapy after the majority of the cancer cells are killed., these studies have led to the possibility that resistance to chemotherapy may be associated with the common characteristics of emt and tumor - initiation abilities. according to recent findings in this review, emt may be a critical process underlying the subpopulation of cancer cells, cscs, that are responsible for tumor initiation at metastatic sites and for regenerating the tumor after initial tumor regression by chemotherapy. this hypothesis may provide insight into current questions about the specific role of emt in tumor metastasis. therefore, identification of cscs as specifically significant characteristics of tumor malignancy facilitated by emt can help to provide an answer for this critical issue. | epithelial - mesenchymal transition (emt) is a multistep process in which cells acquire molecular alterations such as loss of cell - cell junctions and restructuring of the cytoskeleton. there is an increasing understanding that this process may promote breast cancer progression through promotion of invasive and metastatic tumor growth. recent observations imply that there may be a cross - talk between emt and cancer stem cell properties, leading to enhanced tumorigenicity and the capacity to generate heterogeneous tumor cell populations. here, we review the experimental and clinical evidence for the involvement of emt in cancer stem cell theory, focusing on the common characteristics of this phenomenon. |
atopic dermatitis (ad) is a chronic skin disease characterized by pruritus and eczematous skin lesions, and is often associated with other atopic disorders, such as allergic rhinitis and asthma. ad is a widely prevalent disease, as it affects 5% to 15% of children and accounts for 2% to 4% of adults. patients with ad have lower health - related quality of life compared with the general population, and increasing disease severity is related to greater impairment of life quality. the resultant psychological distress is one of the common comorbidities linked to ad, and could negatively affect quality of life. the association between ad and mental health comorbidities in children and adolescents has been well established. recent large population - based studies have demonstrated that ad in children and adolescents is associated with mental health disorders including attention deficit hyperactivity disorder, depression, anxiety, conduct disorder, and autism. additionally, there is a dose - dependent relationship between ad severity and mental health problems. in a prospective cohort study in germany, children with infant - onset eczema were at increased risk for mental health problems in their childhood. suicidal ideation is also more prevalent in adolescents with ad compared with healthy individuals, and both are significantly associated in a multivariate model. in a few studies that use adult samples, it has been reported that patients with ad have higher levels of depression and anxiety than controls. however, these studies have some inherent limitations such as small sample sizes, lack of controlling for potential confounders, or not considering ad severity. a study using a large general population found, after multivariate analyses, that somatization considerably contributes to the association between depression / anxiety and eczema. however, the authors in that study did not separate eczema into atopic and nonatopic, and were unable to show solely the effects of ad. despite mental health problems in a young person potentially leading to many adverse outcomes, including significant impairments in work, education, and social interaction, there are no studies investigating the associations between ad and psychological distress in a large population of young adults. therefore, the aim of the present study was to perform a comprehensive evaluation using a substantial amount of conscription data to examine psychological distress in young adults with ad. in south korea, it is mandatory for all men around 19 years of age to be examined for military conscription by the military manpower administration. each conscript is interviewed and examined to ascertain whether he has physical or mental problems that would impede military service. a conscript with such problems is required to submit medical records from physicians at clinics or hospitals. presence and severity of a problem are determined by the conscription officers, medical specialists, through medical records, face - to - face interviews, and medical work - ups. the present study was conducted using secondary data obtained from a regional military manpower administration between february 1, 2008 and december 31, 2012, therefore institutional review board approval was not needed. the data included information on demographics and medical conditions of conscripts residing in incheon, which is a metropolitan area with a population of approximately 3 million. the present study was conducted with approval from the military manpower administration committee in incheon, south korea. a diagnosis of ad was made clinically and was based on historical and clinical features, such as personal or family history of atopy, morphology and distribution of eczematous skin lesions, and associated clinical signs. a conscription officer in dermatology confirmed ad presence through a medical certificate and physical examination ; ad severity was measured using the eczema area and severity index. the eczema area and severity index involves an assessment of disease extent in 4 body regions (head / neck, upper limbs, trunk, and lower limbs) plus an evaluation of the intensity of a representative area of eczema (erythema, induration / papulation / edema, excoriation, and lichenification). the ad severity was divided into mild and moderate - to - severe. the mental problems of conscripts were evaluated by a psychiatric officer using examinees medical records, an interview, and the korean military multiphasic personality inventory (kmpi). the kmpi is a revised version of the conventional minnesota multiphasic personality inventory, adjusted for korean conscription. neurotic symptoms, psychotic symptoms, suicidal ideation, and other psychiatric symptoms are analyzed using the kmpi ; each scale 's scores are transformed into standardized t - scores. psychological distress included depression, anxiety, and somatization in the neurosis set of the kmpi. since the marked elevation of the t - score implies higher tendency to manifest psychological or psychiatric symptoms compared with the normal range of the score, we determined that an individual had psychological distress if they had a t - score > 65. socio - demographic characteristics such as age, level of education, economic status, and family structure were investigated using a self - administered questionnaire. level of education was categorized into 3 groups according to the duration of education : 6 years of education (elementary school or lower) ; 7 to 12 years (middle or high school) ; and 13 years (college or higher). household economic status was categorized into 5 groups : very low, low, middle, high, and very high. family structure was divided into 3 types by the number of parents residing with the conscripts : nonparent, single parent, and dual parent family. univariate analyses for socio - demographic characteristics of conscripts were performed by independent t test and chi - square test. the differences in the proportion of depression, anxiety, and somatization by ad status and univariate associations were tested using a chi - square test. the linear trends of each psychological distress according to ad severity were analyzed by the cochran - armitage trend test. the multivariate associations between ad status and each type of psychological distress were investigated using logistic regression models. to adjust for potential confounders, covariates such as age, level of education, economic status, and family structure were included in the models. considering that depression, anxiety, and somatization might be associated with each other, we also included the type of psychological distress other than the targeted one as covariates in the models to evaluate whether each type of psychological distress is associated with ad independently of the others. statistical analyses were performed using sas version 9.4 (sas institute, cary, nc). the results were reported as odds ratios (ors), 95% confidence intervals (cis), and p values. a p value of 65. socio - demographic characteristics such as age, level of education, economic status, and family structure were investigated using a self - administered questionnaire. level of education was categorized into 3 groups according to the duration of education : 6 years of education (elementary school or lower) ; 7 to 12 years (middle or high school) ; and 13 years (college or higher). household economic status was categorized into 5 groups : very low, low, middle, high, and very high. family structure was divided into 3 types by the number of parents residing with the conscripts : nonparent, single parent, and dual parent family. univariate analyses for socio - demographic characteristics of conscripts were performed by independent t test and chi - square test. the differences in the proportion of depression, anxiety, and somatization by ad status and univariate associations were tested using a chi - square test. the linear trends of each psychological distress according to ad severity were analyzed by the cochran - armitage trend test. the multivariate associations between ad status and each type of psychological distress were investigated using logistic regression models. to adjust for potential confounders, covariates such as age, level of education, economic status, and family structure might be associated with each other, we also included the type of psychological distress other than the targeted one as covariates in the models to evaluate whether each type of psychological distress is associated with ad independently of the others. statistical analyses were performed using sas version 9.4 (sas institute, cary, nc). the results were reported as odds ratios (ors), 95% confidence intervals (cis), and p values. a p value of < 0.05 was considered significant. among the 120,841 conscripts examined during the study period, 120,508 were included in the analyses. we excluded 333 conscripts from further analysis due to incomplete data in either the kmpi or socio - demographic characteristics. socio - demographic characteristics of the ad population and overall population are shown in table 1. the ad population was younger than the non - ad population (19.8 1.0 and 20.0 1.2, p < 0.001). when compared with the non - ad population, the ad population showed a significantly higher proportion in the categories of low education level (12 years of education) and upper economic classes (high plus very high). characteristics of the study population table 2 shows the differences in the prevalence of psychological distress according to ad presence. the proportion of individuals with at least 1 type of psychological distress was higher in the ad population than in the non - ad population (18.7% and 8.9%, respectively, p < 0.001). the prevalence of each type of psychological distress such as depression, anxiety, and somatization was significantly higher in the ad population. we also found significant relationships between ad and depression (or 2.07, 95% ci 1.752.45), anxiety (or 1.88, 95% ci 1.582.23), and somatization (or 2.09, 95% ci 1.782.45) in the univariate analyses. prevalence of psychological distress according to the presence of atopic dermatitis we then examined the prevalence of depression, anxiety, and somatization according to ad severity category. the prevalence of each type of psychological distress showed significant differences for each ad severity category (table 3). depression (12.0%), anxiety (10.0%), and somatization (12.6%) were most prevalent in individuals with moderate - to - severe ad. there were also linear trends showing that the prevalence of psychological distresses increased with ad severity. prevalence of psychological distress according to the severity of atopic dermatitis table 4 reports the associations between ad and psychological distress according to ad status in the logistic regression models adjusting for potential confounders. depression, anxiety, and somatization showed significant associations with overall ad independently of one another, irrespective of severity (or 1.79, 95% ci 1.402.29, 1.38, 1.081.76, and 1.75, 1.402.20, respectively). after stratifying ad by severity, depression and somatization were still significantly associated with both mild and moderate - to - severe ad. we found that moderate - to - severe ad was related to depression and somatization to a greater extent than mild ad. in the case of anxiety, there was a significant association with mild ad (or 1.42, 95% ci 1.091.84) but not with moderate - to - severe ad (or 1.19, 95% ci 0.622.28). the present study was designed to investigate comprehensively whether psychological distress was related to ad in a large population of young adult males. we found that individuals with ad had an increased prevalence of depression, anxiety, and somatization compared with those without ad. a patient with more severe ad showed a tendency of the higher probability to have such psychological distresses. depression, anxiety, and somatization were significantly and independently associated with ad presence even after controlling for potential confounders. moreover, the associations between ad and depression / somatization correlated with ad severity. there are a few epidemiological studies on relationships between ad and psychological distress in adults. some studies reported that patients with ad have more marked depression than healthy adults did, and that patients with more severe ad are more depressive. yang investigated the association between ad and depression, and the modifying effects of smoking and serum total immunoglobulin e level on the association. they found that the association between ad and depression remained significant after adjusting for the possible modifiers, suggesting that the association is strongly independent of those factors. in terms of anxiety, it has been demonstrated that patients with ad are more likely to be anxious than healthy individuals. however, a previous study reported a nonsignificant difference in the anxiety scale between the ad group and normal controls, and other studies showed no significant correlation between ad severity and anxiety. these discrepancies in the findings on anxiety may result from the cross - sectional study design and complex relationship as determined by a variety of factors, not only ad severity. for these reasons, the results of our study could have a nonsignificant association between anxiety and moderate - to - severe ad. in a population - based study, the association between somatization and nonspecific eczema, which included atopic and nonatopic eczema, was strong and followed a dose - response pattern. the study also found that nonspecific eczema - somatization association is robust for adjustments for depression and anxiety while the associations of nonspecific eczema with depression and anxiety are not significant after adjusting for somatization. these findings are inconsistent with the results of the present study that both depression and anxiety were significantly associated with ad in the models controlling for somatization. this may come from the difference of the characteristics of study subjects such as age group, gender, and the type of dermatitis included in the analyses. the altered immunological responses in ad might be 1 mechanism through which patients with ad often accompany psychological distress. brain barrier directly via active transport or indirectly via vagal nerve stimulation, which facilitates bidirectional communication between the central nervous system and peripheral immune system. therefore, the shared inflammatory mediators might result in the comorbidity of psychological distress in ad patients. pruritus, a major symptom of ad, might be a factor mediating the vicious cycle of ad in individuals with psychological distress. depressed patients often have an elevated level of central corticotrophin - releasing factor, which might decrease the threshold for itching. a recent experimental study showed that depressed patients with ad report higher induced itch than ad patients without depression. moreover, elevated anxiety in ad patients could increase the conditioning of a scratch response to an itching stimulus. the present study, to our knowledge, is the first attempt to comprehensively investigate the relationships between ad and types of psychological distress in young adults using a large dataset. using data from a large population, we could avoid selection biases common in hospital samples, and compare the strengths of potentially weak associations. in the present study, medical specialists evaluated ad and psychological distress based on information such as a conscript 's medical record, interview, physical examination, and validated psychological test, whereas several previous studies have obtained data via self - report. we evaluated the independent effects of depression, anxiety, and somatization by adjusting for the potential confounding effects between each type of psychological distress. most important are the study 's cross - sectional design and chosen study population. using cross - sectional data, a temporal relationship between ad and the development of psychological distress was unable to be established, thereby limiting the interpretation of our findings regarding causality. data used were also confined to young adult males ; this leads to a lack of generalizability of our findings. in a study of the general population, anxiety and depression moreover, a previous study of adolescents aged 17 to 19 years reported that girls with ad do have an increased risk of psychological distress, but with a weaker association than that noted in boys. we can not rule out the underreporting of ad. since a conscription officer performed additional examinations for only conscripts who submitted their past medical records, ad patients could be misclassified in the healthy population if they did not report their disease. despite potential bias weakening the associations, our results were still significant and strong. moreover, the ad prevalence in our study was similar to the prevalence reported in a study of danish conscripts. however, since both the ad and control groups were under the same potential distress during conscription, it is unlikely that this factor is responsible for our observed associations. finally, we included potential confounders such as age, level of education, and economic status in the models nevertheless, residual confounding can not be excluded completely. however, this limitation has probably not produced the main findings of the present study. prospective cohort studies using a general population should be performed in the future to provide additional evidence regarding the causal relationship between ad and psychological distress. in conclusion, the results of our study reveal that depression, anxiety, and somatization are strikingly and independently associated with ad in young adult males. our findings suggest that early treatment of skin inflammation might modify the risk of psychiatric problems, and psychological consultations are needed to prevent the aggravation of ad symptoms. | abstractthe relationship between atopic dermatitis (ad) and psychological distress has been well established for children and adolescents. however, it is unclear whether this relationship exists in young adults. this study aimed to assess the relationship between ad and psychological distress in young male adults in south korea.a cross - sectional study was conducted using regional conscription data from 2008 to 2012. a dermatologist diagnosed ad based on historical and clinical features, and determined severity using the eczema area and severity index. a psychiatrist used medical records, an interview, and a psychological test to examine psychological distress (depression, anxiety, and somatization). the relationship between psychological distress and ad was assessed by multivariate logistic regression analyses.among the 120,508 conscripts, 1517 (1.2%) presented with ad. the odds of having each type of psychological distress were significantly greater for individuals with ad compared with those without ad. the adjusted odds ratios for depression, anxiety, and somatization were 1.79 (95% ci 1.402.29), 1.38 (95% ci 1.081.76), and 1.75 (95% ci 1.402.20), respectively. moderate - to - severe ad was significantly related to depression and somatization to a greater extent compared with mild ad.depression, anxiety, and somatization are strongly and independently associated with ad in young adult males. early treatment of skin inflammation might modify the risk of psychiatric problems. prospective cohort studies are needed to verify causal relationships. |
chondrogenesis is the earliest phase of skeletal development, involving mesenchymal cell recruitment and migration, condensation of progenitors, and chondrocyte differentiation and maturation. mutations or deregulation of these processes lead to skeletal malformation, skeletal malfunction, and/or susceptibility to injury. each step of the chondrocytic differentiation process is characterized by specific morphological change and gene expression profiles. several cytokines, growth factors, extracellular matrix (ecm) components, and transcription factors have important roles in these processes. two groups of transcription factors have been identified that control the key steps of chondrocyte differentiation : the sox and runx families. the sox trio of transcription factors (sox9/sox5/sox6) has important regulatory functions in the early stages of chondrogenesis. sox5 and sox6 are coexpressed with sox9 in pre - chondrocytes and proliferating chondrocytes, and the three sox proteins cooperate with each other to directly activate type ii collagen (col2a1) and aggrecan in vitro. sox9-null mutant cells in mouse embryo chimera are excluded from mesenchymal condensations and fail to express col2a1 and other chondrocyte - specific markers (col9a2, col11a2, and aggrecan), indicating that sox9 is required for mesenchymal condensation and subsequent cartilage formation. sox5 and sox6 have essential but somewhat redundant roles in promoting chondrogenesis, and double - null mice fetuses die at embryonic day 16.5 (e16.5) from heart failure with severe chondrodysplasia. runx2 is mainly expressed when chondrocytes are about to become pre - hypertrophic, and it remains expressed through hypertrophy and terminal differentiation. runx2-null mice have abnormal hypertrophic chondrocyte differentiation in addition to failure of bone formation. however, there is failure of terminal differentiation only in the humerus and femur and delayed terminal differentiation in other long bones, including tibia, fibula, radius, and ulna. the transcription factor c - maf is specifically expressed in late hypertrophic and terminal chondrocytes, making it a candidate for controlling terminal differentiation. in addition, the expression of c - maf and lc - maf, an mrna splicing variant of c - maf, partially overlaps with the expression of both sox9 and runx2. the founding member of maf family, v - maf (musculoaponeurotic fibrosarcoma), was originally identified as a retroviral oncoprotein, and then cellular counterparts, c - mafs, were cloned from a number of vertebrate genomes. the maf family is currently subdivided into two groups, small maf (maff, mafg, mafk, maft, and mafs) and large maf (c - maf, mafa, mafb, and nrl [neural retina leucine zipper ]) proteins based on their domain structure and function (table 1). a transcript variant of c - maf, lc - maf, also has been reported (fig. the large maf proteins contain a conserved n - terminal transactivation domain, whereas the small maf proteins lack this transactivation domain. based on the strong sequence conservation, the members of the maf family are classified under the bzip transcription factor superfamily. binding partners of mafs homo- and heterodimers form between mafs and other bzip family proteins. c - maf and its rna splice variant lc - maf (long form c - maf) contain the same 5 untranslated region. c - maf is transcribed from a single exon, and through mrna splicing, lc - maf has a frame shift that alters the last amino acid of c - maf exon 1 and codes for an additional 10 amino acids (in mouse) or 30 amino acids (in human). the bzip transcription factor superfamily consists of 53 different proteins in humans and has been subdivided into several subgroups, including the hallmark ap-1 complex (c - fos / c - jun), creb / atf, cnc, c / ebp, par, and maf families. bzip transcription factors are activated by a variety of signal transduction pathways to mediate critical aspects of cellular differentiation. the bzip structural motif is a long bipartite -helix of 60 to 80 amino acids in length and comprises a basic region and a leucine zipper. the n - terminal basic region contains an 18amino acid basic peptide responsible for specific dna recognition, whereas the c - terminal leucine zipper domain mediates homo- and heterodimerization with other bzip proteins. the maf family members are unique among the bzip superfamily in that they contain a highly conserved ancillary dna binding domain n - terminal to the typical basic domain (fig. 1), which increases the number of specific dna sequences that can be bound. through this extended dna binding domain, maf proteins bind to relatively long consensus dna sequences termed maf - recognition elements (mares). early in vitro selection studies identified two 13 and 14base pair palindromic sequences, tgctgactcagca (t - mare) and tgctgacgtcagca (c - mare), as the optimal binding sites for c - maf. the t - mare contains a phorbol 12-o - tetradecanoate (tpa)response element (tre : tgactca), and the c - mare contains a cyclic amp - responsive element (cre : tgacgtca) in the middle region, respectively. however, in vitro studies have shown that some base mismatches from the consensus sequences are allowed in maf binding to the mare and that maf homodimers also bind efficiently to mare half - palindromic sites when they are preceded by an at - rich sequence. naturally occurring c - maf binding sites in il-4, crystallin, and other genes are not intact palindromic sequences, as the full - consensus sequences are present only in the 5 half - region. therefore, although there is a degree of specificity in the maf binding to the target dna sequences, there is also considerable flexibility. in the developing mouse embryo, c - maf is strongly expressed in the lens fiber cells, and studies have focused on c - maf s function in lens development. expression of c - maf and mafb was detected at the rna level in limb and rib cartilage as well as eye lens and spinal cord of the e15 rat embryo. the analysis of postnatal rat femur epiphysis in the same study showed that c - maf is expressed strongly in the hypertrophic chondrocytes but low in immature proliferating chondrocytes. a detailed localization study in mouse embryonic cartilage showed that c - maf is expressed mostly in late hypertrophic and terminally differentiated chondrocytes with a maximal expression at e15.5-e16.5 and at low levels in the perichondrium and in the primary spongiosa. on the other hand, lc - maf lc - maf expression is detected only in the perichondrium, with no expression in differentiated chondrocytes. although the differential expression patterns of c - maf and lc - maf during chondrogenesis were described, the functional differences between c - maf and lc - maf are still unknown. thus, the regulation mechanisms that control temporal - spatial expression of the different c - maf isoforms need to be further investigated. the prominent phenotype of the c - maf - null mice is defective lens fiber cell differentiation. direct transactivation of lens - specific crystallin genes by c - maf has been related to this phenotype. although c - maf (+ /) heterozygous mice have normal lenses, naturally occurring heterozygous point mutations in c - maf cause mild cataracts in both mice and humans. these mutations in the basic domain may have a dominant negative effect on wild - type c - maf protein. whether these mutations are related to abnormal cartilage formation it is interesting to note that lens and cartilage share similar gene expression profiles, especially genes involved in ecm such as collagens, other noncollagenous ecm proteins, and matrix metalloproteinases (mmps). the role of c - maf in cartilage was examined in endochondral bone development in mice lacking c - maf. c - maf - null mice have abnormal terminal differentiation of hypertrophic chondrocytes with the most dramatic abnormality at e15.5 to e16.5, which corresponds to the time points of the highest c - maf expression in chondrocytes. terminal differentiation of the hypertrophic chondrocytes is delayed, and the domain of late hypertrophic chondrocytes is expanded in both the e16.5 to e18.5 embryos and 4-week postnatal c - maf - null mice. the expanded hypertrophic zone is associated with delayed vascular invasion and reduced ossification. this suggests that c - maf facilitates both the initiation and the completion of terminal differentiation in chondrocytes. the maf proteins dimerize with themselves and with other bzip proteins through the leucine zipper domain. the maf binding partners that have been identified so far are summarized in table 1, and the various types of c - maf binding interactions are illustrated diagrammatically in figure 2. there is specificity in maf dimerization, and the different binding interactions affect the transactivation function of the maf proteins. however, the circumstances that determine the binding of maf to its partners are not fully understood. the small maf proteins, which do not dimerize with large mafs, generally repress transcription when they form homodimers. in contrast, when they form heterodimers with other members of bzip proteins, they can activate or repress transcription of genes regulating many aspects of cellular differentiation. a heterodimeric partner of small maf, the transcription factor nuclear factor e2 p45-related factor-2 (nrf2), displays decreased expression during chondrocyte differentiation and inhibits numerous aspects of chondrocyte differentiation in vitro. this suggests the possibility that small maf / nrf2 heterodimers may be involved in the negative regulation of chondrocyte differentiation, although no expression data for small mafs are available in chondrocytes. they also have the ability to form heterodimers with ap-1 family members in vitro, although heterodimers have not yet been detected in vivo. in vitro, c - maf and nrl can form heterodimers with c - fos and c - jun. mafa can heterodimerize with c - jun but not with c - fos, whereas mafb can form heterodimers with c - fos but not with c - jun. considering that maf proteins are expressed in a wide variety of cells and tissues, it has been suggested that mixing and matching of dimeric partners might create specific complexes within cells to affect target gene expression and cell differentiation. c - maf can form homodimers as well as heterodimers with other bzip transcription factors. in addition, several factors, including general transcription factors (tbp), cofactors (cbp), and specific transcription factors (sox9), interact with c - maf. mafs can also physically and/or functionally interact with several non - bzip proteins, including general transcription factors, common coactivators, and specific transcription factors (fig. nrl directly interacts with tata binding protein (tbp) in vitro, and tbp is co - immunoprecipitated with the nrl-, c - maf- and mafa - containing complexes in vivo. c - maf recruits the creb binding protein (cbp) and p300 to the promoters of crystallin genes, resulting in the synergistic activation of the genes. this synergistic activation requires histone acetyltransfease activity, indicating that chromatin remodeling is involved in c - maf transactivation. synergy in transcriptional activation by c - maf and other specific transcription factors was also observed. c - maf acts synergistically with sox1, sox2, and sox3, which are important regulators of lens development, to activate the f - crystallin gene. similarly, lc - maf acts synergistically with sox9 to activate the col2a1 and type xxvii collagen (col27a1) gene transcription in chondrocytes. although maf was first identified as a retroviral oncoprotein, the physiological functions of maf family proteins include regulatory roles in a wide variety of cell- and tissue - specific gene expression and in cell differentiation during development. the target genes of maf transcription factors in tissues are well summarized (see review by kataoka). this section focuses on c - maf targets in chondrocytes, summarizing various lines of evidence that c - maf regulates genes related to chondrocyte differentiation and the progression of osteoarthritis. this growing list of genes includes matrix metalloproteinase-13 (mmp-13), connective tissue growth factor (ctgf), col2a1, and col27a1 (fig. schematics of c - maf target genes in chondrocytes and possible pathways regulating c - maf regulation. fgf / mapk / erk pathway may activate and affect the stability of c - maf, based on homology with interactions affecting mafa. some interactions are proposed based on homology of the phosphorylation / ubiquitination sites between mafa and c - maf. these interactions still need to be verified for c - maf, as clarified in the text. mmp-13, along with other matrix metalloproteinases, affects hypertrophic differentiation and vascular invasion through matrix proteolysis. mmp-13 is induced in terminally differentiated chondrocytes in the developing growth plate and is highly upregulated in osteoarthritic cartilage. mmp-13 expression in the hypertrophic chondrocytes region was decreased specifically in the c - maf - null mice embryo, suggesting a regulatory role for c - maf in mmp-13 expression. this is supported by a study of c - maf activity on the mmp-13 proximal promoter. however, there is the possibility that c - maf indirectly regulates mmp-13 expression through the activation or repression of other factors, and further molecular and biological examination will be required. ctgf and c - maf are co - localized in hypertrophic chondrocytes, and interestingly, c - maf - null and ctgf - null mice share common phenotypes, including expanded hypertrophic zones in the e16.5 embryo and reduced ossification of the tibia. ctgf is expressed by proliferating and hypertrophic chondrocytes in the mouse growth plate, and it was identified as a c - maf - responsive gene in chondrocytes. in vitro studies using reporter transfection assays have shown that c - maf and lc - maf activate the ctgf promoter, and chromatin immunoprecipitation assays demonstrated a direct interaction between the ctgf promoter and maf. col2a1 was also upregulated by lc - maf via direct interactions with the intronic enhancer. lc - maf is coexpressed with sox9 and col2a1 in the precartilaginous mesenchymal condensations during mouse embryogenesis, suggesting a role for lc - maf in early chondrocyte differentiation. the same study identified an lc - maf binding site adjacent to the well - known sox9 binding site, although this site does not contain the typical mare consensus sequence. the timing of in vivo expression of the sox proteins and lc - maf during chondrogenesis led to the hypothesis that lc - maf cooperates early with sox9 to activate target genes, including col2a1, during mesenchymal condensation and that sox9/sox5/sox6 function at later steps of chondrocyte differentiation. the enhancers of col27a1 contain a sox9-responsive element similar to that of col2a1, and lc - maf and sox9 synergistically activate the expression of both col27a1 and col2a1. further evidence of the physiological significance of this activation comes from the co - localization of lc - maf and col27a1 in growth plate, with maximal expression of both genes occurring in the proliferating and pre - hypertrophic chondrocytes. c - maf may be involved in chondrocyte apoptosis either in the normal bone formation process or in the progression of pathologic conditions. the targets of c - maf in apoptosis pathways have been identified in nonchondrocyte cells and appear to be dependent on the specific cell context. it has been shown that c - maf interacts with the transcription factor c - myb to downregulate bcl-2 expression and increase apoptosis in peripheral cd4 cells and that c - maf increases apoptosis by direct transactivation of caspase-6 in peripheral cd8 cells. apoptosis through a p53-dependent pathway may also be relevant in both normal chondrocyte maturation and pathologic conditions. the promoter of p53 contains mare consensus sequences, and overexpression of c - maf induces p53-dependent cell death in primary rat embryo fibroblast cells. the p53-null embryonic mice have defects in the skeletal growth, abnormal chondrocyte maturation, and abnormal chondrocyte apoptosis, suggesting that p53 plays a role in normal chondrocyte development. the target genes regulated by c - maf and lc - maf, as well as the phenotypes of c - maf - null mice, generate the general types of abnormalities commonly seen with factors that affect endochondral ossification or chondrocytic differentiation. a causative role for c the maf proteins control multiple biological processes, and they are synthesized at varying levels and in distinct spatial - temporal patterns ; therefore, it is important to determine how they are regulated. there must be regulation mechanisms that control the levels of maf members or splicing variants either on mrna and/or protein levels. several upstream regulators of maf have been identified in the nonchondrocyte cells, and one can speculate that similar maf regulation exists in chondrocytes (fig. the fibroblast growth factor (fgf) pathway is a candidate for regulation of c - maf expression. fgf signaling plays an important role in lens fiber differentiation, which is severely disrupted in c - maf - null mice. deletion of three fgf receptors (fgfr1 - 3) demonstrated that the expression of fgfr is required for normal lens development, and fgfr deletion is involved in reduced expression of c - maf and its target genes, crystallins, in the lens. mapk pathway is activated upon interaction of fgfr and ligand, and the fgf / mapk cascade leads to the activation of erk1/2 kinases, which can phosphorylate mafa. two conserved serine residues (ser14 and ser65) located in the maf transactivation domains are phosphorylated by erk. these phosphorylations affect the stability of maf and are required for maf - dependent transcriptional activation. the transforming activity of mafa is controlled by glycogen synthase kinase3 (gsk-3)dependent phosphorylations, at conserved serine / threonine residues that are also present on c - maf. this phosphorylation resulted in the recruitment of coactivators to increase mafa - dependent transcription, and once released from the coactivators, the phosphorylation induced mafa ubiquitination and degradation. similarly, glucocorticoids can regulate c - maf ubiquitination and proteosomal degradation to negatively regulate c - maf - dependent transcription, although the phosphorylation status of c - maf was not examined. whether these pathways regulate c - maf in chondrocytes or during chondrogenic differentiation remains to be determined experimentally. osteoarthritis (oa) is a degenerative joint disease characterized by erosion of the articular cartilage, hypertrophy of bone at the margins (i.e., osteophytes), subchondral sclerosis, and a range of biochemical and morphological alterations of the chondrocytes. whereas endochondral ossification involves successive steps of chondrocyte maturation resulting in terminal hypertrophy and mineralization, normal articular chondrocytes are constrained from further differentiation, as evidenced by a lack of hypertrophic marker expression. however, during the progression of oa, the articular chondrocytes behave analogously to late hypertrophic and terminally differentiating growth plate chondrocytes with respect to their gene expression and cellular function. increased expressions of hypertrophic chondrocyte markers and osteoblast markers occur in oa, such as type x collagen, osteopontin, osteocalcin, and alkaline phosphatase. moreover, oa chondrocytes actively produce cartilage - degrading enzymes, including mmp-13, which is normally expressed only in the late hypertrophic zone, and cell apoptosis is also frequently observed in oa cartilage. thus, it has been suggested that oa may involve the functional loss of mechanisms that block articular chondrocyte maturation. in this context, understanding the mechanisms that control chondrocyte hypertrophy is important for normal skeletal development and growth, as well as cartilage degeneration during the pathogenesis of oa. microarray analysis of a rat model of oa demonstrated an upregulation of mafb expression in degenerating cartilage. it is noteworthy that the expression patterns of c - maf and mafb in rat embryo cartilage are virtually identical. expression of c - maf was upregulated in oa cartilage compared to normal cartilage by real - time pcr and in situ hybridization. because it has been shown that cells in the chondrocyte clusters in oa have increased expression in both cartilage matrix proteins and matrix - degrading proteases, it is notable that c - maf mrna was detected in proliferating chondrocyte clusters in oa cartilage, whereas almost no c - maf - positive cells were found in normal cartilage. the maf family proteins form a unique subclass of bzip transcription factors. since the identification of v - maf, many aspects of the physiological roles of maf proteins have been elucidated, and currently mafs are widely accepted as regulators of tissue - specific gene expression and cell differentiation. studies of the roles of maf proteins in chondrocyte differentiation and cartilage are under way, as is the identification of genes directly regulated by c - maf. the identification of c - maf binding partners and additional target genes during chondrogenesis and oa will provide insight into molecular functions of c - maf. furthermore, studies to integrate upstream differentiation signal and modulation of c - maf activity at specific differentiation steps will be important to understanding chondrocyte terminal differentiation and oa pathogenesis. | chondrocyte differentiation in the growth plate is an important process for the longitudinal growth of endochondral bones. sox9 and runx2 are the most often - studied transcriptional regulators of the chondrocyte differentiation process, but the importance of additional factors is also becoming apparent. mafs are a subfamily of the basic zip (bzip) transcription factor superfamily, which act as key regulators of tissue - specific gene expression and terminal differentiation in many tissues. there is increasing evidence that c - maf and its splicing variant lc - maf play a role in chondrocyte differentiation in a temporal - spatial manner. this review summarizes the functions of c - maf in chondrocyte differentiation and discusses the possible role of c - maf in osteoarthritis progression. |
polybrominated diphenyl ethers (pbdes) are used as additives in polymers and textiles to prohibit the development of fires. because of the production and use of pbdes, their lipophilic characteristics, and persistence, these compounds have become ubiquitous environmental contaminants. the aim of the present study was to determine potential exposures of pbdes to clerks working full - time at computer screens and personnel at an electronics - dismantling plant, with hospital cleaners as a control group. five pbde congeners--2,2',4,4'-tetrabde ; 2,2',4,4',5,5'-hexabde ; 2,2',4,4',5, 6'-hexabde ; 2,2',3,4,4',5',6-heptabde ; and decabde -- were quantified in blood serum from all three categories of workers. subjects working at the dismantling plant showed significantly higher levels of all pbde congeners in their serum as compared to the control group. decabromodiphenyl ether is present in concentrations of 5 pmol / g lipid weight (lw) in the personnel dismantling electronics ; these concentrations are comparable to the concentrations of 2,2',4, 4'-tetrabde. the latter compound was the dominating pbde congener in the clerks and cleaners. the major compound in personnel at the dismantling plant was 2,2',3,4,4',5',6-heptabde. concentrations of this pbde congener are almost twice as high as for 2,2',4, 4'-tetrabde in these workers and seventy times the level of this heptabde in cleaners. the total median pbde concentrations in the serum from workers at the electronics - dismantling plant, clerks, and cleaners were 37, 7.3, and 5.4 pmol / g lw, respectively. the results show that decabromodiphenyl ether is bioavailable and that occupational exposure to pbdes occurs at the electronics - dismantling plant.imagesfigure 1figure 2 |
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trauma is one of the leading causes of death among people under 40 years of age1. maxillofacial injuries are one of the most common injuries associated with other injuries and adult males are the most common victims. road traffic accidents (rta) are the major cause of maxillofacial injuries in developing countries2. the main aim of this retrospective study was to evaluate the prevalence of maxillofacial injury, etiology, type of injury, site of maxillofacial fractures, and their management. the records of maxillofacial injury patients from may 2014 to november 2015 who reported to the department of dentistry, esic medical college and post graduate institute of medical sciences and research (pgimsr), chennai in india were retrieved. head injury patients with brain involvement who required neural intervention and mortal cases were excluded. the records of 267 patients between the ages of five to seventy - five years were retrospectively analyzed. data including age, gender, mode of injury, etiology, anatomical fracture site, consciousness status, alcohol abuse at the time of injury, and treatment rendered were extracted. the etiology of injury was categorized into four main categories : (1) rta involving automobiles, motorcycles and bicycles, including drivers, pillion riders, passengers, and pedestrians ; (2) fall(s) from heights or while playing or due to systemic illness like epilepsy ; (3) assaults or interpersonal violence ; and (4) sports injuries and other injuries. fractures were grouped and coded according to international classification of diseases (icd)-10 : s02.4, zygomatic complex (zmc) fracture involving malar bone and maxilla ; s02.3, orbital floor fracture ; s02, fractures involving facial and skull bones ; s02.6, mandible fractures (which includes condyle, ramus, angle, body of the mandible, parasymphysis, symphysis) ; s02.5, tooth fracture ; and s02.2, nasal bone fractures. statistical analysis using spss version 16.0 (spss inc. study outcomes were measured using percentages, the mean, standard deviation and tests of proportion as appropriate. the prevalence of injury in a particular age group and gender distribution, etiology, type of fracture, management and influence of alcohol were analyzed. for comparison, the study was approved by the institutional review board of esic medical college and pgimsr (no. out of 267 case records in the age group between 5 to 75 years, maxillofacial fractures accounted for 93.3% of total injuries. the mean age and standard deviation of the patients was 35.011.8 years, with a minimum age of 5 years and a maximum age of 75 years. gender distribution shows that 74.5% (199/267) of subjects were male and 25.5% (68/267) were female, with a male to female ratio of 3:1. table 1) the test of proportion for males and females shows that there was a statistically significantly higher proportion of males involved in accident and injuries (p<0.001). (1) the report shows that the most common cause for maxillofacial injury was rta, accounting for 73.8% of injuries (197/267), among which motorized two - wheelers (mtw) were the major cause of these injuries (90.9%, 179/197), including skids and falls, collision with other vehicles and pedestrians. (2) trauma due to fall accounted for 18.0% of injuries (48/267), mostly involving children who fell while playing, elderly people who fell down due to systemic illness, or men who fell down under the influence of alcohol. (3) assault by a known person constituted for 6.7% of injuries (18/267). (4) three cases of sports injury and one case of industrial injury were reported in our data.(fig. 2, table 1) the test of proportion between rta and other injuries showed that there was a statistically significantly higher proportion of rta compared to other types of injuries (p<0.001). overall results revealed that 41.6% of males (111/267) were under the influence of alcohol at the time of injury. among mtw accidents, 48.6% (87/179) of injuries were on the right side, 31.3% (56/179) were on the left side and 19.6% (35/179) were bilateral. the test of proportion for the side of injury (right vs left) showed that there was a statistically significant difference in the side of injury, indicating that injury on the right side was more common compared to the left side (p<0.05). analysis shows that 41.9% of fractures (112/267) involved the malar and maxillary bone, 33.0% (88/267) were mandibular fractures, 26.2% (70/267) were tooth (dentoalveolar) fractures, 8.6% (23/267) were orbital floor fractures, 7.9% (21/267) were facial bone fractures with skull bone fracture, 6.4% (17/267) were nasal bone fracture, and 6.7% (18/267) were soft tissue injuries.(fig. 3, table 1) the study found that 31.1% of fractures (83/267) were treated conservatively (65 cases of zygomatic maxillary complex, 14 condylar fractures, and 4 nasal bone fractures), close reduction was performed for 27.7% of fractures (74/267), 34.5% of fractures (92/267) were treated by open reduction, and internal fixation was performed wherever indicated. (1) the report shows that the most common cause for maxillofacial injury was rta, accounting for 73.8% of injuries (197/267), among which motorized two - wheelers (mtw) were the major cause of these injuries (90.9%, 179/197), including skids and falls, collision with other vehicles and pedestrians. (2) trauma due to fall accounted for 18.0% of injuries (48/267), mostly involving children who fell while playing, elderly people who fell down due to systemic illness, or men who fell down under the influence of alcohol. (3) (4) three cases of sports injury and one case of industrial injury were reported in our data.(fig. 2, table 1) the test of proportion between rta and other injuries showed that there was a statistically significantly higher proportion of rta compared to other types of injuries (p<0.001). overall results revealed that 41.6% of males (111/267) were under the influence of alcohol at the time of injury. among mtw accidents, 48.6% (87/179) of injuries were on the right side, 31.3% (56/179) were on the left side and 19.6% (35/179) were bilateral. the test of proportion for the side of injury (right vs left) showed that there was a statistically significant difference in the side of injury, indicating that injury on the right side was more common compared to the left side (p<0.05). analysis shows that 41.9% of fractures (112/267) involved the malar and maxillary bone, 33.0% (88/267) were mandibular fractures, 26.2% (70/267) were tooth (dentoalveolar) fractures, 8.6% (23/267) were orbital floor fractures, 7.9% (21/267) were facial bone fractures with skull bone fracture, 6.4% (17/267) were nasal bone fracture, and 6.7% (18/267) were soft tissue injuries.(fig. the study found that 31.1% of fractures (83/267) were treated conservatively (65 cases of zygomatic maxillary complex, 14 condylar fractures, and 4 nasal bone fractures), close reduction was performed for 27.7% of fractures (74/267), 34.5% of fractures (92/267) were treated by open reduction, and internal fixation was performed wherever indicated. wound debridement was performed for 6.7% of soft tissue injuries (18/267).(fig. 4, table 1) the maxillofacial region is the most exposed part of the body and is more vulnerable to trauma. trauma is one of the major causes of death among people under 40 years of age1. major causes for maxillofacial fracture as reported worldwide are interpersonal violence, traffic accidents, falls and sports injuries3. rta contribute significantly to mortality and morbidity throughout the world and in large numbers in developing countries. reports reveal that 20% to 60% of all road traffic injuries involve some form of maxillofacial injury, and 62% involve motorcycles4. the prevalence of maxillofacial injuries varies from 17% to 69%, and this large difference might be due to various environmental factors, socioeconomic conditions, cultural reasons, and traffic rules. in the present study, rta accounted for 73.8% of injuries, and mtw were the major (90.9%) cause in injuries that involved skids and falls in collisions with other vehicles, including riders, pillion riders, and pedestrians. this might be due to differences in the proportion of vehicles registered in india and three highly motorized countries (hmc), the usa, china, and brazil. the car population as a proportion of total motor vehicles is only 13% in india compared to hmcs (56%-80%). on the contrary, the proportion of mtw is much higher (70%) in india compared to hmcs. pedestrians, bicyclists, and mtw riders are very vulnerable to injury and constitute 60% to 80% of all traffic injuries in india5. the number of deaths or injuries caused by mtw is about 15 to 20 times greater than for enclosed vehicles6. in our study, our institution is a referral center, predominantly treating insured employees of low - income groups. nearly (41.6%) of males injured by mtw were under the influence of alcohol at the time of injury. these injuries usually involved a skid or fall from a vehicle or collision with another vehicle or loss of control due to an unexpected encounter with pedestrians or animals. the higher frequency of maxillofacial injuries among males compared to females is a universal finding of previous studies789. in the present study, 74.5% (199/267) were males and 25.5% (68/267) were females, with a male to female ratio of 3:1. studies have reported zygomatic fractures as the most common subtype among midfacial fractures in both children and adults41011. the maxilla (22%), orbit (16%), and nasal (16%) bones were the most frequently fractured facial bones12. in our study, 41.9% of fractures involved the malar bone and the maxillary bone, followed by mandible fractures (33.0%), tooth (dentoalveolar) fractures (26.2%), orbital floor fractures (8.6%), facial bone fracture with skull bone fracture (7.9%), nasal bone fractures (6.4%), and soft tissue injuries (6.7%). in other studies, the incidences of facial bone fractures were 39.3% and 51.8% among children aged 6 to 10 years and 11 to 14 years, respectively, and the most commonly involved age group was 11 to 14 years1314. in other study reports, the incidence of facial fractures in children in india was 5.5%15, and this could be due to the fact that young children are less often involved in occupational or violence related incidents, which are the typical causes of adult facial fractures16. accidental fall is the leading cause of maxillofacial injuries in children, accounting for 43% to 71.42% of injuries13151718. among children below the age of 15 with injury due to falling while playing, the injury was mild, causing only soft tissue laceration or dentoalveolar or tooth fracture, rather than being a severe injury. the literature indicates that as the age of patients increases, the patterns of fractures progressively resemble that of adult patients19. in our study, 2.2% (6/267) of reported injuries were in the 0 to 13 age group, and all were due to accidental fall while playing or fall from a bicycle. out of 6 patients, there were 2 dentoalveolar (tooth) fractures, 3 mandible fractures, and 1 zmc fracture. this low prevalence rate might be because of mild, unnoticed injuries, or subjects may have received treatment in dental clinics outside the study area. in adults, fall was the second most common cause of injury, accounting for 18.0% (48/267) of maxillofacial injury, of which 52.1% (25/48) involved adult males, and 12 were under the influence of alcohol at the time of injury. assault : various reports reveal domestic violence as the cause of maxillofacial injuries among women, with a prevalence between 34% and 73%, representing a worldwide problem that crosses cultural, racial and socioeconomic lines20. in our study, 3.0% (8/267) of females and 3.7% (10/267) of males between 20 to 50 years of age had reported an assault injury by known persons. likewise, the proportion of sports injuries was much lower in the present study, at 1.5% (4/267), and the malefemale ratio was 3:1. patten of injury : the peak incidence (47%) of mid - face fracture was in the age group of 21 to 30 years7. the most common fracture was zmc fracture, ranging from 36% to 62.5%47. in our report, 41.9% of fractures (112/267) involved the malar bone and maxillary bone, 33.0% (88/267) were mandible fractures, 26.2% (70/267) were tooth (dentoalveolar) fractures, 8.6% (23/267) were orbital floor fractures, 7.9% (21/267) were facial bone fracture with skull bone fracture, 6.4% (17/267) were nasal bone fractures, which included 2 cases of isolated nasal bone fracture, and others were associated with lefort ii and iii fractures. reported skull bone fractures (which were referred to our department but not requiring neurosurgical intervention) included depressed frontal bone fractures of the outer table, temporal bone fractures, and sphenoid bone fractures. in addition, 6.7% (18/267) of soft tissue injuries were treated by wound debridement. in the present retrospective study, an interesting point to note was that 61.3% of malar and maxillary bone fractures were on the right side of the face. a few patients were asked about the history of their injury, and they reported that they were right handed and had applied the right - hand brakes at the time of the accident, indicating that the front wheel possibly caused the skid. in future studies, the type of vehicle (mtw) and the mechanism for the side of injury should be further evaluated. management of maxillofacial injuries is a real challenge for oral and maxillofacial surgeons, and demands both skill and expertise. open reduction and internal fixation (orif) were the major types of management performed for patients.(fig. 5) most zmc (s02.6) fractures were treated conservatively, when fractures were nondisplaced and without any functional or esthetic or neurological deficits. dento alveolar fractures, condyle fractures without displacement or dislocation or occlusal derangement were treated by close reduction and indirect fixation by intermaxillary fixation. zmc fractures and zygomatic arch fractures not involving occlusion were elevated by the gillies temporal approach if fractures were stable after elevation, and no direct fixation was done. all panfacial fractures, multiple fractures with occlusal derangement, and displaced fractures that were not reduced by close reduction and unstable zmc fracture after elevation were treated by open reduction and direct fixation by miniplate osteosynthesis.(fig. 6, 7) a study conducted by singh.21 reported that 3.88% of injured patients were intoxicated with alcohol at the time of their accidents and all were male. agnihotri.22 reported that the rate of rta was increased on weekends due to an increase in the number of drivers who were under the influence of alcohol. in our study, 41.6% (111/267) of males when approaching a cross road, in order to turn to the right hand side, the driver needs to change gear, brake, indicate, scan for oncoming traffic, make a decision on when it is safe to turn, and steer and turn the vehicle in a coordinated sequence. prabhu.23 reported that even as alcohol consumption is decreasing in some developed countries, it is on the rise in developing nations, particularly among those aged 21 to 35 years. the monthly incidence of maxillofacial fractures was fairly constant along with seasonal variations, as reported in several previous studies142425. to november is the rainy season, and there is an increased incidence of accidental falls and rta. in the present study, our reports revealed that the main cause of maxillofacial injury was rta, and males aged 20 to 40 years more often sustained maxillofacial injury. fractures of the malar bone and maxilla were the major prevalent fractures in the maxillofacial region, mtw were the major cause of injury, and the influence of alcohol was also one of the important factors responsible for injuries. although the majority of malar and maxillary fractures were treated conservatively, orif was performed wherever indicated. | objectivesthis retrospective study aims to evaluate the prevalence of maxillofacial trauma in a developing country, along with its pattern, etiology and management. data for the present study were collected from the department of dentistry, esic medical college and post graduate institute of medical sciences and research, chennai in india.materials and methodsthe medical records of patients treated for maxillofacial injuries between may 2014 and november 2015 were retrospectively retrieved and analyzed for prevalence, pattern, etiology, and management of maxillofacial trauma. spss software version 16.0 was used for the data analysis.resultsmaxillofacial fractures accounts for 93.3% of total injuries. the mean and standard deviation for the age of the patients were 35.011.8 years and with a minimum age of 5 years and maximum age of 75 years. adults from 20 to 40 years age groups were more commonly involved, with a male to female ratio of 3:1. there was a statistically significantly higher proportion of males more commonly involved in accident and injuries (p < 0.001).conclusionthe most common etiology of maxillofacial injury was road traffic accidents (rta) followed by falls and assaults, the sports injuries seem to be very less. in rta, motorized two - wheelers (mtw) were the most common cause of incidents. the majority of victims of rta were young adult males between the ages of 20 to 40 years. the malar bone and maxilla were the most common sites of fracture, followed by the mandible. the right side of the zygomatic complex was the predominant side of mtw injury. the majority of the zygomatic complex fractures were treated by conservative management. open reduction and internal fixation were performed for indicated fracture patients. |
the calcifying epithelial odontogenic tumor (ceot) was first introduced into scientific literature almost 50 years ago by dr. classified it as a benign odontogenic tumor, which is exclusively epithelial in its tissue of origin. the differential diagnosis for ceot should include adenomatoid odontogenic tumor (aot), calcifying odontogenic cyst (coc), ameloblastic fibro odentoma (afo), odontoma. a 50-year - old male reported to the oral and maxillofacial surgery opd with a swelling in left body of mandible measuring 8 10 cms extending from tooth nos. the swelling was tender and had progressively increased over a period of two years leading to facial asymmetry. orthopantomograph [figure 1 ] revealed a mixed radiolucent - radiopaque lesion, which was multilocular with coarse trabeculae and scattered foci of calcification extending from left lower canine to first molar region with a radio opaque mass representing embedded premolar. the provisional clinical diagnosis of ossifying fibroma, calcifying epithelial odontogenic tumor, ameloblastoma and odontogenic myxoma was made. histopathology findings revealed a neoplasm composed of cells arranged as sheets and anastomosing small and large islands. the neoplastic cells have well defined cell borders, abundant eosinophilic cytoplasm and hyperchromatic mildly pleomorphic nuclei, few bizarre nuclei were seen, however no abnormal mitosis was seen. normal mature lamellar bony trabeculae were seen between tumor islands interspersed with large areas of haemorrhage. the eosinophilic material was confirmed as amyloid upon special staining, diagnosing the lesion to be ceot. orthopantamograph revealing large osteolytic lesion a presurgical computed tomography (ct) scan [figure 2 ] was obtained to ascertain the boundaries of the neoplasm. it [figure 3 ] revealed an osteolytic lesion with foci of calcifications. in view of the extensive involvement, resection of the involved portion of the mandible and reconstruction with an ao unilock the mandible was exposed via an extraoral approach and resection of the left hemimandible from 32 to 37 with safe clinical margins was performed and defect was bridged by 2.4 mm unilock reconstruction plate [figure 4 ]. resected specimen [figure 5 ] was dark brown to grey in color surface measuring 7.55.54 cms. specimen was submitted for histopathological examination where it was confirmed as ceot. a 3d computed tomography (ct) image of the lesion ct scan depicting the large involvement of body of mandible orthopantamograph revealing reconstructing plate bridging the segmental defect neville and colleagues, in their textbook of oral and maxillofacial pathology, assert that the lesion is a distinct entity and probably represents less than 1% of all odontogenic neoplasms. there are differences of opinion within the oral and maxillofacial pathology community regarding degree of differentiation of the odontogenic epithelium which forms the basis for tumor pathogenesis. some authors suggest that the epithelial cells of the pindborg tumor are reminiscent of the sequestered cells in the stratum intermedium layer of the enamel organ.[35 ] this idea is based on the morphological similarity of the tumor cells to the normal cells of stratum intermedium and a high activity of alkaline phosphatase and adenosine triphosphate. it has been suggested that amyloid deposition within pindborg tumor is an immunologic response to these stratum intermedium cells. others insist that it arises from remnants of the primitive dental lamina found in the initial stage of odontogenesis, and these epithelial rests are the more likely true progenitor cell. ceot occurs most commonly between 20 to 60 years of age with mean around 40 years. in 113 cases reviewed by franklin and pindborg, patients ranged from 8 to 92 years of age with mean at 40 years. in 2004, cicconetti and colleagues reported that tumor more frequently affects adults in the age range of 40 to 60 years with peak incidence in the 5 decade with an equal sex distribution. ninety - four percent of the lesions are central and intraosseous and 6% are extraosseous. intraosseous ceot shows a maxilla - to - mandible site ratio of 1:2 and is mainly located in the premolar / molar region. ceot may lead to tooth tipping, rotation, migration, and/or mobility secondary to root resorption. alternatively it may present symptomatically as a slow - growing, painless, expansile, bony swelling with cortical bone resorption and finally perforation, as was seen in the case reported. in the initial stage, it is totally radiolucent, simulating a dentigerous cyst because of its relation with impacted tooth. small intratumoral calcification starts appearing in the second phase, which is characteristic but not diagnostic. the final stages are associated with osseous destruction and the tumoral calcification giving it a honeycomb appearance. quite similarly the radiographic finding in our case showed a loculated / trabeculated honeycomb mixed radiolucent radiopaque finding. the diagnosis of ceot is also based on histopathological examination revealing polyhedral neoplastic cells, which have abundant eosinophilic finely granular cytoplasm with nuclear pleomorphism and prominent nucleoli. an extracellular eosinophilic homogenous material staining like amyloid is characteristic of this tumor with concentric calcific deposits called liesigany ring. numerous surgical treatment modalities have been suggested, and the treatment plan is dependent on multiple factors such as size and location of neoplasm, general condition of patient and operator skill. small, intrabony mandibular lesions with well - defined borders are treated by simple enucleation or curettage followed by judicious removal of a thin layer of bone adjacent to the tumor. large tumors require aggressive approach by segmental resection, hemimandibulectomy and hemimaxillectomy, which causes bone discontinuity requiring reconstruction procedures such as grafting or distraction osteogenesis. patient reported here underwent hemimandibulectomy and there has been no recurrence on 6 month follow - up. numerous surgical treatment modalities have been suggested, and the treatment plan is dependent on multiple factors such as size and location of neoplasm, general condition of patient and operator skill. small, intrabony mandibular lesions with well - defined borders are treated by simple enucleation or curettage followed by judicious removal of a thin layer of bone adjacent to the tumor. large tumors require aggressive approach by segmental resection, hemimandibulectomy and hemimaxillectomy, which causes bone discontinuity requiring reconstruction procedures such as grafting or distraction osteogenesis. patient reported here underwent hemimandibulectomy and there has been no recurrence on 6 month follow - up. | the calcifying epithelial odontogenic tumor (ceot) is a rare entity and represents less than 1% of all odontogenic tumors. dr. j j pindborg (1958) first described four cases of this unusual lesion ; subsequently shafer coined the term pindborg tumor. this lesion is a locally aggressive benign odontogenic neoplasm arising from epithelial tissue. it occurs most commonly in 4th-5th-6th decade of life and bears no gender predilection. a case of ceot in a 50-year - old male arising in the left body region is described. |
its activity accounts for the large chloride conductance of the sarcolemma at rest that is necessary to stabilize the membrane potential. mutations in the clcn1 gene lead to dominant and recessive myotonia (koch., 1992 ; george., 1993 ; steinmeyer the channel is a member of the gene family of clc cl channels and cl / h antiporters (zifarelli and pusch, 2007), which are homodimeric proteins with independent ion permeation pathways in each subunit (dutzler., 2002). in addition to the membrane - embedded part, all mammalian clc proteins possess a large cytoplasmic c - terminal domain that bears two so - called cbs (from cystathionine--synthase) domains (ponting, 1997). the precise role of the cbs domains is still unclear, but several recent reports indicate that cbs domains may be involved in the regulation of protein function by intracellular nucleotides like atp, adp, and amp (scott., 2004 ; bennetts., 2005 ; wellhauser., 2006 ; meyer scott. (2004) showed that isolated cbs domains from clc proteins are able to bind nucleotides. (2006) reported low affinity binding of atp at the c terminus of clc-5 that could be competed by 1 m amp. moreover, nucleotides were found to be bound in the crystal structure of the c - terminal fragment of clc-5 (meyer., 2007). (2005) reported that the activity of clc-1 is regulated by intracellular atp and other nucleotides. they found that the presence of 5 mm atp shifted the open probability (popen) of the slow gate by 50 mv toward positive potentials. moreover, very recently bennetts. (2007) and tseng. (2007) reported that the effect of atp was greatly enhanced by acidification of the intracellular solution. (2005, 2007) were performed on human clc-1 expressed in cultured cells and measured using the whole - cell configuration of the patch clamp technique. first, the effects of different intracellular solutions and their washout are not tested on the same cell. second, indirect effects of atp in the complex cellular environment can not be excluded. third, if large currents are measured, as is the case for clc-1, the whole cell configuration allows only a relatively poor control of the ion concentrations close to the plasma membrane. for example, a reduction of the intracellular chloride concentration close to the plasma membrane may decrease inward currents after long hyperpolarizing pulses because of a reduced driving force for chloride. to find out if atp has a direct effect on clc-1 and if acidification may eventually modulate such an effect, we tested the nucleotide on inside - out patches., we could not detect any significant effect of atp on clc-1 activity even at acidic ph. during the elaboration of this manuscript, tseng. (2007), in a study performed in inside - out oocytes patches, reported, in rough agreement with the findings of bennetts. (2007), that intracellular atp modulates clc-1 activity and that intracellular acidification increases this effect. wt hclc-1 (koch., 1992) was expressed in xenopus oocytes and currents were measured at 18c (some control experiments were performed at 26c to ensure that the temperature did not influence the results), 24 d after injection using the inside - out configuration of the patch clamp technique (hamill., 1981) with an epc-7 (list) amplifier and a custom acquisition program (gepulse, visual c + +, microsoft). data analysis was performed using custom software (written in visual c + +, microsoft) and the origin program (originlab corporation). the control internal solution contained (in mm) 100 nmdg - cl, 2 mgcl2, 10 hepes, 2 egta, ph 7.3. the extracellular (pipette) solution contained (in mm) 100 nmdg - cl, 5 mgcl2, 10 hepes, ph 7.3. some control experiments were conducted with solutions identical to those used by tseng. (2007) (120 mm nmdg - cl, 1 mgcl2, 10 hepes, 1 egta, ph 7.4 or 6.2). in these experiments solutions containing atp (adenosine 5-triphosphate bis(tris) salt dehydrate, sigma - aldrich) were prepared diluting the appropriate volume of atp from a stock solution of 50 mm atp in the internal solution. solutions containing mgatp (sigma - aldrich) were prepared in a similar way but without supplementation of mg. solutions were applied by inserting the patch pipette into perfusion tubes of 0.5 mm diameter. the functionality of the atp solution and the efficiency of the perfusion system were evaluated on the r - cftr construct that was reconstituted from the injection of two separate rnas encoding the n - terminal and c - terminal half of the protein, respectively. the gating of this channel does not require phosphorylation and depends only on atp (bompadre., 2005). the r - cftr was injected in the same batch of oocytes and measured before clc-1. the voltage protocol used to study cftr - mediated currents consists of voltage steps ranging from 80 to 80 mv in 20-mv increments. to study hclc-1mediated currents we used a tail protocol consisting of a 100-ms test pulse to a variable potential ranging from 100 to 160 mv followed by a repolarization to 140 mv for 100 ms. to study in isolation slow gating transitions the test pulses were prolonged to 200 ms, and a 200-s pulse to 160 mv, to fully activate the fast gating, was applied before the 140-mv repolarization. for measurements at ph 6.2 the length of the test pulse was prolonged to 300 ms as in tseng. the presence of this prepulse did not alter the gating parameters derived from the boltzmann fits. the open probability was derived from tail currents fitting the latter with a monoexponential function and extrapolating the current values to the beginning of the tail pulse. open probabilities were derived from the tail currents by fitting a modified boltzmann function (pusch., 1995):(1)\documentclass[10pt]{article } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{pmc } \usepackage[euler]{upgreek } \pagestyle{empty } \oddsidemargin -1.0 in \begin{document } \begin{equation}i(v)=i_{{\mathrm{max}}}(p_{{\mathrm{min}}}+(1-p_{{\mathrm{min}}})/(1+{\mathrm{exp}}[(v_{1/2}-v)/k)])),\end{equation}\end{document}where imax is the (fitted) maximal current, pmin the residual open probability at negative voltages, v1/2 is the midpoint voltage of the open probability, and k the slope factor of the voltage dependence of the open probability. to estimate the apparent kd for atp activation of the r - cftr channel, we perfused inside - out patches with different concentrations of atp and measured the current at 80 mv. not all patches allowed the application of many concentrations and because most patches showed a slow rundown over time we bracketed all measurements by recordings with 1 mm atp and normalized the response to the current measured with 1 mm atp. assuming that, for a single ideal patch without rundown, the atp dependence can be described by\documentclass[10pt]{article } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{pmc } \usepackage[euler]{upgreek } \pagestyle{empty } \oddsidemargin -1.0 in \begin{document } \begin{equation}i(c)=i_{{\mathrm{max}}}\frac{(c / k_{d})^{n}}{1+(c / k_{d})^{n}},\end{equation}\end{document}where c is the concentration of atp, kd is the apparent dissociation constant, and n is the hill coefficient, the normalized response, inorm, is given by(2)\documentclass[10pt]{article } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{pmc } \usepackage[euler]{upgreek } \pagestyle{empty } \oddsidemargin -1.0 in \begin{document } \begin{equation}i_{{\mathrm{norm}}}=\frac{i(c)}{i(1\hspace{.167em}{\mathrm{mm}})}=\frac{(c / k_{d})^{n}}{1+(c / k_{d})^{n}}\frac{1+(1\hspace{.167em}{\mathrm{mm}}/k_{d})^{n}}{(1\hspace{.167em}{\mathrm{mm}}/k_{d})^{n}}\end{equation}\end{document}this equation was used to fit the data shown in fig. we provide supplemental online information on the application of intracellular atp on clc-1 at 26 c (figs. s1 and s2), demonstrating the lack of a significant effect also at this elevated temperature. furthermore, we show the result of sequencing the hclc-1 insert in the expression vector with four primers (fig. the online supplemental material is available at http://www.jgp.org/cgi/content/full/jgp.200709899/dc1. we provide supplemental online information on the application of intracellular atp on clc-1 at 26 c (figs. s1 and s2), demonstrating the lack of a significant effect also at this elevated temperature. furthermore, we show the result of sequencing the hclc-1 insert in the expression vector with four primers (fig. in this work we wanted to study the effect of intracellular atp on hclc-1 expressed in xenopus oocytes using inside - out patches. to test whether our perfusion system and the atp containing solutions were appropriate for this task, we used as a positive control a cftr construct lacking the regulatory domain (r - cftr) (bompadre., 2005). in this construct, activation of the cl conductance depends only on the presence of atp on the cytoplasmic side and does not require activation by protein kinase. we expressed r - cftr in xenopus oocytes and performed inside - out patch experiments, perfusing with control solution and with solutions containing 0.01 (n = 11), 0.1 (n = 14), 1 mm (n = 25), and 5 mm atp (n = 17). in the absence of atp, we observed only very small background currents (fig. 1 a) progressively activated larger cl currents, with 1 mm being already a saturating concentration. switching back to the control solution, devoid of atp, currents returned to background levels (not depicted). (a) current recordings from an inside - out patch upon perfusion with control internal solution (left), and with solutions containing atp as indicated in the panels. (b) atp dependence of currents measured at 80 mv normalized to currents measured in 1 mm atp. error bars indicate sd (the data point at 1 mm has no error bar because it was used for the normalization). 2 with kd = 38 m and n = 0.63. in particular, to make sure that the atp was prepared and delivered at the appropriate concentrations, we performed a dose response curve for atp activation of r - cftr. fig. 1 b shows the atp dependence of currents at 80 mv normalized to the current measured in 1 mm atp. 2, resulting in an apparent dissociation constant of (38 4) m and a hill coefficient of 0.63 0.03. (2005) who reported a kd value of 89 m for the atp dependence of the single channel open probability. these results prove that we have appropriate control of the amount of atp delivered to the patch. using the same recording condition, we then investigated the effect of atp on currents mediated by hclc-1. 2 a. as can be seen, the macroscopic currents do not seem to be largely affected by the presence of 10 mm atp. this was the case also for measurements performed after perfusion lasting up to 5 min. 2 b shows that the apparent open probability, derived from the analysis of the tail currents reported in fig. 2 a, follows a modified boltzmann distribution whose midpoint (v1/2) and minimal open probability (pmin) are only marginally affected by the presence of 10 mm atp as compared with control solution (see legend of fig. (a) macroscopic current recordings from an inside - out patch upon perfusion with control internal solution (left), during application of 10 mm atp (middle), and after washout (right). (b) voltage dependence of the overall open probability obtained from the macroscopic currents in a as described in materials and methods. symbols are : square, control ; circle, 10 mm atp ; triangle, wash. 1 with the following parameters : control, v1/2 = 89 mv, pmin = 0.14 ; 10 mm atp, v1/2 = 88 mv, pmin = 0.13 ; wash v1/2 = 87 mv, pmin = 0.14. data are from the same patch and were obtained from tail currents preceded by a 200-s pulse to 160 mv. 1 with the following parameters : control, v1/2 = 82 mv, pmin = 0.59 ; 10 mm atp, v1/2 = 81 mv, pmin = 0.58 ; wash v1/2 = 78 mv, pmin = 0.63. (2005) reported that the effect of atp was mostly on the slow (or common) gate, which acts simultaneously on both pores of the double - barreled channel (miller and white, 1984 ; accardi and pusch, 2000). to isolate the voltage dependence of the common gate, we adopted the voltage protocol devised by accardi and pusch (accardi., 2001) also in this case, we did not observe a significant difference between currents recorded in the presence and in the absence of atp (traces not shown). in fact, the voltage dependence of the open probability of the slow gate is virtually unaffected by the presence of 10 mm atp as shown for a representative patch (fig. 3 displays the statistical evaluation of the parameters v1/2 and pmin derived as described for the representative measurements in fig. the mean values of v1/2 and pmin are not significantly changed by the presence of atp when the overall gating of clc-1 is considered (fig. the same conclusion holds when only the slow gating mechanism is taken into account (c and d). statistical evaluation of the gating parameters v1/2 and pmin at ph 7.3 obtained in control solution (n = 10) and in solutions containing 1 (n = 3), 5 (n = 5), or 10 mm atp (n = 5). mean values of v1/2 (a) and pmin (b) obtained from the analysis of the overall gating mechanism of hclc-1. mean values of v1/2 (c) and pmin (d) obtained from the analysis of isolated slow gating mechanism of hclc-1. the values are not significantly different (p > 0.06 using student 's unpaired t test). (2007) recently suggested that the atp effect was greatly enhanced by decreasing phint. in particular at ph 6.2, atp was reported to dramatically shift the open probability voltage curve by > 100 mv to more positive voltages, with 1 mm atp being sufficient to reach a maximal effect. to test whether acidification of the intracellular solution could unmask an effect of atp that was not evident at the neutral ph used in the previous experiments, we decreased the phint from 7.3 to 6.2 and assessed the effect of 1 mm atp. 4 a), ph 6.2 (fig. 4 b), and ph 6.2 in the presence of 1 mm atp (fig. 4 c). in agreement with previous observations (rychkov., 1996 ; accardi and pusch, 2000), switching from ph 7.3 to 6.2 slows down the deactivation kinetics and increases the steady - state value of the currents at negative potentials. surprisingly, addition of 1 mm atp had no detectable further effect on the kinetics and voltage dependence of the currents. the changes produced by acidification are quickly reversed upon wash with solution at ph 7.3 (fig. 4 d). the voltage dependence of the popen derived for the representative patch shown in fig. 4 (a d) is practically unaffected by the presence of 1 mm atp (fig. 4 e). this is confirmed by the statistical evaluation of the v1/2 and pmin of the boltzmann distribution describing the popen (fig. 5, a and b), where average values of these parameters are shown. top panel, macroscopic current recordings from an inside - out patch upon perfusion with control internal solution at ph 7.3 (a), ph 6.2 (b), solution at ph 6.2 with 1 mm atp (c), and after washout (d). (e) voltage dependence of the overall open probability obtained from the macroscopic currents in the top panel as described in materials and methods. symbols are : square, internal solution at ph 6.2 ; circle, solution at ph 6.2 with 1 mm atp. 1 with the following parameters : solution at ph 6.2, v1/2 = 94 mv, pmin = 0.50 ; solution at ph 6.2 with 1 mm atp, v1/2 = 95 mv, pmin = 0.43. (f) effect of atp at ph 6.2 on the slow gating mechanism of hclc-1. data are from the same patch and were obtained from tail currents preceded by a 200-s pulse to 160 mv. 1 with the following parameters : solution at ph 6.2, v1/2 = 98 mv, pmin = 0.74 ; solution at ph 6.2 with 1 mm atp, v1/2 = 93 mv, pmin = 0.74. statistical evaluation of the gating parameters v1/2 and pmin obtained with internal solution at ph 6.2 without atp and with 1 mm atp. mean values of v1/2 in the absence and in the presence of 1 mm atp (n = 36 and n = 21, respectively) (a) and pmin (n = 28 and n = 9, respectively) (b) obtained from the analysis of the overall gating mechanism of hclc-1. mean values of v1/2 in the absence and in the presence of 1 mm atp (n = 8 and n = 5, respectively) (c) and pmin (n = 19 and n = 13, respectively) (d) obtained from the analysis of isolated slow gating mechanism of hclc-1. the values in the absence and in the presence of 1 mm atp are not significantly different (p > 0.06 using student 's unpaired t test). the lack of any effect of atp at acidic ph was confirmed by isolating the voltage dependence of the slow gate as already described for ph 7.3 (fig. 4 f) and by the statistical evaluation of the v1/2 and pmin of the boltzmann distribution (fig. 5, c and d). in the experiments described above we had used tris - atp and have added mgso4 at the same concentration, whereas tseng. (2007) and bennetts. whether the discrepancies between our results and theirs could be due to this different source of atp, we performed additional experiments with mgatp. also with mgatp, r - cftr could be reliably activated and no effect on clc-1 currents could be detected (unpublished data), showing that the source of the nucleotide has no influence on our conclusions. we also performed some experiments at 26c to assess whether higher temperature could uncover an effect of atp. in these experiments we employed recording solutions identical to those of tseng. the voltage dependence of opening is slightly shifted by 30 mv to more positive voltages, compared with the recordings at 18c, ph 7.3 (see figs. s1 and s2, available at http://www.jgp.org/cgi/content/full/jgp.200709899/dc1). this shift is probably caused by the combined effect of the higher temperature (bennetts. interestingly, at ph 6.2 almost no effect of temperature on the gating parameters could be observed (fig. most importantly, however, we could not observe any significant effect of 1 mm atp on current magnitude or voltage dependence of gating at ph 7.4 or ph 6.2 (see figs. s1 and s2). in skeletal muscle, cl conductance (gcl) accounts for 80% of the overall membrane conductance at rest (aromataris and rychkov, 2006) and clc-1 is the main determinant of this conductance (lueck., 2007). the cl conductance is required to keep an appropriate resting potential and to repolarize the voltage following an action potential (aromataris and rychkov, 2006). this critical role is underscored by the fact that mutations in the gene coding for clc-1 (pusch, 2002) or aberrant splicing of clc-1 (charlet. several recent reports indicate that intracellular cbs domains may be involved in the regulation of clc proteins by intracellular nucleotides like atp, adp, and amp (scott., 2004 ; bennetts., 2005 ; wellhauser in particular, bennetts. reported that the activity of clc-1 expressed in hek cells and studied with the whole - cell configuration of the patch - clamp technique is regulated by intracellular atp and other nucleotides. at physiological ph (7.2), atp shifted the open probability of the slow gate of clc-1 by 50 mv toward positive voltages with a half - maximal concentration of around 1 mm (bennetts., 2005). moreover, the presence of 5 mm atp produced a sixfold reduction in the number of channels that remained open at negative voltages (bennetts., 2005). on the basis of an in silico docking study of atp binding, using the crystal structure of impdh (inosine - monophosphate dehydrogenase) (zhang., 1999) to model the cytoplasmic region of clc-1, they also suggested residues that modified the effect of atp. in particular, mutating l848, predicted from the model to interact with the adenine base of atp, to alanine, abrogated the effect of atp. very recently, two different publications reported that the effect of atp was greatly enhanced by intracellular acidification, finding that at ph 6.2, addition of 1 mm atp further shifted the open probability toward positive voltages (bennetts. in contrast with previous findings (rychkov., 1996 ; accardi and pusch, 2000), bennetts. (2007) reported also a slight shift toward positive potentials of the popen at acidic ph even in the absence of atp. in stark contrast to these conclusions, we have demonstrated here that direct application of atp to the intracellular side of excised inside - out patches does not exert a significant effect on the current amplitude or on the voltage dependence of the open probability of hclc-1 expressed in xenopus oocytes at neither neutral nor slightly acidic ph (7.3 and 6.2). in particular, we were not able to detect any significant shift in the midpoint of the voltage dependence of the open probability, or on the residual open probability at negative voltages for both fast and slow gate. we observed practically no effect for concentrations of atp up to 10 mm, even after perfusion for up to 5 min. (2007) and in agreement with previous studies (rychkov., 1996 ; accardi and pusch, 2000), we find that acidic ph has at most a small effect on the v1/2 of the popen, and that inward currents are drastically increased at low ph. as already mentioned, bennetts. (2007) reported a slight shift toward positive potentials of the popen at acidic ph even in the absence of atp. the estimated pk for the effect of protons was found to be compatible with the pk of histidine residues. on the basis of this observation and of a structural model, they suggested that a histidine residue (h847) is important to mediate the effect of ph - dependent atp modulation. residue h847 was suggested to interact electrostatically with the phosphate of atp, however the mutational analysis of bennetts. failed to support this hypothesis as changing this residue to either a or to r similarly affected atp dependence. finally, the mutant l848a that was reported in the first work of bennetts. (2005) to have the largest impact on atp effect was not investigated in their latest work, nor in the work of tseng. we proved that we efficiently apply active atp because we could robustly and reversibly activate currents mediated by r - cftr, a channel that is gated by intracellular atp (bompadre., 2005). moreover, we quantitatively controlled the concentration of atp delivered to the patches by performing a dose response curve of the atp activation of r - cftr, obtaining a kd in agreement with published results (bompadre., 2005). to exclude possible sequence variations of the cdna construct we fully sequenced the open reading frame of our hclc-1 clone and found it to be in complete agreement with the published hclc-1 cdna (see fig. s3, available at http://www.jgp.org/cgi/content/full/jgp.200709899/dc1). to exclude any influence of the type of atp salt used, we tested both tris salt and mg salt without observing any difference. moreover, we used pulse protocols of variable length (see materials and methods) to ensure that the current reached a steady state during our experiments. finally, we proved that temperature in the range between 18 and 26c did not influence the lack of effect of atp on clc-1induced currents. hence, we conclude that intracellular atp does not directly affect the function of clc-1 at both neutral and acidic ph (2005, 2007) might result from the different electrophysiological methods used and/or the different expression system adopted. in this respect, it is important to note that the whole - cell configuration used by bennetts. most importantly, in the whole - cell configuration it is normally not possible to change the internal solution, implying that the comparison between different atp concentrations can not be performed directly on the same cell. furthermore, the interior of the cell, even after internal access has been established by the patch pipette, remains probably a complex and biochemically active milieu in which atp can potentially activate signaling cascades that may indirectly modulate channel function. for example, clc-1 activity could be regulated by protein kinases (chen and jockusch, 1999 ; rosenbohm., 1999). to assess whether the difference in the apparent atp sensitivity of clc-1, is caused by the different expression systems, we attempted to measure clc-1 currents from inside - out patches of transfected hek cells. unfortunately, as also reported by bennetts. (2007), we were not able to obtain a sufficient expression level to allow reliable measurements. it is more difficult to explain the difference with the study of tseng. (2007) in which the same expression system (xenopus oocytes) and excised inside - out patch clamp recordings were used. we are not able to provide any reasonable explanation for the discrepancy between our study and the one of tseng. our results have implications for the interpretation of recent results from x - ray crystallography of clc proteins and for the physiological role of clc-1. in accordance with the lack of functional effects on clc-1, meyer and dutzler (2006) could not detect atp binding to the cytoplasmic domain of the highly homologous clc-0 channel, and no nucleotides could be detected in the crystal structure of the c terminus of the clc - ka channel (markovic and dutzler, 2007). interestingly however, the same group found that nucleotides bind with 100 m affinity to the cbs domains of c terminus of the cl / h antiporter clc-5 (meyer., 2007), in agreement with biochemical data on isolated c - terminal protein fragments (scott., 2004 ; wellhauser., 2006). of course our results do not exclude that nucleotides can bind to the c terminus of clc-1. however, the lack of direct functional alteration of the channel by atp shows that if nucleotides have any effect in the physiological regulation of the skeletal muscle chloride conductance, it is of indirect nature. regarding the physiological role of clc-1, an important finding is that macroscopic gcl is reduced by intracellular acidification (pedersen., 2004, 2005), an effect that, in principle, could be mediated by a direct ph dependence of clc-1. ; accardi and pusch, 2000) have shown that low phi activates rather than inhibits clc-1, a finding that we plainly reproduce (see current traces in fig. in contrast to these findings, from apparent shifts of activation curves, bennetts. (2007) deduced that clc-1 currents are inhibited by low phi, and speculated that acidosis directly inhibits clc-1 and leads to hyperexcitability. moreover, they speculated that their finding of increased effect of atp on clc-1 upon intracellular acidification (bennetts., 2007) would explain the so far elusive link between acidification, decrease in gcl, and recovery of muscle excitability (nielsen., 2001 ; pedersen., 2004 ; pedersen., 2005) because acidification, potentiating the effect of atp, which is to shift the popen toward positive potentials, would greatly decrease gcl facilitating muscle excitation (bennetts., 2007). our measurements do not support any of these speculations and point to an indirect role of clc-1 and a more complicated mechanism of regulation of gcl that can probably be thoroughly understood only in the context of active skeletal muscle tissues. | clc-1 belongs to the gene family of clc cl channels and cl/h+ antiporters. it is the major skeletal muscle chloride channel and is mutated in dominant and recessive myotonia. in addition to the membrane - embedded part, all mammalian clc proteins possess a large cytoplasmic c - terminal domain that bears two so - called cbs (from cystathionine--synthase) domains. several studies indicate that these domains might be involved in nucleotide binding and regulation. in particular, bennetts. (j. biol. chem. 2005. 280:3245232458) reported that the voltage dependence of hclc-1 expressed in hek cells is regulated by intracellular atp and other nucleotides. moreover, very recently, bennetts. (j. biol. chem. 2007. 282:3278032791) and tseng. (j. gen. physiol. 2007. 130:217221) reported that the atp effect was enhanced by intracellular acidification. here, we show that in striking contrast with these findings, human clc-1, expressed in xenopus oocytes and studied with the inside - out configuration of the patch - clamp technique, is completely insensitive to intracellular atp at concentrations up to 10 mm, at neutral ph (ph 7.3) as well as at slightly acidic ph (ph 6.2). these results have implications for a general understanding of nucleotide regulation of clc proteins and for the physiological role of clc-1 in muscle excitation. |
cadmium taken up from lung and gastrointestinal tract is transported via blood to liver and kidney. on long - term exposure to cadmium, renal tubular dysfunction develops in humans and experimental animals. data from animal experiments demonstrate that initially after exposure cadmium in blood is bound to albumin and proteins with higher molecular weight. such cadmium is mainly taken up in liver. for a few days after exposure cadmium exists as metallothionein in plasma and blood cells. after both single and long - term administration of cadmium bound to metallothionein, cadmium is taken up by the kidney. the concentration of metallothionein - bound cadmium in plasma is quite low due to continuous renal clearance. cadmium from metallothionein is taken up in renal tubules by pinocytosis and subsequently degraded in lysosomes, thereby releasing cadmium which stimulates de novo synthesis of metallothionein but also binds to reabsorbed metallothionein. catabolizing and rebinding are continuous and prevent excretion of cadmium. because of differences in transport, renal metabolic handling forms of cadmium are also different for different forms of cadmium administered and rate of administration. a single dose of metallothionein - bound cadmium given intravenously is almost immediately and completely taken up in the renal tubule. under such conditions, resynthesis and rebinding processes are insufficient to sequester cadmium from sensitive tissue receptors, and renal damage occurs at total tissue concentrations much lower than when renal cadmium concentrations rise slowly. this explains the wide range (10 - 200 micrograms cd / g wet weight) of cadmium in the renal cortex that associated with renal tubular dysfunction in experimental animals.imagesfigure 5. afigure 5. bfigure 5. cfigure 6. afigure 6. bfigure 6. c |
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we studied whether the categorization of spoken words can still trigger task - relevant motor plans during early sleep stages. one main difficulty in addressing this issue consists in instructing a new task to sleeping subjects, arguably because prefrontal regions dealing with executive functions are then particularly suppressed in comparison to other cortical regions [8, 9 ]. one potential solution is to rely on the induction approach commonly used by studies on implicit perception in awake participants. this research reveals that the processing stream involved in making a semantic classification can, through explicit practice, be automatized and bypass prefrontal regions. under those conditions, the categorization of visual words and numbers can lead to the covert activation of motor cortex even when those stimuli are masked and presented below the threshold of consciousness [10, 11 ]. in the current study, we extend this task induction strategy to track the ability of sleepers in extracting task - relevant information from speech and preparing for the appropriate motor plan. we recorded the electroencephalogram (eeg) of human participants while they were awake and instructed them to classify spoken words as animals or objects (figure 1). this procedure allowed us to compute lateralized readiness potentials (lrps)a neural marker of response selection and preparation by mapping each specific semantic category to a specific motor plan (e.g., animals with the right hand and objects with the left hand, counterbalanced across participants). this design allows for the assessment of lateralized response preparation toward the side associated with the appropriate semantic category. thus, it allows for testing of whether sensory signals are processed beyond semantic levels by probing how the meaning extracted from external words can lead to the covert selection and preparation of context - dependent actions. testing conditions encouraged the transition toward sleep while remaining engaged with the same task set : subjects received explicit allowance to fall asleep and were sitting in a dark room, eyes closed, in a reclining chair, listening to several repetitions of the same list of stimuli with a long intertrial interval of 69 s. crucially, participants received an entirely new list of words (n = 48) during sleep to ensure that their responses were based on the extraction of word meaning rather than a mere reactivation of stimulus - response associations established during the wake stage. sleep onset was assessed online both behaviorally, by ensuring the absence of overt responses for at least 2 min of stimulation, and electrophysiologically, through sleep markers (i.e., disappearance of low - amplitude alpha / beta rhythms and development of high - amplitude delta / theta rhythms [see figure s1 available online ], presence of slow eye movements and other sleep graphoelements such as vertex sharp waves, and regular spontaneous and evoked k complexes or sleep spindles) before and after the presentation of each word. participants underwent the transition from full wakefulness to light sleep and then oscillated primarily between the non - rapid eye movement 1 (nrem1) and nrem2 stages. note that trials were only considered as nrem1 when there was a complete lack of alpha rhythm accompanied by sleep markers. in order to discard epochs comprising brief awakenings and microarousals (i.e., reappearance of a wake - like eeg activity for less than 3 s ; percentage of trials : mean = 11.6, sd = 8) and to ensure that each trial included in the sleep conditions genuinely reflected a state of sleep, we performed an offline and conservative evaluation of sleep stages relying on strict criteria. scoring was performed here by two trained neurophysiologists blind to experimental conditions who additionally verified that participants remained asleep after stimuli onset by tracking any electrophysiological signs of arousals (reappearance of a wake - like eeg activity for more than 3 s, whether the trial was associated with a button press), or any microarousals. details and statistics about sleep scoring are provided in the supplemental experimental procedures (see also figure s1 for individual sleep architectures). lrps constitute a direct and sensitive measure of response selection and preparation toward the target side, which is maximal in amplitude at scalp sites over the motor / premotor cortices contralateral to the responding hand [14, 15 ]. lrps, traditionally computed by reference to response onset, can also be measured by reference to stimulus onset [16, 17 ], making them suitable to measure cortical responses in the absence of overt motor responses (i.e., during sleep). we first characterized the main (i.e., state - independent) effect of response preparation by collapsing sleep and wake trials and computing stimulus - locked lrps using cluster - based permutation analysis (see the supplemental experimental procedures). this analysis revealed a first negative deflection corresponding to the lrp, with two significant peaks at 660 and 1,620 ms, primarily over central (c3/c4) and central posterior (cp3/cp4) electrodes (figure 2a). interestingly, after 2,000 ms, the lrp returned to baseline for several seconds until the emergence of a second negative deflection peaking around 5,570 ms. second, to test the difference between wake and sleep states, we subtracted the wake condition from the sleep condition (figure 2b). remarkably, we found no significant difference for the first lrp deflection but a clear significant effect afterward, during the opposite deflection, around 2,920 ms for c3/c4 and 3,800 ms for cp3/cp4. restricted analysis for each vigilance state confirmed the significant early lrp deflection for wake trials and, crucially, also for sleep trials separately (figures 2c and 2d). however, the opposite and later positive deflection was present only during wake trials. as shown in figure 2c, the distribution of response times during wake trials suggests that the initial lrp reflects the preparation of the motor plan, while the inversion of potential appears to follow manual responses. this interpretation was confirmed by performance of a similar analysis on readiness potentials now time locked to the actual response showing the classical lrp deflection at response onset, followed immediately by the opposite deflection after the manual response (figure s2). as discussed below, this opposite deflection in the wake condition is likely to reflect a postresponse checking mechanism that is exacerbated under conditions of drowsiness. these results suggest that task - relevant motor preparation can be triggered during sleep. our procedure for assessing sleep involved both online scoring and waiting for at least 2 min of absent responses before shifting to the new list of words, subjects sometimes pressed buttons either spontaneously or in response to auditory stimulation during the sleep list (14% of trials, not included in the analyses). those button presses could be regarded as temporary arousals whereby the subjects might wake up for one or two trials, or even microarousals (i.e., less than 3 s). however, they might also reflect a nonconscious triggering of motor actions in responses to a sensory stimulation, as it is well - known in the literatures on visual masking (e.g., subliminal action priming) and blindsight patients [19, 20 ]. in addition, past studies have shown that motor reflexes can be triggered during sleep. finally, these button presses might reflect, more simply, the fact that subjects during early sleep stages are prone to perform small movements considered in the literature as peripheral motor activations (i.e., unrelated to task or environmental contexts), such as muscle twitches. inspection of the data revealed that in most cases button presses were associated with microarousals, although there were cases where button presses were not accompanied by any signs of arousal. importantly, we computed our sleep lrps not only by excluding any trial with button presses, but also after performing a conservative evaluation of their vigilance state. indeed, in order to be fully confident that the trials that we included in our analysis genuinely reflect a state of sleep, microarousals and arousals (associated with button presses or not) were detected and trials in the direct vicinity of these events were discarded, although they may be considered as sleep trials according to established guidelines. a related issue concerns the fact that our participants received the sleep list from the onset of nrem1, and thus our sleep condition reflects a mixture of nrem1 and nrem2 stages. yet, contrary to nrem2, the nrem1 stage is sometimes regarded as an ambiguous transitory state in which awareness and responsiveness might be partially preserved [1, 7, 23 ]. our data set did not allow us to reliably separate the two sleep stages due to a lack of power, as the 48 items were distributed across both stages. it thus remains possible that, even controlling for electrophysiological and behavioral markers of arousal, participants may have somehow remained conscious of the stimuli during trials scored as nrem1. to account for this potential issue and ensure that task - relevant responses can genuinely be triggered during sleep, we implemented a more stringent control of vigilance in the second experiment, where only nrem2 brain activity was considered in the sleep condition. another potential issue concerns the use in our study of a specific scoring method developed by hori and collaborators for protocols with short epochs and focusing on hypnagogia [24, 25 ]. one might argue that this method, which is less commonly used, might underestimate the level of sleepiness and/or miss potential contaminations by microarousals in comparison to the standard scoring approach. we thus rescored our semantic decision data using the widely used guidelines of the american academy of sleep medicine (aasm). we observed that the two scoring methods largely matched in terms of classifying trials in the wake or sleep state (93.1% overlap across participants ; sd = 4.1%). crucially, reanalyses of our data using the aasm scoring revealed a very similar pattern with a significant lrp deflection for sleep trials (see the supplemental experimental procedures and the results in figure s3), confirming the presence of task - relevant responses during sleep even when a more conventional method for scoring sleep was used. finally, regarding the comparison with the wake state, a potential issue might be that the strong positive deflection that we observed with a reversal of the lrp response after an overt motor response might reflect specific conditions of drowsiness. indeed, participants were tested while falling asleep and reaching a certain level of drowsiness, which might increase the reliance on postresponse checking mechanisms, leading to the reconfiguration and amplification / reduction of ipsi-/contralateral motor areas [26, 27 ]. hence, a more direct comparison between wake and sleep states would thus not only exclude nrem1 trials as described above, but also compare sleep with conditions of full wakefulness (i.e., avoiding the drowsiness period where subjects are in the process of falling asleep). we performed a second experiment in which we instructed participants to perform a lexical decision on spoken material. participants classified auditory stimuli as words versus pseudowords (i.e., items that do nt exist in the lexicon but share the same phonological properties as real words) with their left versus right hand (counterbalanced across subjects). this second experiment, in addition to dealing with the potential issues mentioned above regarding the wake - sleep transition, allowed us to verify whether the induction approach can be generalized to other classification tasks on external stimuli. here, the nap was preceded by a session in which participants received the first list of stimuli under full wakefulness while sitting upright and not being allowed to fall asleep. participants were then presented repeatedly with the same list while being reclined and allowed to fall asleep under similar testing conditions as in the semantic decision group. in addition, participants in this second experiment received the second list of stimuli (n = 72) only after the onset of the nrem2 stage (i.e., after the first appearance of a spontaneous k complex or sleep spindle). this design allowed contrasting lrps during consolidated sleep versus full wakefulness rather than during the transition, where subjects are either drowsy or in a labile (nrem1) sleep stage. since the hori scoring is optimized for evaluating the hypnagogic period (primarily nrem1), we decided to apply aasm scoring for this second experiment while also controlling for microarousals (see the supplemental experimental procedures and figure s4 for individual hypnograms). trials associated with a button press and microarousals dropped to 2.3% (sd = 1.7%) and 0.3% (sd = 0.6%), respectively, and were excluded from further analysis. analysis of the main (i.e., state - independent) effect of response preparation revealed two lrp clusters, with an early effect peaking at 1,276 ms and a later and more sustained effect peaking at 5,016 ms and extending from 3,508 ms until the end of the epoch at 8,000 ms (figure 3a). separate analyses for each vigilance state showed that the early lrp component was mostly driven by the wake condition, whereas the later and more sustained cluster was primarily driven by the sleep condition (figures 3c and 3d). indeed, whereas the lrp in the wake condition was rather transient and overlapped with the reaction times distribution, the lrp during sleep corresponded to a large and sustained response developing slowly over time. as a consequence, the contrast of wakefulness (i.e., the difference between wake and sleep trials ; figure 3b) revealed a trend for an early negativity, suggesting a stronger early lrp under wake conditions, and a significant and sustained positivity for the late component, reflecting a delayed lrp during sleep. these results confirm the presence of covert task - relevant responses to speech during sleep and extend the finding in the semantic decision from experiment 1 to the classification of lexical properties during the nrem2 state. notably, the opposite deflection found in experiment 1 under drowsy conditions was not observed here under conditions of full wakefulness. it is also interesting to observe that the lrp during sleep was further delayed in time compared to experiment 1. we interpreted this finding as the involvement of slower mechanisms of evidence accumulation during the n2 stages in experiment 2, compared to the mixture of n1 and n2 responses in experiment 1. one might still argue that participants in our study were somewhat aware of the spoken stimuli, with fleeting microarousals that are difficult to detect in the eeg, resulting in a state of transient arousal / drowsiness not allowing them to perform an overt behavioral response. in order to directly address this issue, through an operational measure of stimulus awareness, we instructed participants to perform an explicit recognition task right after the lexical decision experiment, after regaining full consciousness. they were presented with the stimuli from the wake list, from the sleep list, or from a new list of completely novel items (counterbalanced across participants) and were instructed to classify each stimulus as either old or new and then rate their confidence about their decision on a scale ranging from 1 (completely guessing) to 7 (completely sure). results of the posttest (see figure 4) revealed that participants could distinguish new words from words presented during the wake period (performance = 81.5%, d = 2.16, both p < 0.0001) but, crucially, not from words presented during sleep (performance = 51.2%, d = 0.13, both nonsignificant [n.s. ]). consistently, the postdecision confidence estimates also did not differ between the new and sleep lists (mean confidence = 4.79 versus 4.80, respectively ; n.s.), whereas they were significantly higher for the list presented during the preceding period of wakefulness (5.80, both p < 0.001). overall, these results add strong evidence supporting the fact that participants did not have explicit access to the stimuli presented during sleep and confirm that the lrp obtained during sleep most likely reflects a nonconscious form of speech processing. there is now converging evidence that environmental stimuli can still be processed during sleep, at least to a certain degree. for instance, meaningful stimuli (e.g., own names, own baby s cry, and fire alarm) are more likely to lead to awakening [2831 ]. furthermore, sleeping participants, while in rem or nrem stages, can create novel sensory associations between tones and odors or reactivate existing semantic associations as evidenced by the presence of an n400 component in eeg [47 ]. besides, sleepwalkers are able to re - enact recently learned sequences of movements. thus, there is evidence, albeit scarce, that sleep does not preclude meaning extraction or the activation of learned associations and sensorimotor mappings. however, to date, no study has directly tested the possibility that environmental stimuli are processed in a flexible manner, all the way up to the preparation of task - relevant responses. here, using lrps, we show that sleeping participants are still able to prepare for the appropriate response on semantic and lexical decision tasks practiced before falling asleep. the current design, using single - word presentations, does not directly test for meaning extraction (unlike classical n400 paradigms using word pairs or sentences). however, our study reveals speech processing through semantic and lexical categorization by demonstrating the preparation of motor plans conditional on the meaning of spoken words. these results not only confirm previous findings showing that semantic information can still be extracted during sleep, but further show that this nonconscious meaning extraction can be routed by the task context and reach higher processing levels, up to motor preparation stages. this suggests that when processing environmental information during sleep, at least during early nrem stages, only the final stages related to action execution might be suppressed. an important remaining question, therefore, is where in the neural stream ranging from motor preparation to action execution lays the bottleneck responsible for the lack of behavioral responses. previous studies revealed that sleep is associated with both the inhibition of dorsolateral prefrontal cortex, a crucial area for executive functions [8, 9 ], and the functional breakdown in thalamocortical connectivity, associated with the loss of wakefulness and sensory awareness. on the contrary, neural activity in other cortical regions, including sensorimotor areas, does not importantly differ from the wake stage [9, 34, 35 ]. one might even expect that, as long as a given task has been induced during the wake stage, almost any processing stream could potentially remain activated during sleep. future studies will be necessary to address this issue and, in particular, whether even higher - order regions dealing with executive functions such as cognitive control or task switching can be triggered using a task - induction strategy. it remains to be elucidated whether this finding would generalize to other sleep stages, and in particular to rem sleep, in which there is an almost complete muscular paralysis but electrophysiological activity is closer to that of wakefulness. on the one side, because the strong inhibition of motor neurons during rem sleep involves only subcortical structures (such as the locus coeruleus, which targets motor neurons in the spinal cord), and given the relatively preserved information processing capabilities during this stage, one might still expect similar covert responses as found here. on the other side, these findings might be restricted to the initial stages of sleep, during which the thalamus is mostly deactivated whereas large parts of the cortex remain active. future studies relying on full - night protocols will be necessary to address whether the integration of semantic and decision processes can bypass early sleep stages. beyond revealing unsuspected processing capabilities in the sleeping brain, this study uncovers a promising avenue to study nonconscious processes. research investigating the distinction between conscious and nonconscious mechanisms (the so - called contrastive approach) generally focuses on the notion of contents of consciousness. in this framework, the participant can be nonconscious of a specific content as in a typical situation of visual masking but remains fully conscious in the intransitive sense of being aroused and vigilant. for instance, although previous studies using subliminal priming have shown that invisible primes can trigger lateralized readiness potentials [10, 39 ], participants in these studies were still having conscious access to their goal - directed behaviors in order to perform a specific task on target stimuli. here, although sleeping participants may continue to process information in a goal - oriented manner, this task set is presumably maintained without the participant being conscious of it. moreover, our experimental approach relying on levels rather than contents of consciousness not only allows examination of the neural consequences of perceptual processes when the subject is nonconscious in any possible respects, but also offers the opportunity to use sensory stimuli that are not degraded in any manner. indeed, the strong degradation of sensory signals typically used to achieve robust unawareness in masking studies, either in the visual or the auditory modality, unavoidably decreases the strength of neural responses, especially in brain regions dealing with high - level information. hence, studying sleep in this context allows pushing further the limits and extents of nonconscious processes and establishing the properties of a broader and more natural type of cognitive unconscious. | summaryfalling asleep leads to a loss of sensory awareness and to the inability to interact with the environment [1 ]. while this was traditionally thought as a consequence of the brain shutting down to external inputs, it is now acknowledged that incoming stimuli can still be processed, at least to some extent, during sleep [2 ]. for instance, sleeping participants can create novel sensory associations between tones and odors [3 ] or reactivate existing semantic associations, as evidenced by event - related potentials [47 ]. yet, the extent to which the brain continues to process external stimuli remains largely unknown. in particular, it remains unclear whether sensory information can be processed in a flexible and task - dependent manner by the sleeping brain, all the way up to the preparation of relevant actions. here, using semantic categorization and lexical decision tasks, we studied task - relevant responses triggered by spoken stimuli in the sleeping brain. awake participants classified words as either animals or objects (experiment 1) or as either words or pseudowords (experiment 2) by pressing a button with their right or left hand, while transitioning toward sleep. the lateralized readiness potential (lrp), an electrophysiological index of response preparation, revealed that task - specific preparatory responses are preserved during sleep. these findings demonstrate that despite the absence of awareness and behavioral responsiveness, sleepers can still extract task - relevant information from external stimuli and covertly prepare for appropriate motor responses. |
dependence on addictive drugs spread all over the world and the side effects of addiction, it is necessary to study the function of drugs in animal trials especially in placenta. many behavioral problems in addicted mother s infants indicated the effects of opioids on embryo (1, 2). the majority of studies focused on the embryo, whereas they neglected to study the placenta as an important organ. disruptive effects of consumption of opioids in human samples and laboratory animals were well conducted. the research showed that consumption of opiate materials by pregnant mothers cause delay in embryonic development and malfunctions, such as spinabifida (1, 3). in accordance to previous studies, high blood corticosteron concentration of pregnant mother attenuate placenta and embryo. the capacity of placenta for displacement and releasing food materials depends on the placenta s shape, size and transferring factors. as morphine is small and imploring molecule, it can easily pass through blood barrier and placenta and then become effective on embryonic cells (4 - 6). as placenta in mammals is the most important part to exchange materials between embryonic and maternal blood, the size of the placenta is directly related to food material transporting (simple and active transport) (2, 4, 6). the morphine effects were presented with mio, kappa and delta opioids receptors and activating of these receptors caused several changes, including decrease in the camp, an increase in output of -k+ ion and a decrease in input of ca - ion (7, 8). on the other hand, the ca - ion has important role in secretion of estrogen and progesterone hormone from placenta, stableness and embryonic development (9, 10). by progress of pregnancy, placenta can act as a gland that secrets progesterone, estrogen and other enzymes that are needed for embryonic development and considered as an alternative for ovary secretion hormones (6, 11). therefore, morphine can interfere and causes dysfunction in placental secretion operating and delay in placental development (5, 10). several experiments have shown that morphine administration cause the decrease of placenta weight in rabbits (12, 13). on the other hand, because the placenta is a protective barrier, it can prevent the input or output of some materials. placental barrier as a protective mechanism is often considered against pathogenic factors (2, 14, 15). disorders in the development of placenta cause placental disability in exchanging endocrine and protective acts in embryo function (6). the importance of mother s blood corticosteron concentration in placenta development and the effects of morphine on delay of placental development are the major reasons for the present research that consider the oral morphine administrations effect on the placenta of addictive mothers on 10 and 14 days of pregnancy. the animals were housed 2/cage to a temperature of (24 10c) and controlled environment with a 12-h light / dark cycle and were provided with food and water. this experimental study was accomplished with financial support of baqyiatallah (a.s.) university of medical sciences as a thesis project. all experiments were conducted in accordance with standard ethical guidelines and approved by the local ethical committee (the baqiyatallah (a.s.) university of medical sciences committee on the use and care of animals, 86/143, apr 15, 2007). in this study, prepared morphine sulphate from iran temad co, was used orally. twenty - four female wistar rats were divided into four equal groups consisting of six animals each. the female rats were mated into 2 groups with one adult male rat. after pregnancy (with the observation of vaginal plug and existence of sperm in vagina), they were separated from male rats the next morning and kept in the same coed - groups. thereafter (0 day of pregnancy), experimental group received a daily dose of 0.05mg / ml morphine (5 mg per 1000 ml potable water from city pipeline for 12 rats) (15). also, 1ml bloods were collected from retro - orbital sinus at 13 days of pregnancy. finally plasma corticosterone was assessed by rat corticosterone elisa kit (eia-4164 ; drg instruments gmbh, germany) in both experimental groups. in 10 and 14 days of pregnancy, the animals were anesthetized with chloroform and placenta were removed and transferred to 10% formalin solution for 10 days, then, placenta were put in tissue processing molding, then blocks were sagittaly sectioned with 5um thickness and serially by microtome (15, 16). these slices were put on slides and stained with hematoxylin and eosin methods (h&d). after staining, slides were studied microscopically. differences between all groups were calculated by a one - way analysis of variance (anova) and post - hoc duncan test by using the spss / pc computer program (version 9.1). statistical significance between the two measurements result were considered statistically significant when p < 0.05. the thickness of portion placenta, blood cisterns surface, and number of cells in the experimental and control groups was measured with motic software. the system used included a microscope connected to a computer and a monitor with software which could take photos from slides. subsequently, the number of cells on each layer was counted randomly and compared with that of the experimental groups. our results showed that the oral morphine consumption in pregnant rats increased the placenta concentration of corticosteron at day 13 of pregnancy in comparison with control group. in addition morphine administration in pregnant rats placenta showed, thickness of maternal part has increased in 10 and 14 days placentae (table i). in contrast, significant decrement in thickness of placenta in embryonic part was shown (table i). also, increment in cells number of placenta in maternal and embryonic part in experimental groups was assessed (table i), thereby decrement in blood pools surface of placenta in maternal and embryonic part in 10day experimental groups was shown in this study (table i). indicates effect of administration of oral morphine on placenta and plasma corticosterone concentration in rats result were considered statistically significant when p < 0.05. effect of administration of oral morphine on the10, 14 days of placenta portions thickness and cell number and lacuna area development furthermore, plasma corticosterone concentration in rats which received oral morphine on the 13 day of pregnancy and comparison of control and experimental groups. changes in thickness of the placenta portion in experimental(b1) and control(a1) group in the 10-day old placenta by 40 (two arrowheads). morphologic changes on 10-day old placenta in experimental group (b2) indicates placenta cells and blood lacuna of placenta shuch as indicates placenta cells and blood lacuna in control group (a2) in 10 days placenta with zoom 100, (a one head arrow). the changes of thickness in placenta layer in 14-day old placenta in experimental(b3) and control(a3) groups zoomed 40 (two heads arrow). the changes of the blood cisterns and the number of placenta portion cells in 14-day old placenta in experimental (b4) and control (a4) groups, zoomed 100 (one head arrow). results of the present study showed that morphine consumption during pregnancy can cause delay in the development of embryonic and maternal portion of placenta. on pregnant mother, concentration of corticosteron at blood plasma will increase at second period of pregnancy so we measured corticosteron concentration at 13 days after pregnancy (17, 18). in agreement with this subject, our data also indicated the oral morphine administration increased blood plasma corticosteron concentration on addictive pregnant mothers (table i). increase in corticosteron density of the blood plasma of the pregnant female mice can justify the side effects in embryos of these mothers. in addition the results of these studies were consistent with previous ones, which indicated that cortisol administration could delay differentiation of placental cells (1, 5). regarding the fact that embryonic development is the result of placental natural function and due to the essential function of that is the hormone secretion and material exchange, any disorder in normal function of fetal and maternal portion of placenta can cause abnormality on embryo growth (3, 16, 19). morphologic studies have shown that if physiologic changes of spiral veins occur during pregnancy, trophoblast cells will attack placenta and blood flow will increase at this site and finally placental villi disrupting will appear (6, 20, 21). morphologic and morphometric results have shown the oral morphine effect on both embryonic and maternal parts of placenta (table i). also our study indicated that oral morphine administration in pregnant mouse caused corticosteron secretion in experimental group (5, 10, 17). fowden and forhead declared, the increase of placing embryo and placenta in exposure of glococorticoides cause attenuation of embryo and placenta, and this will directly change the cell cycle from mitotic to differentiated state (5, 6, 10). also corticosteron induces proliferation of cytotrophoblast cells of maternal and fetal portion of placenta (6, 17, 22) by stimulation of procytotrophoblastic cells to shortening of interphase (5, 17, 23), so cells do not have enough time for growth, protein synthesize, replication, and enough growth, and finally cause disorder in normal function of placental fetal cells (6, 7, 23), in contrast lead to late differentiation of placental cell and embryonic development (5, 7, 10, 19). results of present research together with above data showed high level of corticosteron by morphine administration raised maternal portion thickness and number of these cells in placenta of day 10 and 14 of pregnancy. in addition morphine consumption has decreased more placenta fetal portion in contrast to maternal portion and this increment cause severs abnormality at embryo (table i). the previous studies indicated oral morphine administration in the 9 day of pregnancy attenuate neural tube and neural plate evolution and development of frontal cortex in embryo were reduced in the 17 day of pregnancy (15, 16, 24, 25). the major role of placenta is material exchange between maternal blood and placenta and secretory substances such as steroids, peptides, cytokine and glycoproteins release from mother blood and enter to embryo by placenta. otherwise because consumption and production of nutrition material were determined by placenta any disorder in functionality of fetal placental portion (5, 6, 11) cause delay in development of embryo and placenta. during pregnancy in parallel with vessel angiogenesis, thickness of fetal placenta portion will decrease, so material exchanges become more and finally morphine uptake increases and decrease in thickness of fetal placenta portion is the effect of this increment (14, 20, 23). so, speed of morphine transition and disruptive effects have direct ratio with decrease of fetal layer thickness. in present research, effect of morphine resulted decrease in abnormal thickness in embryonic unit of experimental group compared to control group in 10 and 14 day old placenta, and decrease of blood cisterns surface, also number of cell in both 10 and 14 day old placenta in experimental group compared to control group are consistent with increase of corticosteron concentration (table i). the other researches of morphine effect on placenta fetal portions of both groups indicated, morphine causes decrease in blood cisterns area and these data not only are consistent with koulin s and doppler s researches, but also they are valuable to research in human. in other researches, it was shown ; that the morphine consumption orally and by injection indicated the same effect (20, 26, 27). the effective factors on blood vessel contraction are corticosteron and opioides receptors on the membrane of placental cells (2, 3, 7), that located on placental villi and be contracted by morphine stimulation and resulted decrease in blooding, embryonic hypoxia and delay in embryonic development (20, 28) embryonic portion of placenta basically give rise to cyncytiotrophoblast cells and these cells play important role in embryonic development through secretory function, like estrogen and progesterone hormone (10, 17) and disorder in the secretion of these cells cause delay in placental and embryonic development (6, 19) and can aggravate embryonic abortion. according to studies, morphine administration caused embryonic abortion and decrease in babies weight in pregnant rabbits (12). in total, these results indicated oral morphine consumption causes the unusual increment in plasma corticosteron density and delay in placenta fetal and maternal portion development in wistar rat but it was not identified if present study results are due to morphine or corticosteron effect or both of them. although behavioral disorders in infants or embryonic abortion from addictive pregnant mothers need to be studied more. | background : previous studies have shown that morphine consumption during pregnancy may delay embryo development or cause abnormal nervous system function. objective : the present study focused on the effect of maternal morphine consumption on development of placenta and blood corticosteron concentration in addictive pregnant mothers. materials and methods : 24 female rats, 170 - 200 g weight, were used. the experimental groups after pregnancy received an oral dose of 0.05 mg / ml of morphine by tap water while the control group received only tap water. on 10th and 14th day of pregnancy, rats were anesthetized and placenta removed surgically, 1ml blood was collected from each pregnant mother from retro - orbital sinus, the concentration of blood corticosteron was determined by corticosteron elisa kit after centrifugation. the fixed tissue was processed, sectioned and stained with hematoxylin and eosin. placenta was studied microscopically according to the thickness of layers, area of blood cisterns, and the number of cells. results : comparing the plasma corticosteron concentration of the treatment and the control groups, not only a severe increase in the treatment group was detected, but also the thickness of maternal and embryonic portions of the placenta at day 10th and 14th of gestation was different significantly (p0.05). furthermore, an increase in number of cells in maternal and embryonic portion of placenta and a decrease in blood cistern area were demonstrated in both the experimental and the control groups. conclusion : the effects of morphine, including an increase in blood concentration of corticosteron, in dependent pregnant mothers were seen. development of placenta in the experimental group was delayed. |
it is a traumatic event that can have psychological consequences for the couples (1). threatened abortion, as demonstrated by vaginal bleeding with or without abdominal cramps, is a common complication of pregnancy. it occurs in 20 percent of women during early gestation and approximately half of these pregnancies will abort (2, 3). even if abortion does not follow early bleeding, these fetuses are at increased risk for preterm delivery, low birth weight and prenatal death (4). a number of recent studies suggest that progesterone can reduce pregnancy loss in women with threatened abortion (510). by progression of pregnancy, the role of insufficient progesterone level declines and uterine structural malformation with or without cervical incompetency is responsible more than hormonal deficit. in this case, some approaches like cervical cerclage can be effective more than medical interventions (11). progesterone plays a crucial role in the maintenance of pregnancy. in the presence of sufficient progesterone levels during pregnancy, lymphocytes synthesize a mediator called progesterone induced blocking factor (pibf), which is anti - abortive in mice (12, 13). besides inducing secretary changes in the endometrium and supporting early pregnancy, it modulates the maternal immune response to prevent fetal rejection and relaxes the uterine smooth muscles (14). despite this physiological evidence, which has led to progestogens being used in management of threatened abortion for many years, there is little data available to support their routine use in this issue (15). the aim of this study was to determine whether progesterone is effective in allowing pregnancy to proceed beyond week 20 in women with threatened abortion. this clinical trial study was done on 60 pregnant women with threatened abortion from april 2009 to march 2012 at taleghani hospital affiliated to shahid beheshti university of medical sciences. this trial was a single - blind study, in which the researchers did not have any placebos for the control group and were unaware which patient had received progesterone. the study was approved by the bioethics committee (400.11199/30 may 2012) of shahid beheshti university of medical sciences after the approval of the research group in the obstetrics and gynecology department. verbal consent of all the pregnant women participating in the trial study was received as well. pregnant women, who had vaginal bleeding until 20 weeks of their pregnancy, were assessed for inclusion. participants underwent a general and pelvic examination, a pelvic ultrasound and a complete measurement of their blood count. in order to be included in the study, the women were required to have no systemic disease or fever and no loss of conception tissue. the presence of singleton pregnancy and detection of fetal heart activity, besides gestational age of less than 20 weeks was verified by ultrasound. women were excluded if they had reaction to cyclogest, multiple gestation, absence of fetus or fetal heart tone, uterine anomaly or fetal anomaly. participants were divided into two groups ; the control group, which did not undergo any treatment and the case group. the case group was given 400 mg of vaginal progesterone suppository (cyclogest, actavis, uk) each day until their bleeding stop - ped for several days, mostly less than one week. progesterone in suppository and injection form and in a short time usage did not have any adverse effect on mother or fetus (4, 6). those who had slight vaginal bleeding received progesterone sup - pository only for 2 days. however, for those with moderate or severe vaginal bleeding, it continued up to one week. the sample size was 60 based on p1 = 80%, p2 = 50% expected rate of abortion in threatened abortions, = 0.05 and = 20%. both groups consisted of 30 participants and were kept under standard care in terms of hydration and rest. qualitative and quantitative variables were analyzed statistically by chi square and t - test respectively. qualitative and quantitative variables were analyzed statistically by chi square and t - test respectively. a total number of 60 pregnant women, with threatened miscarriage, participated in the study. all of them were referred to the department of obstetrics and gynecology at taleghani hospital. the total age range was from 18 to 37 years with a mean value of 274. there was no statistically significant difference between the case group and the control group in terms of mothers ages, gestational ages and parity score. the mean value of mothers ages was 274 years in the case group and 275 years in the control group. average gestational age was 9 weeks3 days in the case group and 10 weeks3 days in the control groups. moreover, there were 20 nulliparous women in the case group and 18 in the control group, with the rest being multiparous. five patients in the case group and nine patients in the control group had moderate to heavy uterine bleeding. overall, 9 patients in the case group and 7 patients in the control had abdominal cramp with vaginal bleeding, but the outcome of ongoing pregnancy or abortion did not differ in both groups. the number of abortions in the case group was lower than the control group (6 cases, 20% against 10 cases, 33.3% respectively). however, the difference was not statistically significant. as demonstrated in table 1, 80% of women in the case group and 66.7% of women in the control group had a successful term pregnancy. approximately 66.7% of patients with successful delivery in the case group and 60% in the control group were nulliparous (p = 0.592). moreover, 70% of the patients with successful delivery in the case group and 76.7% of them in the control group had no abdominal cramp (p = 0.559). accordingly, neither parity nor abdominal cramp had any effect on the outcome of pregnancy. this study was conducted in order to assist pregnant women with threatened abortion, since miscarriage is a deeply distressing condition for couples (1). the study demonstrated that the rate of abortion was reduced clinically but not statistically in women treated with progesterone suppository as compared to women who received only supportive care. these findings support recent studies on women with threatened abortion that have shown a reduction in pregnancy loss with progesterone treatment (3, 710, 21). pro - inflammatory cytokines associated with mis - carriage and progesterone- induced blocking factor (pibf) had inhibitory effect on immune reaction and shifting of cytokines from type 1 to type 2 cytokines caused an increase in the production of cytokines type 2 (22). pregnancy is often hampered by immunological factors, luteinic and neuroendocrine deficiencies and myometrial hyper - contractility. this may explain the reduction in abortion in women treated with prophylactic progesterone (3, 6, 9, 19). there are some studies, however, that show insufficient data on the effect of progestogens on threatened miscarriage (2, 5, 6, 15). according to one study from el - zibdeh, miscarriage rates were significantly lower in the group treated with dydrogesterone as compared to the untreated group (16). progestogens also have a direct pharmacologic effect by reducing the synthesis of prostaglandins, thereby relaxing uterine smooth musculature and preventing inappropriate contractions that may result in miscarriage and preterm labor pain (5, 1820). some pregnant women have lower abdominal pain followed by missed period, but if lower abdominal pain is associated with uterine bleeding, it may predict impending abortion. in this study, however, most of the pregnant women had no abdominal pain in either the case group or the control group and the difference between two groups was not statistically significant. a study has shown that the use of progesterone is effective in both pain relief and decreasing the frequency of uterine contractions after 5 days of progesterone usage (8). effect of progesterone on different gestational ages was also evaluated in this study. in gestational age of 8 weeks or less, 80% of participants in the case group and 50% of participants in the control group had successful delivery. in gestational age of 8 to 16 weeks, 80% of pregnant women treated with progesterone had successful delivery as compared to the 60% in the control group. in gestational age of more than 16 weeks, 80% of pregnant women treated with progesterone and 50% of women in the control group had successful delivery. in previous studies, there has been no report of comparison between case and control groups with respect to different gestational ages. there are some studies which have reported some problems during the course of pregnancy in women with threatened abortion (4, 16). however, in this study, all pregnant women who passed their course of threatened abortion had normal term pregnancies. the study demonstrated that the rate of abortion was reduced in women treated with progesterone, irrespective of their gestational age. nevertheless, its effect on prevention of abortion was not statistically meaningful, which may be due to the study 's small sample size. the use of large sample sizes, double - blind and randomized controlled trials are recommended for future studies on this issue. this article is extracted from the obstetrics and gynecology resident thesis in shahid beheshti university of medical sciences. | backgroundthreatened abortion is a common complication of pregnancy. in order to prevent miscarriage in the cases with threatened abortion, this study was conducted to determine whether progesterone suppository is effective in allowing pregnancy to proceed beyond week 20 in women with threatened abortion.methodsthis single - blind clinical trial study was done on 60 pregnant women with threatened abortion. pregnant women, who had vaginal bleeding until 20 weeks of their pregnancy, were assessed for inclusion. participants were divided into two groups by random allocation ; the control group, which did not undergo any treatment and the case group. the case group was given 400 mg of vaginal progesterone suppository (cyclogest) each day until their bleeding stopped in less than one week. participants were followed up until the end of their pregnancy. the treatment was considered successful if pregnancy continued beyond 20 weeks of gestation. qualitative and quantitative variables were analyzed statistically by chi square and t- test respectively. the p - values of less than 0.05 were considered significant.resultsthere was no statistically significant difference between the case and the control groups in terms of background variables. the number of abortions in the case group (6 cases, 20%) was lower than the control group which had 10 abortions (33.3%).conclusionthe study demonstrated that the rate of abortion was reduced in women treated with progesterone suppositories. however, the difference was not statistically significant. |
interferons (ifn) are a linchpin of inflammatory signaling, assisting in host defense against pathogens, antigen presentation, and immunomodulation. there are two main classes of interferons : type i (ifn-/) and type ii (ifn-). interferons bind to their respective transmembrane receptors, inducing dimerization and regulation of inflammatory gene expression through the jak / stat signaling pathway. janus kinases (jaks) are tyrosine kinases that interact with interferon receptors, resulting in recruitment and phosphorylation of signal transduction and activator of transcription (stat) proteins. the jak / stat association in turn promotes transcription of pro - inflammatory genes including inducible nitric oxide synthase (inos). interferons coordinate the inflammation response in concert with other innate immune pathways, particularly toll - like receptor (tlr) signaling. tlrs are pattern recognition receptors that respond to infectious markers and induce a pro - inflammatory response. these two pathways synergistically interact in macrophages to elicit an immune response toward infective threats. macrophage priming with ifn- improves the inflammatory response to tlr ligands, such as lipopolysaccharide (lps) for tlr4. in turn, tlrs upregulate type i interferons, and nf-b assists in transcription of multiple interferon - inducible genes. of particular interest, the inos promoter has binding sites for both stat1 and nf-b. transcription of inos may be activated by multiple inflammatory factors, including lps, type i, and type ii interferons. stat1 activation by ifn- has an autocrine / paracrine mechanism preceding inos activation and serves as a necessary transcription factor for synthesis. it has been demonstrated that knockdown of the interferon-/ receptor 1 (ifnar1) mitigates inos expression, even in the presence of lps. thus, activation of the tlr and interferon interrelated pathways can be regulated at their convergence in jak / stat signaling. nonetheless, it has been a significant challenge to regulate individual pathways with high specificity and selectivity. generally, inflammatory signaling is beneficial and may protect the host against infection. however, in autoimmune pathologies such as systemic lupus erythematosus and multiple sclerosis, an overabundance of interferon signaling can have deleterious effects. indeed, it has been noted that among centenarian women, polymorphisms that result in decreased ifn- may contribute to longevity. additionally, a small molecule modulator of ifn signaling may provide a useful tool in the study and treatment of autoimmune diseases. here, we report a small molecule that is able to inhibit the interferon - induced jak / stat1 signaling pathway without compromising the effectiveness of other components of the innate immune system such as tlrs. we started our search for anti - inflammatory agents by looking for inhibitors of lps - induced tlr4 activation. the c2d2 pilot library consists of 2200 drug - like compounds that represent a variety of diverse and commercially available scaffolds. the initial screen was performed using lps - activated raw 264.7 cells in a 96-well plate format to monitor nitric oxide (no) production. this assay uses a sandmeyer reaction to convert 2,3-diaminonapthalene to fluorescent napthalenetriazole in the presence of no. as no is produced in the tlr inflammatory response, this readout provides information on the extent of tlr signaling. initial screening yielded 31 hits, representing 5 scaffolds (figure s1, supporting information). we selected the scaffold with an isothiazolone 1,1-dioxide core for further development as it produced the most numerous and potent hits. compounds 1 and 2 (figure 1) represent two of the more potent hits. generic scaffold of hit molecules selected from the screening of the colorado center for drug discovery (c2d2) pilot library. both variable functionalities (ar = substituted benzene ring, r= various amide moieties) shown in these representative structures have been subsequently optimized. we have developed an efficient synthetic route for the generic scaffold in figure 1. as an example, the synthesis of the most potent hit molecule, 1 (scheme 1), began with the oxidative dimerization of commercially available 3 to give dithiodipropionic acid 4. conversion to the dithiodipropionyl chloride was completed with thionyl chloride, and subsequent treatment with anhydrous ammonia gave dithiodipropionamide 5 as a mixture of diastereomers. as previously described by lewis and co - workers, oxidative cyclization was performed with sulfuryl chloride to give an inseparable mixture of 6 and 7. this mixture of isothiazolinones was then deprotonated with sodium hydride, and alkylated with benzyl chloride 9. the alkylation yields two synthetically useful intermediates 12 and 13, which are easily separable by column chromatography. chloro - intermediate 12 was oxidized with meta - chloroperoxybenzoic acid (m - cpba) to give the isothiazolone 1,1-dioxide core, 14. (i) i2, ki, naoh, h2o, (4 = 99%) ; (ii) (1) socl2, (2) nh3, ch2cl2 (5 = 66%) ; (iii) so2cl2, etoac (6 and 7 = 51%) ; (iv) piperidine or furfurylamine, dipea, ch2cl2 (9 = 95%, 10 = 99%) ; (v) boc2o, dmap, thf (11 = 81%) ; (vi) (1) nah, dmf, (2) 9 (12 = 70%, 13 = 67%) ; (vii) m - cpba, ch2cl2 (14 = 49%) ; (viii) arb(oh)2, pd(dppf)cl2ch2cl2, k2co3, 1,4-dioxane (1 = 47%, 15 = 32%) ; (ix) 4-bromopyridinehcl, pd(oac)2, koac, dma (16 = 54%) ; (x) m - cpba, ch2cl2 (17 = 84%). chloro - intermediate 14 was coupled to 3,4-dimethylphenylboronic acid, using previously reported conditions to give the parent library hit, 1. these conditions failed, however, when 4-pyridinylboronic acid was used. making use of intermediate 13 with heck conditions and the coupling product 16 was fully oxidized at both sulfur and the pyridyl nitrogen to give 17. the synthesis of compounds 15 and 17 provide analogues that exhibit both higher and lower calculated log p values, 4.70 and 1.29 respectively, as compared to 1 (4.46). however, 15 only showed a small improvement in potency, and 17 showed greatly reduced potency as compared to 1 (table 1). ic50 values were obtained using raw 264.7 cells treated with 20 ng / ml lps and varying concentrations of compound. a cell viability assay was used to determine cytotoxicity at each tested concentration. the purity of tested compounds was evaluated via h nmr (> 95% sample purity). the 2-benzyl-4-methyl-5-phenylisothiazol-3-one 1,1-dioxide core of 1, 15, and 17 was sensitive to a variety of mild reaction conditions. the few successfully synthesized analogues exhibited only modest activity, so we thought it best to make more drastic structural changes. in an effort to simplify synthesis and increase stability, we took inspiration from saccharin 18 (scheme 2). (xi) (1) nah, dmf, (2) 9 (19 = 65%) or 11 (103 = 62%) ; (xii) tfa, ch2cl2 (20 = 87%). saccharin inspired analogues, 19 and cu - cpd103 (103), were easily synthesized from commercially available saccharin and previously synthesized benzyl chlorides 9 and 11. a significant improvement in activity was observed with intermediate 103, so we sought related analogues 2643 (scheme 3 ; detailed syntheses can be found in the supporting information). compounds 19, 20, 103, and 2643 contain the same piperidine or furfuryl amide moieties (figure 1) that were present in our initial library screen so we could have a consistent basis for comparison. the lithium aluminum hydride reduction of saccharin 18, previously described by porter and co - workers, provided 2,3-dihydro-1,1-dioxo-1,2-benzisothiazole 21. alkylation of this sultam with 9 and 11 provided analogues 26 and 27. commercially available 1-isoindolinone 22, phthalimide potassium salt 23, 1,2-benzisothiazol-3(2h)-one 24, and 3-hydroxybenzisoxazole 25, were alkylated with 9 and 11 to give analogues 29, 30, 32, 33, 35, 36, 38, and 39 (scheme 3). treatment of analogues 35 and 36 with m - cpba at 0 c gave analogues 38 and 39, respectively, as racemic mixtures. all boc protected analogues were treated with trifluoroacetic acid in methylene chloride to give deprotected analogues 28, 31, 34, 37, 40, and 43. (xiv) (1) nah, dmf, (2) 9 (26 = 84%) or 11 (27 = 81%) ; (xv) tfa, ch2cl2 (28 = 92%) ; (xvi) (1) nah, dmf, (2) 9 (29 = 57%) or 11 (30 = 10%) ; (xvii) tfa, ch2cl2 (31 = 71%) ; (xviii) 18-crown-6, dmf, 9 (32 = 84%) or 11 (33 = 81%) ; (xix) tfa, ch2cl2 (34 = 81%) ; (xx) (1) nah, dmf, (2) 9 (35 = 48%) or 11 (36 = 27%) ; (xxi) tfa, ch2cl2 (37 = 97%) ; (xxii) m - cpba, ch2cl2, 35 (38 = 85%) or 36 (39 = 79%) ; (xxiii) tfa, ch2cl2 (40 = 92%) ; (xxiv) (1) nah, dmf, (2) 9 (41 = 55%) or 11 (42 = 14%) ; (xxv) tfa, ch2cl2 (43 = 87%). the removal of the saccharin series 3-position carbonyl gave us the sultam series 26, 27, and 28 (table 2). as we had seen with the parent saccharin series, the boc protected amide 27 was the most potent of the series. for this reason, we believe the 3-position carbonyl to be somewhat important to either binding or cell permeability. replacement of the saccharin series 1-position so2 moiety with a methylene unit gave us the isoindolinone series 29, 30, and 31. unlike others, the analogue bearing a boc protected amide 30 shows the lowest potency.. this led us to believe that the 1-position so2 moiety makes a significant interaction, perhaps as a hydrogen bond acceptor. as previously discussed, the 3-position carbonyl of 103 appears less significant, so the carbonyl of the isoindolinone 30 may be making a weaker hydrogen bond interaction as a poor bioisostere of the so2 moiety. ic50 values were obtained using raw 264.7 cells treated with 20 ng / ml lps and varying concentrations of compound. the purity of tested compounds was evaluated via h nmr (> 95% sample purity). to further investigate the role of the so2 moiety, the 3-position carbonyl of 103 was maintained and the so2 moiety replaced with an additional carbonyl, as shown with the phthalimide analogues 32, 33, and 34. this suggests that a carbonyl is a suitable bioisostere to the so2 moiety so long as the 3-position carbonyl is intact, unlike 30. furthermore, a loss of one, or both, oxygen atoms from the so2 moiety of 103 results in a complete loss of activity, as seen with 36 and 39. as seen before as expected, the piperidine amide analogue, 38 shows a further decrease in potency. however, 35 inexplicably shows comparable potency as compared to 103 and is a great improvement over the related analogue 26. when the unoxidized sulfur of 36 is replaced with a smaller oxygen atom 42, the activity is once again comparable to that of 103. again, the deprotected analogue 43 shows significantly lower activity, although 41 shows some modest activity. we further investigated the role of the so2 moiety by synthesizing the fully reduced and partially reduced variants, 3540. if the so2 moiety does play the role of hydrogen bond acceptor, then these reduced analogues would be less efficacious. as expected, we observed either a loss in activity or no change in activity from analogues 3640. the fully reduced analogue 35, however, inexplicably shows activity comparable to that of 103. this modification resulted with a drop in activity for 41 as compared to 35 and a great improvement with 42 as compared to 36. in fact, the potency of 42 is comparable to that of 103, 27, and 33. the steric constrains of this position seem flexible, given two of the more active analogues in this series, 103 and 42, are the largest and smallest of the series, respectively. the less active of this series are of varying size, but are consistently less basic than the more active analogues. from this series of molecules, there are five analogues that have ic50 values of less than or roughly equal to 10 m. four of these five analogues have the same boc - protected furfurylamide (103, 27, 33, and 42). this implies that there is an important interaction(s) being made with the boc group, and/or that an amide n analogues 50, 52, 54, 55, 56, 57, and 58 were synthesized from the corresponding carboxylic acid 45 (scheme 4). (xxvi) (1) nah, dmf, (2) 4-bromomethylbenzoic acid tert - butyl ester (44 = 62%). (xxiv) procedure a, 46 in ch2cl2, then ammonia in thf (49 = 92%), or methylamine in thf (51 = 79%), or 2-furan-2-yl - ethylamine and dipea (53 = 85%), or n - methylfurfurylamine and dipea (55 = 86%) ; procedure b, (1) tert - butylcarbamate, lihmds, thf, (2) 46 in ch2cl2 ; procedure c, (1) nah in dmf, then 47 or 48, (2) 46 in ch2cl2 (57 = 41%, 58 = 17%). interestingly, intermediate ester 44 showed modest activity (table 3) while intermediate carboxylic acid 45 shows no activity. this may imply that the previously successful analogues bearing boc groups might be benefiting from hydrophobic interactions with the tertiary butyl moiety. boc protected amides 52 and 56, however, have shown very poor activity. initial efforts to synthesize 56 produced the double - boc protected amide 50, which also has poor activity, suggesting that the furan substituent was necessary. we also investigated a very minor change by synthesizing the extended linker analogue 54. ic50 values were obtained using raw 264.7 cells treated with 20 ng / ml lps and varying concentrations of compound. the purity of tested compounds was evaluated via h nmr (> 95% sample purity). to test if the presence of an acidic amide n h could be causing a negative effect on activity, we synthesized 55. however, we observed significantly lower potency, perhaps suggesting that the rotational constraints of a tertiary amide are not conducive to activity. given this observation, we speculated that perhaps a carbonyl component was still required for activity. however, the n - methylcarbamate and n - acyl analogues, 57 and 58, were significantly less potent than even 20. we concluded that the furfurylamide component was essential, and that boc protection was the optimal substituent. on the basis of these results, 103 was selected as our lead compound. our initial screen identified compounds that could inhibit the lps - induced inflammatory response. to determine if 103 specifically targets tlrs, lps (tlr4), poly i : c (tlr3), and pam2csk4 (tlr2/6) were chosen to encompass the most variety in signaling, including differences in tlr localization and adaptor proteins (figure s3, supporting information). as figure 2 shows this suggests that 103 does not bind specifically to an individual tlr but rather inhibits a common downstream factor of these tlrs. the dose response curves for all three ligands show comparable inhibition, with minor deviations due to tlr expression levels and the effectiveness of the ligand to induce inflammation. primary macrophage cells demonstrated the same behavior as raw 264.7 cells, with 103 inhibiting lps signaling with an ic50 value of 9.61 1.45 m (figure s2, supporting information). it is important to note that 103 shows no cytotoxicity at concentrations up to 100 m (figure s4, supporting information). raw 264.7 cells were treated with lps (tlr4 ligand), poly i : c (tlr3 ligand), or pam2csk4 the ic50 values are 2.61 0.40, 10.9 0.74, and 1.69 0.43 m, respectively. treatment with 103 decreased no production with all tlr ligands in a dose - dependent fashion. these results demonstrate that 103 does not specifically inhibit a particular tlr but rather, a common downstream effector. data was normalized [(raw data - untreated cells)/(tlr agonist + solvent control - untreated cells) ] such that tlr agonist + solvent is 100% activation, and untreated cells are 0% activation. data points shown are the average of nine replicates, with error bars represented as the standard deviation. to further confirm that inos was being down regulated by treatment with 103, quantitative real - time polymerase chain reaction (rt - pcr) and western blot experiments were performed. rt - pcr data was obtained using raw 264.7 cells treated with lps and varying concentrations of 103. figure 3 demonstrates that treatment with 103 decreases inos mrna in a dose - dependent fashion. western blots were performed with a pan nos antibody and again inos is seen to decrease in a dose - dependent fashion (figure 4), indicating that 103 suppresses inos at mrna, protein, and cell signaling levels. while lps was used as the inflammation - inducing ligand in the no production assay to maintain consistency, its effects are indirect. therefore, a secondary assay that monitors ifn- induced mrna changes was carried out to confirm the validity of the no assay as the primary readout for understanding sar and compound optimization (figure s7, supporting information). these results demonstrated the same trend of inhibition for 103, 36, and 49 (49 is commercially available from enamine ltd.). compound 103 is able to inhibit greater than 70% of ifn- induced mrna, whereas 36 and 49 show a weaker potency, with maximum inhibition of 50%. importantly, these results corroborate the ic50 values determined in the previously described no production assay. 103 treatment decreases inos mrna in a dose - dependent fashion. raw 264.7 cells were incubated with 20 ng / ml lps and varying concentrations of 103 for 20 h. data is shown with ligand - induced cells normalized to a fold change of 1. data shown is the average quantification of three biological replicates, each in technical duplicate, with error bars represented as the standard deviation. (a) 103 treatment reduces inos protein expression in a dose - dependent fashion. the inos protein is induced by lps treatment and decreases with compound treatment, suggesting that compound reduces the inflammation that results in inos expression. the image shown is a representative image, with brightness and contrast adjusted for clarity. data shown is the average quantification of three biological replicates, with error bars represented as the standard deviation. p 0.001. regardless of their ligand or localization, all tlrs activate nf-b (figure s3, supporting information). to test the inhibitory effects of 103 on nf-b activity, a secreted embryonic alkaline phosphatase (seap) assay was performed in hek 293 t cells. when tested at concentrations up to 100 m, the compound did not down - regulate nf-b activation through tlr3 or tlr4 (figure s5, supporting information). to determine if any modulation occurs through other nf-b pathways, tnf- was used to activate nf-b signaling. as seen in the figure s4 in the supporting information, this data suggests that 103 does not directly modulate the tlr signaling pathway at any point, as nf-b is essential to all tlr signaling. we next sought to confirm this result through observation of nf-b - induced cytokines, particularly tnf-. a commercially available enzyme - linked immunosorbent assay (elisa) was used to measure tnf- in raw 264.7 cells. figure s6 in the supporting information shows that there was no change in tnf- cytokine levels with compound treatment. these results confirm in two cell types that there is no modulation of nf-b by 103, regardless of ligand or signaling pathway. however, previous results demonstrated that tlr - induced no activation is inhibited by 103. the inos promotor has binding sites for both nf-b and stat1. because nf-b activation is not being affected with 103 treatment, the inhibition of inos was therefore likely to occur within the jak / stat1 pathway. as no direct antagonism was observed through tlrs and nf-b, additional tests were carried out to identify the potential anti - inflammatory mechanism of 103. (ifn-/) and interferon ii (ifn-) pathways cause upregulation of inos, which results in production of no. as such, we speculated that observed no inhibition might occur through inhibition of the jak / stat signaling pathway. to test this hypothesis, inhibition of no occurred in a dose - dependent fashion with treatment of 103. the ic50 value with ifn- is 7.88 1.25 m, which corroborates the ic50 value of lps (figure 5). this indicated that the jak / stat1 pathway is involved in the inhibitory function of 103. additionally, as tlr activation results in production of type i interferons, synonymous inhibition with ifn- proposes a shared target between these two pathways. thus, it is likely that the molecular target of 103 lies in the interferon - induced stat1 pathway. raw 264.7 cells were treated with 5 ng / ml ifn- to activate jak / stat1 signaling. data was normalized [(raw data - untreated cells)/(tlr agonist + solvent control - untreated cells) ] such that tlr ligand + solvent is 100% activation, and untreated cells are 0% activation. data points shown are the average of nine replicates, with error bars represented as the standard deviation. additionally, a commercial jak / stat rt - pcr array was used to determine if jak / stat pathway signaling is by and large modulated with 103 treatment. the modified genes, including nos2, cebpb, and gtp1, suggest that 103 modulates stat1 signaling. additional validation is required to target the specific site of action for 103. taken together, our results provide consistent evidence that 103 functions through the interferon - induced jak / stat1 signaling pathway, suppressing inos. in summary, we report the identification of a group of novel ifn inhibitors based on a saccharine core. extensive sar studies have shown a narrow tolerance for change at the lone amido position. a boc protected furfurylamide has proven to be the most consistently successful substituent. our results demonstrate that 103 is a potent inhibitor of inos on both an mrna and protein expression level. however, this occurs without nf-b modulation, indicating a tlr - independent mechanism. further biochemical studies imply potential inhibition in the stat1 pathway, as this is shared between type i and ii interferons, and associated genes were observed to change via pcr array. 103 may provide therapeutic insight for inflammatory diseases such as systemic lupus erythematosus and multiple sclerosis. | a series of novel, saccharin - based antagonists have been identified for the interferon signaling pathway. through in vitro high - throughput screening with the colorado center for drug discovery (c2d2) pilot library, we identified hit compound 1, which was the basis for extensive structure activity relationship studies. our efforts produced a lead anti - inflammatory compound, tert - butyl n-(furan-2-ylmethyl)-n-{4-[(1,1,3-trioxo-2,3-dihydro-16,2-benzothiazol-2-yl)methyl]benzoyl}carbamate cu - cpd103 (103), as a potent inhibitor using an established nitric oxide (no) signaling assay. with further studies of its inhibitory mechanisms, we demonstrated that 103 carries out this inhibition through the jak / stat1 pathway, providing a drug - like small molecule inflammation suppressant for possible therapeutic uses. |
nitric oxide (no) is a pleiotropic signaling molecule produced endogenously by no synthases in diverse biological systems. s - nitrosylation is the addition of a no group to a specific cysteine (cys) residue of a protein to form a s - nitrosothiol. this prototypic, redox - based post - translational protein modification process has been increasingly recognized as a key cellular and molecular mechanism for no signaling and plays an important role in physiological conditions and in diseases such as diabetes, asthma, heart failure, cancer, and neurodegenerative disorders.s - nitrosylation of critical cys residues on specific proteins regulates their activities and has been demonstrated in protein misfolding resulting in interference of normal cellular functions. in order to investigate mechanisms underlying no signaling, a number of methods have been established for identifying s - nitrosylated proteins (sno - proteins) and mapping their s - nitrosylated cysteine (sno - cys) sites. the biotin switch technique (bst) developed by jaffrey. it involves first blocking free thiols with a sulfhydryl - reactive reagent, such as s - methyl methanethiosulfonate (mmts), then selectively reducing the s - nitrosothiols with ascorbate, and labeling with n-[6-(biotinamido)hexyl]-3-(2-pyridyldithio) propionamide (biotin - hpdp). the biotinylated sno - proteins can be enriched via avidin - agarose affinity capture, and then proteins of interest are detected by immunoblotting. despite its popularity, this method is relatively low - throughput, and it is difficult to locate the modification sites. additionally, the biotin affinity enrichment may introduce false positive signals due to the existence of endogenous biotin - like molecules. to specifically assess sno - cys modification status and increase the throughput of detection, several mass spectrometry (ms)-based methods have been developed by modifying the bst protocol, including sno - cys site identification (snosid), s - nitrosothiol resin - assisted capture (sno - rac), s - alkylating labeling strategy, and cystmt switch assay. however, none of these modified bst methods are able to achieve all of the following features desired for comprehensive characterization of sno - cys modification : a simple, robust workflow for sno - protein identification, an irreversible thiol linkage for effective peptide digestion and for unambiguous sno - cys mapping, as well as sample multiplexing for increased throughput and relative quantification of sno - proteins under various conditions. in this study, we present a novel ms - based modified bst, the iodotmt switch assay (isa), using cys thiol - reactive iodoacetyl tandem mass tag (iodotmt) sixplex reagents to identify and quantify protein s - nitrosylation. with a simple workflow, isa accomplishes irreversible labeling of sno - cys sites, s - nitrosylated peptide (sno - peptide) enrichment using high affinity anti - tmt chromatography with competitive elution, and multiplex quantification of protein s - nitrosylation via six unique tmt mass reporter ions. notably, this workflow is capable of detection and quantification of sno - cys modifications under physiological and pathological conditions. in this study, we applied this approach to the investigation of protein s - nitrosylation in a neuroinflammation modelendotoxin lipopolysaccharide (lps)-stimulated immortalized murine bv-2 microglial cells representing a convergent nitrosative stress and neuroinflammatory response in the pathogenesis of age - associated neurodegenerative diseases. we further evaluated the effects of s - allyl cysteine (sac), an active nutritional compound of aged garlic extract (age), on lps - stimulated s - nitrosylation in bv-2 cells. the iodotmtsixplex reagents, anti - tmt antibody, immobilized anti - tmt antibody resin, bicinchoninic acid (bca) protein assay kit, c18 tips, and primary antibodies for protein validation were obtained from thermo fisher scientific (rockford, il). sep - pak tc18 1 cc vac cartridges were purchased from waters (dublin, ireland). amersham ecl prime western blotting detection reagent was purchased from ge healthcare (buckinghamshire, uk). trypsin (modified, sequencing grade) was obtained from promega (madison, wi). dulbecco s modified eagle s medium (dmem), penicillin - streptomycin, and l - glutamine were obtained from gibco (grand island, ny). all other reagents including sac (a garlic active component) were from sigma - aldrich (st. louis, mo). the purity and stability of sac were validated prior to use through the nutrition core of the center for botanical interaction studies at the university of missouri. the immortalized murine bv-2 microglial cells were cultured in dmem containing 5% (v / v) heat - inactivated fbs, 25 u / ml penicillin, and 25 g / ml streptomycin at 37 c in a saturated humidity atmosphere containing 95% (v / v) air and 5% (v / v) co2. cells were plated at a density of 1 10/ml medium in 35 mm or 100 mm plates and grown to 70% confluence prior to testing. bv-2 cells were starved for 4 h in fbs - free dmem (without phenol red) and then exposed to 100 ng / ml lps for 16 h in the presence or absence of 5 mm sac or 0.5 mm n--nitro - l - arginine methyl ester hydrochloride (l - name). after lps stimulation, conditioned medium was collected and mixed with an equal volume of griess reagent [1% (w / v) sulfanilamide and 0.1% (w / v) n-(1-naphthyl) ethylenediamide in 5% (v / v) phosphoric acid ]. after a 10 min incubation at room temperature (rt), absorbance was read at 543 nm using a biotek synergy-4 microplate reader (biotek instruments, inc., a series of sodium nitrite dilutions (0100 m) was used to generate a nitrite standard reference curve. after treatment, 0.5 mg / ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2h - tetrazolium bromide (mtt) was added to the cell culture. after incubation at 37 c for 4 h, the formed formazan crystals were collected and dissolved in dimethyl sulfoxide (dmso). the absorbance at 540 nm was read using the synergy-4 microplate reader. for in vitro s - nitrosylation, 400 g of bv-2 cell lysates in hents buffer [250 mm hepes - naoh, ph 7.4, 1 mm edta, 0.1 mm neocuproine, 1% (v / v) triton x-100, 0.1% (w / v) sds, 1% (v / v) protease inhibitor cocktail ] were exposed to a final concentration of 200 m s - nitrosocysteine (snoc) for 30 min at rt in the dark. in vivo treated bv-2 cells were rinsed twice in cold phosphate - buffered saline followed by lysis in hents buffer. free sulfhydryl groups in the protein sample were first blocked with 20 mm mmts for 30 min at 50 c with frequent vortexing in hen (250 mm hepes - naoh, ph 7.4, 1 mm edta, 0.1 mm neocuproine) buffer containing 2.5% (w / v) sds. to remove excess mmts, proteins were precipitated using 4 volume of cold acetone for 30 min at 20 c. proteins were then redissolved in hen buffer containing 1% (w / v) sds and labeled with 1 mm iodotmt reagent in the presence of 5 mm sodium ascorbate for 2 h at rt. for ms analysis, after acetone precipitation, iodotmt - labeled proteins were redissolved with tris - hcl, ph 8.0, containing 0.1% (w / v) sds. six different protein samples individually labeled by iodotmtsixplex reagents were combined before tryptic digestion. the iodotmt - labeled proteins were reduced with 10 mm dithiothreitol (dtt) at 55 c for 1 h and then alkylated with 25 mm iodoacetamide at 37 c for 1 h in the dark. after acetone precipitation, the protein pellet was resuspended with 50 mm ammonium bicarbonate containing 20 ng/l trypsin and digested overnight at 37 c. undigested proteins were removed by centrifuge at 2500 g, rt for 10 min. the peptides were then desalted with 50 mg waters sep - pak tc18 columns and eluted with 50% (v / v) acetonitrile (acn) and 0.1% (v / v) trifluoroacetic acid (tfa). a small portion of this unfractionated sample was retained for future analysis of the unenriched sample. for peptide enrichment, the above samples were lyophilized and redissolved in 600 l of tbs buffer (25 mm tris - hcl, ph 7.5, 0.15 m nacl). typically, 100 l of settled immobilized anti - tmt antibody resin is employed for enrichment of every 1 mg of s - nitrosylated sample. in our experiments, the peptide sample was incubated with 200 l of settled anti - tmt antibody resin overnight at 4 c with end - over - end rocking. after the supernatant was removed, the resin was washed three times with one column volume of tbs and three times with one column volume of milli - q water. peptides were finally eluted with four column volumes of elution buffer [10 mm cis-2,6-dimethylpiperidine (2,6-dmpp)/500 mm tetraethylammonium bicarbonate (teab), ph 8.5 ]. a easy - nlc 1000 hplc system and easyspray source (thermo scientific) with easy - spray pepmap rslc c18 25 cm 75 m i d column (thermo scientific) were used to separate peptides with a 525% (v / v) acn gradient in 0.1% (v / v) formic acid over 120 min at a flow rate of 300 nl / min. samples were analyzed on orbitrap elite and ltq orbitrap xl mass spectrometers (thermo scientific) using top 15 fourier transform (ft) ms / ms with higher - energy collision dissociation (hcd) or top 3/3 ion - trap collision - induced dissociation (cid)/ft hcd experiments. proteome discoverer software version 1.4 (thermo scientific) was used to search ms / ms spectra against the swiss - prot mouse database using the sequest ht or mascot 2.3 search engines. dynamic modifications included carbamidomethylation (c), iodotmtsixplex (c), and methionine oxidation. resulting peptide hits were filtered for a maximum 5% false discovery rate using the percolator. the iodotmtsixplex quantification method within proteome discoverer software was used to calculate the reporter ratios with a mass tolerance 10 ppm. anti - tmt antibody affinity was determined using a tmt - derivatized cm5 chip on a biacore 3000 instrument (ge healthcare). for competitive elution buffer scouting, anti - tmt antibody was first bound to tmt - derivatized surface followed by a timed injection of tmt analogue compounds. for sds - page, 4 sds sample buffer with 1% (v / v) 2-mercaptoethanol was added to the protein samples. proteins were then resolved on 10%, 1 mm sds - page gels. for immunoblotting the membrane was first blocked in phosphate - buffered saline plus 0.1% (v / v) tween-20 (pbst) containing 5% (w / v) nonfat milk at rt for 1 h followed by incubation with primary antibody in pbst at 4 c overnight. after being washed three times with pbst, the membrane was incubated with secondary antibody (anti - mouse igg - peroxidase antibody produced in goat or anti - rabbit igg - peroxidase antibody produced in goat, sigma) in pbst at rt for 1 h. immuno - reactive bands were visualized using amersham ecl prime western blotting detection reagent and detected with las-4000 imaging system (fujifilm). degree of freedom and z score for each iodotmt modification site was calculated by a special student s t - test, known as welch s t - test. we further used a t - distribution implemented in the r statistical package to calculate the p - value based on degree of freedom and z score. s - nitrosylation level change of a sno - cys was considered as significant if its fold change is greater than 1.3 and p - value is less than 0.05. motif consensus sequences for protein s - nitrosylation was analyzed by the motif - x algorithm. sno - proteins with significant s - nitrosylation level changes were subjected to functional annotation by ingenuity pathway analysis (ipa ; http://www.ingenuity.com) and the in - house multicom - pdcn software. iodotmtsixplex reagents are commercially available and were used in the study to determine protein s - nitrosylation. each isobaric iodotmtsixplex reagent within a set has the same nominal mass and consists of a thiol - reactive iodoacetyl group, a ms - neutral spacer arm, and a ms / ms reporter. the chemical structure of iodotmt-126 (one of the sixplex reagents ; 126 refers to the mass of the reporter ion) is shown in figure 1a. during ms / ms analysis to derive fragment ions and sequence information, the isobaric mass tags are cleaved generating reporter ions with unique m / z of 126131. in isa, after blocking free thiols in the proteins with mmts, an iodotmt reagent is used to irreversibly label nascent thiols generated by the selective reduction of sno - cys bonds by ascorbate (figure 1b). the irreversibility of iodotmt labeling was validated under our tested conditions (supplemental figure s1, supporting information). to test the specificity of iodotmt labeling, bv-2 cell lysates were s - nitrosylated in vitro by exposure to a physiological no donor snoc (200 m) and followed by labeling with iodotmt-126. as shown in figure 1c, iodotmt - labeled samples showed much stronger signals than control conditions without snoc and/or ascorbate on the anti - tmt immunoblot. to test the sensitivity of the iodotmt labeling assay, bv-2 cell lysates were treated with different concentrations (10, 50, 100, 200 m) of snoc. after switch labeling with iodotmt-126, sno - protein signals in snoc - treated samples exhibited a dose - dependent increase compared to the untreated control sample (figure 1d). these results suggested that iodotmt switch labeling is feasible and reliable for detecting sno - proteins and is sensitive to as low as 10 m snoc treatment. (a) the chemical structure of iodotmt-126 reagent. the iodotmtsixplex reagents including iodotmt-126 illustrated here consist of a cys thiol - reactive iodoacetyl group, a mass normalizing spacer arm, and a mass reporter. (b) reaction scheme for the labeling of sno - protein by iodotmt reagents. sno - cys residues are then selectively released by ascorbate and labeled by thiol - reactive iodotmt reagents. s - nitrosylated by snoc (200 m) were switch - labeled by iodotmt-126. this sample exhibited strong signals on the anti - tmt immunoblot, whereas controls omitting snoc and/or ascorbate displayed much lower signal levels. (d) bv-2 cell lysates exposed to snoc (0, 10, 50, 100, 200 m) were switch - labeled by iodotmt-126. as the concentration of snoc increased, more sno - proteins were detected on the anti - tmt immunoblot. the protocol allows both sno - protein quantification and sno - cys site - mapping. furthermore, up to six different protein samples can be simultaneously analyzed with iodotmtsixplex reagents, iodotmt126131. after individually labeling of protein samples with iodotmtsixplex reagents as shown in figure 1b, samples are combined before trypsin digestion (figure 2a). since the labeling of cys thiols by iodotmt reagents is irreversible, reduction with dtt and alkylation with iodoacetamide are possible for more efficient digestion than that of the reversible thiol reactions. digested peptides are then subjected to enrichment with anti - tmt antibody resin following the protocol described in materials and methods. because of the low abundance of sno - proteins and sno - cys modifications, it is essential to enrich the iodotmt - labeled peptides before ms analysis. during lc ms / ms analysis of enriched peptides, sno - cys are identified by the site of iodotmt labeling and quantified based on ratios of the iodotmtsixplex reporter ions. after switch labeling with iodotmtsixplex reagents as shown in figure 1b, the six samples are combined for in - solution trypsin digestion and peptide enrichment with anti - tmt resin. ms / ms for sno - cys site identification and quantification based on iodotmt reporter ions. (a) surface plasmon resonance binding curve of anti - tmt binding to tmt - derivatized biacore chip with (gray line) and without (black line) addition of a competitive tmt analogue compound. (b) graph of competitive tmt analogue compounds screened using surface plasmon resonance. binding data is normalized to mock injections and tmt antibody off rate steady state kinetics at 10 mm concentration of the testing compounds in 500 mm teab buffer, ph 8.5. dmac, dimethylacetamide ; dmpz, dimethylpiperizine, tmp, tetramethylpiperidine, mp, methylpiperidine, dmpp, 2,6-dmpp, dipea, diisopropylethylamine. (c) comparison of acidic elution buffer eb1 and competitive elution buffer eb2 for enrichment of iodotmt - labeled sno - peptides. bv-2 cell lysates treated or untreated with snoc (200 m) were processed following the isa workflow. ms / ms analysis shows that iodotmt - labeled peptides account for 0.15%, 0.22%, 6.13%, and 21.36% of total peptides in the unenriched, flow through, enriched by eb1 elution, and enriched by eb2 elution, respectively. a unique feature of the isa workflow is the use of an immobilized anti - tmt resin for labeled peptide enrichment. since an iodotmt reagent is used to label peptides in the switch assay instead of a biotinylation reagent, endogenously biotinylated proteins are not pulled down during enrichment, resulting in improved specificity of detecting sno - proteins. previously, an acidic elution buffer [50% (v / v) acn/0.4% (v / v) tfa ; eb1 ] was used to elute tmt - labeled peptides from the anti - tmt resin after enrichment. however, this buffer can also elute a certain amount of nonspecific binding peptides along with the specific tmt - labeled peptides. in order to further improve the specificity of the peptide enrichment while retaining the efficiency of the anti - tmt affinity precipitation, a new elution buffer containing a tmt structural analogue that could specifically compete for tmt - labeled peptide binding was developed. we screened a series of compounds representing various structural motifs of the tmt tag for testing anti - tmt binding affinity using a biacore competition assay (figure 2b, a). we found that structural analogues of the mass tag reporter region had the greatest ability to compete for anti - tmt antibody binding (figure 2b, b). comparing the original acidic elution buffer eb1 with the newly developed competitive elution buffer (10 mm 2,6-dmpp/500 mm teab, ph 8.5 ; eb2) for enrichment of the iodotmt - labeled sno - peptides revealed a significant increase in labeled peptide percentage of total identified peptides from 6.13% to 21.36% (figure 2c). these data show that eb2 works more effectively than that of eb1 and was therefore used in isa for subsequent studies. to demonstrate the efficiency of the isa method, triplicates of untreated and snoc (200 m)-treated bv-2 microglial cell lysates were analyzed by isa. figure 3a shows a representative ms / ms spectrum of an identified sno - peptide (left ; peptide sequence : yvdiaipcnnk ; protein reference : 40s ribosomal protein sa ; sno - cys modification site : c8). the quantification of this peptide is shown as well (figure 3a, right). in total, 134 sno - cys sites were identified, and they were detected in all of the three replicates. ninety of them from 68 proteins showed significant changes in s - nitrosylation level (fold change > 1.3, p 1.3, p 1.3, p 1.3, p 1.3, p 1.3, p < 0.05) responding to snoc, lps, and lps + sac treatments in bv-2 cells, respectively (figure 6a). there were 14 sno - proteins shared by snoc- and lps - treated samples, suggesting common and distinct signaling pathways were triggered by these two treatments. twelve common sno - proteins were found between lps and lps + sac treatments (figure 6a), indicating sac partially attenuated the effect of lps on protein s - nitrosylation but also had its own distinct targets. nineteen out of 46 sno - proteins responding to lps + sac - treatment were in common with the snoc - treatment. the six sno - proteins shared by all of the three groups include lcp1, pkm2, slc25a5, cfl1, enoa, and ldha. (a) the overlaps between the three subsets of sno - proteins identified from different treatment conditions in this study. (b) subcellular location of the 46 sno - proteins responding to sac treatment in lps - stimulated bv-2 cells. (c) top 10 ipa canonical pathways targeted by sac in lps - stimulated bv-2 cells are presented : (a), glycolysis i ; (b), gluconeogenesis i ; (c), eif2 signaling ; (d), sucrose degradation ; (e), 14 - 3 - 3-mediated signaling ; (f), tca cycle ii (eukaryotic) ; (g), regulation of eif4 and p90s6k ; (h), mtor signaling ; (i), remodeling of epithelial adherent junctions ; (j), glycine biosynthesis i. these pathways were ranked according to their log (p - value) (black bars). a ratio (gray square) indicates the number of identified sno - proteins found in each pathway over the total number of proteins in that pathway. (d) the top protein network associated with sac - treatment in lps - stimulated bv-2 cells functions in neurological disease as predicted by ipa. twenty - seven identified sno - proteins are involved in this network and displayed in gray. the color intensity indicates the degree of down - regulation of protein s - nitrosylation. solid lines in the network imply direct interactions between proteins, and dashed lines indicate indirect interactions. geometric shapes represent various general functional protein groups (diamond for enzyme, oval for transcription regulator, trapezoid for transporter, inverted triangle for kinase, double circle for complex / group, and circle for others). proteins in white shapes are not part of our data set but have relationships with our proteins in the network. in order to learn more about the molecular mechanisms of no signaling under microglial activation and of sac to modulate lps - induced s - nitrosylation in bv-2 microglial cells, we conducted functional annotation and pathway analysis of the sno - proteins identified. ipa analysis shows the 68 sno - proteins significantly altered by snoc treatment are mainly associated with neurological disease, immunological disease, and inflammatory disease, while the 30 sno - proteins in response to lps - stimulation are mostly involved in immunological disease and inflammatory disease / response (data not shown). other functional annotation results for snoc and lps treatments, including molecular and cellular functions (supplemental tables s4 and s5), subcellular locations, and top canonical pathways (supplemental figures s5 and s6) are shown in supporting information. specific effects of sac were assessed using an in - house multicom - pdcn analysis. the results of this analysis shows that the 46 sno - proteins modulated by sac are mainly located in the cytoplasm and the nucleus (figure 6b), and they are involved in diverse molecular and cellular functions including carbohydrate metabolism, cellular assembly and organization, cellular function and maintenance, molecular transport, and rna trafficking as predicted by ipa (table 1). top canonical pathways in which these proteins participate include glycolysis i, gluconeogenesis i, eif2 signaling, sucrose degradation, 14 - 3 - 3-mediated signaling, tca cycle ii (eukaryotic), regulation of eif4 and p90s6k, mtor signaling, remodeling of epithelial adherent junctions, and glycine biosynthesis i (figure 6c). moreover, sac mediated a protein protein interaction network that functions in neurological disease (figure 6d). twenty - seven out of the identified 46 sno - proteins are involved in this network. these findings illustrate that sac exhibits multimodal action on protein s - nitrosylation under microglial activation, which may further contribute to the reversing effects in neurological diseases. s - nitrosylation is regarded as a major mechanism for no bioactivity with participation in cellular processes, including vasodilation, neurotransmission, apoptosis, cellular trafficking, and cell cycle regulation. to facilitate efficient investigation of s - nitrosylation under physiological and pathophysiological conditions, we have developed a novel ms - based isa method based on the conventional bst assay for the detection, identification, and multiplex quantification of sno - cys residues. we demonstrated in both in vitro and in vivo studies under physiological conditions that isa is an effective tool for investigation of protein s - nitrosylation with several unique features : (i) irreversible labeling of sno - cys residues for specific ms detection and site - mapping ; (ii) anti - tmt enrichment and competitive elution to improve efficiency of identification of sno - peptides ; and (iii) multiplex iodotmt reagents for unbiased quantification of sno - proteins from complex biological samples under different conditions. there is substantial evidence indicating that s - nitrosylation is a highly conserved process and is precisely regulated in cells. despite the possibility of multiple cys residues in a protein, only specific sites can be s - nitrosylated in response to no signaling and subsequently regulate protein activities. unlike other ptms in proteins (e.g., phosphorylation, glycosylation, and sumoylation), the linear consensus motif and structural environment that promote protein s - nitrosylation are currently under intensive discussion. increasing evidence shows a consensus acid base motif containing an acidic or basic residue flanking the sno - cys sites in the primary amino acid sequence or tertiary structure plays a critical role in protein s - nitrosylation. proteome - wide studies also show that hydrophobicity of flanking residues, sulfur atom exposure, and cys relative surface accessibility are potential factors contributing to the specificity of s - nitrosylation. since there are multiple molecular mechanisms for protein s - nitrosylation, it seems that the environments surrounding modified cys residues possess diverse features rather than a general rule that applies to all sno - cys sites. in our study, we identified a total of 133 sno - cys sites from 105 proteins in bv-2 cells responding to different stimulations (snoc, lps, and lps + sac ; supplemental table s6, supporting information). motif analysis by the motif - x algorithm using the 133 sno - cys sites identified here demonstrated six consensus sequences (supplemental figure s7, supporting information). in motifs # 2, # 3, and # 6, modified cys residues are flanked by an acid or basic amino acid, which is in line with the previously found acid motifs # 4 and # 5 are consistent with the previously predicted motifs containing hydrophobic amino acids. the most statistically significant consensus sequence, mxxc in motif # 1, has not been observed previously, suggesting that the proximal polar amino acids may also play a role in selective cys s - nitrosylation. given the complexity, prediction of s - nitrosylation sites by computational approaches remains challenging. therefore, determining the locations of sno - cys sites still relies on proteomic analyses to provide large informative data sets. in previously established snosid and sno - rac methods, the labeling of sno - proteins with biotin - hpdp or thiol - reactive resin is a reversible process, and therefore the tags can be lost during peptide elution from the resin at the affinity enrichment step. without labeling, it is difficult to locate the sno - cys sites on peptides with multiple cys residues by ms analysis. in addition, it is difficult to exclude nonspecific peptides, thus introducing false positive signals. the peptides linked to sno - peptides by intermolecular disulfide bonds could be one of the nonspecific sources, but a reduction step to break the disulfide bonds in protein samples before affinity enrichment is not feasible in these methods due to their reversible nature of sno - cys labeling. in contrast, the iodoacetyl reaction of iodotmt reagent with s - nitrosothiol used in the isa method is irreversible resulting in a stable tagging modification even under reducing environments. the iodotmt tag on sno - cys easily removes the ambiguity of site assignment and excludes potential false positive signals from nonspecific peptides in ms analysis. for example, the gapdh peptide ivsnascttnclaplak, corresponding to residues 144160, was previously identified by the snosid method as endogenous sno - cys - containing peptide, although the position of the cys residue undergoing s - nitrosylation was unknown. in this study using the isa approach, we successfully determined cys-150 as the s - nitrosylation site but not cys-154 in response to lps treatment (supplemental table s2 and figure 4c, supporting information). it has been reported that s - nitrosylation of gapdh at cys-150 leads to its binding to siah1 (an e3 ubiquitin ligase) which further initiates apoptotic cell death. on the basis of this example, unambiguous sno - cys site - mapping by the isa approach can provide clues and directions for future functional analysis of sno - proteins. sensitivity is an important criterion when investigating s - nitrosylation due to the labile nature and low abundance of this modification. however, most existing methods were assessed under very different conditions (e.g., concentrations of no donors, cell or tissue types, or treatment conditions), resulting in difficulty in comparing sensitivity. particularly, high doses of no donors, beyond the range of physiological no concentrations, were used in previous proteomics investigations. for instance, s - alkylating labeling strategy determined 586 sno - cys sites of 384 sno - proteins from 1 mm s - nitroso - n - acetylpenicillamine / l - cysteine - treated mouse ms-1 endothelial cells, and sno - rac identified 396 sno - peptides using 500 m snoc treatment in hek293 cells. even though a large number of sno - proteins were identified, the biological relevance of these data and the sensitivities of these methods under in vivo conditions remain uncertain. methods using physiological or pathological conditions to induce s - nitrosylation have previously identified up to 90 sno - cys sites. in particular, a recent report using a sequential iodotmt switch strategy to monitor multiple cys modifications identified 13 sno - cys on 12 proteins exhibiting significant level changes in s - nitrosoglutathione - treated heart myoblast h9c2 cells under hypoxia conditions. with similar experimental conditions, we identified 90, 38, and 46 sno - cys sites significantly altered by snoc, lps, and lps + sac treatments in bv-2 microglial cells, respectively. the good sensitivity of isa results from its robust workflow with irreversible iodotmt labeling for investigation of protein s - nitrosylation only and the use of competitive elution for efficient anti - tmt peptide enrichment. as shown in figure 2c, the new volatile competitive elution buffer containing a tmt analogue (2,6-dmpp) significantly increased the percentage of the iodotmt - labeled peptides in total identified peptides (21.36%) compared to that eluted by the nonselective acidic elution buffer (6.13%), which was used in previous studies. ms / ms is a powerful method for simultaneous analysis of samples from biological replicates, time courses, or different experimental conditions such as healthy versus disease or various drug treatments. previously, ms quantification of s - nitrosylation has been accomplished using isotope - coded affinity tags (icat) labeling, s - nitrosothiol capture (snocap) reagents, isobaric tag for relative and absolute quantitation (itraq)-coupled sno - capture techniques, or cystmt switch labeling. icat and snocap are stable isotope labeling methods for comparing relative peptide abundance at the ms level but are limited to the analysis of two samples at a time. moreover, the quantification of peptide ratios using ms peak intensities may be unreliable due to the shift of elution time between the light and heavy labeled counterparts on the rp - lc separation by icat and snocap. in contrast, isobaric tags with the identical mass and chemical structures (e.g., itraq and tmt) allow for multiplexing up to 8 or 10 samples in a single lc ms / ms analysis. amine - reactive itraq tags have been used for labeling sno - peptides, but as there is no mass signature on sno - cys, the sno - rac workflow is unable to localize sno - cys sites despite ms quantification of the enriched peptides. utilizing isobaric cystmtsixplex reagents in the cystmt however, the output of this method is relatively low for cell - based study, as only 25 sno - cys sites were detected from snoc (200 m)-treated cells. since cystmt labeling of sno - proteins is reversible, it is not compatible with reduction / alkylation, which is required for complete protein denaturation before trypsin digestion. this may result in incomplete digestion in complicated protein samples therefore hindering the efficiency of ms detection. unlike cystmt, the iodotmt reagents irreversibly label cys thiols for improved labeling of sno - proteins. coupled with the improved competitive elution buffer for anti - tmt enrichment, the isa workflow allows for multiplex s - nitrosylation sample analysis with high efficiency and specificity. emerging evidence indicates that protein s - nitrosylation exerts critical roles in a variety of cellular processes by regulating protein folding, ubiquitination, mitochondrial dynamics, and signal transduction. many neurodegenerative diseases involve nitrosative stress and proinflammatory responses including protein s - nitrosylation with activation of microglial cells. our investigation of the s - nitrosylation proteome using isa in lps - stimulated bv-2 cells provided insights into the no signaling pathways under microglial activation and shed light on possible molecular mechanisms contributing to neurological diseases. ipa results showed that several mitochondrial metabolic pathways (e.g., glycolysis i, gluconeogenesis i, and tca cycle ii) are altered by s - nitrosylation in lps - stimulated bv-2 microglial cells (supplemental figure s6, supporting information). these results are consistent with a recent report in which a number of enzymes that regulate these metabolic pathways were found to be s - nitrosylated in different mouse organs. there are also other studies showing that glycolysis was increased in lps - induced bv-2 cells, and monocyte - derived inflammatory dendritic cells committed to glycolysis to maintain atp levels when oxidative phosphorylation was inhibited by no. it is possible that s - nitrosylation of the enzymes in glycolysis and other mitochondrial metabolic pathways identified here are involved in the regulation of these pathways during inflammatory response. administration of sac to cells before lps induction of s - nitrosylation was able to restore the effect of lps on glycolysis i and gluconeogenesis i (figure 6c), suggesting a protective effect for sac on mitochondrial metabolism. to our knowledge, this is the first time sac has been linked to glycolytic / gluconeogenic metabolism regulation. our data also showed that sac altered protein s - nitrosylation in the eif2 (eukaryotic initiation factor 2) signaling pathway, which is known to be an important pathway controlling translation initiation of cellular recovery genes in response to various stresses in eukaryotic cells and is associated with pathogenesis of neurodegenerative diseases including alzheimer s disease. sac may act through eif2 signaling as well as other neurological disease - related pathways identified here (figure 6c), such as mtor- and 14 - 3 - 3-mediated signaling, to take part in nitrosative stress defense. collectively, our data demonstrate that the isa method is a powerful tool to profile protein s - nitrosylation under physiological / pathological conditions. isa provides a simple solution to the multiple problems existing in previous methods for s - nitrosylation analysis. with high specificity, high sensitivity, and site - mapping / quantification ability, isa well meets many of the current demands for the investigation of protein s - nitrosylation. this approach could have broad applications in the determination and comparison of s - nitrosylation proteome under various conditions. it can be used to help further our understanding of molecular mechanisms for no signaling and progression of diseases. moreover, it may also facilitate identifying potential therapeutic targets for disease treatment and elucidating effects of drugs. | s - nitrosylation is a redox - based protein post - translational modification in response to nitric oxide signaling and is involved in a wide range of biological processes. detection and quantification of protein s - nitrosylation have been challenging tasks due to instability and low abundance of the modification. many studies have used mass spectrometry (ms)-based methods with different thiol - reactive reagents to label and identify proteins with s - nitrosylated cysteine (sno - cys). in this study, we developed a novel iodotmt switch assay (isa) using an isobaric set of thiol - reactive iodotmtsixplex reagents to specifically detect and quantify protein s - nitrosylation. irreversible labeling of sno - cys with the iodotmtsixplex reagents enables immune - affinity detection of s - nitrosylated proteins, enrichment of iodotmt - labeled peptides by anti - tmt resin, and importantly, unambiguous modification site - mapping and multiplex quantification by liquid chromatography tandem ms. additionally, we significantly improved anti - tmt peptide enrichment efficiency by competitive elution. using isa, we identified a set of sno - cys sites responding to lipopolysaccharide (lps) stimulation in murine bv-2 microglial cells and revealed effects of s - allyl cysteine from garlic on lps - induced protein s - nitrosylation in antioxidative signaling and mitochondrial metabolic pathways. isa proved to be an effective proteomic approach for quantitative analysis of s - nitrosylation in complex samples and will facilitate the elucidation of molecular mechanisms of nitrosative stress in disease. |
the nomenclature system designed by the who / oie / fao h5n1 evolution working group to classify the a / goose / guangdong/1/1996 lineage of eurasian highly pathogenic h5n1 avian influenza viruses has been in place since early 2008. based on the criteria used to distinguish variant groups of the h5 hemagglutinin (ha) gene, the system has formally identified 20 distinct clades of the virus., using phylogenetic characterization and nucleotide sequence divergence, h5n1 virus clades have been defined based on sharing of a common ancestral node and monophyletic evolution with a bootstrap value of 60 at the cladedefining node (after 1000 neighborjoining bootstrap replicates) and maintaining the average percentage pairwise nucleotide distances between and within clades of > 15% and 90% of the fulllength open reading frame). a nucleotide alignment containing 2947 ha sequences a large neighborjoining tree was initially constructed from the alignment using a gtr+i+g model in paup v4.0b10, and clade assignments (using previously established nomenclature parameters) were made for new sequences based on their location within a clade (figure s1). cladespecific alignments were then used to generate neighborjoining trees with 1000 bootstraps and rooted to either a / goose / guangdong/1/96 or a common outlier to that clade (figure s2a g). monophyletic groups within each of the clades defined in the 2009 update containing more than four nonredundant sequences with bootstrap values of 60 were selected as potential candidates for new order clade designation. to determine whether a clade had undergone a sufficient divergence to exceed the preestablished limit (i.e. required splitting), the average withingroup nucleotide pdistance for sequences was determined using mega4 ; clades having withingroup distances 15% were analyzed further. only groups containing samples with a collection date of 2007 or later were subjected to this analysis. new order clade designations were made for those groups that had betweengroup divergences of 15% and withingroup divergences of 15%. as described previously, the new clade number consisted of the name of the originating clade followed by a period and a single numeric digit added on the right (figure s2a g). following the identification of new higherorder clades, neighborjoining and bayesian trees of 196 representative sequences were constructed using paup v4.0b10 and mrbayes, to verify the overall h5n1 ha tree topology and for statistical inferences (figure 1). phylogenetic relationships of recently diverged a / goose / guangdong/1/1996like h5 hemagglutinin (ha) genes. neighborjoining tree of 196 ha gene sequences from h5n1 viruses constructed with 1000 bootstrap replicates (above branches) and bayesian posterior probabilities (below branches). the tree was constructed using paup v4.0b10 with a gtr+i+g model and rooted to a / goose / guangdong/1/1996. solid triangles denote ha clades of viruses that have not been in circulation since 2008 or earlier ; the year of the most recent isolation is shown in parenthesis. the sequence data set used to generate this figure is provided as supporting information in fasta format (supplementary data s2). hemagglutinin sequences of the highly pathogenic avian influenza a (h5n1) viruses within the eurasian a / goose / guangdong/1/1996 lineage were obtained from the epiflu database in gisaid, which contains all of the h5n1 ha sequences available in the influenza virus resource of ncbi, as well as shared data from individual laboratories within the who global influenza surveillance and response system (gisrs) and oie / fao global network of expertise on animal influenza (offlu). only sequences with at least 1600 nucleotides of the ha gene were included (> 90% of the fulllength open reading frame). a nucleotide alignment containing 2947 ha sequences a large neighborjoining tree was initially constructed from the alignment using a gtr+i+g model in paup v4.0b10, and clade assignments (using previously established nomenclature parameters) were made for new sequences based on their location within a clade (figure s1). cladespecific alignments were then used to generate neighborjoining trees with 1000 bootstraps and rooted to either a / goose / guangdong/1/96 or a common outlier to that clade (figure s2a g). monophyletic groups within each of the clades defined in the 2009 update containing more than four nonredundant sequences with bootstrap values of 60 were selected as potential candidates for new order clade designation. to determine whether a clade had undergone a sufficient divergence to exceed the preestablished limit (i.e. required splitting), the average withingroup nucleotide pdistance for sequences was determined using mega4 ; clades having withingroup distances 15% were analyzed further. only groups containing samples with a collection date of 2007 or later were subjected to this analysis. new order clade designations were made for those groups that had betweengroup divergences of 15% and withingroup divergences of 15%. as described previously, the new clade number consisted of the name of the originating clade followed by a period and a single numeric digit added on the right (figure s2a g). following the identification of new higherorder clades, neighborjoining and bayesian trees of 196 representative sequences were constructed using paup v4.0b10 and mrbayes, to verify the overall h5n1 ha tree topology and for statistical inferences (figure 1). phylogenetic relationships of recently diverged a / goose / guangdong/1/1996like h5 hemagglutinin (ha) genes. neighborjoining tree of 196 ha gene sequences from h5n1 viruses constructed with 1000 bootstrap replicates (above branches) and bayesian posterior probabilities (below branches). the tree was constructed using paup v4.0b10 with a gtr+i+g model and rooted to a / goose / guangdong/1/1996. solid triangles denote ha clades of viruses that have not been in circulation since 2008 or earlier ; the year of the most recent isolation is shown in parenthesis. the sequence data set used to generate this figure is provided as supporting information in fasta format (supplementary data s2). surveillance for the continuing circulation of highly pathogenic avian influenza a (h5n1) in poultry and wild birds in asia, the middle east, europe, and africa has led to an increase in the number of sequences available for analysis. the current analysis was applied to 2947 ha gene sequences consisting of at least 1600 nucleotides, of which 1637 sequences (primarily from 2008 to 2010) were added since the previous nomenclature update. phylogenetic analysis of the neighborjoining tree generated from this data set revealed that the addition of these new sequence data resulted in the generation of one or more monophyletic groups with high bootstrap support within each of the currently circulating clades of h5n1 (figure s1). in addition, many of these groups had long branches separating them from the nearest node in the tree, indicating further nucleotide divergence. each of the currently circulating clades (1, 2.1.3, 2.2, 2.2.1, 2.3.2, 2.3.4, and 7) was then examined independently, to measure the average withingroup pairwise nucleotide distances. all were found to have > 15% withingroup divergence indicating the need to split these groups into new order clades. after generation of cladespecific trees, monophyletic groups with bootstrap values more than 60 were selected for further testing (figure s2a g). based on the previously defined nomenclature, 12 new second, third, and fourthorder clades were identified (table 1). updates to the h5n1 highly pathogenic avian influenza virus clade nomenclature system to summarize these findings and to confirm the consistency of clade topology using a smaller data set, a small tree containing 196 ha sequences was generated using both nj and bayesian methods (figure 1). this tree is annotated with the 12 new clades presented here and displays older, noncirculating, and putatively noncirculating clades as collapsed nodes (figure 1). both nj bootstrap support and bayesian posterior probabilities were calculated to demonstrate confidence in clade assignments when using smaller data sets. surveillance for the continuing circulation of highly pathogenic avian influenza a (h5n1) in poultry and wild birds in asia, the middle east, europe, and africa has led to an increase in the number of sequences available for analysis. the current analysis was applied to 2947 ha gene sequences consisting of at least 1600 nucleotides, of which 1637 sequences (primarily from 2008 to 2010) were added since the previous nomenclature update. phylogenetic analysis of the neighborjoining tree generated from this data set revealed that the addition of these new sequence data resulted in the generation of one or more monophyletic groups with high bootstrap support within each of the currently circulating clades of h5n1 (figure s1). in addition, many of these groups had long branches separating them from the nearest node in the tree, indicating further nucleotide divergence. each of the currently circulating clades (1, 2.1.3, 2.2, 2.2.1, 2.3.2, 2.3.4, and 7) was then examined independently, to measure the average withingroup pairwise nucleotide distances. all were found to have > 15% withingroup divergence indicating the need to split these groups into new order clades. after generation of cladespecific trees, monophyletic groups with bootstrap values more than 60 were selected for further testing (figure s2a g). based on the previously defined nomenclature, 12 new second, third, and fourthorder clades were identified (table 1). updates to the h5n1 highly pathogenic avian influenza virus clade nomenclature system to summarize these findings and to confirm the consistency of clade topology using a smaller data set, a small tree containing 196 ha sequences was generated using both nj and bayesian methods (figure 1). this tree is annotated with the 12 new clades presented here and displays older, noncirculating, and putatively noncirculating clades as collapsed nodes (figure 1). both nj bootstrap support and bayesian posterior probabilities were calculated to demonstrate confidence in clade assignments when using smaller data sets. the highly pathogenic avian influenza virus a (h5n1) lineage was first identified in 1996 and has subsequently spread over much of the eastern hemisphere. as these viruses have established geographically and ecologically isolated enzootic foci of infection, new clades of viruses continuously evolve. the who / oie / fao h5n1 evolution working group has previously identified 20 such clades and established specific criteria for naming h5n1 clades. since the most recent recommendations of the h5n1 evolution working group in 2009, h5n1 viruses have continued to spread and evolve locally and regionally. to address the question of whether this continued evolution has led to the appearance of new clades, we initially constructed a phylogenetic tree composed of nearly 3000 ha sequences from either public databases or ongoing surveillance programs. as expected, the addition of more than 1500 new sequences to the previous h5n1 nomenclature update tree produced a number of monophyletic groups within each of the currently circulating clades of h5n1. after measuring within and betweengroup average nucleotide pdistances, it was observed that all extant clades of h5n1 required splitting into one or more newly defined second, third, and/or fourthorder clades. the current analysis, therefore, proposes the designation of 12 new clades that meet the criteria of the h5n1 nomenclature system. the designation of 12 new h5n1 clades (in contrast with the previous nomenclature update in which only one new clade was identified) is not surprising, considering the length of time since the last nomenclature update and improved surveillance / reporting of viruses in recent years, increasing the number of sequences available for analysis. interestingly, the clades that had already diverged into thirdorder groups in previous analyses required the largest number of new clade designations. previous assignment to thirdorder clades indicates that the viruses had already evolved considerably, presumably because of short generation times and large population sizes in enzootic areas. the subdivision of these clades suggests the continued evolution and divergence in regions where h5n1 is considered entrenched in poultry. for example, clade 2.3.4 viruses circulating in china, vietnam, laos, and neighboring regions were split into 2.3.4.1/2.3.4.2/2.3.4.3, whereas clade 2.1.3 in indonesia was split into 2.1.3.1/2.1.3.2/2.1.3.3. the egyptian thirdorder clade 2.2.1 also required splitting into new clade 2.2.1.1, indicating further divergence of contemporary strains of h5n1 circulating in egyptian poultry. in addition, clade 2.3.2 enzootic in china was subdivided to accommodate the new clade 2.3.2.1, which has been identified in poultry populations as well as wild birds in many diverse regions outside of china., while these groups have been identified as sufficiently divergent from the previously defined clades, their ancestral populations have not been detected in recent years and are likely to no longer circulate. one new thirdorder clade, termed 2.2.2, comprising viruses from india and bangladesh was identified. this clade diverged from clade 2.2 after its introduction into this region in approximately 2006. finally, firstorder clades 1 and 7 required designation of secondorder clades as a result of their continued divergence in the mekong delta regions of southern vietnam / cambodia and china, respectively. while clade 1.1 appears to represent a continuum of evolution in the relatively wellsampled mekong delta region,, clade 7.1 and 7.2 viruses remain widely diverse groups (average betweengroup divergence of 45%), suggesting that these groups represent undersampled virus populations with only sporadic detection (figure s2a g)., antigenic analysis of viruses from each of these new clades will inform decisions on whether to update the library of prepandemic vaccine candidates (see http://www.who.int/influenza/resources/documents/characteristics_virus_vaccines/en/index.html). in addition to the emergence of novel clades of h5n1, a number of previously circulating clades of h5n1 have not been detected for several years (since at least 2008 or earlier). although we can not rule out the possibility that the lack of detection of viruses in these clades may in some cases be related to gaps in surveillance, it is more likely that many of them have been supplanted by new clades and become inactive. as shown in figure 1, 13 clades (shown as collapsed nodes in the tree with the last year of detection shown in parentheses) have not been detected since 2008 or earlier (0, 2.1.1, 2.1.2, 2.3.1, 2.3.3, 2.4, 2.6, 3, 4, 5, 6, 8, and 9). therefore, although 12 new clades have been identified and characterized in this study, 13 older clades may now be considered out of circulation. again, this is not unexpected given the time since the last nomenclature update. as observed previously, the majority of viruses cluster with others of similar geographic and temporal origin, with few exceptions that might indicate viral spread over longer distances (i.e., poultry trade and/or bird migration)., for example, clade 2.3.2.1 viruses have now been detected in 9 countries since 2007. in addition, while the majority of the novel ha gene clades have approximately similar levels of within (15%) group diversity, the clade 2.3.2.1 viruses have a higher withingroup mean nucleotide divergence (17%). this result suggests that the clade 2.3.2.1 ha genes have diversified to a greater extent than those in other clades, perhaps owing to the adaptation to different bird species and introduction into multiple geographic regions by migratory birds. although this average withingroup nucleotide pdistance exceeds the < 15% withingroup mean criterion originally proposed as a cutoff for clade determination,, designation of a single fifthorder clade awaits additional data to determine its significance. despite very successful efforts to eliminate or control the spread of h5n1 in poultry, viruses persisting in enzootic regions seed virus epizootics in new areas via poultry trade or wild birds. periodic population sweeps in enzootic and epizootic areas with sustained viral circulation may lead to the extinction of some clades and extensive divergence of viruses within more successful clades. as a consequence, the classification of h5n1 viruses based on ha evolution requires periodic updating, making classification dynamic as the virus has expanded within several disparate ecosystems and along distinct evolutionary trajectories. although this report focuses strictly on genetic divergence as a measure of h5n1 classification, clearly additional studies on h5n1 antigenicity and crossclade protective immunity are needed to inform prepandemic vaccine strain selection as well as risk management policies for both veterinary and human health. as always, continued surveillance for h5n1 in animals and humans is crucial for this process to be most effective. data s2. the nucleotide sequence alignment used to generate the tree shown in figure 1 (a g) cladespecific neighborjoining trees with 1000 bootstrap replicates were generated for each clade and rooted to either agooseguangdong196 or a common outlier to that clade. | please cite this paper as : who / oie / fao. (2012) continued evolution of highly pathogenic avian influenza a(h5n1) : updated nomenclature. influenza and other respiratory viruses 6(1), 15. background continued evolution of highly pathogenic avian influenza a (h5n1) throughout many regions of the eastern hemisphere has led to the emergence of new phylogenetic groups. a total of 1637 new h5n1 hemagglutinin (ha) sequences have become available since the previous nomenclature recommendations described in 2009 by the who / oie / fao h5n1 evolution working group. a comprehensive analysis including all the new data is needed to update ha clade nomenclature. methods phylogenetic trees were constructed from data sets of all available h5n1 ha sequences. new clades were designated on the basis of phylogeny and pdistance using the preestablished nomenclature system (emerg infec dis 2008 ; 14:e1). each circulating h5n1 clade was subjected to further phylogenetic analysis and nucleotide sequence divergence calculations. results all recently circulating clades (clade 1 in the mekong river delta, 2.1.3 in indonesia, 2.2 in india / bangladesh, 2.2.1 in egypt, 2.3.2, 2.3.4 and 7 in asia) required assignment of divergent ha genes to new second, third, and/or fourthorder clades. at the same time, clades 0, 3, 4, 5, 6, 8, 9, and several second and thirdorder groups from clade 2 have not been detected since 2008 or earlier. conclusions new designations are recommended for 12 ha clades, named according to previously defined criteria. in addition, viruses from 13 clades have not been detected since 2008 or earlier. the periodic updating of this dynamic classification system allows continued use of a unified nomenclature in all h5n1 studies. |
the emerging health care system increasingly values patient engagement and shared decision - making between patients and their providers. the practice of these values is gaining importance as the patient - centered medical home model and personalized medicine come into greater use. exploration of patient preferences about personal health data use for research and quality improvement is a fundamental element of the provider - patient relationship. giving patients an explicit opportunity to discuss their options about use of their data and implementing a process that allows patients to receive desired communications about how their information is used can help build patient trust, a requirement for successful care partnerships. working to change organizational cultures that exclude patients from participation in important decisions related to personal health information use promotes a strong patient - provider relationship and, ultimately, lays the foundation for improved health care through expanded use of patient data. in the rapidly evolving age of precision medicine, there is much excitement about the use of patient data for research. genomic medicine offers new opportunities to identify beneficial treatments previously not known or not recognized as appropriate for a patient. the growing shift to the patient - centered medical home model of care delivery facilitates increased practice of personalized medicine,1 as well as expanded opportunities to bring genomic medicine into broader, primary care - based practice.2 the national institutes of health s precision medicine initiative cohort program3 will enable a more thorough understanding of the biological, environmental, and behavioral influences on disease development and will position investigators to discover and develop treatments targeted to specific individuals. these new advances in treatment will bring with them greater quantities of personal health information (phi) and clinical data than ever before seen in medicine. the emergence of precision medicine and the technologies that make it possible bring new challenges in clinical care ; research innovation ; and ethical, legal, and social issues. policies that govern data sharing and use will evolve too as the foremost concerns of patients and health care professionals come to light. as these technological advances become integrated into the cycle of practice innovation, implementation, evaluation, and improvement, patients and providers will need to forge new relationships based on mutual trust and shared decision - making. nowhere will that requirement become more pressing than with regard to data use and sharing. this commentary considers the evolving use of phi in clinical care ; explores key issues in provider - patient relationships that underlie data sharing ; and describes an approach to phi sharing that facilitates patient engagement, relationship building, and shared decision - making. to date, patients opportunities to choose how their phi is used outside of clinical encounters have often been limited, and in some cases providers too have had little to say about secondary uses. for example, health care institutions frequently review electronic health records to identify opportunities to improve quality of care, an activity that can benefit both patients and providers and that may be disclosed in general terms in facility registration forms. similarly, during hospital admission or clinic sign - in, patients sign an agreement permitting details of their visit to be shared with payers or perhaps to forego care. research involving biospecimens may not entail patient consent at all, a situation that affords efficient tissue processing but can leave patients feeling marginalized and mistrustful of clinicians motivation when ordering tests. patients do not always understand information about genomic sequencing and, even when presented with information about what is involved, a significant number consider the benefits unclear.4 psychosocial distress occurs in some individuals who undergo testing,5 and oncology nursing best practice includes patient education and support before, during, and after biospecimen collection and testing.6,7 the use of phi in initiatives such as comparative effectiveness research and decades - long monitoring and reporting offers significant opportunities to improve care. use of phi is also critical in identifying unmet needs, such as the lack of adequate housing, and in addressing health care inequities. for example, provisions of the patient protection and affordable care act provide for standardized data collection related to disability, which will enable the health care system to identify barriers to care and improve the care received by people with disabilities.8 in addition, emerging technologies such as wearable devices and home sensors for remote monitoring, as well as more established technologies such as social media and mobile health (mhealth) applications, are expanding the opportunities for patients to generate data about themselves. patient - generated health data can help patients and their care teams facilitate health management and optimize quality of life.9 as use of these new technologies becomes routine, clinical practices and researchers will find secondary uses for such data and will need processes for managing it. at the same time, use of phi in such data - driven efforts raises multiple challenges, including privacy - related concerns, issues associated with obtaining informed consent for current and future data uses, difficulties related to patient participation in precision medicine - focused initiatives, and matters of equity in who receives care and how data are used with regard to varying ethnic and socioeconomic groups.10 more broadly, the issue of equity is also of concern in low- and middle - income countries, which frequently lack not only the resources for precision medicine but also the resources for routine procedures and medications under patent.11 this list is likely to grow as greater uses for data are developed and embedded within clinical practice and health care research. the evolution of medicine to this more personalized, complex form brings with it the need for a richer and more nuanced relationship between patients and their care teams, and the need for new strategies that promote greater security, transparency, and appreciation of patient contribution and perspectives. reaping the benefits of precision medicine, however, will require greater patient engagement with the health care system than is the norm in many settings currently. not only will patients be seen in clinical and online settings, as they are today, but also patient information will be reviewed and reused in clinical and research settings in which patients are not present. growing pressure on health care institutions to track and publish outcomes is expanding data analysis performed routinely, much of which relies on retrospective record review. the ongoing scarcity of research funding has motivated researchers to find new uses for existing data, and current regulations do not require disclosure of all such uses to patients. data on patient perspectives and priorities is integral to developing new therapies and practices that will result in improved outcomes. achieving these goals relies in large measure on patients confidence that what they are told about how their information will be used is true, and will be truly of benefit to them and other patients. numerous research and survey efforts indicate that patients are willing to share their health information for both therapeutic and research uses when data users and managers take adequate and appropriate actions such as encryption and data access auditing to protect patients privacy.1215 building trust related to the use of even de identified data is critical,16 as is having the ability to control one s information. by gathering pertinent details about patient preferences related to permission seeking, de identification, notification of data use, and ongoing engagement at the time that patients establish a relationship with their primary provider, clinicians can begin building the relationship needed to support lifelong data use and sharing. preferably this conversation would occur at an early visit (e.g., the patient s first or second clinical encounter with the provider), but could occur later if the patient wished to develop a stronger relationship with the provider or care team before making decisions about data sharing. ideally, the patient s decisions would be recorded in the electronic health record as well as given to the patient in hard copy or electronic form, as preferred. this initial detail - gathering process also offers a nonthreatening way for providers to start a conversation about the value of shared decision - making if a patient does not wish to participate in making choices about the use of his or her data, an effort that may benefit both parties when difficult care decisions must be made in the future. for example, patients who feel it is disrespectful to question a provider s advice may be reluctant to state any preference regarding use of personal data. similarly, patients who are very ill may lack the energy or interest to read patient education materials about the benefits and risks of data sharing, preferring to leave this seemingly less important decision to the provider. health literacy and numeracy skills also affect patients willingness to make decisions about their care,1719 adding to the challenge of talking through care decisions with providers. though patient engagement in decision - making is becoming a core value in health care, some patients prefer the more traditional provider - centric approach,20 and their preferences need to be respected and accommodated. patients have varying degrees of sensitivity toward sharing their phi, and willingness to provide that information may vary between demographic and financial data, anthropometric measurements, lifestyle and nutrition practices, diagnosis and treatment specifics, and genomic data. these types of data are associated with a varying desire for confidentiality, as well as a variable commercial value beyond their use in management of patients health and mandated public health reporting. some individuals fear the sharing of information that has resulted in, or that they believe will, result in stigmatization such as about their obesity or their history of mental illness or having had an abortion. others fear the revelation of information that could disrupt their lives or put them at financial peril, such as documentation of marital infidelity or substance abuse, or the discovery of a life - shortening condition. information that may be predictive of an individual s future health status, such as genetic data or a history of life - threatening conditions, may by its nature spur confidentiality concerns. the unintended sharing of different types of data may elicit varying degrees of concern among patients, e.g., sharing of general demographic data may be less distressing to patients than sharing of medication history. thus, an approach that facilitates informed consent, management of data use permissions, and data sharing by data type will offer patients and their care team the greatest flexibility in managing health data for research. an application that provides opportunities for clinicians and researchers to identify data relevant to specific investigations and for patients to choose the types of data they wish to share and the purposes for which sharing may occur can be an effective bridge between the need for privacy and the desire to innovate. this application should facilitate mandated public health reporting as well as institutional and commercial purposes of specific value to the populations whose data are collected. for example, in a region with a high incidence of vehicular - related injuries and deaths, the application might include items about motorcycle and automobile ownership, seatbelt use, and frequency of driving after drinking alcohol. though implementing a data - sharing mechanism requires efforts not commonly included in current processes, implementing an approach that meets the data - sharing preferences of each patient with regard to privacy, security, and transparency need not be onerous, financially ruinous, or technically overtaxing. when a patient provides the personal demographic and historical information necessary to set up the electronic medical record initially, the patient can be asked a series of additional questions related to his or her understanding of how what data are collected and how they can be used, such as for retrospective analysis for quality improvement, for medication adverse effect identification and reporting, for identification of eligible clinical trial registrants, and others. for instance, patients can be asked if they wish to evaluate opportunities to participate in research involving only records review, research involving some additional data collection (e.g., blood tests beyond those they normally undergo), or other research that may help their care partners to better understand their health and, potentially, identify therapies or care that may help maintain their health. patient education materials developed for this effort should encompass the principles used in creating other patient - directed materials, e.g., text that is readable by individuals with moderate levels of health literacy, accurate illustrations, use of multimedia to illustrate complex concepts, etc. at the initial encounter, patients can also be offered the option of making decisions about use of their data at a future clinical encounter, after they have had time to think about their preferences and, if appropriate, develop a list of questions to ask their provider. this opportunity for reflection and consultation with family, friends, and trusted associates (e.g., culture brokers) is critical for two reasons. first, it provides patients with education and an opportunity to think through the many issues related to decisions about the use of their personal data. second, it demonstrates the shared nature of the decision - making that will occur between patients and the members of their care teams. in this way clinicians, researchers, and patients can move forward together in a true spirit of nothing about me without me.21 this clear and present effort to partner with patients rather than act unilaterally on their behalf strengthens the dynamic of patient engagement that will underlie provider - patient relationship in years to come. if a patient wishes to share his or her data, the clinician can then inquire as to whether the patient wishes to receive notifications when the data are used in clinical- or research - focused initiatives. if the patient does wish to receive such communications, he or she can provide an email address where notifications can be sent that contain a link to a secure patient portal or protected website where study information may be accessed. in this way patients data, and even the fact that an individual s data have been used, remain in a protected environment. including an additional field containing the patient s email address in the record is unlikely to place an undue burden on database administrators or system requirements. annual requests to the patient to confirm the email address, as well as a simple procedure for updating the address outside the annual cycle, will help patients make more active decisions regarding their information and engage proactively with the health care system without adding substantially to the organization s workload. providing summaries of analyses to patients whose data were used, when desired by patients, will promote a positive view of data sharing and will strengthen relationships by helping patients to understand the importance and safety of using phi in research. return of raw genomic data has already generated interest among potential research participants;22 though such activity may not be immediately feasible, it points to patient desire for a greater role in health management. given the highly publicized possible drawbacks to sharing genomic information discrimination in employment and insurance procurement, disruption of family relationships when individuals are made aware of health information they wished not to know, and anxiety over predisposition to health conditions for which there is yet no cure, among others the fact that individuals still seek to obtain genetic information about themselves reflects a more proactive to personal health. such a system could also offer patients the option to share their data without receiving notifications of data use and to change their notification preference at a future time. in addition, a process like the one described here can facilitate data access management at multiple levels based on purpose and personnel. for some data uses, such as public health surveillance, federal and state laws will determine the type and degree of data sharing required without explicit decision - making by patients and their care team. for other uses, such as clinical trial recruitment and health goal tracking, patients are well placed to identify who providers, researchers, family members, durable powers of attorney for health care, etc.should be able to access each data type. in an environment of shared decision - making, a process that manages patient preferences and permissions can flexibly support the needs of patient, care teams, and researchers. new approaches that require changes to existing workflows may be among the most difficult to implement. although technical issues are real, the primary challenges to change may be attitudinal and cultural, as in the case of the sharing of genomic information with patients.23 processes such as the one previously described in this paper could facilitate shared decision - making and help health care providers address emerging concerns such as data use policy development and implementation, data management and protection, and compliance with federal and state regulations such as the health insurance portability and accountability act. applications that facilitate shared decision - making can help health care providers address emerging concerns such as the monitoring of patients receiving investigational therapies outside clinical trials;24 growing use of telemedicine in the provision of routine care ; and the shift to care management through patient portals, mobile health applications, and other digital tools. systems that support greater patient decision - making will benefit not only patients, but also providers and health care organizations today and in the future. | background : the emerging health care system increasingly values patient engagement and shared decision - making between patients and their providers. the practice of these values is gaining importance as the patient - centered medical home model and personalized medicine come into greater use.opportunity for improvement : exploration of patient preferences about personal health data use for research and quality improvement is a fundamental element of the provider - patient relationship. giving patients an explicit opportunity to discuss their options about use of their data and implementing a process that allows patients to receive desired communications about how their information is used can help build patient trust, a requirement for successful care partnerships.practice advancement : working to change organizational cultures that exclude patients from participation in important decisions related to personal health information use promotes a strong patient - provider relationship and, ultimately, lays the foundation for improved health care through expanded use of patient data. |
vogt - koyanagi - harada (vkh) disease is characterized by bilateral granulomatous uveitis with neurologic, auditory, and dermatologic manifestations. a 50-year - old chinese han woman presented with difficulty walking, numbness on the left side of the body, and difficulty with urination. the patient was diagnosed with incomplete vkh disease and received corticosteroid treatment prior to the neurological presentation. clinicians should consider acute myelitis as a rare possible neurological manifestation in vkh disease patients, and early systemic administration of corticosteroids will suppress the acute inflammatory process and prevent recurrences. this report raises the possibility that vkh disease and acute myelitis share common pathogenic pathways. vogt - koyanagi - harada (vkh) disease is an idiopathic, multisystem autoimmune disorder characterized by its affects on pigmented tissues in the ocular, auditory, integumentary, and central nervous systems. the prevalence of vkh disease varies markedly, and the risk of developing at least one neurological manifestation exceeds 50%.1 certain neurological manifestations - including aseptic meningitis, encephalitis, encephalomyelitis, and cranial nerve neuropathy - are occasionally associated with this disorder, but acute myelitis has rarely been reported. early systemic administration of corticosteroids will suppress the acute inflammatory process, and prevent recurrences and the development of complications. we present a case of vkh disease accompanied by acute myelitis, and review two previously published case reports in an attempt to elucidate the pathogenesis. a 50-year - old chinese han woman presented with sudden onset of difficulty walking, numbness on the left side of the body, and difficulty with urination for 6 days. twelve days prior to the presentation she had been diagnosed with incomplete vkh disease by an ophthalmologist based on blurred vision in both eyes, bilateral nontraumatic granulomatous iridocyclitis, retinal edema, and the presence of exudates. she had received corticosteroid treatment (500-mg intravenous methylprednisone for 3 days followed by 300-mg intravenous methylprednisone for 3 days and a tapering course of 80-mg prednisone for 6 days). neurological manifestations (headache, tinnitus, difficulty climbing stairs, numbness on the left side of the body, and dysuria) emerged during the tapering of steroid treatment. she recalled a history of upper respiratory tract infection a month previously but denied any history of vaccination. neurological examinations showed a normal mental status and cranial nerves except for a visual acuity of 20/200 bilaterally and papilledema of both eyes in a fundus examination. the babinski sign was present bilaterally, and her response to the finger - to - nose test was impaired. screening for connective - tissue disease, including c reactive protein, rheumatic factor, antinuclear antibody, c - antineutrophil cytoplasmic autoantibody, anti - double - strand dna, anti - smith, anti - ribonucleoprotein, anti - sjogren 's syndrome a, and anti - sjogren 's syndrome b antibodies, produced normal or negative results. cerebrospinal fluid (csf) contained 128 wbcs / mm (normal < 10/mm) with 94% mononuclear cells and 4.83 g / l protein (normal 1.5 - 4.5 optical coherence tomography performed independently by two ophthalmologists revealed bilateral disc edema accompanied by serous retinal detachment (fig. 1c and d) revealed a hyperintense signal in the t2-weighted sequence with significant gadolinium enhancement between the c1 - 3 vertebrae in the cervical cord. the patient was diagnosed as aseptic meningitis with acute myelitis complicating vkh disease, and was given pulse steroid therapy again (intravenous administration of 1000 mg of methylprednisolone for 3 days, 500-mg intravenous methylprednisolone for 10 days, 250-mg intravenous methylprednisolone for 10 days, and a tapering course of 60-mg prednisone over a 6-month period). her visual acuity recovered to 50/200 and her numbness and dysuria also improved significantly. at the 1-year follow - up she was back to her baseline overall condition and showed no recurrence of any visual or neurological symptoms. the neurological manifestations and spinal - cord mri findings of the patient were consistent with acute myelitis. the differential diagnosis for the etiology of acute myelitis primarily included behet disease, sarcoidosis, infections (e.g., syphilis, toxoplasmosis, and viruses), systematic diseases (e.g., lupus erythematosus and sjogren 's syndrome), and neuromyelitis optica. behet disease, sarcoidosis, infections, and systematic diseases were excluded by the absence of disease - related clinical manifestations and negative serological tests. neuromyelitis optica was excluded based on previous ophthalmological examinations indicating that the visual impairment was caused by retinal edema and exudates, and the igg index and vep being normal.2 to our knowledge there are only two mri - documented cases of myelitis in vhk disease in the literature,3,4 and our patient (the third case) has the oldest onset age of 50 years (table 1 and 2). women were affected in all three cases (one was from jordan and the other two were from china) with a wide range for the onset age (37, 16, and 50 years, respectively), which is consistent with reports that vkh disease occurs mostly in native americans, east indian, asian, middle eastern, and hispanic populations, people aged between 20 and 50 years, and women.5,6 spinal shock was not seen in the three cases, and is possibly less common in myelitis with vkh disease than in idiopathic acute transverse myelitis. spinal - cord mri revealed cervical lesions in all three patients, while thoracic changes are more frequent in idiopathic acute transverse myelitis cases. however, too few patients with vkh disease and acute myelitis have been reported to allow firm conclusions to be drawn about potential differences in disease expression. in contrast to our patient, the other two cases did not present with headache and showed normal csf cells or mild pleocytosis, which may be due to variations in the extraocular appearance, such as meningismus, tinnitus, vitiligo, and alopecia.7 moreover, the other two patients had already been off steroids before the neurological presentation, which may be explained either as part of the normal course of the disease or adequate corticosteroid therapy not being introduced early enough.7 although the clinical profile of vkh disease is well - established, little is known about its pathogenesis. triggering of cd4 t cells (th1, t helper 17, and regulatory t cells) reactive to melanocyte - specific proteins [e.g., tyrosinase, tyrosinase - related protein 1, and trp-2 ] by an infectious agent is proposed to be involved in the pathogenesis.8 in addition, genetic factors, including hla - dr4, hla - dr1, and hla - drb10405, may also play an important role.8 - 11 the absence of melanocytes in the spinal cord means that the precise mechanism by which vkh disease leads to acute myelitis is unclear. the effectiveness of steroid therapy in the three cases suggests underlying immunological pathogenic mechanisms, which might involve myelin basic protein.12 the history of upper respiratory tract infection of our patient suggests that infectious factors were involved in the pathogenesis, and the specific geographic distribution of these three cases suggests that genetic background also influences the development of vkh disease with acute myelitis. in summary, new insights into immune responses and genetic abnormalities will help to clarify the pathogenic mechanisms underlying vkh disease with acute myelitis. | backgroundvogt - koyanagi - harada (vkh) disease is characterized by bilateral granulomatous uveitis with neurologic, auditory, and dermatologic manifestations. however, acute myelitis complicating vkh disease has rarely been reported.case reporta 50-year - old chinese han woman presented with difficulty walking, numbness on the left side of the body, and difficulty with urination. the patient was diagnosed with incomplete vkh disease and received corticosteroid treatment prior to the neurological presentation. acute myelitis was diagnosed based on both clinical and spinal - cord mri findings.conclusionsclinicians should consider acute myelitis as a rare possible neurological manifestation in vkh disease patients, and early systemic administration of corticosteroids will suppress the acute inflammatory process and prevent recurrences. this report raises the possibility that vkh disease and acute myelitis share common pathogenic pathways. |
gastrointestinal stromal tumors (gist) are the most common mesenchymal tumor of the human gastrointestinal tract, which arise from intestinal cells of cajal. before the imatinib (glivec, novartis korea, seoul, korea) era, surgery was the only therapeutic treatment for gist. even after the complete resection of gist, however, most patients with advanced disease relapsed making the prognosis of patients with metastatic and/or recurrent gist extremely poor. although a number of experiences have been reported with imatinib mesylate (i m) in patients with unresectable or metastatic gists without operation, there were few reports about neoadjuvant experience in those cases. we report a case of unresectable gastric gist who underwent neoadjuvant therapy followed by surgical resection. a 53-year - old female was admitted to our department of general surgery because of dyspepsia and vomiting during the previous month. for her past history, contrast - enhanced abdominal computed tomography (ct) and f-18 fluorodeoxyglucose positron emission tomography (fdg - pet) ct showed an approximately 12 cm sized heterogeneous mass located from fundus to mid body of stomach, which seemed to invade pancreas, spleen and left adrenal gland (fig. endoscopic biopsy performed in our institution and pathologic report revealed a spindle cell tumor consistent with gist (figs. 2, 3). results of immunohistochemical stain : the tumor cell was positive for cd34 and c - kit (cd117), weak positive for smooth muscle actin ; whereas s-100 was negative (fig. two weeks later, we performed laparotomy, but the mass was huge and invaded the pancreas and retroperitoneum very tightly so that it could n't be resected. ten days after the operation, the patient was started on imatinib therapy, a daily dose of 400 mg, maintained for 4 months. a follow - up ct, 2 months after the start of imatinib, showed a dramatic reduction in the size of the tumor. and 4 months after follow - up ct, there was no evidence of mass lesion in the stomach (fig. 5) ; on the gastroduodenoscopy, the mass lesion was invisible with only an ulcerative lesion observed in the same location (fig. although the mass lesion was not revealed clearly at the follow - up imaging study (fig. 5 months after the treatment with imatinib, the patient underwent total gastrectomy and splenectomy. the stomach was adhered to the spleen, tightly, and the lesion was found between stomach and spleen, firm and white, having irregular margin. pathologically, very few tumor cells were in the fibrotic tissue ; residual tumor size was 0.3 0.2 cm and 0.2 0.2 cm, respectively. the tumor consisted of spindle cells with a mitotic activity index of 1/50 hpf on microscopy. around the residual tumor, hyalinized fibrosis, inflamed granulation tissue, dystrophic calcification, and necrosis and abscess gist is the most common mesenchymal tumor of the gastrointestinal tract. up to two - thirds of gists show malignant behavior with high recurrence rates. therefore, improving the rate of complete resection is a key challenge in the treatment of gists. neoadjuvant therapy with tyrosine kinase inhibition has potential usefulness in primary gist, although not yet as a standard of care. several retrospective studies suggested that resection of residual lesions could prolong progression - free survival if it is done while the tumors are under control with imatinib. it is also emphasized that imatinib should be continued even after complete resection of all visible disease. however, the role of resection of residual tumors after imatinib therapy has not been established, and several phase iii clinical trials to investigate the role of surgical resection in this setting are ongoing or planned worldwide. there is no disagreement on the benefit of treatment with imatinib for malignant gists, but the clinical outcome of neoadjuvant treatment with imatinib is not well established. in our case, we could perform complete resection of the gist by down - sizing of the tumor after treatment with imatinib. the duration of neoadjuvant therapy with imatinib may vary according to response to the treatment, but surgery may be performed after sufficient shrinkage of tumors is observed ; typically after 4 to 6 months and within 12 months of imatinib treatment. the standard initial dose of imatinib is 400 mg per day in patients with unresectable or metastatic gists. in a randomized phase iii trial that compared 400 mg daily with 800 mg daily, the 800 mg daily was a more toxic but not more effective dose. the goal of surgery is complete resection of tumor, possibly avoiding the occurrence of tumor rupture and achieving negative margins. in our case, there was no tumor rupture during the operative procedure and pathologically negative margins were obtained. radiographic tumor response to preoperative imatinib correlated significantly with complete resection, with 91% of patients with partial responses achieving complete resection, versus 4% of patients with progressive disease. radiographic and metabolic complete response based upon fdg - pet are not always concordant with a pathologic complete response ; therefore, it should be born in mind that the pathological evaluation on the surgically resected materials obtained from patients treated with i m might be indispensable for the elucidation of the therapeutic effect of i m on gist. in our case, follow - up pet showed a hypermetabolic lesion around medial side of the spleen ; residual tumor was observed in that site on microscopy, but follow - up ct showed no evidence of mass lesion in the stomach. in the surgical findings, the lesion had extensive areas of fibrotic changes and there were severe adhesions between the lesion and spleen, so it was suspected to be invasion, thus we performed total gastrectomy and splenectomy. first, the patient 's follow - up period is short and will not be an accurate prognostic assessment. suspected to be in an inoperable state, neoadjuvant chemotherapy should take precedence over surgical procedure. in summary, we report a case of gist in an unresectable state, initially, who underwent complete surgical resection following imatinib treatment. in cases of advanced gists, neoadjuvant treatment with imatinib may provide an opportunity for surgeons to perform a complete resection. | a 53-year - old woman was diagnosed with gastrointestinal stromal tumor (gist) of the stomach. computed tomography (ct) revealed a huge mass (12 cm in diameter), likely to invade pancreas and spleen. in the operation field, the tumor was in an unresectable state. the patient was then started on imatinib therapy for 4 months. on follow - up imaging studies, the tumor almost disappeared. we performed total gastrectomy and splenectomy upon which two small - sized residual tumors were found on microscopy. in this paper, we describe a case of clinicopathologic change in unresectable gist after neoadjuvant imatinib mesylate. |
the prospective investigation by engstrm and colleagues in a previous issue of critical care sets out to determine if apneic oxygenation via a pharyngeal catheter during endotracheal intubation would prevent or increase the time to life - threatening hypoxemia in an animal model with acute lung injury. in this investigation, eight anesthetized piglets with induced lung collapse were randomized to receive either 10 l of oxygen per minute via a pharyngeal catheter or no oxygen. the investigators found that pharyngeal oxygenation markedly prolonged the time to severe systemic arterial oxygen desaturation, defined as oxygen saturation (spo2) of less than 60%. emergency endotracheal intubation in the critical care unit and in the emergency department can be fraught with complications. difficulty with oxygenation can be challenging in the critically ill as they have much less reserve and are more prone to desaturate during the brief apneic period that occurs while the patient is being intubated. traditionally, patients typically do not receive supplemental oxygen as attempts are made to pass the endotracheal tube through the vocal cords. in one prehospital study, for example, it was found that among 54 trauma patients who had been intubated following a rapid sequence induction technique, 57% experienced significant desaturation with a median duration of 160 seconds. the specific premise of the current investigation by engstrm and colleagues was to use an animal model involving induced acute lung injury in order to demonstrate that even in underlying conditions at greater risk for life- threatening hypoxemia, the oxygen desaturation can be minimized by using the pharyngeal catheter supplemental oxygen technique. one limitation of the study was that the authors chose a threshold saturation level that was markedly low (60%). in other studies of critically ill adults, ' severe hypoxemia ' was defined as a spo2 of less than 80% and ' hypoxemia ' was defined as a spo2 of less than 90%. it would be helpful to know when the piglets ' spo2 fell below 90%, a point at which traditional concerns for hypoxia during intubation typically begin for most clinicians. though less contentious, the investigators chose a continuous 10-minute period of apnea as their cutoff point for desaturation. one prospective cohort study of endotracheal intubation attempts in critically ill adults demonstrated that 10.3% of intubations did require 10 or more minutes to achieve. however, these patients had multiple intubation attempts in which they were ventilated with a bag - valve - mask (bvm) between intubation attempts. accordingly, it is likely that none of these patients had a continuous 10-minute period of prolonged apnea. several reports in the anesthesia literature support the use of nasopharyngeal oxygenation following pre - oxygenation in human subjects, but all in relatively healthy, cooperative persons. for example, teller and colleagues conducted a randomized placebo controlled trial in 12 uncomplicated patients undergoing endotracheal intubation during scheduled surgery. an 8 fr catheter was inserted through a nasal airway and insufflated with oxygen (3 l / minute). they found that the arterial oxygen saturation (sao2) never fell below 97% during the entire 10 minutes of apnea. how the results of these studies would then translate to critically ill human subjects undergoing emergent intubation remains to be seen, particularly among those with underlying pulmonary impairment. it makes intuitive sense that if one provides supplemental oxygenation using a method that allows simultaneous intubation procedures to take place, the time to desaturation would be prolonged, and the experimental evidence certainly supports that contention. although the presence of a pharyngeal catheter may provide a barrier to obtaining a good seal when using modern bvm devices, it appears (as observed with previous studies) that such a device could be rapidly replaced whenever the bvm is removed. overall, the authors succeeded in demonstrating the benefits of pharyngeal oxygen administration in reducing the risk of desaturation during intubation in the presence of acute lung injury using an animal model. although the study design implemented in the current investigation would be difficult to repeat in human subjects, this study supports the intuitive contention that pharyngeal oxygenation should be considered during emergent intubation of critically ill patients, particularly during periods of apnea. severe life - threatening hypoxemia is a well - known adverse consequence of emergent intubations and any reasonable measure used to minimize this risk is a reasonable conclusion. the authors should be congratulated for taking this first step in the methodical process of proper evidence - based approaches in clinical care of the critically ill. | critically ill patients requiring emergent endotracheal intubation are at risk for life - threatening hypoxemia during the intubation procedure, particularly when the patient is apneic and not receiving any supplemental oxygen. in a current study, engstrm and colleagues investigated the effect of nasopharyngeal oxygenation in eight anesthetized pigs with induced acute lung injury. the investigators confirmed, even in this model, that pharyngeal oxygenation significantly prolonged the time to desaturation during periods of apnea. recognizing the limitations of directly extrapolating these experimental results to critically ill human subjects, the findings do support the contention that, until proven otherwise, nasopharyngeal oxygenation should at least be considered as one technique to diminish hypoxemic complications in very sick patients, particularly those with underlying pulmonary impairment. |
a 58-year - old male presented with a lesion on the medial aspect of left lower eyelid near the punctum. he had a small elevated nodular swelling at the margin of the eyelid for the past two decades. there was a gradual increase in the size of the swelling for past 4 years, however, during past 6 months it became more visible associated with discomfort, itching, and a noticeable mass. physical examination revealed a solitary 2 cm 2 cm size brownish firm fleshy growth with hyperkeratotic surface covered with crusts in some areas, involving the skin of the left lower eyelid [fig. 1 ]. it did not involve the underlying deeper tissues also and there was no regional lymphadenopathy. fleshy growth with hyperkeratotic surface covered with crusts in some areas, involving the skin of the left lower eyelid the mass was excised along with a 3 mm margin of healthy skin, and the resultant skin defect was repaired with a median frontoglabellar pedicle skin flap. the final pathologic diagnosis of excision biopsy specimen confirmed syringocystadenoma papilliferum. on gross examination, skin covered tissue measuring 1.3 cm 1.1 cm 1 cm. this showed a nodular lesion 0.7 cm in its greatest axis 0.3 cm away from the nearest peripheral surgical cut margins. on microscopic examination, section showed a nodular lesion lined by epidermis with varying degree of papillomatosis [fig. 2 ]. these were lined by two rows of cells. the luminal row consisted of columnar cells with evidence of active decapitation secretion and outer row of cells composed of small cuboidal cells. on gross examination, skin covered tissue measuring 1.3 cm 1.1 cm 1 cm. this showed a nodular lesion 0.7 cm in its greatest axis 0.3 cm away from the nearest peripheral surgical cut margins. on microscopic examination, section showed a nodular lesion lined by epidermis with varying degree of papillomatosis [fig. 2 ]. these were lined by two rows of cells. the luminal row consisted of columnar cells with evidence of active decapitation secretion and outer row of cells composed of small cuboidal cells. syringocystadenoma papilliferum is a rare benign tumor that is believed to be derived from the apocrine or the eccrine sweat glands. in a recent report, immunohistochemistry proved the apocrine nature of the benign, noncystic lesion by virtue of its nuclear androgen receptor and cytoplasmic gross - cystic disease fluid protein-15 positivity, along with its smooth muscle actin - positive myoepithelial layer. this is predominantly a childhood tumor ; however presentation at a later stage like our case has been described in literature. in half of those who are affected, it is present at birth, and in a further 1530%, the tumor develops before puberty. the lesion is frequently misdiagnosed as basal cell carcinoma or cyst or squamous cell carcinoma. in a series of 14 patients, this is unlike other benign eyelid lesions, where histopathological diagnosis confirms clinical diagnosis in 95.9% cases. tumor is usually described as a skin colored to pink, hairless, firm plaque of grouped nodules or as a solitary nodule. cauliflower like, verrucous, papillary, hyperkeratotic, or sometimes moist fleshy excrescences have also been described. most of the lesions develop and enlarge slowly, although a few can increase to significantly within a short period. the plaque type that presents a hairless area of the scalp is commonly associated with a sebaceous nevus of jadassohn. in about one - third of the case, appearance of the lesion in the face and neck region is seen in the linear type ; however, a solitary nodular type shows predilection for the trunk. syringocystadenocarcinoma papilliferum is a malignant counterpart of syringocystadenoma papilliferum. the diagnosis is clinically suspected and histologically confirmed. ulceration or a rapid enlargement of an existing tumor is indicative of a malignant transformation. we were suspicious in the index case because of the recent increase of size. in about one - tenth of cases of syringocystadenoma papilliferum squmaous cell carcinoma may also develop, but much less frequently. because of this, surgical excision is the treatment of choice. in our case, excision of the tumor was done followed by repair of skin defect with median frontoglabellar pedicle skin flap. | syringocystadenoma papilliferum can rarely affect eyelid skin. the lesion is frequently misdiagnosed as basal cell carcinoma or cyst or squamous cell carcinoma. we are presenting a case that was clinically diagnosed as basal cell carcinoma of eyelid but was later histologically diagnosed as syringocystadenoma papilliferum. |
the optimization of ventilatory support is a crucial issue in the management of patients with acute respiratory failure, aiming at improving gas exchange and patient - ventilator interaction while preventing ventilator - induced dysfunction of the respiratory system. a very important study by levine. shows in humans, as previously observed in animal models, that the combination of diaphragm inactivity and mechanical ventilation is associated with marked diaphragm atrophy. diaphragm atrophy, which may occur after just 18 hours of controlled mechanical ventilation as a result of increased myofibril proteolysis, may contribute to prolongation and/or failure of the weaning process. this study, performed in brain - dead organ donors, underlines the importance of using modes of partial ventilatory support whenever possible and not contraindicated, in order to reduce the time spent on mechanical ventilation and its side effects. relevant clinical research has investigated the complex interaction between patient and ventilator during partial support. compared three different strategies conventionally aimed to optimize patient - ventilator interaction in pressure support ventilation (psv) : reduction of support level, reduction of insufflation time and application of positive end - expiratory pressure (peep). the reduction of pressure support succeeded in eliminating ineffective triggering in two - thirds of the patients without increasing the work of the respiratory muscles or altering gas exchange, while the effects of reducing insufflation time and increasing peep were less important and not significant. the improvement in patient - ventilator synchrony becomes relevant when tidal volume (vt) is reduced to 6 ml / kg, which is therefore suggested by the authors as a target for adjustment of inspiratory pressure support level. neurally adjusted ventilatory assist (nava) is a new form of partial support wherein the ventilator applies positive pressure throughout inspiration in proportion to the electrical activity of the diaphragm, as assessed by transoesophageal electromyography. allowing ventilator functioning and cycling to be under direct control of the patient 's respiratory drive and rhythm, nava aims to enhance patient - ventilator interaction and minimize the risk of over - assistance. a recent study by colombo. compares the physiological response to different levels of assistance delivered with nava and psv. as opposed to nava, psv was prone to the risk of over - assistance, as identified by higher vt, significant mismatch between neural and flow - based timing and frequent occurrence of ineffective efforts. the study indicates that, compared to psv, nava has the potential to limit the risk of over - assistance, prevent patient - ventilator asynchrony, and, overall, improve patient - ventilator interaction. two recent trials have evaluated the impact of different ventilator settings in the prevention of ventilator - induced lung injury (vili) in patients with acute lung injury and acute respiratory distress syndrome (ards). meade. compared the effects of a ventilatory strategy combining low vt, recruitment manoeuvres and high levels of peep with the established ventilatory strategy. in the experimental group, vt was set at 6 ml / kg, with plateau airway pressures (paw - plateau) 40 cmh2o. each trial started with a 40-second breath - hold at 40 cmh2o paw recruitment manoeuvre on a fraction of inspired oxygen (fio2) of 1.0, which could be repeated up to four times daily. the control group underwent the ards network ventilation protocol (volume - assist control mode, vt 6 ml / kg and paw - plateau 30 cmh2o, with peep determined according to fio2, no recruitment manoeuvre). results showed a significant difference in peep levels associated with lower incidence of hypoxemia and fio2 requirement, less recourse to rescue oxygenation therapies, and fewer deaths due to refractory hypoxemia in the intervention group, as opposed to the control group, while no significant difference in mortality rate and duration of mechanical ventilation was found. evaluated a strategy using high peep levels to increase alveolar recruitment while avoiding excessive hyperinflation by limiting paw - plateau and using low vt. in the control group, paw - plateau was kept as low as possible without falling below oxygenation targets, setting peep between 5 and 9 cmh2o. in the experimental group, peep was adjusted based on paw and kept as high as possible without increasing paw - plateau above 28 - 30 cmh2o, regardless of its effects on oxygenation. the experimental strategy significantly improved oxygenation and respiratory system compliance and reduced the time spent on mechanical ventilation and with organ failures, but failed to reduce mortality. in an intriguing study, consider vili a result of excessive lung stress and strain, as assessed by transpulmonary pressure and inflated volume to functional residual capacity ratio, respectively. as stress and strain can not be measured in clinical practice, the study aims to determine the accuracy at which the two parameters can be inferred by analyzing their commonly utilized surrogates, that is, paw - plateau and vt / kg. currently, prevention of vili is based on recognizing the harmful threshold for these surrogates, identified as 28 - 30 cmh2o (paw - plateau) and 6 ml / kg (vt / kg). in a varied population ranging from postoperative patients with intact lung function to patients with ards, paw - plateau and vt interestingly, the stress to strain relationship was the same in all the subgroups, being linear in the ranges of pressures and volumes explored. the specific lung elastance was also similar in the various subgroups, remaining constant at approximately 13.5 cmh2o, regardless of ventilator settings. because stress and strain were demonstrated to be linked by a constant proportionality factor, that is, the specific lung elastance, stress can be calculated as 13.5 cmh2o strain. as a strain greater than 2, corresponding to an end - inspiratory lung volume in the range of total lung capacity, has been identified as critical for the risk of development of lung injury, the harmful threshold of stress will consequently be 2 13.5 cmh2o ; therefore the recommended paw - plateau threshold of 28 - 30 cmh2o seems reasonable for most acute lung injury / ards patients. the identification of diaphragmatic atrophy after just 18 hours of controlled mechanical ventilation suggests the importance of adopting modes of partial ventilatory support as soon as possible. the optimization of patient - ventilator synchrony should be based either on the reduction of the assistance provided with the commonly used modes of partial support or on the application of new forms of mechanical assistance designed to improve the interaction between the patient and the ventilator, such as nava. in patients with ards, ventilatory strategies for optimizing peep with the aim of increasing alveolar recruitment while limiting hyperinflation succeeded in improving lung function and reducing the duration of mechanical ventilation, but failed to reduce mortality. the introduction of the measurement of lung stress and strain into daily clinical practice could allow a better identification of the safe limits of mechanical ventilation in the presence of lung injury. our institution received an icu ventilator with nava software, a portable pc, and dedicated data acquisition software from maquet critical care, used in the study conducted by colombo.. | recently, several studies have been performed to better outline the pathophysiology of acute respiratory failure and evaluate the therapeutic profile of different modes of ventilation and ventilator settings. here we briefly report those we consider most relevant for daily intensive care unit clinical practice. |
helicobacter pylori infection in humans is associated with the development of numerous gastric pathologies such as peptic ulcer, chronic gastritis and gastric cancer. once established, the infected individuals carry h. pylori in their stomach yet if untreated with antibiotics, they can persist in the body for a lifetime. during the last decade, the rate of multidrug resistance among clinical h. pylori isolates has increased, which could be a result of widespread and non - appropriate use of various antibiotics (1, 2). by evaluation of resistance to clarithromycin, metronidazole, quinolones, amoxicillin and tetracycline, the investigators found that point mutations, redox intracellular potential, efflux pump systems and membrane permeability were the most common mechanisms of resistance in h. pylori (3). among the mechanisms involved in multidrug - resistance, active pumping out of the drugs by efflux pumps may be an important mechanism by which the gram negative bacteria may be protected from toxic effects of antibiotics (4 - 6). in general, the efflux pumps of gram - negative bacteria consist of an inner - membrane pump, a periplasmic adaptor protein and an outer - membrane channel in which the first one (inner membrane protein) acts with the second (periplasmic protein) and third (outer membrane protein). these efficiently structured complexes can cause the multidrug - resistance phenotype, which provides an efficient means for the export of structurally unrelated drugs (7, 8). by analyzing h. pylori 26695 genome, 27 putative translocase have been identified that include atp - binding cassette (abc) super family, major facilitator super family (mfs), resistance - nodulation - cell division (rnd) family and multidrug and toxic compound extrusion (mate) proteins (9). one of such translocases, which is found in h. pylori 26695 genome is hp1184 that belong to the mate family (10). as proposed by van amsterdam., and johnson., mutation in hp1184 can result in the susceptibility of h. pylori to etbr (8, 9). hp1181 is another putative translocase, which may be related to the major facilitator super family. analysis of hp1181 gene product in h. pylori has revealed that hp1181 could be an integral membrane protein, which can contribute to resistance via a multi - drug resistance efflux protein (11, 12). the results of each basic study on the mechanisms of multidrug resistance in the case of h. pylori may be beneficial for designing more suitable antibiotic combinations for successful treatment of infection. the aim of the present study was to evaluate the association between presence of two predicted efflux - related genes, hp1181 and hp1184, in mdr clinical strains of h. pylori and their role in the active - efflux of antibiotics. the mdr strains employed in this study were selected from screening of the h. pylori strains isolated by culturing of biopsy specimens from children admitted to the medical center of tehran for peptic disease problem (13). the isolated strains were plated onto modified campy blood agar containing brucella agar base (merck, germany), supplemented with 5% defibrinated sheep blood and antibiotics. the grown colonies were identified by gram staining, standard biochemical tests and pcr using h. pylori specific primers for 16srrna and urec (14, 15). helicobacter pylori strain 26695 (from cnrs, bordeaux, france) for which complete sequence and predicted proteins are available, was used as a positive control for presence of genes studied in pcr analysis (12). atcc43504 strains (from cnrs, bordeaux, france) was the reference strain for quality control of antibiotic susceptibility testing (16). determination of minimal inhibitory concentration (mic) for ethidium bromide (etbr) was performed by the disc diffusion method. for this purpose, three - day - old bacterial cultures with a mc - farland opacity of 2.0 were cultured on muller hinton agar (merck, germany) plates supplemented with 7% fresh sheep blood. plates were dried for five minutes, and the blank discs containing etbr (merck, germany), with concentration of 4 to 64 g / ml were placed on their surfaces ; the inhibition zones of resistant strains were compared with the susceptible ones (13). the cut off for mic determination of etbr was considered 4 g / ml. to select the mdr - strains, h. pylori isolates were screened for resistance to amoxicillin (amx), ampicillin (amp), ciprofloxacin (cip), erythromycin (ery) (zakaria and exir, iran), metronidazole (mtz), and tetracycline (tet) (sigma, usa), using the minimal inhibitory concentration method. for this purpose, plates were prepared with mueller hinton (merck, germany) agar plus 7% fresh sheep blood and an appropriate concentration of antibiotics from 0.5 to 64 g / ml. the plates containing antibiotics were then spread with bacterial suspension and incubated for two to three days at 37c under microaerobic conditions. the cut off values for mic determination was 4, 4, 4, 4, 8 and 8 g / ml for amp, amx, tet, cip, mtz and ery, respectively, according to the breakpoints previously described (13, 16). the mic for all strains was compared in the presence and absence of cccp, as previously described (13, 16, 17). accumulation assays were performed according to a previously described protocol with some modifications (13). briefly, h. pylori strains were grown on campy blood agar for two days harvested and resuspended in pre - warmed brucella broth (37c) to an optical density of 0.5. the cell suspension was incubated for 10 minutes at 37c, and then centrifuged, washed and resuspended in 15 mm sodium phosphate buffer (ph 7.2). uptake assays were initiated by the addition of an antibiotic or etbr to the bacterial cell suspension at a final concentration of 10 g / ml. eight minutes after antibiotic or etbr addition, cccp was added to one - half of the reaction mixture at a final concentration of 200 m and the other half was used as the control. after 15 minutes, each of the collected samples were placed at 4c for one minute (to stop the function of the efflux pumps), centrifuged at 6000 g for 15 minutes, and the supernatants were used to measure the fluorescence of the respective antibiotics with a shimadzu rf 5000 spectrofluorometer (shimadzu scientific instruments) at excitation and emission wavelengths of 400 and 450 for amp, amx and tet ; 300 and 350 for ert ; 279 and 447 for cip ; and 544 and 590 for etbr, respectively. these wavelengths have previously been selected by testing various experimental conditions (13). the pellet was used for measurement of the dry weight of bacteria by subtracting the initial weight of the cell - containing vial. concentration of antibiotics in the supernatants were calculated using a standard curve of the respective antibiotics (concentration ranging from 100 to 1000 ng) in 0.1 m of glycine hydrochloride ph 3.0.the results were expressed as ng of antibiotics and etbr per mg (dry wt) of bacteria. dna was extracted using the phenol - chloroform method, as previously described (14). based on the sequence alignments of the genes from h. pylori 26695 strain in gene bank, specific hp1181 primers were designed as follow : f, 5'aag aag aag agc gca ccaa3 ' and r, 5'gct cta agg tgg caa aac c3 '. the reaction consisted of 32 cycles, with each cycle composed of one minutes at 95c, three minutes at 60c and four minutes at 72 c. after a final extension of 15 minutes at 72c, the amplicons were electrophoresed on 1.2% agarose gel. the gene specific hp1184 primers were prepared according to the primers described by van amsterdam as follow : f, 5'acg ctc tag act caa agc acg gca gaa ttt3 ' ; r, 5'acg cct cga gga act ttt ggt gcg ttg gat3 ', which predicted to amplify a 445-bp amplicon. the reaction consisted of 32 cycles, with each cycle composed of one minute at 95c, four minutes at 59c and seven minutes at 72c. for each pcr reaction, a pcr control without template dna was used. after a final extension of 15 minutes at 72c rt - pcr was performed to compare the difference in expression of hp1181 between 26695 susceptible (standard) strain and mdr clinical strains. for this purpose, the concentration of high - pure rna, prepared according to the manufacturer s recommendations (roche, germany), was measured at 260 nm and the cdna was synthesized using first - strand cdna synthesis kit for rt - pcr (roche, germany). the reaction for cdna synthesis the rt - pcr - products were visualized by electrophoresis on 1% agarose gel and 15% acrylamide gel with a 100-bp ladder size - marker (cinnagene, iran). the rt - pcr analysis of hp1181 was performed for mdr strains, susceptible and 26695 h. pylori strains. the size of products for the 26695 susceptible strain was compared with that of mdr strains and was calculated by comparison with standard of mw. the mdr strains employed in this study were selected from screening of the h. pylori strains isolated by culturing of biopsy specimens from children admitted to the medical center of tehran for peptic disease problem (13). the isolated strains were plated onto modified campy blood agar containing brucella agar base (merck, germany), supplemented with 5% defibrinated sheep blood and antibiotics. the grown colonies were identified by gram staining, standard biochemical tests and pcr using h. pylori specific primers for 16srrna and urec (14, 15). helicobacter pylori strain 26695 (from cnrs, bordeaux, france) for which complete sequence and predicted proteins are available, was used as a positive control for presence of genes studied in pcr analysis (12). atcc43504 strains (from cnrs, bordeaux, france) was the reference strain for quality control of antibiotic susceptibility testing (16). determination of minimal inhibitory concentration (mic) for ethidium bromide (etbr) was performed by the disc diffusion method. for this purpose, three - day - old bacterial cultures with a mc - farland opacity of 2.0 were cultured on muller hinton agar (merck, germany) plates supplemented with 7% fresh sheep blood. plates were dried for five minutes, and the blank discs containing etbr (merck, germany), with concentration of 4 to 64 g / ml were placed on their surfaces ; the inhibition zones of resistant strains were compared with the susceptible ones (13). to select the mdr - strains, h. pylori isolates were screened for resistance to amoxicillin (amx), ampicillin (amp), ciprofloxacin (cip), erythromycin (ery) (zakaria and exir, iran), metronidazole (mtz), and tetracycline (tet) (sigma, usa), using the minimal inhibitory concentration method. for this purpose, plates were prepared with mueller hinton (merck, germany) agar plus 7% fresh sheep blood and an appropriate concentration of antibiotics from 0.5 to 64 g / ml. the plates containing antibiotics were then spread with bacterial suspension and incubated for two to three days at 37c under microaerobic conditions. the cut off values for mic determination was 4, 4, 4, 4, 8 and 8 g / ml for amp, amx, tet, cip, mtz and ery, respectively, according to the breakpoints previously described (13, 16). the mic for all strains was compared in the presence and absence of cccp, as previously described (13, 16, 17). accumulation assays were performed according to a previously described protocol with some modifications (13). briefly, h. pylori strains were grown on campy blood agar for two days harvested and resuspended in pre - warmed brucella broth (37c) to an optical density of 0.5. the cell suspension was incubated for 10 minutes at 37c, and then centrifuged, washed and resuspended in 15 mm sodium phosphate buffer (ph 7.2). uptake assays were initiated by the addition of an antibiotic or etbr to the bacterial cell suspension at a final concentration of 10 g / ml. eight minutes after antibiotic or etbr addition, cccp was added to one - half of the reaction mixture at a final concentration of 200 m and the other half was used as the control. after 15 minutes, each of the collected samples were placed at 4c for one minute (to stop the function of the efflux pumps), centrifuged at 6000 g for 15 minutes, and the supernatants were used to measure the fluorescence of the respective antibiotics with a shimadzu rf 5000 spectrofluorometer (shimadzu scientific instruments) at excitation and emission wavelengths of 400 and 450 for amp, amx and tet ; 300 and 350 for ert ; 279 and 447 for cip ; and 544 and 590 for etbr, respectively. these wavelengths have previously been selected by testing various experimental conditions (13). the pellet was used for measurement of the dry weight of bacteria by subtracting the initial weight of the cell - containing vial. concentration of antibiotics in the supernatants were calculated using a standard curve of the respective antibiotics (concentration ranging from 100 to 1000 ng) in 0.1 m of glycine hydrochloride ph 3.0.the results were expressed as ng of antibiotics and etbr per mg (dry wt) of bacteria. dna was extracted using the phenol - chloroform method, as previously described (14). based on the sequence alignments of the genes from h. pylori 26695 strain in gene bank, specific hp1181 primers were designed as follow : f, 5'aag aag aag agc gca ccaa3 ' and r, 5'gct cta agg tgg caa aac c3 '. the reaction consisted of 32 cycles, with each cycle composed of one minutes at 95c, three minutes at 60c and four minutes at 72 c. after a final extension of 15 minutes at 72c, the amplicons were electrophoresed on 1.2% agarose gel. the gene specific hp1184 primers were prepared according to the primers described by van amsterdam as follow : f, 5'acg ctc tag act caa agc acg gca gaa ttt3 ' ; r, 5'acg cct cga gga act ttt ggt gcg ttg gat3 ', which predicted to amplify a 445-bp amplicon. the reaction consisted of 32 cycles, with each cycle composed of one minute at 95c, four minutes at 59c and seven minutes at 72c. after a final extension of 15 minutes at 72c, the amplicons were electrophoresed on 1.2% agarose gel (7). rt - pcr was performed to compare the difference in expression of hp1181 between 26695 susceptible (standard) strain and mdr clinical strains. for this purpose, the concentration of high - pure rna, prepared according to the manufacturer s recommendations (roche, germany), was measured at 260 nm and the cdna was synthesized using first - strand cdna synthesis kit for rt - pcr (roche, germany). the reaction for cdna synthesis was held at 25c for ten minutes, 42c for 60 minutes and the rt - pcr - products were visualized by electrophoresis on 1% agarose gel and 15% acrylamide gel with a 100-bp ladder size - marker (cinnagene, iran). the rt - pcr analysis of hp1181 was performed for mdr strains, susceptible and 26695 h. pylori strains. the size of products for the 26695 susceptible strain was compared with that of mdr strains and was calculated by comparison with standard of mw. multi - resistance or resistance patterns of the strains, according to previously described representation (12), are shown in table 1. these mdr strains have been classified into three groups, according to their behavior toward active efflux inhibition by cccp. as demonstrated in table 2, in the presence of cccp, the mics of eight mdr strains to etbr was decreased by two to four folds ; five of them also showed a two - fold reduction in their mic for antibiotics (amx, amp, tet, cip and mtz). accumulation assay for etbr, exhibited active efflux for eight mdr strains (s3, s11, s13, s1, s2, s4, s9 and s12), of which five demonstrated active efflux for all of the antibiotics except ery (table 3). in the presence of cccp, the increase in the amounts of accumulated etbr for the strains of group i and ii was two folds and the increase in those of antibiotic for the strains of group ii, was between 1.4 and 1.8 folds, which was lowest for -lactam, and highest for tet (table 3). in the presence of cccp, no difference in mic for etbr or antibiotics, as well as no difference in the amount of this group comprised of the following strains, s7, s10 and s13, which were only resistant to -lactams and strains, s5, s6, s8 and s14, which were mdr. two strains, s16 and s17, susceptible to all of the antibiotics and etbr, demonstrating no efflux phenotype for them, amplified neither for hp1184, nor for hp1181. the relationship between the presence of active efflux and presence of two genes hp1181 and hp1184, are demonstrated in table 2. the results of pcr for the detection of hp1184 (a product of 445 bp) and hp1181 (a product of 579 bp) are shown in figure 1a - d. standard 26695 and atcc43504 strains, which were both susceptible to the entire antibiotic tested in this work, showed no efflux for etbr or antibiotics. to understand the status of hp1181 gene in the mdr strains as well as in 26695 standard strain, they were analyzed by rt - pcr. the product of rt - pcr for hp1181 in the mdr strains was approximately 100 bp shorter than standard 26695 susceptible strain (figure 1c - d). cut off values for mic determination was 4, 4, 4, 4, 8 ; 8, and 4 g / ml for amp, amx, tet, cip, mtz, ery, and etbr. values are expressed as ng of antibiotics and etbr per mg (dry wt) of bacteria. groups i - iv are classified as the table 2, st : strain 26695. a, detection of hp1181 : 1 : 100-bp ladder ; 2 : s17 ; 3 : s16 ; 4 : s12 ; 5 : 26695 standard strain, 6 : s4 ; 7 : s2 ; 8 : s1 ; 9, control (no template) ; b, detection of hp1184 : 1 : 26695 standard strain, 2 : s3 ; 3 : s2 ; 4 : s1 ; 5 : 100-bp ladder ; c, rt - pcr detection of hp1181 gene on 1% agarose gel. at the left : 100-bp standard ; 1 - 2 : 26695 standard strain ; 3 - 4 : s12 ; d, rt - pcr detection of hp1181 gene on 15% polyacrylamide gel. at the left : 100-bp standard ; 1 : control negative for rt - pcr ; 2 : s16, 3 : s12, 5 : 26695 standard strain. we compared the relationship between resistance to non - related antibiotics and their active efflux by evaluation of antibiotic accumulation in the bacterial cells using cccp as a protonophore. this compound reduces the transmembrane electrochemical gradient and inhibits the efflux pumps (5, 7, 16). although in our previous work (13), we demonstrated the efflux properties of the strains used in this work, the differences of antibiotic accumulation in the presence and absence of cccp, was higher with the optimized protocol used in the present work. we found that decrease in mic to antibiotics, as well as decrease in mic to etbr, however, observation of a positive hp1184 status in only six strains demonstrating efflux phenotype for only etbr, suggested the more specific role of hp1184 in the active efflux of etbr (table 2). we observed that decreases in mic to etbr and antibiotics, associated with increase in their accumulation amounts, was associated with the presence of hp1181 (100%) and hp1184 (50%) among the strains of group ii (tables 2 - 3). this observation indicated the involvement of hp1181 and hp1184 in their active efflux - phenotype. however, only five out of 12 mdr - strains, which demonstrated active efflux phenotype for -lactam, tet, mtz and cip, had a positive status for hp1181, suggesting the more specific role of hp1181 gene in the efflux of these antibiotics (9, 10). nevertheless, despite resistance to cla, no changes either in mic or accumulation amount was observed in the presence or absence of cccp. using phe - arg - b - naphthylamide (pabn), as an efflux pump inhibitor, hirata., proposed the contribution of efflux pumps to cla resistance in helicobacter pylori (18). they suggested that the rnd - type multidrug efflux pump was involved in cla resistance. is related to the nature of efflux pumps involved in the resistance to cla ; which is rnd - type multidrug efflux pump for cla and mate for the hp1184 and hp1181 mdr efflux pumps (10). furthermore, as rnd family would be responsible for macrolide intrinsic resistance in h. pylori, the nature of its specific inhibitors would also be different since it was demonstrated that various inhibitors influence the efflux pump behaviors (19, 20). consistent with the results discussed above, an association was observed between the absence of active efflux either for etbr or for antibiotics and absence of either hp1184 or hp1181 genes in the strains of group iv, as well as in the susceptible strains. therefore, acquisition of multidrug resistance in mdr strains is due to their active efflux phenotypes and may be associated with presence of hp1181 and hp1184 genes. however, 26695 h. pylori strain, which was susceptible to all of the antibiotics, contained both of hp1181 and hp1184 genes. the results of rt - pcr performed for the mdr strains and for 26695 control h. pylori strain could explain this question. the rt - pcr for hp1181 was positive for both mdr strains containing hp1181, and for 26695 strain, however, the size of rt - pcr product in the mdr strain was approximately 100-bp smaller than that of the 26695 strain. a similar result has been previously observed in the expression of hp1165 gene in tetracycline - resistant strains of h. pylori strains (21). according to the hypothesis postulated in our objective, we could show the presence of an association between hp1184 gene and the active efflux of etbr. also, we demonstrated the association of hp1181 gene expression with the specific efflux of non - related antibiotics among clinical mdr strains of h. pylori. in conclusion, expression of the genes hp1184 and hp1181 is associated with the active efflux of etbr and non - related antibiotics, respectively. however, for displaying these phenotypes, a post - transcriptional regulation step may be required | backgroundduring the last decades the rate of multidrug resistance among clinical helicobacter pylori isolates has increased. active pumping out of the drugs may be an important mechanism for multidrug resistance in h. pylori strains.objectivesthe aim of this study was to evaluate the association of two h. pylori efflux - genes, hp1181 and hp1184 with the active - efflux phenotype in mdr clinical - strains of h. pylori.materials and methodsminimal inhibitory concentration (mic) and drug accumulation for -lactames, tetracycline (tet), erythromycin (ery), metronidazole (mtz), ciprofloxacin (cip) and ethidium bromide (etbr) was performed in the presence and absence of carbonyl cyanide m - chlorophenyl hydrazone (cccp). presence of hp1181 and hp1184 genes was detected by the polymerase chain reaction (pcr). rt - pcr was performed to compare expression of efflux genes by mdr strains, demonstrating active efflux with the strains without active efflux.resultstwo- to four - fold decrease in minimum inhibitory concentration (mic) and two - fold increase in accumulation were observed for etbr in the presence of cccp for 67% (8) of 12 mdr strains. with cccp, two- to four - fold decrease in mic and 1.4- to 1.8-fold increase in the accumulation of -lactames, tet, cip and mtz were obtained for 42% (5) of the mdr strains. six, five and three of the 12 mdr strains amplified hp1184, hp1181, and both of them, respectively. the rt - pcr product for expression of hp1181 by mdr strains was approximately 100 bp shorter than that of the 26695 susceptible standard strain.conclusionsexpression of the genes hp1184 and hp1181 are associated with the specific active efflux of etbr and non - related antibiotics, respectively. for displaying these phenotypes, a post - transcriptional regulation step may be required. |
systemic sclerosis (ssc) is a multiorgan disorder, and progressive interstitial lung disease (ild) and scleroderma renal crisis (src) are serious complications in ssc patients. cyclophosphamide (cyc) is the recommended therapy for ssc - associated ild (ssc - ild), but its effectiveness is not sufficient. we report a src patient who underwent treatment with cyc for progressive ild and whose src was successfully treated with angiotensin - converting - enzyme inhibitor (ace - i) and plasma exchange (pe). a 67-year - old female was referred to our hospital for stiffness in her hands and a high titer of antinuclear antibody (ana) of 1:640. the patient had a history of hypertension, and her blood pressure (bp) at first consultation was 110/66 mmhg with a medication of 5 mg amlodipine besylate a day. physical examination revealed scleroderma extending bilaterally to the upper arms, nailfold bleeding, and fine crackles on her back. her ana titer was positive at 1:1280 with homogeneous and nucleolar patterns, and anti - topoisomerase i antibody titer examined by immunodiffusion was positive at 1:2. serum krebs von den lungen-6 (kl-6) level was 331 u / ml, which was within the normal range. in addition to blood cell counts and urinalysis, other clinical data were unremarkable (table 1). electrocardiogram demonstrated no arrhythmia, and ventricular dysfunction and pulmonary arterial hypertension were not revealed by echocardiography. chest computed tomography (ct) presented bilateral slight ground - glass opacity (ggo) predominantly around the dorsal area (figure. 1a), and gastroduodenal endoscopy showed atrophic gastritis and mild reflux esophagitis. from her clinical findings, the diagnosis of diffuse - type ssc was made, and 20 mg / day of prednisolone (psl) was initiated for the newly onset edematous scleroderma. laboratory data on first examination chest ct presented ggo shadow around dorsal area at first examination(a), got worse before ivcy(b), persisted before pe(c), improved right after pe(d), and still stabilized 2 years after pe(e). seven months after psl therapy initiation (4 months before pe), she was aware of dyspnea on effort and worsening of hand stiffness. the ggo shadow in her lung field in the chest ct had deteriorated (figure. 1b), and arterial oxygen pressure under room air was decreased to 67.1 mmhg. percentages of forced vital capacity (fvc) for the predicted values also decreased to 52.4% (table 2). progressive ild was suspected, and therefore, 13.7 mg / kg body weight intravenous cyc (ivcy) pulse therapy was initiated for every 4 weeks. however, after four courses of ivcy therapy, clinical improvement could not be obtained, and she began to complain of appetite loss, nausea, and general fatigue. on second admission (19 days before pe), her bp was 106/70 mmhg and body temperature (bt) was 36.8c. laboratory data revealed that total bilirubin (t - bil) and lactate dehydrogenase (ldh) levels had increased to 1.8 mg / dl and 345 u / l, respectively. c - reactive protein (crp) level was 0.73 mg / dl, which slightly exceeded the normal upper limit of 0.5 mg / dl. the serum kl-6 level had progressively increased to 767 u / ml. urinalysis revealed mild proteinuria, but no hematuria. the ggo shadow of her lung field in a chest ct, which presented a nonspecific interstitial pneumonia (nsip) pattern, had become slightly worse after four courses of ivcy therapy (figure. 1c). cardiac dysfunction and pulmonary arterial hypertension were not observed again by echocardiography. by a few weeks (5 days before pe), the serum t - bil, ldh, and creatinine (cr) levels the patient s hemoglobin levels had decreased to 10.5 g / dl, and her haptoglobin level was below 10 mg / dl with a negative coombs test, and schistocytes were observed under the microscope. further examination of the blood revealed an international normalized ratio prothrombin time of 0.88, an activated partial thromboplastin time of 30.8 s, and fibrinogen / fibrin degradation products (fdp) was 7.1 g / ml. the fdp level was slightly elevated, and her fibrinogen level was 388 mg / dl, which was within the normal range. the plasma renin activity (pra) and aldosterone (ald) levels were elevated to 18.8 ng / ml / hr and 289 pg / ml, respectively. her blood pressure was 123/65 mmhg, which was within the normal range, but slightly elevated over her usual level. a disintegrin - like and metalloproteinase with thrombospondin type-1 motifs 13 (adamts13) level was 32.4%, which was below the lower limit, but still detectable. tests for the perinuclear and cytoplasmic antineutrophil cytoplasmic antibodies (anca) and anti - rna polymerase iii antibody were negative. taking these clinical findings into consideration, we diagnosed the patient with src with thrombotic microangiopathy (tma) and hemolytic anemia. although we did not get the result of adamts13 level when serum cr levels progressively elevated, we could not deny the involvement of thrombotic thrombocytopenic purpura (ttp). therefore, we initiated enalapril treatment accompanied by pe with 73.4 ml / kg body weight frozen fresh plasma for nine courses. renal biopsy was not performed because of the need for immediate therapeutic induction and avoiding progressive renal damage. upon pe completion her blood pressure returned to the normal level of 110/70 mmhg, and abnormal laboratory data had improved (figure. 1d and e), and the worsening of pulmonary function had stopped and improved at 2 years after pe (table 2). change in pulmonary function before and after pe clinical course of serum creatinine, lactate dehydrogenase, and platelet. after ace - i accompanied with pe therapy, the abnormal laboratory data suggesting tma and renal damage had improved. src is a severe complication, which mainly occurs in approximately 5% of diffuse - type ssc patients. typical manifestations of src are progressive bp elevation and renal damage, which sometimes lead to poor prognoses. our case did not present severe hypertension or renal damage, but her bp and serum cr were elevated above normal levels. tma, which occurred in half of the src patients, was identified in our case. in addition, the highest risk group for src are patients with early diffuse - type ssc and rapidly progressive skin sclerosis, but majority of ttp associated with ssc was reported to be of the limited type. src results from endothelial injury and intimal proliferation in the renal arteries, followed by activation of the renin - aldosterone system. the pra and ald levels in our patient s case were high, which possibly indicated src. although our patient should be diagnosed with src, it is difficult to rule out ttp at an acute phase of src because of the similarity between these two disorders and absence of adamts13. to avoid therapeutic delay for the suspected disorder, initiation of pe one of the reasons for a normal bp was the patient s long - term use of antihypertensives, and the other was slowly elevation rate of bp. considering the clinical course, the serum cr and ldh levels rapidly elevated from 5 days before pe (figure 2). at this time pra had elevated because of the decrease in renal blood flow, followed by progressive hypertension. we supposed that early initiation of ace - i and pe interrupted the elevation of bp. if we had not started the therapy for src immediately, hypertension would have become apparent in our case. this idea is based on the hypercoagulative state of the blood brought about by the corticosteroid. on the other hand, some reports have mentioned that corticosteroid administration is not a risk factor of src, but rather is the result of therapy for severe ssc. steen. reported that a significantly greater proportion of src patients than ssc control patients received high - dose (> 15 mg / day) corticosteroid for 6 months prior to src onset, but there was no difference in the patients who had been taking corticosteroid continuously for more than 6 months. in our case, 11 months had passed from initiation of steroid therapy to src onset, which was a long interval for the development of steroid - induced src. therefore, the contribution of corticosteroid to the development of src is unclear in this case. typical renovascular histopathological findings of src include so - called onion skin lesions in the arteries ranging from the arcuate to cortical radiate arteries and fibrinoid thrombosis of the small vessels. these changes are caused by endothelial damage to the small vessels, leading to an increase in vascular permeability, endothelial cell proliferation, and stenotic change because of platelet aggregation. the increased production of th2 cytokines, such as interleukin-4, -13, and transforming growth factor- (tgf-) in ssc patients cause angiofibrosis and intimal stenosis by the activation of fibroblasts. other mechanisms of vascular damage in ssc are antibody - dependent cell - mediated cytotoxicity by anti - endothelial autoantibody and hyper - excretion of endothelin-1 (et-1). although the only recommended therapy for src is ace - i, the efficacy of pe combined with ace - i has also been reported. the therapeutic mechanism of pe is considered to be via the removal of cytokines that induce tissue fibrosis ; vasoconstrictive factors, such as et-1 ; and autoantibodies. ssc - ild is also suspected to be caused by vascular stenosis and interstitial fibrosis due to advanced cytokine production. currently, the only recommended therapy for progressive ssc - ild is cyc ; however, the therapeutic efficacy of cyc is not sufficient. in our case, the progressive interstitial shadow of pulmonary fibrosis stopped after pe, and stable radiographic findings had been maintained and pulmonary function had improved. renovascular stenosis by endothelial injury, observed in the pathology of ssc patients, is also observed in the pulmonary arteries. therefore, we speculate that pe has contributed to the clinical improvement of ssc - ild to some extent. the serum cr level at diagnosis is reported to be the prognostic indicator for src.. immediate initiation of ace - i and pe combination therapy is thought to be effective for the amelioration of renal function and treatment of progressive ild. | abstractsystemic sclerosis (ssc) is a multiorgan disorder involving the skin, heart, lungs, kidneys, and intestines. progressive interstitial lung disease (ild) is a serious complication in ssc patients, and cyclophosphamide (cyc) is the only recommended therapy for this condition;1) however, its clinical effectiveness is not sufficient. scleroderma renal crisis (src) is a rare complication, characterized by acute renal failure and progressive hypertension. angiotensin - converting - enzyme inhibitor (ace - i) is a widely accepted therapy for src. we report an ssc patient with src and progressive ild who underwent treatment with cyc and successful treatment with ace - i and plasma exchange (pe). src and ild are significant contributors to morbidity and mortality among ssc patients, and the therapy for these disorders is of great interest to rheumatologists. this study presents the possibility of favorable effects of pe for ssc - associated ild and src. |
we examined a 68-year - old man who had been suffering from diplopia for a week. the patient had been diagnosed with a nasopharyngeal squamous cell carcinoma 8 months prior and had received cisplantin - based chemotherapy with radiotherapy. on the first examination, 8-prism diopter right esotropia in the primary position and a remarkable limitation in abduction in his right eye were observed. the marginal reflex distance 1 (mrd1) was 3 mm in the right eye and 5 mm in the left. in addition to right mild ptosis, anisocoric pupils with a smaller right pupil were found and the anisocoria increased in the dark. after topical application of 0.5% apraclonidine (iopidine ; alcon, fort worth, tx, usa), the patient 's right pupil dilated from 2.5 mm to 5 mm in bright light and the right mrd1 increased from 3 mm to 5 mm (fig. brain magnetic resonance imaging (mri) revealed newly appeared gadolinium - enhanced mucosal thickening in both sphenoid sinuses combined with bony destruction of the skull base. these lesions extended to the right cavernous sinus with encasement of the internal carotid artery (ica) (fig. seven weeks later, the esotropia worsened and the limitation of abduction, anisocoria, and right ptosis persisted. neither abducens nerve palsy nor horner 's syndrome provides an indication of the location of the lesion. however, when both are present, it is important to localize the site of the lesion. when these rare clinical features are manifested, we have to consider paratrigerminal syndrome or a lesion of the posterior cavernous sinus. sympathetic nerve fibers travel over the wall of the carotid artery and in the cavernous portion the fibers leave the ica and accompany the abducens nerve for only a few millimeters in the posterior portion of the sinus (fig. 5). therefore, we could consider a posterior cavernous sinus lesion due to combined abducens nerve palsy. furthermore, incomplete horner 's syndrome without anhydrosis, as in the case reported here, indicates the involvement of the periarterial sympathetic plexus at the third angulation point. to date, only 13 cases with this combination of symptoms secondary to metastatic neoplasm within the cavernous sinus have been reported [4,10 - 16 ]. regarding the primary lesions in these cases, there were 1 small cell lung cancer, 1 parotid cancer, 1 breast cancer, 1 gastric cancer, 1 uterine cancer, 3 undetermined origins and 3 nasopharyngeal cancers. unlike in other three nasopharyngeal cancer cases reported by hirao., the cancer only invaded the 6th nerve and sympathetic fiber without involvement of the 5th nerve in this case. malignant tumors that affect the cavernous sinus commonly result in the 5th and the 6th nerve injuries. the more medial location of the abducens nerve has been postulated as the reason for its frequent involvement when confronted with the compressive forces in a confined space or direct invasion. in this case, the nasopharyngeal cancer extended through the sphenoidal sinus demonstrated by brain mri and positron emission tomography (fig. therefore, the cancer affected only the 6th nerve without involvement the 3rd nerve, which occupies a superior position at the lateral wall of the sinus, in addition to the 4th and 5th nerve. although autopsy was not permitted and there was a limitation of anatomical access, we suggest that tumor cells in the sphenoidal sinus had spread to the 6th nerve in the cavernous sinus, and then perineurally metastasized to the oculosympathetic nerve fibers before joining the first division of the 5th nerve. a plausible alternative to cocaine or hydroxyamphetamine in the diagnosis of horner 's syndrome is apraclonidine, a direct -receptor agonist with strong 2 and weak 1 activity. in patients with horner 's syndrome, reversal of anisocoria and improvement of ptosis with respect to the length of survival in patients with tumor invasion of the cavernous sinus region as reported in the literature, most individuals live no longer than six months and the ultimate outcome is a dismal 75% to 85% expected mortality within 2 years. the patient described in this report died 5 months after the presentation of combination abducens nerve palsy with horner 's syndrome. in other words, this manifestation may be a significant predictor of survival in addition to the location of the lesion. this is the first reported case of abducens nerve palsy with horner 's syndrome due to tumor invasion into the cavernous sinus in a korean patient. the presence of horner 's syndrome and the 6th nerve palsy should be cautiously investigated to confirm the existence of the cavernous sinus lesions. | a 68-year - old male patient presented with a week of sudden diplopia. he had been diagnosed with nasopharyngeal cancer 8 months prior and had undergone chemotherapy with radiotherapy. eight - prism diopter right esotropia in the primary position and a remarkable limitation in abduction in his right eye were observed. other pupillary disorders and lid drooping were not found. after three weeks, the marginal reflex distance 1 was 3 mm in the right eye and 5 mm in the left eye. the pupil diameter was 2.5 mm in the right eye, and 3 mm in the left eye under room illumination. under darkened conditions, the pupil diameter was 3.5 mm in the right eye, and 5 mm in the left eye. after topical application of 0.5% apraclonidine, improvement in the right ptosis and reversal pupillary dilatation were observed. on brain magnetic resonance imaging, enhanced lesions on the right cavernous sinus, both sphenoidal sinuses, and skull base suggested the invasion of nasopharyngeal cancer. lesions on the cavernous sinus need to be considered in cases of abducens nerve palsy and ipsilateral horner 's syndrome. |
arguably, the most appropriate and patient - centred outcome for patients who have moderate or severe pain deriving from any painful condition, acute or chronic, is that of no worse than mild pain (moore., 2013). rapid absorption and speed of onset in acute pain may represent one way of achieving this goal. with ibuprofen, plasma concentration and pain intensity are inversely correlated after 1 h (laska., 1986). fast - acting oral analgesic formulations typically involve rapidly dissolving salts such as ibuprofen sodium, lysine or arginine, or additional agents such as surfactants (polymeric surface active agent poloxamer 407 in this case) to increase tablet dissolution. systemic administration of non - steroidal anti - inflammatory drugs is not necessarily better than the oral route (tramr., 1998). fast - acting oral formulations generally have lower (better) numbers needed to treat (nnts) than standard formulations (moore., 2011). analysis of a single data set demonstrated a strong relationship between the speed of reduction of pain intensity and overall pain relief, and that early pain intensity reduction was associated with longer term outcomes of good overall pain relief and less need for additional analgesic (moore., 2014). as has been pointed out, these results were derived from only one data set and should be validated by data from other trials in acute pain, and ideally in other pain conditions (peloso, 2014). individual patient data analysis of a different clinical trial in third molar extraction comparing ibuprofen sodium, ibuprofen - poloxamer, paracetamol and placebo has therefore been used to verify the relationship between speed of onset of pain relief over the first hour with overall analgesic experience and duration of action. the published trial report demonstrated significant graded differences between placebo, paracetamol and fast - acting ibuprofen formulations. relationships between speed of onset and overall analgesia can therefore be tested for particular interventions and for all patients together. the data for the analyses were at the individual patient level, supplied as 38 pdf files of data listings for study nl0406, a published clinical trial using standard methods in third molar extraction (daniels., 2009). briefly, eligible patients were 1640 years of age with a primary diagnosis of at least one mandibular third molar (with full bony impaction and an impaction score of 4 on a 5-point scale) indicated for removal, or two ipsilateral third molars with a combined total impaction score no greater than 6. after extraction, and for entry into the trial, patients had to have moderate or severe baseline pain intensity as assessed using a 4-point categorical pain intensity scale and confirmed with a visual analogue scale (vas) score of 50 mm but 85 mm (where 0 = no pain and 100 mm = worst pain). there were four treatment arms in this randomized and double - blind trial, which was designed to test the efficacy of two fast - acting formulations of ibuprofen against paracetamol and placebo. sodium ibuprofen delivers maximum plasma drug concentrations at about 3040 min, compared with around 90120 min for standard ibuprofen acid formulations (moore., 2014). the second investigational ibuprofen formulation contained ibuprofen acid plus the surfactant poloxamer 407 from the poloxamer family of polymeric non - ionic surface - active agents to increase the rate of dissolution of the tablet and enable more rapid absorption relative to standard ibuprofen formulations. there is inadequate evidence that ibuprofen - poloxamer delivers very much faster peak plasma concentrations than standard ibuprofen acid (moore., 2014). the four treatment arms included 8082 patients receiving : a single data set in acute pain has shown that rapid reduction of pain intensity in the first hour is associated with better overall pain relief and longer lasting analgesia with reduced need for re - medication. a single data set in acute pain has shown that rapid reduction of pain intensity in the first hour is associated with better overall pain relief and longer lasting analgesia with reduced need for re - medication. validation that rapid reduction in pain intensity is associated with good overall and longer lasting pain relief. validation that rapid reduction in pain intensity is associated with good overall and longer lasting pain relief. placebo : two matched placebo for sodium ibuprofen tablets plus two matched placebo for ibuprofen - poloxamer tablets plus two matched placebo for 500 mg paracetamol caplets ; paracetamol 1000 mg : 2 500 mg paracetamol (tylenol extra strength) caplets plus two matched placebo for sodium ibuprofen tablets plus two matched placebo for ibuprofen - poloxamer tablets ; ibuprofen sodium 400 mg : 2 256 mg ibuprofen sodium dihydrate tablets (each tablet equivalent to 200 mg ibuprofen acid) plus two matched placebo for ibuprofen - poloxamer tablets plus two matched placebo for 500 mg paracetamol caplets ; ibuprofen - poloxamer 400 mg : 2 200 mg ibuprofen acid tablets, each tablet incorporating 60 mg of the surfactant poloxamer 407, plus two matched placebo for sodium ibuprofen tablets plus two matched placebo for 500 mg paracetamol caplets. among other measurements, visual analogue scale pain intensity (vaspi) and categorical pain relief scores were measured at baseline, and every 545 min, and then at 60, 90, 120, 180, 240, 300 and 360 min. information was also available on the time after dosing when any re - medication occurred. the slope (mm / min) of the regression line of vaspi against time over the period of 060 min was calculated for each patient using least - squares regression. the mean value of vaspi was computed for the periods 06 and 26 h. for the calculation of total pain relief (totpar), data from a 5-point categorical pain relief scale (none = 0, slight = 1, moderate = 2, good = 3 and complete pain relief = 4) were used to calculate the sum of pain relief scores over 6 h. if a patient had complete pain relief immediately after taking an analgesic and maintained that level of pain relief for 6 h, he / she would have a 6-h maximum totpar of 24 (4 6). for each patient, totpar was converted to percentage of maximum totpar (% maxtotpar) by division into the calculated maximum value (cooper, 1991). for any patient who re - medicated, pain intensity returned to its initial level and pain relief to zero for all subsequent time points (baseline observation carried forward, bocf). the nnt with 95% confidence intervals was calculated by the method of cook and sackett (1995). statistical differences between nnts were examined using the z - test (tramr., 1997). analyses were performed according to treatment, comparing average results with those for individuals for changes in vaspi over time, and for patients from all four treatment groups combined to repeat the assessments of association of speed of pain intensity reduction and overall pain relief as well as need for re - medication. a single data set in acute pain has shown that rapid reduction of pain intensity in the first hour is associated with better overall pain relief and longer lasting analgesia with reduced need for re - medication. a single data set in acute pain has shown that rapid reduction of pain intensity in the first hour is associated with better overall pain relief and longer lasting analgesia with reduced need for re - medication. validation that rapid reduction in pain intensity is associated with good overall and longer lasting pain relief. validation that rapid reduction in pain intensity is associated with good overall and longer lasting pain relief. trial data were available for all 322 patients ; 62% were women and the mean age was 21 years. patients given the fast - acting ibuprofen formulations had more rapid and profound median reductions in pain intensity using vaspi than did those given paracetamol or placebo (fig. 1). using categorical pain relief scores, for the outcome of 50%maxtotpar, both ibuprofen formulations were significantly better than paracetamol 1000 mg (table 1). nnts compared with placebo were 1.6 and 1.7 for ibuprofen 400 mg rapid formulations for achievement of 50%maxtotpar over 6 h, and 4.1 for paracetamol 1000 mg. evolution of median visual analogue scale pain intensity (vaspi) and patients remaining in the study without re - medicating. placebo = purple ; paracetamol 1000 mg = red ; ibuprofen sodium 400 mg = light blue ; ibuprofen - poloxamer 400 mg = dark blue. numbers needed to treat (nnts) for the outcome of 50%maxtotpar compared with placebo calculated from individual patient data for different treatments each ibuprofen formulation significantly better than paracetamol, p < 0.001, z - test. 2, where bocf imputation used initial pain intensity from the time of use of additional medication, and early re - medication is seen as a series of parallel horizontal lines in consequence. most patients reported consistent results over time whether vaspi was high or low. for placebo and paracetamol,, vaspi scores fell rapidly during the first hour and were then typically maintained until later re - medication. evolution of individual patient visual analogue scale pain intensity (vaspi) scores over 6 h for all four treatments. the relationship between speed of onset of pain relief and overall pain experience was investigated by seeking an association between the slope of change in pain intensity over 060 min and % maxtotpar over 06 h. in these analyses, all patient data were examined together, irrespective of treatment. figure 3 shows the relationship, in individual patients, between the overall pain relief over 06 h measured as the percentage of maximum totpar obtained, and the speed of pain intensity reduction over the first hour, measured as the slope of the regression line fitted to vaspi data, compared with the previous analysis (moore., 2014). the degree of consistency in these two relationships is remarkable with equations to the regression line of : relationship between speed of reduction of visual analogue scale pain intensity (vaspi) over 060 min and overall pain experience measured as % maxtotpar over 06 h in individual patients, for the analysis in this paper (nl0406) and a previous analysis (moore., 2014). rapid initial fall in pain intensity in individuals (higher negative values) corresponded to high overall levels of pain relief (% maxtotpar) over 6 h. as with the previous analysis, slopes of vaspi reductions of 0.5 mm / min or more negative (roughly equivalent to a vaspi reduction of about 30 mm or more in the first hour) over the first hour resulted in a high proportion of patients with 50%maxtotpar (fig. 4). relationship between speed of reduction of visual analogue scale pain intensity (vaspi) over 060 min and proportion of patients achieving the standard individual pain relief outcome of 50%maxtotpar over 06 h, for the analysis in this paper (nl0406 ; red) and a previous analysis (moore. 147 required additional analgesia within 6 h. for these, the mean initial rate of pain intensity reduction was 0.22 0.56 (sd) mm / min. for the 175 patients who did not require additional analgesia within 6 h, the mean initial rate of pain intensity reduction was 0.72 0.50 mm / min. this statistically significant difference (p < 0.001, student 's t - test) was similar to that of the previous analysis (moore., 2014). faster onset of pain relief was associated with a lesser chance of requiring additional analgesia within 6 h. where the slope was 0.5 or more negative, the chance of a patient needing additional analgesia was 3 in 10. where the slope was more positive than this, the chance was above 6 in 10. patients given the fast - acting ibuprofen formulations had more rapid and profound median reductions in pain intensity using vaspi than did those given paracetamol or placebo (fig. 1). using categorical pain relief scores, for the outcome of 50%maxtotpar, both ibuprofen formulations were significantly better than paracetamol 1000 mg (table 1). nnts compared with placebo were 1.6 and 1.7 for ibuprofen 400 mg rapid formulations for achievement of 50%maxtotpar over 6 h, and 4.1 for paracetamol 1000 mg. evolution of median visual analogue scale pain intensity (vaspi) and patients remaining in the study without re - medicating. placebo = purple ; paracetamol 1000 mg = red ; ibuprofen sodium 400 mg = light blue ; ibuprofen - poloxamer 400 mg = dark blue. numbers needed to treat (nnts) for the outcome of 50%maxtotpar compared with placebo calculated from individual patient data for different treatments each ibuprofen formulation significantly better than paracetamol, p < 0.001, z - test. 2, where bocf imputation used initial pain intensity from the time of use of additional medication, and early re - medication is seen as a series of parallel horizontal lines in consequence. most patients reported consistent results over time whether vaspi was high or low. for placebo and paracetamol,, vaspi scores fell rapidly during the first hour and were then typically maintained until later re - medication. evolution of individual patient visual analogue scale pain intensity (vaspi) scores over 6 h for all four treatments. the relationship between speed of onset of pain relief and overall pain experience was investigated by seeking an association between the slope of change in pain intensity over 060 min and % maxtotpar over 06 h. in these analyses, all patient data were examined together, irrespective of treatment. figure 3 shows the relationship, in individual patients, between the overall pain relief over 06 h measured as the percentage of maximum totpar obtained, and the speed of pain intensity reduction over the first hour, measured as the slope of the regression line fitted to vaspi data, compared with the previous analysis (moore., 2014). the degree of consistency in these two relationships is remarkable with equations to the regression line of : relationship between speed of reduction of visual analogue scale pain intensity (vaspi) over 060 min and overall pain experience measured as % maxtotpar over 06 h in individual patients, for the analysis in this paper (nl0406) and a previous analysis (moore., 2014). rapid initial fall in pain intensity in individuals (higher negative values) corresponded to high overall levels of pain relief (% maxtotpar) over 6 h. as with the previous analysis, slopes of vaspi reductions of 0.5 mm / min or more negative (roughly equivalent to a vaspi reduction of about 30 mm or more in the first hour) over the first hour resulted in a high proportion of patients with 50%maxtotpar (fig. 4). relationship between speed of reduction of visual analogue scale pain intensity (vaspi) over 060 min and proportion of patients achieving the standard individual pain relief outcome of 50%maxtotpar over 06 h, for the analysis in this paper (nl0406 ; red) and a previous analysis (moore. of the 322 patients in the study, 147 required additional analgesia within 6 h. for these, the mean initial rate of pain intensity reduction was 0.22 0.56 (sd) mm / min. for the 175 patients who did not require additional analgesia within 6 h, the mean initial rate of pain intensity reduction was 0.72 0.50 mm / min. this statistically significant difference (p < 0.001, student 's t - test) was similar to that of the previous analysis (moore., 2014). faster onset of pain relief was associated with a lesser chance of requiring additional analgesia within 6 h. where the slope was 0.5 or more negative, the chance of a patient needing additional analgesia was 3 in 10. where the slope was more positive than this, the chance was above 6 in 10. these analyses have validated a previous finding using patient level data from acute pain trials that the rate of pain intensity reduction over the first hour after dosing is strongly related to overall pain relief over 6 h, and that fast initial pain reduction is strongly associated with better overall pain relief and lesser need for additional analgesia. the level of agreement was remarkably high, and it is probably therefore safe to conclude that, for acute pain following third molar extraction, faster acting analgesic formulations provide earlier onset of pain relief, better overall pain relief, and a lesser need for additional analgesia, indicating longer lasting pain relief. the presentation of longitudinal pain intensity scores from individual patients, as performed here, is novel and supports the consistency of the results. they demonstrate that, irrespective of treatment, patients either do consistently well and have low pain intensity scores, or do badly and have high pain intensity scores. what we do not see is any of the variability predicted for responders or non - responders (dworkin., 2014). confidence about this result for third molar extraction should not be extrapolated to all acute pain conditions. it needs replication in other acute pain settings, such as other post - surgical pain, in acute pain from other causes, and independently in conditions like tension headache or migraine. post hoc analysis of trials in these setting requires the availability of individual patient data in trials that have recorded pain intensity and pain relief frequently in the first hour as well as overall. alternatively, direct comparison of fast - acting and standard formulations of analgesics at the same dose might serve to demonstrate superiority of fast - acting formulation. the active participation of the pharmaceutical industry is needed to drive knowledge in this area, as well as to investigate the impact of formulation in chronic pain (peloso, 2014). the implication that equivalent analgesic effect can be delivered at a lower dose of drug (moore., 2014) has important safety as well as efficacy implications for individuals and populations because the effect of a fast - acting formulation can be equivalent to doubling the dose (mcquay and moore, 2007 ; moore., 2014). in acute pain following third molar extraction, faster acting analgesic formulations provide earlier onset of pain relief, better overall pain relief and a lesser need for additional analgesia, indicating longer lasting pain relief. r.a.m. wrote the original draft, and all authors contributed to the development of interim and final drafts. | backgroundprevious analysis of a single data set in acute pain following third molar extraction demonstrated a strong relationship between the speed of reduction of pain intensity and overall pain relief, as well as need for additional analgesia.methodsindividual patient data analysis of a single randomized, double - blind trial of placebo, paracetamol 1000 mg, ibuprofen sodium 400 mg and ibuprofen - poloxamer 400 mg following third molar extraction. visual analogue scale pain intensity (vaspi) and other measurements were made at baseline, every 545 min, and at 60, 90, 120, 180, 240, 300 and 360 min.resultsmost patients produced consistent vaspi results over time. for placebo and paracetamol, few patients achieved low vaspi scores and maintained them. for both ibuprofen formulations, vaspi scores fell rapidly during the first hour and were then typically maintained until later re - medication. analysis of all patients showed that rapid vaspi reduction in the first hour was strongly correlated with good overall pain relief (high total pain relief over 06 h), and with lesser need for additional analgesia within 6 h. results for this analysis were in very good agreement with a previous analysis, validating the relationship between fast initial pain intensity reduction and overall good pain relief in this setting.conclusionsin acute pain following third molar extraction, faster acting analgesic formulations provide earlier onset of pain relief, better overall pain relief and a less frequent need for additional analgesia, indicating longer lasting pain relief. |
according to recent publications on cancer incidence in korea by the korea central cancer registry,1 the incidence of hepatocellular carcinoma (hcc) and its proportion among total carcinomas has decreased slightly over the past several years. however, hcc is still the fifth most common carcinoma in the korean population as of 2008. while it is true that early detection of hcc has become easier due to increasing interest in regular checkup in high risk patients and upgraded imaging techniques, considerably enlarged tumors are not infrequently encountered in clinical practice. huge hcc, which is generally defined when its greatest diameter is 10 cm or more, although there is a variation according to literatures, has a poorer prognosis than that of smaller hcc due to higher incidence of vascular invasion and more aggressive tumor biology. unfortunately, patients with huge hcc are not eligible for other treatment modalities such as liver transplantation, percutaneous ethanol injection therapy (peit), radiofrequency ablation (rfa), and while some centers tries transarterial embolization (tae), the 5-year survival rate is less than 10%. consequently, surgical resection remains the only treatment option that offers opportunities for long - term survival or complete cure. however, surgical resection of huge hccs is a great challenge to the liver surgeons, because surgery for these tumors entails prolonged operative time and has an increased risk for massive bleeding or liver failure after major hepatectomies in patients with chronic hepatitis or early stage cirrhosis ; furthermore, rapid recurrence after surgery is not infrequent. an increasing number of studies have reported favorable outcomes after hepatectomy for huge hcc,234567 but the results of surgical intervention in huge hccs still remain poor compared to that of small hccs. to maximize the effect of surgical resection through prolongation of survival duration and improving the quality of life in patients with huge hcc, adequate selection of candidates for surgery is of great importance. this study was performed to investigate the effect of hepatectomy for huge hcc by comparing the surgical outcomes of patients with huge hcc to those of patients with small hcc, and to establish surgical strategy for preoperative patient selection by identification of independent prognostic factors in patients with huge hcc. between july 1994 and february 2009, 51 patients with hcc underwent partial hepatectomy without gross residual tumor with intent of radical surgery at dankook university hospital. retrospective analysis was made by reviewing the medical records and if required, by telephonic communication. patients with hcc10 cm were classified in large (l) group, and others were classified in small (s) group. both groups were compared with respect to the following preoperative demographic and clinicopathologic characteristics, such as age, gender, presenting symptoms, etiology of hcc, child - pugh classification, platelet count, prothrombin time, serum total bilirubin, serum alpha - fetoprotein (afp), aspartate transaminase (ast), alanine transaminase (alt), serum albumin, and preoperative tae ; histopathological factors, such as gross classification of tumor, tumor size, tumor cell differentiation, presence of satellite nodules, presence of macroscopic and microscopic vascular invasion, presence of capsular invasion, ruptured tumor, radical resection, resection margin, tumor tnm stage, and presence of liver cirrhosis ; factors related to surgery, such as type of hepatectomy, operative time, intraoperative blood loss, perioperative transfusion, postoperative hospital stay, morbidity rate, and mortality rate ; surgical outcomes, such as postoperative disease - free survival and overall survival. then these factors were also analyzed to identify significant prognostic factors for patients with huge hcc. gross classification of hcc was determined according to the classification suggested by baer.8 in 1989, which is the most widely employed classification at present, well predicts the resectability of hcc, and divides hcc into 3 types ; hanging, pushing, and invasive type. modified uicc stage (4 ed) of the liver cancer study group of japan, which was adopted in the practice guidelines for management of hepatocellular carcinoma 2009 of the korean liver cancer study group, was used for tumor staging.9 surgery was determined by taking into consideration the degree of liver cirrhosis and disease progression simultaneously. candidates with hcc selected for surgery were those in whom all the tumors, regardless of tumor size and number and vascular invasion, would be included in the extent of resection estimated by liver function, and there was no extrahepatic metastasis nor tumor thrombosis in the main portal vein and inferior vena cava on preoperative imaging studies. liver function was assessed by using the child - pugh classification, serum total bilirubin, the 15-minute retention rate for indocyanine green (icg r15), presence of portal hypertension, and liver volumetry. in principle, surgery was not performed in patients with huge hcc who were in portal hypertension or child - pugh class b. type of hepatectomy was divided into major resection ; which was the resection of more than 3 segments according to the couinaud classification of liver anatomy, and minor resection ; in which less than 2 segments were removed. laparoscopic surgery was not separately classified and was analyzed together with open surgery in this study. intraoperative ultrasonography was willingly used to assess the resection plane related to tumor location, the relationship between the tumor and vessels, and the additional tumors not preoperatively detected. the method for parenchymal division was different according to the period of surgery, and consisted of the fracture technique with either kelly clamps or fingers and resection with waterjet or cusa (cavitron ultrasonic surgical aspirator). whether to use and how to perform the intraoperative temporal block of the hepatic inflow differed according to the period of surgery and the surgeon 's preference. curative resection was defined as grossly and microscopically complete removal of the tumor. according to the classification suggested by dindo.10 in 2004, postoperative complications which required surgical, endoscopic, or radiologic intervention, or were life - threatening were categorized into major complications, and the rest were into minor complications. operative mortality was defined as death before discharge from the hospital or death within 30 days after surgery. factors that showed significant statistical difference between the two groups and those factors that have frequently been described as significant in the previous literature were selected as prognostic factors for overall survival in patients with huge hcc. the factors subsequently selected were gender (male vs. female), age (60 years vs. 5 cm) hccs were similar to those of small hccs. different gross classifications of tumor have been employed between researchers ; eggel.28 classified hccs into three categories, nodular type, massive type, and the diffuse type according to tumor size and morphology in 1901, while in 1984 okuda.29 classified hccs according to pattern of tumor growth - expanding type, spreading type, and the multifocal type. kanai.30 suggested another classification in 1987, which is presently adopted by the liver cancer study group of japan, in which four types have been described according to tumor morphology ; single nodular type, single nodular type with extranodular growth, confluent multinodular type, and the infiltrative type. thus, it can be seen, that there had been no uniform consensus on the gross classification of hccs, which had led to inconsistent research and assessment. in 1989, baer.8 presented a classification - hanging type, pushing type, and the invasive type, that predicts resectability of liver tumors, including hccs, and which is widely being adopted at present. as the present study is an investigation of hepatectomy for huge hccs, the classification suggested by baer.8 was incorporated. while several studies have reported the clinical characteristics and surgical outcomes of the whole hccs with respect to gross morphology, studies limited to huge hccs have only recently emerged.1218 regardless of kind of gross classification, non - invasive huge hccs demonstrate a more favorable prognosis, and this study was also able to confirm that patients with non - invasive gross type showed longer survival, despite the limitation of small number of study cases. taniai.17 and pawlik.11 concluded that the presence of liver cirrhosis in patients with huge hccs was a significant prognostic factor, and taniai.17 further postulated that huge hccs with gross vascular invasion or multiple tumors in cirrhotic background were not appropriate for surgery. since liver cirrhosis itself is a risk factor for recurrence of hcc in the remnant liver, it is an independent prognostic factor for survival, irrespective of tumor size. in this study, despite the fact that liver cirrhosis was present in 4 (36.4%) patients with huge hccs, there was no significant difference in survival according to liver cirrhosis. this may be due to mild cirrhosis with relatively preserved liver function of child - pugh classification a. another possible reason is that survival was determined by other more significant factors related to tumor, rather than cirrhosis. it is the common expectation of all liver surgeons to maximize the surgical outcomes by appropriate selection of patients with huge hccs for surgery, based upon studies of independent prognostic factors for survival. in this context, poon.2 and shimada.21 emphasized that surgical outcomes could be maximized in patients of a single nodular huge hcc without gross vascular invasion or tumor thrombosis. to date, recent trends have concentrated on the presence of vascular invasion and multiplicity of tumors as the guideline for establishing the selection criteria for surgery in hcc patients, rather than tumor size.12 to improve the prognosis of patients with huge hcc, major hepatectomies, which resect 3 or more segments according to the couinaud 's classification of liver anatomy, are mostly performed. these hepatectomies may sometimes be combined with other resections of neighboring organs or tissues for radical surgery. while reports on such combined resections are rare, chen.19 performed combined resections in 17.2% of hcc patients, and partial resection of diaphragm was conducted in 2 (18.2%) patients of this study. although the number of cases was small and further studies are required in future, considering the results of this study that there was no significant statistical difference in overall survival after surgery in both groups, as the similar results of the previous studies on surgical treatment for huge hcc, surgical resection for huge hccs should be actively attempted, irrespective of tumor size, as long as the patient 's condition is acceptable. furthermore, to enhance surgical outcome of huge hcc and to improve quality of life in these patients, careful and appropriate selection of candidates for surgery should be based on the independent prognostic factors, such as satellite nodules. | purposein spite of the recent improved results of hepatectomy for huge hepatocellular carcinomas (hcc), the prognosis of patients with huge hccs is still poor compared to that of patients with small hccs. this study was performed to compare the results of hepatectomy between patients with huge hccs and those with small hccs, to identify the prognostic factors in patients with huge hccs, and to determine the preoperative selection criteria.methodswe retrospectively analyzed 51 patients who underwent hepatectomy, between july 1994 and february 2009 at dankook university hospital. patients with hcc10 cm were classified in large (l) group and others were classified in small (s) group. the clinicopathological features, operative procedures, and postoperative outcome were compared between both groups and various prognostic factors were investigated in group l.resultseleven patients were classified in group l. tumor size, vascular invasion, and tumor stage were higher in group l. postoperative morbidity was higher in group l, but mortality was not different between the groups. disease - free survivals were significantly lower in group l than in group s (36.4%, and 24.2% vs. 72.0%, and 44.0% for 1- and 3-year), but overall survival rates were similar in both groups (45.5%, and 15.2% in group l vs. 60.3%, and 41.3% in group s for 3- and 5-year). presence of satellite nodules was the only prognostic factor in multivariate analysis after surgery for huge hcc.conclusionregardless of tumor size, huge hccs deserve consideration for surgery in patients with preserved liver function. furthermore, the effect of surgery could be maximized with appropriate selection criteria, such as huge hcc without satellite nodules. |
sodium hydroxide, sodium lauryl sulphate, cetylpyridinium chloride, tween 80, polyethylene glycol 400, 2000, and 6000 were purchased from merck, darmstadt, germany. witepsol h15 (wh15) and witepsol w35 (ww35) were procured from daruphakhsh pharmaceutical company, tehran, iran. different types of witepsols are often used in commercial formula as main fatty bases and various grades of polyethylene glycols (peg) are used as the main water soluble bases. lipophilic and hydrophilic base suppositories were prepared using wh15, ww35, and different combination of peg 400, 2000 and 6000, respectively. suppository formulations were produced manually on a small scale by homogenizing npx (500 mg / suppository) into the melted suppository bases. accurately weighed quantities of the respective suppository bases, after mixing with peg 400, 2000 and 6000 (in the case of hydrophilic suppository according to table 1) were melted on a water bath at an appropriate temperature for each base. finally npx powder (500 mg) and/or additive were incorporated using the displacement values calculations into the melted base along with continuous stirring. the melted mass was poured into the appropriate suppository mould (3 g capacity, erweka - type 126 alc, germany) and allowed to cool at room temperature. excess base was scraped off after solidification. before molding of some hydrophilic suppositories (b, c and d combinations), it was necessary to lubricate the moulds with liquid paraffin. displacement value was calculated based on the following equation : f=(100(eg))/(gx)+1. where f is the displacement value, e is the weight of the suppository without active substances (the calibration value of the mould for the certain base), g is the weight of the suppository with active substances and x is the active substance content in percentage. composition of different formulations of hydrophilic bases the weight of five separate suppositories was checked and means weight value was calculated. melting point was measured by the collodion tube method for three suppositories (erweka ssp, german). hardness was measured by the resistance to crushing using hardness tester (erweka sbt, german). the uniformity of drug content for each base (20 suppositories) was determined by dissolving peg base suppositories in phosphate buffer at ph 7.2 in 37, after 30 min stirring, the absorbance was measured by spectrophotometry (shimadzu 120a, japan) at a wavelength of 230.2 nm after dilution. the procedure was repeated to determine the uniformity of drug content of lipophilic base suppositories using repeated extraction with phosphate buffer (ph 7.2). to ensure complete extraction of the drug from the bases, blank suppositories without the drug were prepared and subjected to the same analytical procedure to serve as the blank for spectrophotometric determination. a number of in vitro dissolution techniques for determination of the dissolution rate of drug substances from suppositories such as dialysis method and through a flow cell method were described in the literature. in this study, the in vitro dissolution rate of npx from the suppositories was examined using the basket method (apparatus i, erweka - type dt6r, germany). the temperature was maintained at 37 and the stirring rate was kept constant at 50 rpm. one thousand millilitre phosphate buffer (ph 7.2), was used as a dissolution medium, and samples of 5 ml test solution were collected manually at specified time intervals for up to 3 h and 4 h for lipophilic and hydrophilic bases, respectively. the withdrawn volume of the sample was replaced with the equal volume of fresh dissolution medium contained at the same temperature. the us pharmacopeia describes a uv spectrophotometric method for the assay of npx tablets and for a dissolution study. in this study, calibration curve data were generated using phosphate buffer (ph 7.2), in the concentration range of 0.3125 - 5 g / ml (r=0.999). for investigation of the effect of nonionic, anionic and cationic surfactants on the release rate of npx, tween 80, sodium lauryl sulphate, and cetylpyridinium chloride with concentrations of 0.5 and 1% w / w were used in formulations. different types of witepsols are often used in commercial formula as main fatty bases and various grades of polyethylene glycols (peg) are used as the main water soluble bases. lipophilic and hydrophilic base suppositories were prepared using wh15, ww35, and different combination of peg 400, 2000 and 6000, respectively. suppository formulations were produced manually on a small scale by homogenizing npx (500 mg / suppository) into the melted suppository bases. accurately weighed quantities of the respective suppository bases, after mixing with peg 400, 2000 and 6000 (in the case of hydrophilic suppository according to table 1) were melted on a water bath at an appropriate temperature for each base. finally npx powder (500 mg) and/or additive were incorporated using the displacement values calculations into the melted base along with continuous stirring. the melted mass was poured into the appropriate suppository mould (3 g capacity, erweka - type 126 alc, germany) and allowed to cool at room temperature. excess base was scraped off after solidification. before molding of some hydrophilic suppositories (b, c and d combinations), it was necessary to lubricate the moulds with liquid paraffin. displacement value was calculated based on the following equation : f=(100(eg))/(gx)+1. where f is the displacement value, e is the weight of the suppository without active substances (the calibration value of the mould for the certain base), g is the weight of the suppository with active substances and x is the active substance content in percentage. melting point was measured by the collodion tube method for three suppositories (erweka ssp, german). hardness was measured by the resistance to crushing using hardness tester (erweka sbt, german). the uniformity of drug content for each base (20 suppositories) was determined by dissolving peg base suppositories in phosphate buffer at ph 7.2 in 37, after 30 min stirring, the absorbance was measured by spectrophotometry (shimadzu 120a, japan) at a wavelength of 230.2 nm after dilution. the procedure was repeated to determine the uniformity of drug content of lipophilic base suppositories using repeated extraction with phosphate buffer (ph 7.2). to ensure complete extraction of the drug from the bases, blank suppositories without the drug were prepared and subjected to the same analytical procedure to serve as the blank for spectrophotometric determination. a number of in vitro dissolution techniques for determination of the dissolution rate of drug substances from suppositories such as dialysis method and through a flow cell method were described in the literature. in this study, the in vitro dissolution rate of npx from the suppositories was examined using the basket method (apparatus i, erweka - type dt6r, germany). the temperature was maintained at 37 and the stirring rate was kept constant at 50 rpm. one thousand millilitre phosphate buffer (ph 7.2), was used as a dissolution medium, and samples of 5 ml test solution were collected manually at specified time intervals for up to 3 h and 4 h for lipophilic and hydrophilic bases, respectively. the withdrawn volume of the sample was replaced with the equal volume of fresh dissolution medium contained at the same temperature. the us pharmacopeia describes a uv spectrophotometric method for the assay of npx tablets and for a dissolution study. in this study, the amount of dissolved npx was detected at 230.2 nm on a uv spectrophotometer. calibration curve data were generated using phosphate buffer (ph 7.2), in the concentration range of 0.3125 - 5 g / ml (r=0.999). for investigation of the effect of nonionic, anionic and cationic surfactants on the release rate of npx, tween 80, sodium lauryl sulphate, and cetylpyridinium chloride with concentrations of 0.5 and 1% w / w were used in formulations. variations in weight for the suppositories without surfactant and for the suppositories containing surfactant were in the range of 97 - 112%, which met the standard criteria (85.0 - 115.0%). the mean content of each suppository preparation was in the range of 97 - 104% and 97 - 105% of the labeled value for the suppositories without and containing surfactant, respectively (sd14.86), which is in the acceptable range (95 - 105%). mean liquification time for suppositories without surfactant was between 7.7 and 8.7 min (sd1.57). the respected range in the presence of surfactants was 4.66 - 8.66 min (sd1.15). hardness test results showed that hardness of all formulations were more than 5400 g (proper hardness). the effect of different suppository bases on the in vitro release of drugs has been described in several investigations. in general, drug release from a number of suppository bases depends on the drug solubility in the base, the chemical composition of the base and drug particle size. numerous studies have shown that drug release from suppository bases is influenced by the presence of other additives in the formulation and may result in an increase or decrease in the rate of release depending on not only the nature of the base but also the additive and its concentration considering the safety, efficacy, and compatibility with other ingredients of the formulation. results of dissolution tests in the present study showed that drug release profiles from both lipophilic bases were approximately the same (fig.. furthermore dissolution rates of five tested hydrophilic bases did not show considerable difference (fig. 1b) and in all formulations the total amount of drug released in 240 min. npx is a lipophilic drug and it has low solubility in hydrophilic bases like pegs. therefore, it is expected to have a tendency to diffuse out the hydrophilic bases into the dissolution medium. however, the findings of this study revealed slow release of npx from both fatty bases and pegs. in a study about the crystallization behavior and microstructural evolution of solid dispersions prepared by co - melting method, it was found that for the fast crystallizing drugs like npx, the interaction between the drug and the peg matrix slowed down the crystallization rate of npx and made larger drug domains. it was also found that, drugs with strong interaction in peg (e.g., npx / peg) favored the inter - lamellar incorporation of npx in peg matrix before crystallization of npx. however, it is obvious that because of long time required for drug release, none of the investigated bases is ideal for npx suppository formulation per se. obtained results of release kinetic investigation showed that the release rates of all above formulations were best described with peppas model (r>0.96, 0.50.94) which indicates that the release mechanism alters from diffusion to erosion (table 2). 3 depicts the effect of mixing of surfactants into the ww35 base on drug release. it is evident that only cetylpyridinium chloride could enhance dissolution rate of npx from ww35. in formulations containing sodium lauryl sulphate and tween 80 no significant amount of npx it is known that ww35 is a fatty base with higher hydroxyl value compared to wh15 (40 - 50 vs. 5 - 15), having higher monoglyceride contents. the different level of interaction between monoglcerides and polar group of dissolution medium may be added to surfactant addition effect leading to a different drug release rates from the investigated fatty base suppositories. on the other hand, it is possible that sodium lauryl sulphate and tween 80 cause interactions between npx and ww35 base, which could result in decreased drug release. fig. 4 compares the release profiles of hydrophilic bases and effect of addition of tween 80 and sodium lauryl sulphate into these bases. release profiles showed that addition of these surfactants had no significant effect on drug release rate. this result could be a consequence of the presence of surfactants in concentrations around the critical micelle concentration, which prolong drug release because of micellar entrapment of drug. in summary, it can be concluded that hydrophilic bases are not suitable bases for npx suppository formulation. results indicated that formulations consisted of wh15 with 0.5% w / w of tween 80 and ww35 with 0.5% of cetylpyridinium chloride were most suitable bases for npx rectal dosage form. this conclusion is more confirmed by the fact that fatty bases are better from a patient comfort perspective, since, the peg bases are known to cause rectal mucosa irritation, due to their osmotic effects. however to optimize npx release from different bases, further investigations using more enhancers and more characterizing techniques like differential scanning calorimeter, infrared spectroscopy are necessary. | the aim of this work was to develop the best formulations for naproxen suppositories. the effects of different bases and surfactants on the physicochemical characteristics of the suppositories were determined by several tests such as weight variation, melting point, assay, hardness, and release rate. all formulations met the standard criteria for tested physicochemical parameters ; weight variation (97 - 112%), content uniformity (97 - 105%), melting point (4.66 - 8.7 min) and hardness tests (> 5400 g). based on release rate studies, hydrophilic, and lipophilic bases without surfactants were not suitable bases for naproxen suppository. amongst the formulations containing surfactants only witepsol h15 with 0.5% w / w of tween 80 and witepsol w35 with 0.5% of cetylpyridinium chloride were suitable and released nearly complete drug during 30 and 60 min, respectively. this study demonstrates the effects of incorporation of known agents on the in vitro release characteristics of naproxen suppository. |
antibody - mediated rejection (abmr) is a recalcitrant entity with great impact on patient and graft survival [1, 2 ]. in the past decade, improvements in hla technology along with the recognition of the role of c4d in abmr have revolutionized the understanding of this important entity, and significant advances have occurred in the treatment of abmr [35 ]. however, the mechanism of abmr is far from being fully elucidated, and the long - term survival of these allografts is greatly reduced when compared to that of grafts without rejection or history of t cell - mediated rejection (tcmr) [2, 6 ]. microcirculation inflammation, including glomerulitis and peritubular capillaritis (ptcitis), has been recognized as a cardinal feature in the diagnosis of abmr [9, 10 ]. peritubular capillary dilation is another important side of microcirculation changes, and although it has been noticed in the abmr for years, it is far from being clearly demonstrated and its pathogenesis remains unclear. t - bet is a member of the t - box family of transcription factors regulating th1 lineage commitment. in a recent study, we found that transplant glomerulopathy, a principal form of late abmr, had a significant increase in t - bet expression in peritubular capillaries (ptc), and this expression was strongly correlated with the count of intra - ptc inflammation cells. furthermore, ptc dilation was also strongly correlated with the intra - ptc inflammation. in a previous study, we found intraglomerular inflammation correlated with in situ expression of t - bet in patients with abmr. we hypothesize that t - bet expression might be also correlated with ptc injury in early abmr. hif-1 is a transcription factor which acts as a master regulator coordinating oxygen homeostasis, and the hif system is ubiquitous which is instantaneously up - regulated upon hypoxia. in abmr, whether ptc injury can cause tissue damage via hypoxia is unknown. in this study, we explored the dilation of ptc in relation to inflammation, t - bet expression, and hypoxia. the patients were retrospectively selected from among 226 renal allograft recipients who had performed renal biopsy between june 2008 and may 2012 at jinling hospital, nanjing university school of medicine, nanjing, china. among them, 18 recipients were diagnosed as having c4d - positive acute rejection episodes according to clinical manifestations and histological features. the diagnosis was based on the following : (1) clinical evidence of acute rejection, manifested as rapid renal dysfunction and/or decrease of urine volume ; (2) c4d deposition in the ptc area ; and (3) pathologic features that met banff 's 1997 criteria for acute rejection grade i, ii, or iii. additionally, rejection episodes occurring beyond the first month after transplantation are more likely to offer a mixed histologic picture, often demonstrating acute and chronic, vascular and tubulointerstitial, pathology. this group was compared with a group of tcmr patients who were diagnosed within the same time period. since ptcitis (ptc) and glomerulitis (g) are often associated with abmr and g + ptc = 0 was confirmed to be a useful diagnostic algorithm for tcmr exclusion, we excluded all the recipients with ptcitis and glomerulitis in tcmr group. these patients were randomly matched with a group of recipients with stable graft function who received protocol biopsies as controls. in addition, we also included a tg group to compare ptc variation with abmr. we applied the following inclusion criteria : (1) biopsy confirmation of the presence of a duplication of the glomerular basement membrane on periodic acid - schiff or silver stain, and (2) 1 year of follow - up after the diagnosis. an electron microscopic evaluation was performed to exclude membrane duplication that was caused by recurrent or de novo glomerular disease. informed consent was obtained from all patients, and the human subjects committee of jinling hospital, nanjing university school of medicine approved all of the study protocols. protocol biopsies were performed between days 12 and 17 posttransplantation (so - called 2-week protocol biopsy), and diagnostic biopsies were performed upon clinical indication and according to local standard of practice. two needle biopsy cores were obtained from each renal allograft for morphologic study : one for formalin fixation and the other for quick - freezing. hematoxylin and eosin, periodic acid schiff, methenamine - silver, and masson stains were routinely used on the formalin - fixed tissue. fresh - frozen tissues were analyzed by immunofluorescence microscopy using a conventional panel of antibodies against igg, igm, iga, c3, c4, c1q, and c4d. c4d staining was routinely performed on frozen slides using an indirect immunofluorescence technique with a primary affinity - purified monoclonal antibody (mouse anti - human ; quidel, san diego, ca, usa) and an fitc - labeled affinity - purified secondary rabbit anti - mouse igg antibody (dako, denmark). positive c4d staining was defined as a bright linear stain along the capillary basement membranes that involved over half of the sampled capillaries in accordance with the 2001 banff meeting. cd4, cd8, cd68, and cd20 were regularly detected when a biopsy was performed. the intragraft expression of hif-1 and cd31 was retrospectively studied via immunohistochemistry using stored residual biopsy tissues. the antibody regimens were conducted as follows : mouse monoclonal antibody hif-1 (novus biologicals, littleton, co, usa) ; mouse monoclonal antibody against t - bet (santa cruz biotechnology, santa cruz, ca) ; and mouse monoclonal antibodies against cd4 (novocastra, newcastle upon tyne, uk), cd8 (novocastra), cd68 (dako, carpinteria, ca, usa), cd20 (dako), and cd31 (dako). the sections were reviewed by two pathologists, and the results are expressed as the total number of positive cells per glomerulus or per square millimeter in the cortex. to assess the number and size of ptcs, a cd31 costaining was used as previously described [18, 19 ]. the ptc density was calculated by counting the total number of ptcs within the confines of each of 10 random 0.25 mm fields (each of these fields was delineated by a 1 cm ocular grid that was viewed at 400 magnification), and the result is expressed as the mean per field. the diameter of these ptcs was also measured, and their mean was calculated in micrometers. to measure the ptc spaces, slides from each case were examined using an olympus ix70 inverted system microscope (olympus america, melville, ny, usa) connected to a hewlett packard computer with image - pro plus software (media cybernetics, silver springs, md, usa). the intra - ptc area is expressed as the proportion of total ptc spaces over the entire cortex field. intraglomerular capillaries were measured for their quantity (capillary counts per glomerulus) and diameter on cross - section of the glomeruli. to calculate the intracapillary inflammation cells, costaining of cd31 and inflammation markers (e.g., cd4, cd8, and cd68) were performed, and the results are expressed as the mean per ptc or glomerulus. glomerular cross - sectional area was also determined using the image - pro plus software. statistical analyses were conducted using spss (v16.0) and graphpad prism (v5) software. pairwise comparisons of variables based on proportions were done by fisher 's exact test with bonferroni correction for p value. continuous variables were presented as mean s.d. and compared using one - way analysis of variance (anova) followed by post hoc pairwise comparisons using lsd tests, or analyzed using nonparametric method if the data were not normally distributed. ordered categorical data were presented as median (25th75th percentiles) and compared using the nonparametric kruskal - wallis anova on ranks for global comparison, followed by duncan 's analysis for multiple comparisons. the level of statistical significance was set at p 0.05 (two - sided). forty - five renal allograft recipients were included in this study, including 18 cases of abmr, 13 cases of tcmr, and 14 cases of stable grafts as controls. there were no significant differences among the three groups with respect to patient age, gender, time of prior transplantation, time of biopsy, or incidence of positive panel - reactive antibody. each patient received anti - il-2 receptor monoclonal antibody for the induction of immunosuppressive therapy and was subsequently maintained on a similar immunosuppressive protocol after transplantation (table 1). a comparison of histological lesions in the three groups is given in tables 2 and 3. recipients with lymphocytes infiltration in ptcs or glomeruli were excluded from tcmr group while ptcitis was observed almost in all patients in abmr group. tubulitis was seen in all patients of acute rejection enrolled in the study, compared with 42.9% of stable graft. significantly more patients (88.9%) have intimal arteritis in the abmr group compared with either of the other two groups. we used immunohistochemistry to detect cd4, cd8, cd68, and hla - dr expression. in the abmr group, the average values for cd4, cd8, cd4/cd8 and cd68, were all similar to the tcmr group. however, when compared with stable grafts group, every kind of lymphocyte is significantly higher in both abmr and tcmr groups. labeling the endothelial cells for cd31 made it possible to calculate the number and diameters of the capillaries. figures 1(a) and 1(b) show that cd31 staining labeled the capillaries notably well. overall, abmr was correlated with an increased ptc diameter and a decreased ptc density. we compared the ptc density and capillary diameter among the three groups (figures 1(c) and 1(d), table 4). in the abmr group, the density of ptcs was significantly lower compared with the stable graft group (22.22 2.51 versus 25.64 1.82/field, resp., p < 0.001), whereas the diameter of the existing ptcs was significantly larger (2.34 0.81 versus 1.09 0.29 m, resp., p < 0.001), suggesting a rarefaction and dilation of the capillaries (figure 1(a)). however, in the tcmr group, we found no such rarefaction and dilation of ptcs as observed in the abmr group (figure 1(b)). the capillary density was similar between the tcmr group and protocol biopsies (27.23 2.49 versus 25.64 1.82/field, resp. in addition, image - pro plus revealed a significantly larger intra - ptc area in the abmr group (0.035 0.018) compared with the tcmr (0.023 0.012, p = 0.026) and stable graft (0.020 0.006, p = 0.006) groups (figure 1(e)). in addition to ptcs, capillary dilation was also observed within the glomeruli. however, there were no significant differences between the three groups in the diameter of the intraglomerular capillaries and intraglomerular capillaries ' area. in the abmr group, we detected a significant correlation between ptc dilation and microcirculation inflammation. the dilation of ptcs was strongly correlated with the quantity of intra - ptc infiltrating cells (r = 0.664, p = 0.004), as shown in figure 2(a). in contrast, as shown in tcmr group (figure 1(b)) and sg group (not shown), capillary dilation was rare, and it was also found to mostly coexist with luminal inflammatory cells. we also calculated the ratio of the total intra - ptc area within the cortex to the area of the entire cortex in abmr group, and found that this ratio was also strongly correlated with intra - ptc cell counting (r = 0.578, p = 0.006) (figure 2(b)). a significant increase in t - bet expression was detected in the ptcs, and the majority of t - bet+ cells were typically located within the capillary lumen. intra - ptc t - bet expression was also detected in the tcmr group ; however, its expression was much lower than that in the abmr group (1.34 0.56 versus 0.15 0.22/capillary, p < 0.001) (figure 3(a)). in the abmr group, the t - bet expression correlated well with the quantity of infiltrating cells (r = 0.768, p < 0.001) (figure 3(b)) moreover, since ptc dilation is correlated with microcirculation inflammation as above mentioned, we also find a good correlation between the intra - ptc expression of t - bet and the ptc diameter (r = 0.491, p = 0.038) (figure 3(c)). similar to the findings in experimental acute renal failure, the strongest and most abundant signals were detected in the medulla, and signal intensity and abundance increased from the outer medulla to the deep papilla. moreover, cortical tubular immunostaining for hif-1 was positive and even signals can be detected in some glomeruli. according to the results of double staining of cd31 and hif-1, capillary dilation and hif-1 expression occurred somewhat in parallel both in abmr and tcmr. it is noteworthy that, as shown in figure 4, ptc dilation could be observed where hif-1 was up - regulated while hif-1 expression was not necessary for capillary dilation. in order to compare the capillary variation between abmr and tg, 32 cases of tg were selected as control group (the baseline characteristics previously described in). we found that the ptc loss is similar in two groups, while the ptc diameters were significantly larger in tg group (2.34 0.81 versus 6.35 2.16, p < 0.001), leading to much more severe ptc dilation. moreover, the diameters of capillary loops within glomerulus were also increased, and the intraglomerulus area was also significantly increased and showed an enlarged glomerulus. anyway, the sizes of glomerulus as well as capillary loops within glomerulus were similar between abmr and tcmr cases. we confirmed that ptc dilation is correlated with microcirculation inflammation, and ptc inflammation is in turn strongly correlated with in situ t - bet expression. these data suggest that t - bet plays an important role in the pathogenesis of microcirculation injury in abmr. these findings shed a new light on the pathogenesis of abmr. though it is the general belief that abmr is associated with peritubular capillary dilation [2325 ], it is not until this study that dilation has been demonstrated clearly in this setting. our study clearly demonstrates that the ptc diameter and intra - ptc area were significantly increased in the abmr. shortly after the development of abmr, the diameters of ptcs are increased to twice of the control group, which leads to significantly increased intra - ptc area. nevertheless, the dilation is diffused and can be seen in most of the ptcs, no matter in cortex or medulla. this variation can be regarded as a feature of abmr as it can not be observed in tcmr. as the diameter of ptc is easy to measure and evaluate throughout the whole specimen, it may be applicable to biopsy interpretation and may be of good diagnostic utility for abmr. moreover, our data also revealed a rarefaction of ptc during abmr. to our knowledge, this is the first report on ptc loss when abmr occurs. although the diameter of each ptc is increased, the count of ptc is significantly decreased (figure 1, table 4) which can be observed in early biopsies immediately after the abmr occurs. the quick rarefaction of ptc seems to be unique in early abmr as it is not observed in tcmr or tg. ptc loss has been demonstrated in models involving ageing, cyclosporine, angiotensin ii infusion, chronic catecholamine infusion, glomerulonephritis, radiation - induced injury, and potassium depletion, which mostly are involved in a local alteration in the balance between angiogenic and angiostatic factors [27, 28 ]. however, the quick ptc rarefaction during the rejection suggests a dramatic process, which is most probably caused by direct damage to the capillary wall. ptc loss after renal transplantation usually associates with increased interstitial fibrosis / tubular atrophy, and predicts reduced renal function ; thus the dramatic ptc loss during abmr is an important process to graft dysfunction. the pathogenesis of ptc variation during abmr is not clear ; however, as the dilation can be observed quickly when abmr occurs, it should be a response to the antibody activity. our data revealed that the ptc dilation is strongly correlated with intra - ptc inflammation. although interstitial inflammation might cause quick vessel dilations in the early phase, the ptc dilation is more likely to be related to intra - ptc inflammation, as the dilation can not be observed in tcmr cases, which have more severe interstitial inflammation. moreover, the dilation of the ptc lumen was strongly correlated with the degree of microcirculation inflammation, whereas ptc dilation was mostly observed in areas with ptc inflammation. this phenomenon can be seen in tg as well and it is quite likely that the capillary dilation is caused by in situ inflammation. those data suggest a central role of inflammation in the development of microcirculating variation during abmr. furthermore, we find that intra - ptc inflammation is strongly correlated with in situ expression of t - bet. this correlation is unique to the abmr group, as the tcmr and stable graft groups, which rarely have microcirculation inflammation, only rarely exhibited t - bet expression in the ptc areas. t - bet is a member of the t - box family of transcription factors and is a key determinant of t - helper cell differentiation into th1. our previous study showed that t - bet expression is correlated with glomerulitis in abmr, further study revealed that both glomerulitis and ptcitis are correlated with t - bet expression in tg, which is a chronic form of abmr. this study proved that both kinds of microcirculating inflammation, ptcitis and glomerulitis, are correlated with t - bet expression, no matter in acute abmr or chronic type. higher t - bet expression is correlated with more ptc inflammation, and in turn is correlated with larger ptc diameters. this study suggests that t - bet pathway might be a potential target in the management of abmr. as capillary damage may lead to local hypoxia, it is possible that hypoxia is involved in the pathogenesis of abmr. at experimental level, there has also been described an intense dilation of the peritubular capillaries in chronic allograft dysfunction [26, 30 ], and the morphological changes were thought to be related to chronic ischemia. we hypothesized that hypoxia takes part in the development of ptc dilation. since hif allows for hypoxia detection at a single cell resolution [3133 ], and as previously described in vivo, renal hif-1 immunostaining was almost exclusively found in tubular segments, hif-1 immunostaining had been performed in this cohort. somewhat surprising, ptc dilation could be observed where hif-1 was up - regulated while some dilation had nothing to do with hif-1 expression, which suggests that hypoxia is not necessary for ptc dilation in abmr. obviously the ptc dilation in abmr has a pathway that is different to chronic allograft dysfunction. current data also showed that the microcirculating variation in abmr is similar to its chronic pattern, tg. both abmr and tg have significant ptc dilation, which is correlated with ptc inflammation, and in turn with in situ t - bet expression. it suggests that both acute and chronic patterns of humeral rejection share a common mechanism of ptc variation ; t - bet expression might account for the development of microcirculation inflammation. however, in tg, the ptc dilation is much more severe than in abmr, and there are enlarged glomerulus loops and glomerulus size, which are not shown in the abmr group. the diagnosis of abmr has largely depended on the c4d staining in the past decade. however, in spite of high specificity, c4d is lacking sensitivity, and many cases of rejection with anti - hla are c4d negative [7, 3537 ]. the variation of ptc during abmr, including ptc dilation and rarefaction, differs to tcmr and may be used in the diagnosis of abmr. moreover, our previous study had reported that the predominance of t - bet over gata-3 may distinct abmr from tcmr ; the current data even showed that the predominance of t - bet expression in ptc is a feature of abmr and can also be regarded as a diagnosis marker. in summary, this study shows that abmr is associated with ptc dilation and the latter is correlated with microcirculation inflammation, and microcirculation inflammation is strongly correlated with in situ t - bet expression. these results suggest that inflammation may take part in the pathogenesis of ptc dilation, and they warrant further investigation. | antibody - mediated rejection (abmr) remains one of the major causes of graft loss after renal transplantation. it is dominated by endothelial damage in microcirculation. clarifying the mechanism of microcirculating damage is obviously a key step to understand the pathogenesis of abmr. here we characterized capillary variation in abmr and its possible mechanisms. compared with t cell - mediated rejection and stable grafts, there was a significant dilation and rarefaction in peritubular capillaries (ptcs) of the abmr group ; image - pro plus revealed a significantly larger intra - ptc area. interestingly, the dilation of ptcs was strongly correlated with the intra - ptc cell counting. moreover, peritubular capillary inflammation is correlated with in situ t - bet expression, and there was a good correlation between the intra - ptc expression of t - bet and the ptc diameter. hif-1 up - regulation could be observed in abmr but it was not necessary for capillary dilation. in general, abmr is characterized with early capillary dilation and rarefaction ; our data confirmed that the dilation is strongly correlated with intracapillary inflammation, which in turn is correlated with in situ t - bet expression. t - bet plays an important role in the development of microcirculating injury, and thus it is a potential target for the treatment of abmr. |
a 48-year - old female with complaints of prominence of left forehead and asymmetric left - sided hemifacial atrophy, alopecia, asymmetric thinning of left lower leg, without any history of trauma, presented to the dermatology department of our hospital. she also had slight skin pigmentation over the left eyebrow and the left palpebral conjunctiva. her symptoms started around 6 years ago with mild hemifacial atrophy on the left forehead, and progressed slowly with alopecia. the asymmetric thinning of her left lower leg had started around one year before. no facial motor and sensory deficits were noted physically with normal tongue movements, without any enophthalmus or ptosis of the left upper eyelid. the left - sided facial and forehead asymmetry was detected mainly due to subcutaneous fatty loss with prominent bony ridges on the left forehead and left maxillary region. intra and extraoral examination revealed normal physical yields without any atrophy of chewing muscles and/or tongue. paranasal sinus x - ray examination also presented a normal frontal and maxillary sinus on the left side. there was asymmetric prominent subcutaneous fat and tissue loss in the left cruris region on inspection (figures 1a & 1b, figures 2a & 2b). she had several normal brain ct scans and electromyographic approaches during the first 6 years. following consultation, she was admitted as inpatient admission to the surgery department, then referred to egerad private medical imaging center for brain mri. the brain mri revealed asymmetric, prominent fatty loss in the dural and galeal parts of the left frontal region. no cortex or deep white matter involvement, including white matter hyperintensities - cortical atrophy was indicated (figures 3a - c). an image showing : a) general physical appearance of the patient ; and b) left sided facial hemiatrophy and left forehead prominence with bony ridges. an image of the patient showing ; a) asymmetric fatty loss and thinning of left lower leg ; and b) non - homogenous alopecia. an image showing : a) fatty loss of the scalp and galeal subcutaneous fatty tissue of the left frontal area, seen in t2 weighted (w) axial image ; b) tissue loss of the scalp of the left frontal region, presented in t2w coronal image ; and c) calvarial fat and soft tissue loss in left frontal side, shown in t1w axial image. laboratory results for a complete blood count and biochemistry revealed hemoglobin : 13.58 [normal value (nv) : 12 - 16 g / dl ], hematocrit : 41.3 [nv : 37 - 50 g / dl ], mean corpuscular volume : 90.2 [nv : 80 - 99 g / dl ], mean corpuscular hemoglobin 28.5 [nv : 33 - 37 g / dl ], white blood count (4700/lt [nv : 4800 - 10800/lt ], c - reactive protein 2.23 mg / dl [nv : 0 - 10 mg / dl ], sedimentation 38/hour [nv : 0 - 20/hour ], and mild thrombocytosis (430.000 g / dl) were indicated. prothrombin time, and activated partial thromboplastin time were normal with negative hepatitis b surface antigen, anti hepatitis b surface antibodies, hepatitis c virus, and human immunodeficiency virus. urine analysis was normal, csf analysis showed mildly elevated protein (16 mg / dl ; nv : 0 - 10 mg / dl), normal glucose and no pleocytosis. in the differential diagnosis, scleroderma or connective tissue disorders were considered, however, she had negative hla - b27 with normal glucose and electrolyte levels in serum blood. based on the clinical findings and history, a diagnosis of parry - romberg syndrome was made. alloplastic aesthetic surgery with autologous fat injection to the atrophied left forehead was suggested to our patient by a plastic surgeon, but due to financial difficulties, she declined any surgical management. the treatment plan excluding the surgical management of our patient was physical and clinical follow - up in our research hospital ; the follow up included physical rehabilitation including sports and exercise, use of a chin mask, and/or analgesic use. it is characterized by a slow progressive unilateral atrophy of the face, including skin - soft tissue, muscles and bones, which were present in our case.1 - 5,9 the extension of the atrophy is frequently limited to one side of the face, and ipsilateral facial involvement is rare (10 - 20% of cases were reported bilaterally).2 - 5,8,9 the early hemifacial asymmetry progresses over years to extreme loss of subcutaneous tissues, resulting in a sunken and wrinkled face on one side.5,8,9 the incidence of prs is not well - established, the average onset of disease is around 10 years of age ; however, onset can be found as late as 40 - 50 years in some patients;8 - 10 our patient was a 48-year - old female. the severity of prs with late onset, including facial atrophy - asymmetry, neurological and ocular findings and so forth, may be less than prs in younger ages, probably due to the complete development of craniofacial and nervous structures.5,8 - 10 to date around 150 cases of prs had been reported in the literature.1 - 10 this syndrome is more prevalent in females without any precise geographical distribution. it mostly manifests on the left side of the face, and can occur in any members of the same family.1 - 5,8 characteristically, hemifacial atrophy is usually self - limited and stabilizes in most of the patients after 5 - 10 years duration.8 - 10 guerrerosantos classified prs into 4 types : type 1 and 2 involve a decrease in the facial soft tissue, type 3 and 4 also involve thinning of soft tissue, as well as bone and cartilage atrophy. type 1 is the mildest form, and type 4 is the most severe, and most serious form.10 our patient is considered to be type 3 due to thinning of the subcutaneous soft tissue in the left face and cruris region. electromyography, blink reflex, and trigeminal evoked potential abnormalities may indicate false implications through the brain stem as the etiology of the disease, and hyperactivity of the brain stem sympathetic centers, which may be caused by autoimmune processes, may lead to the cutaneous and subcutaneous atrophy in prs.1,5,10 clinically, dry and hyperpigmented skin may be the first sign, also present in our patient, and in some cases, a demarcated line between normal and abnormal skin can be seen, known as the en coup de sabre that means cut of the sword, can also be shown in a localized form of scleroderma.1 - 3 ocular involvement is common, such as progressive enophthalmos, pseudoptosis, uveitis, pupillary disturbances, heterochromia, and restrictive strabismus. the most frequent opthalmic manifestation is enophthalmos due to loss of fat around the affected orbit, which was not observed in our patient.2 - 5,9 alopecia and thinning of the ear due to fat atrophy on the involved side are also frequent signs ; alopecia was present in our case.1,9,10 neurological complications of prs are trigeminal neuralgia, facial paresthesia, migraine, and seizures. epilepsy, especially contralateral epilepsy may also occur occasionally,5,10 however, except for headache, other findings were not found in our patient. atrophy of the tongue on the affected side, deviation of the mouth and nose to the affected side, atrophy of the upper lip leading to an exposition of teeth, small teeth with short roots on the involved side are the other important features of prs, which were absent in our patient.8 - 10 although the pathophysiology of this disease is not well known, some authors have proposed that the primary cause is neurologic disturbance of fat metabolism, resulting from a trophic malformation of the cervical sympathetic nervous system.5,9,10 parry - romberg syndrome may have strong correlation with autoimmune disorders, endocrine, and hereditary disturbances, scleroderma, and rasmussen encephalitis.1,5,10 neuroimaging findings of prs may be intracranial calcifications, cerebral and cerebellar atrophy, deep white matter lesions, encephalomalacia, heterogeneous meningeal enhancement and so forth.5,10 the brain mr imaging of our patient showed asymmetric, prominent fatty loss in the dura and galea of the left frontal side without any deep white matter and cortical involvement of the cerebrum and cerebellum. there is no curative therapy for prs and treatment options are limited, symptomatic treatment including pain relief, anticonvulsant drugs, and reconstructive surgery are the main management modalities ; steroid and immunosuppressive therapy have also been tried in some cases.3,10 surgical treatment is usually based on the reposition of fat on the atrophied side, autogenous fat and cartilage grafts, silicon injections and prostheses, bovine collagen and inorganic implants are the alternatives of aesthetic surgical correction of fatty atrophy.3,9,10 our case had left face asymmetry with slight left facial hemiatrophy, a prominent left forehead with bony ridges due to subcutaneous fatty tissue loss, slight pigmentation over the left eyebrow and conjunctiva, and alopecia, which are the classical features of prs in previous reports.1 - 10 she had normal neurologic and ocular findings, with normal psychomotor functions, and brain mri revealed an asymmetric fatty loss in the left frontal side without any cortex or deep white matter involvement. she also had subcutaneous fatty loss in the left lower leg, resulting in an asymmetric thinning of the left cruris, which on literature review, is a previously unknown feature of prs.1 - 10 due to the non - existence of a precise cure, affected patients should be managed by a multidisciplinary team of physicians, surgeons, dentists, and psychologists to alleviate all possible difficulties related to prs. fat and dermis grafts are the most appropriate for type 1 and 2 prs, whereas panfacial volumization with autologous fat - cartilage and bone graft - galeal flap injection is an excellent tool for replacing volume and restoring contour to the aging face in type 3 and 4 prs.3,10 alloplastic aesthetic surgical implantation with lipoinjection was suggested for our patient, but she declined. in conclusion, prs is a rare disease that manifests with facial hemiatrophy and subcutaneous fatty loss on the affected side ; its pathophysiology remains unknown. no definite curative treatment exists for prs, and the most widely available treatment approach is based upon plastic aesthetic surgery. | progressive hemifacial atrophy also known as parry - romberg syndrome is an acquired, slowly progressive disorder, occurring more in women, primarily affecting one side of the face, mainly characterized by unilateral atrophy, and loss of skin and subcutaneous tissues of face, muscles, and bones. ocular and neurologic involvements are common. the possible etiology is unclear without any known cure. we report a rare case of parry - romberg syndrome with classical features. the clinical features, radiological imaging findings, differential diagnosis, and available treatment options are discussed in this report. |
an anastomotic leak can be a very difficult problem to treat in an inflamed, infected area, especially in a physically compromised patient. methods of treatment have included primary repair, esophageal resection or diversion, or both, and various types of patches including pleural, mucomuscular, and vicryl mesh. we report here the use of decellularized human skin for patch repair of an anastomotic esophageal leak. a 60-year - old male with symptomatic, biopsy proven, esophageal cancer underwent a laparoscopic - assisted distal esophagectomy with right thoracotomy (ivor lewis esophagectomy). the patient presented from an outside hospital with a history of progressive solid and later liquid dysphagia. the laparoscopic gastric mobilization was performed via a 10-mm umbilical port, two 10-mm ports in the right lateral abdomen, two 10-mm ports in the left lateral abdomen, and a 5-mm subxiphoid port for liver retraction. the distal esophagus was also mobilized well into the mediastinum. through a right sixth intercostal minithoracotomy incision, the proximal third of the stomach was transected laparoscopically by using a laparoscopic gia stapler. the proximal stomach was pulled into the chest, and the esophagus was transected about 2 cm proximal to the gross tumor margin and passed to the pathology department for margin evaluation. due to a microscopically positive proximal margin, an anastomosis was created in an end - to - side fashion between the distal esophagus and the mobilized distal stomach. the anastomosis was fashioned in a single - layer technique using 3 0 interrupted full - thickness silk sutures. a nasogastric (ng) tube was left in the stomach ; a feeding jejunostomy tube was placed laparoscopically in the proximal jejunum. the right chest was closed with a straight 28f and an angled 28f chest tube in place. on postoperative day # 3 the ng tube was removed, and the patient was started on a clear liquid diet the following day. on pod # 4, one chest tube was removed and due to minimal output the second chest tube was removed on pod # 8. on pod # 9, the patient was found to have a new pleural effusion that was drained. the following day, pod # 10, the patient complained of acute onset of back pain. a ct scan with oral contrast was performed and showed a leak into the right chest. the leak was not immediately apparent, and a gastroscope was passed into the proximal esophagus and used to evaluate the leak. the majority of the anastomosis appeared intact, with a small 2-mm dehiscence at the lateral anastomotic edge. due to the friable nature of the surrounding tissue alloderm was used as a patch onlay over the site of the breakdown. a piece 2x3 cm with a thickness of 0.031 inches to 0.077 inches was fashioned. it was sewn into place with four 3 0 prolene simple interrupted corner sutures, covering the point of leak. one more, simple interrupted suture was placed between the corner sutures to reinforce the patch. the patient was extubated in the recovery room and admitted to the intensive care unit. on postoperative day (pod) # 1, jejunal tube feeds were initiated. on pod # 3, chest x - rays were obtained daily and showed no effusion. on pod # 6 after his second surgery, the patient underwent a gastrografin swallow that showed no sign of leakage. on the same day, he was started on a liquid diet. on pod # 7 and # 8, five days later, the patient was discharged to a rehabilitation hospital, tolerating a full liquid diet with nightly jejunal tube feeds. one month after surgery, the patient was readmitted to the hospital due to nausea and vomiting. he underwent an esophagogastroduodenoscopy, which showed a small ulceration at the anastomosis site, but no sign of stenosis. currently, the patient is at home, tolerating an oral diet with supplemental tube feedings and receiving ongoing oncological treatment for his esophageal cancer. one of the most feared complications of esophageal reconstruction surgery is a leak at the anastomotic site. alanezi reported a 7.5% rate of esophageal anastomotic leak, with an associated 35% mortality. junemann - ramirez noted an anastomotic leak rate of 5.1%, with 30- day mortality significantly higher in the leak group vs. nonleak group (35.7% vs 4.2%). treatment of esophageal leaks depends on time to diagnosis of the perforation and whether the perforation is walled - off or not. a walled - off perforation with minimal symptoms and no sepsis may be treated conservatively. this includes nothing - by - mouth status, antibiotics, and an alternative feeding source. the reinforcement can be gastric fundus as a partial or total fundoplication, a pleural or muscle flap, or as we are suggesting alloderm mesh. if there is free perforation and early diagnosis, this primary repair and reinforcement is also recommended. studies report that this technique may also be used for free perforation after a delay of 48 hours to 72 hours. other techniques used when a diagnosis of perforation has been delayed include resection, cervical esophagostomy, gastrostomy, and jejunostomy. these include reconstruction of complicated abdominal wound defects, reconstruction of dural defects, pharyngeal reconstruction, vaginal repairs, and multiple plastics applications. isch successfully applied an alloderm patch repair in a canine cervical esophageal leak model. in their study, no occurrence of wound infection, sepsis, or anastomotic leak was noted with the use of this material. furthermore, on evaluation by barium esophagram, no leak, stricture, or diverticulum formation was found. our hypothesis is that alloderm provides the properties needed for a successful esophageal leak repair. alloderm provides an acellular matrix for regeneration and healing even in a contaminated field. due to its properties it also has been shown to minimize the inflammatory reaction associated with prosthetic graft repair. we have successfully applied alloderm to patch an esophageal intrathoracic leak in a patient with esophageal carcinoma without any resulting leak or stenosis. to our knowledge, this is the first report of successful alloderm use in an esophageal anastomotic leak. we feel that alloderm is currently the optimal option in complicated esophageal repairs, adding to the surgeon 's armamentarium of repair techniques. | multiple surgical techniques have been described for repair of esophageal leaks. none of the repairs are optimal, and the morbidity and mortality of an esophageal leak remains high. to our knowledge, this is the first case report of use of alloderm (lifecell corp, brachburg, nj) to successfully repair a thoracic anastomotic esophageal leak. |
four papers concentrated on the effects of innovative therapeutic strategies on the macrocirculation and microcirculation in animal models of sepsis or hemorrhage, thereby focusing on arginine vasopressin (avp), erythropoietin (epo) and inducible nitric oxide synthase (inos). three further articles concentrated on metabolic homeostasis, acid base status, energy expenditure and gastrointestinal motility ; and the two final articles report studies concerning basic supportive measures (that is, fluid resuscitation and the control of shivering during core temperature reduction). low - dose avp infusion is increasingly used to treat sepsis - related vasodilatation and to decrease vasopressor requirements in patients with refractory septic shock. the encouraging effects of low - dose avp infusion such as restored vascular tone, increased blood pressure, reduced catecholamine needs, and improved renal function reported in animal studies however, are counterbalanced by data on adverse events related to a markedly reduced systemic blood flow and oxygen transport. furthermore, despite a reduced mortality in a subgroup of patients with less severe septic shock, low - dose avp did not improve the outcome in the recently published vasopressin versus norepinephrine in septic shock trial (vasst) when compared with the standard - treatment control group receiving noradrenaline. any safety issue possibly limiting the clinical use of avp is therefore a matter of concern. in this context, the effect on hepatosplanchnic blood flow assumes particular importance given its possibly crucial role for both the initiation and aggravation of sepsis. krejci and colleagues investigated the effect of low - dose avp on the microcirculation and regional blood flow during early, short - term, normotensive and normodynamic fecal peritonitis - induced porcine septicemia, a study complementary to their simultaneous report on the effects on the gastrointestinal circulation. avp (0.06 iu / kg / hour) reduced the liver blood flow, mainly due to a decrease in portal venous flow, and reduced microcirculatory perfusion in the pancreas. renal macrocirculatory and microcirculatory perfusion decreased as well, while the urine output remained unaffected most probably as a result of the increased blood pressure. while the rise in hepatic arterial flow most likely reflects the well - maintained hepatic arterial buffer response already shown for terlipressin during long - term, hyperdynamic porcine endotoxemia, the overall data reported by krejci and colleagues are in contrast with previous reports in well - resuscitated shock models characterized by a sustained increase in cardiac output. based upon their findings an accompanying commentary underscored the crucial importance of the experimental design, concluding it mandatory to transfer experimental data on avp infusion in shock models into the clinical scenario that is, the duration, the underlying hemodynamic status, the necessity of adequate fluid resuscitation and the strict adherence to low - dose infusion regimens demonstrated to be safe by other research. in addition to septic shock, uncontrolled hemorrhagic shock is a primary focus of the research on avp. the main objective during resuscitation from severe hemorrhage comprises increasing oxygen delivery to vital organs without concomitant augmentation of bleeding. current guidelines for hemodynamic stabilization of critically injured patients with uncontrolled hemorrhage recommend fluid administration, while there is an ongoing debate on the time involved and the volume and type of fluid solutions used. vasopressors also allow restoration of blood pressure, while limiting the amount of volume infused, and several experimental studies and individual case reports have shown promising effects of avp under these circumstances [11 - 13 ]. the putative advantage of avp over fluid resuscitation alone in this context relates to its potent vasoconstrictive properties, resulting in increased coronary and cerebral perfusion pressure as well as a redistribution of cardiac output to these organs at the expense of the skeletal muscle, cutaneous and splanchnic vascular beds, thus consecutively increasing vital organ perfusion and reducing further blood loss, even in severe acidosis and distinct vasoplegia. to clarify the possible impact of avp on hemodynamics and short - term survival during potentially lethal hemorrhage, stadlbauer and colleagues compared avp infusion with fluid resuscitation and a saline placebo during abdominal vascular injury and subsequent hemorrhagic shock in swine. when the mean arterial blood pressure decreased below 20 mmhg due to a blood loss of 2 l, either avp (bolus, 0.4 iu / kg ; following infusion, 0.08 iu / kg / min) or fluid resuscitation (25 ml / kg lactated ringer 's solution and 25 ml / kg gelatine solution) was initiated. all untreated control animals died within 15 minutes. while initially the mean arterial blood pressure increased with both treatments, it subsequently decreased more rapidly in the fluid resuscitation group due to a higher total blood loss, which resulted in death in all but one animals in that group within the first 30 minutes before surgical intervention and supplementary fluid therapy the authors therefore persuasively repeated their previous results in an uncontrolled hemorrhagic shock model due to liver trauma. nevertheless, as the authors correctly mention, data are lacking on regional blood flow, visceral organ function and integrity, and the authors also highlight the ambiguity of their results with respect to neurological function or long - term survival. consequently, the advantage of the avp treatment alone during uncontrolled hemorrhage has to be investigated in comparison with a fluid vasopressor combination in particular because another experimental study in porcine liver damage - induced uncontrolled hemorrhage did not show any superior effect of avp compared with noradrenaline when combined with small - volume resuscitation. based upon its stimulating effect on erythroid progenitors within the bone marrow, epo is predominantly used to treat anemia in various clinical settings and thus to reduce the need for blood transfusions. in addition, despite unchanged transfusion requirements, a recently published study on the use of epo in critical illness showed an unexpected mortality benefit, which was referred to protective nonhemopoietic properties. in fact, epo a type i cytokine with antiapoptotic functions was demonstrated to reduce systemic inflammation and/or organ injury in several preclinical shock models. in particular, epo is protective for many organs after ischemia / reperfusion, among which the brain, the heart and the kidney seem to be the most promising targets. such a protective effect has already been shown in patients after acute ischemic stroke, and was recently confirmed for the kidney and the spinal cord in a clinically relevant model of porcine thoracic aortic occlusion mimicking surgery for thoracic aortic aneurysm. in this model epo also reduced the vasopressor requirements needed to maintain blood pressure during the early reperfusion period, thus also suggesting a beneficial effect on vasoconstrictor responsiveness. in the context of vasoconstrictor properties of epo due to direct effects on smooth muscle cells and increased circulating levels of endothelin-1, kao and colleagues investigated the effect of epo (400 u / kg ; that is, doses similar to those used to reduce transfusion needs in critically ill patients) on skeletal muscle capillary perfusion and tissue oxygenation 18 hours after induction of murine sepsis with cecal ligation and puncture. while epo did not affect the systemic hemodynamics or lactate levels, the initially impaired microcirculatory perfusion and increased bioenergetic impairment assessed by functional capillary density and by nicotinamide adenine dinucleotide fluorescence using intravital microscopy of the extensor digitorum longus muscle was restored to the levels of the sham control mice. six hours after drug administration, the epo - treated septic mice still presented with increased capillary perfusion, which coincided with significantly lower skeletal muscle tissue nicotinamide adenine dinucleotide fluorescence. the authors concluded that epo improved mitochondrial oxidative phosphorylation and pyruvate metabolism as a result of attenuated tissue hypoxia due to a rapid normalization in the perfused capillary density. nevertheless, other possible effects of epo contributing to preserved mitochondrial integrity and subsequent enhanced oxidative phosphorylation that is, prevention of mitochondrial membrane depolarization and cytochrome c release through antiapoptotic mechanisms may also assume importance in this context. nitric oxide (no) is an important regulator of microvascular homeostasis by modifying the vascular tone, leukocyte and platelet adhesion to endothelial cells, and capillary leakage. its overproduction due to activation of the cytokine - inos as a response to infection, however, is thought to assume major importance in sepsis - related microcirculatory failure, contributing to organ dysfunction and failure in severe sepsis. several nitric oxide synthase inhibitors have consequently been developed, but clinical investigations failed probably based upon the attenuation of the protective effects of constitutive no formation due to the use of a nonselective nitric oxide synthase inhibitor. moreover, despite the well - established deleterious effects of its excess release, no is known to block leukocyte adhesion and to scavenge reactive oxygen species, and thus to be an important protector for the endothelium against oxidative stress and subsequent damage. in line with this rationale, preclinical investigations using selective inos inhibitors or inos - deficient mice yielded promising results. using a well - established and clinically relevant murine model of resuscitated hyperdynamic cecal ligation and puncture - induced sepsis, hollenberg and colleagues investigated the impact of both genetic deletion (inos) and selective pharmacological inhibition of inos (1400 w) on leukocyte dynamics and on microvascular permeability. rolling and adhesion of labeled leukocytes and leakage of fitc - conjugated albumin was assessed by intravital fluorescence microscopy in the cremaster muscle 15 to 20 hours after sepsis induction. both genetic deficiency and pharmacological inhibition of inos attenuated vascular leakage, while the sepsis - related aggravation of leukocyte dynamics could not be prevented. the authors therefore concluded that inos activation seems to play an essential role in the modulation of vascular permeability, but that this regulation occurs independently of its action on leukocytes. consequently, to sustain the protective effects of the constitutive no formation, hollenberg and colleagues suggest selective inos inhibition rather than nonselective nitric oxide synthase blockade although the impact of this approach remains to be elucidated in the proper clinical studies. metabolic acidosis is common during hemorrhagic shock, and hyperlactatemia is conventionally considered the main cause. respecting the physicochemical fundamentals of the acid base balance (that is, the dissociation equilibrium, the necessity of electrical neutrality, and the principle of mass conservation), bruegger and colleagues in a highly standardized canine model of hemorrhagic shock concentrated on the profile of unmeasured anions in relation to other acid base parameters in order to characterize the potential contributors to the unmeasured anions. in addition to the traditional parameters used to identify the presence of unmeasured anions (for example, the anion gap and the strong ion difference), the strong ion gap was calculated. the strong ion gap is defined as the difference between the apparent strong ion difference, derived from measuring strong cations and anions and summing their charges, and the effective strong ion difference, which is estimated from the carbon dioxide partial pressure and the concentrations of the weak acids (for example, albumin, phosphate). in the current study, both the anion gap and the strong ion gap increased after the induction of shock, which was associated with significantly increased lactate, citrate, acetate and urate serum levels, measured with the help of capillary electrophoresis. while not detectable at baseline, fumarate and -ketoglutarate were both found in all animals from the induction of shock until the end of the experiment. the authors concluded that mitochondrial dysfunction may be responsible for their finding, since acetate, coupled with coenzyme a, is normally consumed during krebs cycle in the mitochondria, and citrate, fumarate and -ketoglutarate represent intermediate substrates of this cycle. although direct proof of mitochondrial dysfunction and its correlation with the strong ion gap is not yet available, bruegger and colleagues ' findings support the concept of early mitochondrial dysfunction and energy debt during hemorrhagic shock : in fact, in critically ill patients, the strong ion gap was a strong predictor of mortality if it was the major source of metabolic acidosis. consequently, strategies decreasing cellular energy expenditure by modulating mitochondrial respiration, such as cooling down or hydrogen sulfide - induced suspended animation, may prove beneficial in hemorrhagic shock. although the impact of fluid resuscitation on the acid base status must not be overlooked, the strong ion gap might present an attractive bedside parameter in critical illness resulting from hemorrhagic shock, based on the assumption that it is indeed directly related to the degree of mitochondrial dysfunction in hemorrhagic shock. several factors seem to be associated with feeding intolerance and dys - motility of the gastrointestinal tract, such as admission diagnosis and ongoing therapy with sedatives and/or cate - cholamines, but although our understanding has markedly improved in recent years, the precise mechanisms remain unclear. it is well established that plasma concentrations of cholecystokinin and peptide yy plasma levels are elevated in fasted states as well as in anorexia nervosa or malnutrition. nguyen and colleagues studied the relation between peptide yy and cholecystokinin concentrations and gastric emptying in 39 mechanically ventilated intensive care unit patients, two - thirds of whom presented with delayed gastric emptying as assessed with the c - octanoate breath test. plasma cholecystokinin and peptide yy concentrations were significantly higher in these latter patients both at baseline and after gastric feeding. furthermore, while fasting and postprandial cholecystokinin and peptide yy plasma levels and gastric emptying were inversely related, the feeding - induced rise of the blood concentrations of these two hormones was directly related to gastric emptying. the latter finding is complementary to a simultaneous report from the authors ' group that baseline and duodenal feeding - induced plasma cholecystokinin levels are higher in critically ill patients than in healthy control individuals. the authors suggest there is a complex interaction between hormonal release, nutrients and gastric emptying, and consequently they emphasize the role of enterogastric hormones in the pathogenesis of disturbed gastric emptying and gastrointestinal passage during critical illness. this proposition may assume particular importance given the high incidence of a disturbed gastroenteral motility, which often limits enteral nutrition, while there is multiple evidence that successful early enteral feeding is associated with improved outcome in critically ill patients. in this context, the assessment of gastric emptying and/or gastrointestinal passage at the bedside remains a challenge, and at present it is unclear which co2 breath test will allow overcoming this problem. jeschke and colleagues examined the putative association between the percentage of total body surface area burn and the inflammatory response, body composition, metabolism and organ function. for this purpose, 187 severely burned children (mean age, 7 to 8 years) were divided into four groups according to burn size : total body surface area 80%. larger burn size was associated with a higher presence of third - degree burns, inhalation injury, ventilator dependency and number of surgical interventions, as well as with a higher incidence of infection and sepsis leading to an increased length of stay and increased mortality. while hypermetabolism, expressed as a percentage of the predicted resting energy expenditure, was present in all groups from admission to discharge, it only persisted in the two most severely burned groups. the highest serum il-6 and il-8 levels were seen in > 80% total body surface area, most probably due to the fact that more than one - half of these patients presented with infection or sepsis. in addition to the ongoing debate of whether crystalloid or colloid solutions should be used for fluid resuscitation during critical illness, the individual qualities of the various colloid solutions have been the focus of research. colloids are reported to have various nononcotic properties that may influence vascular integrity, inflammation and pharmacokinetics. in a prospective clinical trial, gombocz and colleagues therefore compared the effects of perioperative 6% dextran-70 infusion on the inflammatory response and myocardial ischemia - reperfusion injury after cardiac surgery using cardiopulmonary bypass with those of 5.5% oxypolygelatin. dextran-70 infusion was associated with lower peak plasma levels of procalcitonin, il-8, il-10, endothelial leukocyte adhesion molecule-1 and intercellular adhesion molecule-1, thus suggesting attenuated endothelial damage and leukocyte activation. this reduced inflammatory response coincided with improved clinical and laboratory markers of cardiovascular function : higher stroke volume and, consequently, higher cardiac index, and lower peak troponin - i levels than in the oxypolygelatin - treated patients. by contrast, the postoperative drainage volume was higher in the dextran-70 group which did not assume clinical importance, however, since neither hematocrit nor transfusion requirements significantly differed. these authors ' findings are in good agreement with experimental data that dextran-60 prevented leukocyte / endothelial cell interaction after extracorporeal circulation, while 10% hydroxyethyl starch affected only adherent white cells. within the limits of the relatively small number of low - risk patients rather than high - risk patients, who are probably more susceptible to benefit from these measures the study by gombocz and colleagues adds an interesting piece to the exciting puzzle of cardiac surgery - related systemic inflammation. mild hypothermia represents one of the most challenging aspects of prevention of organ failure, since it can improve outcome but may also be associated with marked side effects. depending on the technical device used, some of the side effects limiting the initiation of hypothermia due to the inherent increase of whole body oxygen consumption are vasoconstriction and shivering. several drugs are known to lower the thresholds for shivering or vasoconstriction, among which meperidine has been shown one of the most effective. kimberger and colleagues therefore investigated the impact of a skin warming system and/or a medium dose of meperidine on thermoregulatory thresholds in healthy volunteers infused with 4c lactated ringer 's solution to decrease the core temperature by 2.4c / hour until shivering started. both skin surface warming and meperidine administration reduced the vasoconstriction and shivering thresholds, and combining the two approaches reduced the shivering threshold below 34c without the occurrence of adverse effects such as respiratory depression. combining external warming to prevent vasoconstriction with meperidine administration might therefore prove effective for the induction and maintenance of mild therapeutic hypothermia. it must be noted, however, that healthy volunteers rather than critically ill patients were studied, so any impact of disturbed neurological function, the neuroendocrine axis and/or the autonomous nervous system either related to the disease per se or caused by the ongoing treatment with sedatives, catecholamines, and so forth remains open. avp : arginine vasopressin ; epo : erythropoietin ; il : interleukin ; inos : inducible nitric oxide synthase ; no : nitric oxide. pr received consultant fees from ferring pharmaceutical a / s (kbenhavn, denmark) for help with designing preclinical experiments. | the research papers on shock published in critical care throughout 2007 are related to three major subjects : the modulation of the macrocirculation and microcirculation during shock, focusing on arginine vasopressin, erythropoietin and nitric oxide ; studies on metabolic homeostasis (acid base status, energy expenditure and gastrointestinal motility) ; and basic supportive measures in critical illness (fluid resuscitation and sedation, and body - temperature management). the present review summarizes the key results of these studies and provides a brief discussion in the context of the relevant scientific and clinical background. |
the siv h1n1 and h3n2 subtypes have been circulating for more than 30 years, and the siv subtype h1n2 has been circulating since it was first isolated in great britain in 1994 [2, 3 ]. in april 2009, a new influenza a virus of subtype h1n1 emerged in the human population in mexico and the united states. this was a multiple reassortant virus containing genes from north american and eurasian lineages [4, 5 ]. human - to - pig transmission was first reported in canada in late april 2009, then in european pig population in early september 2009, and has since been reported in several countries all over the world [69 ]. in october 2009, siv was reported for the first time in norway when an integrated pig herd tested positive for pandemic (h1n1) 2009 virus after showing mild clinical signs of respiratory disease. the clinical picture of pandemic (h1n1) 2009 infections in experimentally and naturally infected pigs was described as mild respiratory illness, increased temperature, and decreased appetite [6, 11, 12 ]. in some infected herds, clinical signs were not detected. studies have shown that immunological nave pigs experimentally infected with pandemic (h1n1) 2009 virus could transmit the virus efficiently to other nave pigs [11, 14, 15 ]. although pandemic (h1n1) 2009 virus contains gene segment genetically related to other swine influenza virus strains circulating in europe and north america, it shows antigenetic differences in the major glycoproteins of the virus. however, it has been shown that pigs infected or vaccinated with h1n1 european avian - like swine influenza virus could produce cross - reactive antibodies against pandemic (h1n1) 2009 virus which protected pigs against the infection of pandemic (h1n1) 2009 virus [13, 16 ]. the pig production in norway consists of approximately 2500 herds, mainly small family farms, with about 1.5 million slaughtered pigs in 2010. national surveillance and control program shows that the pig population is free for aujeszky 's disease (ad), transmissible gastroenteritis (tge), porcine respiratory corona virus (prcv), porcine reproductive and respiratory syndrome (prrs), and mycoplasma hyopneumoniae [17, 18 ]. the norwegian food safety authority has been running annual surveillance programs to document the status since 1994. blood samples from about 500 randomly selected herds are collected annually for specific disease surveillance. the surveillance has never detected siv infection in norway until october 2009, except for pigs in one herd that tested seropositive for subtype h3n2 in 1998 without showing clinical signs or further spread of the infection. this paper consists of studies that describe the initial spread of pandemic (h1n1) 2009 virus amongst norwegian pig herds, the control measures initiated, and the infection status 20 months after the introduction. the paper also discusses the manifestation of the infection in the norwegian pigs given that they had no prior immunity to any influenza a viruses and are free from most other viral respiratory diseases. following the primary outbreak in october 2009, an intensified surveillance was immediately initiated to investigate the extent of siv infection in the pig population in norway, referred to as study 1 in the results section. the study targeted the following categories of herds : (i) herds where pigs with influenza - like signs were observed, (ii) herds where the pigs had been exposed to humans with verified pandemic (h1n1) 2009 virus infection or influenza - like illness (ili), and (iii) herds with a history of contact with or in close proximity to infected herds through transfer of animals, usage of common equipment, for example, transport vehicles to slaughterhouse, and visitations from farmers of infected farms. in herds suspected to have an active infection, nasal swabs were collected from 20 pigs at different ages, with priority of sampling pigs with clinical symptoms. in herds without clinical signs, blood samples were collected from at least 20 pigs (sows or slaughtered pigs) for serological testing. calculation of sample size was done assuming 100% test sensitivity and a within - herd prevalence > 15%. the sample size implies that the risk of having 20 nonreactors from a positive farm is less than 4%. during the follow - up study, there was a special focus on the prevalence in nucleus and multiplier herds, hereafter referred to as study 2. the nucleus herds are closed herds meaning that they have no contact with other herds except via semen from the norsvin ai station. the multiplying herds are either closed or obtain replacement sows from a single nucleus herd. serological surveillance was carried out to investigate the possible spread of pandemic (h1n1) 2009 to these herds in the early phase of the outbreak. between 1st of october 2009 and 31st of january 2010, blood samples were collected from 20 slaughtered pigs from 112 nucleus and multiplier herds. a third study was initiated to follow the course of the pandemic (h1n1) 2009 infection. from the herds that tested positive for pandemic (h1n1) 2009 in 2009, 38 herds consisting of 13 nucleus herds, 20 multiplier herds, and five conventional integrated herds were selected for the study. from each herd, blood samples from 20 slaughtered pigs were tested for antibodies against pandemic (h1n1) 2009. blood samples were taken on the condition that 6 to 12 months had elapsed since the herd first tested positive and that the pigs tested were born at least 2 months after that time. an additional testing was performed in 9 herds about 18 months after the pandemic (h1n1) 2009 introduction. study 4 consists of data from the national surveillance programme for si in norwegian pigs in 2009 and 2010. the program includes all nucleus and multiplier breeding herds and the nucleus unit of all sow pools. in addition, a random selection of conventional pig herds, 300 integrated and piglet - producing herds and 60 fattening herds, was selected from a population of approximately 2500 herds. ten pigs in each herd were tested. with the exception of pigs from finishing herds, which are sampled at the abattoirs, clinical and epidemiological information recorded by the food safety authority was available from 43 pig herds suspected to be infected. the information was collected, while the herd was being sampled during the early phase of the outbreak between 10th of october 2009 and 31st of december 2009. the size of these herds ranged in numbers from 5 to 2000 pigs (average 550 pigs). the investigation used a structured questionnaire consisting of questions on epidemiological information including clinical picture within the herd, likely source of infection, contact with people diagnosed with pandemic influenza or had ili symptoms and being in contact with pigs while sick, movement of animals in or out of the farm, and handling and biosecurity measures. initially, serum samples were analyzed for specific antibodies against a / sw / belgium/1/98(h1n1) and a / sw / flanders/1/98(h3n2) using hemagglutination - inhibition (hi) assays according to the method described in the oie manual of diagnostic tests and vaccines for terrestrial animals. following introduction of pandemic (h1n1) 2009 virus in the norwegian pig population, serum samples were tested for influenza - a - specific np antibodies using an elisa kit (i d screen influenza a antibody competition test, i d vet, montpellier, france). a herd was considered seropositive if three or more blood samples from the herd were positive for influenza - a - specific np antibodies. in case one or two of the 20 blood samples from a herd were elisa positive, the herd was retested with samples from another 20 pigs, and the herd concluded positive if at least one of these samples was seropositive. positive and/or inconclusive samples were retested for hemagglutinin - specific antibodies using hi assay with a / sw / belgium/1/98(h1n1), a / sw / flanders/1/98(h3n2), and a / california/07/2009 (pandemic (h1n1) 2009) as antigens. the antigens for the hi tests were produced in chicken eggs at the norwegian veterinary institute in oslo. nasal swabs (copan innovation ltd, brescia, italy), individual or in pools of two, were placed in 1 ml of transport medium (emem 2% ibfs / tris) before they were shipped to the laboratory. total rna was extracted from 200 l of the sample material using the automatic extraction instrument nuclisens easymag (biomrieux, norge as, oslo, norway) according to the manufacturer 's instruction. detection of influenza a virus was performed as described by the world health organization, the who collaborating centre for influenza at cdc atlanta. specific detection of the ha gene of the pandemic (h1n1) 2009 subtype was carried out on influenza - a - positive samples as described by robert koch institute. amplification was performed on a stratagene mx3500p (lajolla, ca, usa) with superscript iii platinum one - step quantitative rt - pcr system (invitrogen, paisley, uk). in addition, some samples were analyzed at dtu veterinary (national veterinary institute, technical university of denmark) using an inhouse real - time rt - pcr for detection of pandemic (h1n1) 2009 virus (ref statens serum institut, denmark, unpublished). one rt - pcr - positive sample was sufficient to confirm a herd as positive for pandemic (h1n1) 2009 virus. full genome sequencing of isolate a / sw / norway/02_11342/2009(h1n1) was carried out through a modified version of the who sequencing primers and protocol provided by the who collaborating centre for influenza in cdc, atlanta, usa (28 april 2009). briefly, 1 l extracted rna was amplified using 46 primer pairs and a one - step rt - pcr kit (qiagen, oslo, norway). the resulting overlapping amplicons, carrying primer - derived m13 terminal sequences, were sequenced with m13 primers using the abi bigdye terminator v1.1 cycle sequencing kit and the abi 3130 genetic analyzer (applied biosystems, warrington, uk). sequences were assembled using sequencher v.4.9 (genecodes corporation, ann arbor, mi, usa) and compared to related h1n1 virus sequences using bioedit software. descriptive statistics and calculation of true population prevalence using confidence intervals were used. to measure the association between county prevalence and the pig density by county after the introduction of pandemic (h1n1) 2009, the intensified surveillance (study 1) shows that 54 of the 114 herds tested were positive for pandemic (h1n1) 2009 virus by rt - pcr, and 55 of the 140 herds tested by serology were positive for antibodies against pandemic (h1n1) 2009. altogether 91 herds tested positive from 10th of october to 31st of december 2009. in the 55 seropositive herds, 59% (range 5% to 100%) of the individual samples from each herd were positive. from the 54 rt - pcr - positive herds, an average of 65% (range 5% to 100%) of the individual samples from each herd were positive. geographically, the infected herds were found throughout norway (figure 1). in 2010, two out of nine herds tested were positive for pandemic (h1n1) 2009 by rt - pcr. during the first six months of 2011, these herds were sampled on suspicion of infection with siv based on clinical signs in the pigs. in study 2, a total of 2349 blood samples from 112 nucleus and multiplier herds were tested for antibodies against influenza a virus. testing of the nucleus herds was completed by the 18th of december 2009, and the multiplier herds were finished on the 31st of january 2010. the surveillance shows that 27.1% of the nucleus herds and 34.4% of the multiplier herds were seropositive for pandemic (h1n1) 2009. between six and 12 months after a herd was seropositive for antibodies against pandemic (h1n1) 2009 virus, blood samples were collected from slaughtered pigs born at least two months after the initial detection (study 3). in total, 1127 pigs from 38 herds were tested. three of the 38 herds tested positive for antibodies against pandemic (h1n1) 2009 virus. the seropositive herds were multiplier herds located in the south west region where pig farming density is highest (figure 1). pigs from all the 13 previously positive nucleus herds selected for the study were found negative at this time. however, the intensified surveillance is still ongoing in the nucleus herds. by the end of july 2011, nine of the 13 nucleus herds pigs from five of these herds were positive for antibodies against pandemic (h1n1) 2009 virus, between 12 to 20 months of time after the primary infection in the herds. within the 2009 routine surveillance of study 4, 4724 samples from 452 herds were tested for antibodies against siv (table 1). in total, 16 herds were found seropositive for pandemic (h1n1) 2009. retrospectively, the earliest seropositive samples were collected from a herd in the county of rogaland on the 30th of september 2009, suggesting that pandemic (h1n1) 2009 has been circulating in the norwegian pig population since september 2009. the results from the surveillance program in 2010 show that 41% of 459 tested herds were seropositive (table 1). the proportion of seropositive herds in different counties varied from 21% in the low pig density counties of vestlandet to 57% in the high pig density county of rogaland and agder. simple regression analysis shows that herd prevalence is positively correlated (r = 0.013, p = 0.023) with the pig density by county (figure 1). moreover, the surveillance program in 2010 shows that 42% (19 positive/45 tested herds) of nucleus and 44% (30 positive/68 tested herds) of multiplier herds were seropositive. questionnaires with epidemiological information were available from 35 pig herds confirmed positive by laboratory testing (serology or pcr). in 60% (21/35) of these infected herds, there was evidence to suggest that humans were the most likely source of infections. in these herds, the farmer, farm worker, or the farmer 's family members were diagnosed with pandemic (h1n1) 2009 (33% or 7/21) or had ili just before or during the outbreak (66% or 14/21). twenty percent (7/35) reported contact with another infected farm through humans or movement of infected animals as source of infection. one farmer of a positive herd had bought pigs from a positive herd and also had one farm worker with ili during this period. forty - nine percent (17/35) had reported that clinical signs were observed in the pigs. the most common clinical signs reported were coughing, fever, and loss of appetite, lasting for less than two weeks. nineteen percent (8/43) of the herds from which epidemiological information was gathered tested negative although 3 of these herds had histories of contact with humans with ili. full genome sequence analysis was carried out on nasal swab rna from an infected pig in the index herd, as well as from the virus isolated from a sick staff member at the same farm. the sequences of these two full viral genomes were virtually identical (99.9985% identity) (acc. no jq253790-jq253797). moreover, the ha sequence showed at least 99.8% identity to other pandemic (h1n1) 2009 circulating in humans in norway at the same time. the complete sequence further indicated that the virus had not acquired known resistance mutations in the neuraminidase gene. pandemic (h1n1) 2009 virus was first detected in norwegian pigs on the 10th of october 2009. as it was reported that the pandemic (h1n1) 2009 virus was transmitted rapidly between humans and also from humans to pigs, the norwegian food safety authority and the pig industry initiated biosecurity measures such as use of gloves and gauze masks to protect the norwegian pig population from this virus. following the detection of the index case, an intense screening based on detection of virus and antibodies against the virus, a total of 91 herds had tested positive for pandemic (h1n1) 2009 virus and/or antibodies against the virus. our investigations showed that despite rapid implementation of control measures, the virus had in a short period of time been spread to pig herds throughout norway, including herds that had high biosecurity and to closed herds. the first confirmed human case of pandemic (h1n1) 2009 virus in norway was in may 2009. it was followed by a first minor wave of infected people during the summer and a second major wave in the autumn. shortly after the onset of the autumn wave, the first pig herd positive for pandemic (h1n1) 2009 virus was detected. hence, the farmer and family members must care for the pigs even when being ill. many of the first positive pig herds had been in contact with people that were confirmed with pandemic (h1n1) 2009 virus infection or people with ili. herd - to - herd transmission was ruled out because there was no history of contact between herds that were positive for the virus or were seropositive. it is also known that many people could be infected with pandemic (h1n1) 2009 without showing clinical symptoms. this suggests that infected humans, with or without clinical symptoms, were an important factor in the introduction and transmission of pandemic (h1n1) 2009 virus to the norwegian pig population. sequencing analysis of virus isolated from humans and from pigs indicated that almost identical viruses had infected both species. furthermore, the high prevalence in nucleus and multiplying herds, that normally have no or few contacts with other herds, indicates that humans, and not trade with pigs, were responsible for introduction of pandemic (h1n1) 2009 virus to norwegian pigs. the national surveillance program for siv in norway shows that the first herd positive for antibodies against pandemic (h1n1) 2009 virus was sampled on the 30th of september 2009. as shown in table 1, almost four percent of the herds that had been tested in this program in 2009 were seropositive. the results also show that in areas with high pig density, a high proportion of the herds were seropositive. this suggests virus transmission between pig herds, either by movement of pigs and people visiting infected herds. however, air transmission can not be excluded. the farm size and density of pigs in the area around the farm experimental studies have shown that pandemic (h1n1) 2009 virus is efficiently transmitted between pigs [11, 14, 15 ]. it is also known that other siv subtypes are efficiently spread between pigs and herds. norway has previously been free of siv as documented by a national surveillance program for swine. although one multiplier herd was tested seropositive in 1998 for antibodies against influenza a h3n2 virus, follow - up studies for many years in this herd did not reveal new seropositive pigs, nor any spread of the virus in contact herds. based on this, it was concluded that the pigs in this herd had probably been infected by a human - adapted h3n2 virus that did not have the capacity to be transmitted further between pigs. since the norwegian pig population has been free of siv, there are no cross - reactive antibodies to pandemic (h1n1) 2009 virus or any of the other siv subtypes. this has probably contributed to the high prevalence of pandemic (h1n1) 2009 virus in the norwegian pig population. it has been shown that european pigs naturally infected or vaccinated with european sivs have cross - reactive hemagglutinating antibodies against pandemic (h1n1) 2009 virus [15, 16 ]. moreover, natural infection with influenza virus stimulates mucosal immunity and cellular immune responses, responses that give partial protection against infection with an antigenetically unrelated strain [27, 28 ]. surveillance of siv in europe prior to the emergence of pandemic (h1n1) 2009 virus showed that the three siv subtypes avian - like human - like h1n2 were most often found in animals between 3- and 6 months old. it should be noted that norway is carrying out surveillance for siv, including 45005000 samples from between 450 and 500 herds per year. consistent with other studies, there were mild or no clinical signs observed in herds that tested positive for pandemic (h1n1) 2009 virus. for example, in australia, pigs naturally infected with pandemic (h1n1) 2009 virus only showed mild clinical signs. it was suggested that a lower infection pressure in natural infection as compared to experimental infection, as well as absence of other respiratory pathogens, could result in milder clinical signs. in norway, only a few pigs showed clinical signs like coughing, fever, and loss of appetite which are typical for siv. as norway is free from most other viral respiratory diseases, presence of clinical signs like coughing would alert farmers who would notify their veterinarian or the authorities. herds positive for pandemic (h1n1) 2009 virus in 2009 were tested again in 2010. the results show that there was no seroconversion in pigs born at least two months after the virus was first detected, indicating that there was no ongoing infection in the herds. a possible explanation may be that the norwegian pig herds are small, and many herds practice batch farrowing of three, five and a half, or seven weeks intervals, with strict sectioning of the herds with all - in all - out system. however, results from the surveillance in 2011 show that some pigs born in 2010/2011 are seropositive, indicating an ongoing spread of the infection. it is not clear to what extent this is caused by new introduction from humans or continued virus circulation between susceptible pigs since 2009. continuing surveillance is important to show whether the virus has become enzootic in the norwegian pig population. there have been several reports of reassortant virus that have genes from pandemic (h1n1) 2009 virus and other siv subtypes [3033 ]. as long as norway is free of other siv subtypes, however, reassortant viruses evolving in humans might be a risk for the norwegian pig population. it is therefore important to continue the surveillance of siv and genetic characterization of positive samples. given the potential long - term presence of influenza virus in norwegian pigs, it is also interesting to evaluate the economic impact of this disease and assess the cost effectiveness of new biosecurity measures or change in husbandry practices. pandemic (h1n1) 2009 virus was introduced to the norwegian pig population in september / october 2009. the virus spread rapidly to a large proportion of all categories of herds all over the country, including closed, high - biosecurity nucleus herds. the clinical impact was mild, and in many infected herds, no clinical signs were observed. the ongoing national surveillance program will reveal whether the virus will become enzootic in the norwegian pig population. | pandemic (h1n1) 2009 influenza a virus was detected in norwegian pigs in october 2009. until then, norway was regarded free of swine influenza. intensified screening revealed 91 positive herds within three months. the virus was rapidly transmitted to the susceptible population, including closed breeding herds with high biosecurity. humans were important for the introduction as well as spread of the virus to pigs. mild or no clinical signs were observed in infected pigs. surveillance of siv in 2010 revealed that 41% of all the norwegian pig herds had antibodies to pandemic (h1n1) 2009 virus. furthermore, this surveillance indicated that pigs born in positive herds after the active phase did not seroconvert, suggesting no ongoing infection in the herds. however, results from surveillance in 2011 show a continuing spread of the infection in many herds, either caused by new introduction or by virus circulation since 2009. |
18f - fluorodeoxyglucose (fdg) positron emission tomography (pet) has become an integral part of the management protocol for patients with cancer, where it is used for initial staging, assessment of response to therapy, as well as for detection of recurrent disease. as is seen with other imaging techniques, there are certain pitfalls in the interpretation of pet and pet / computed tomography (ct) studies. it is important that physicians interpreting pet / ct are aware of the potential pitfalls, so that the impact of these pitfalls is minimized and the interpretation is accurate. awareness of the potential causes that may lead to false positive or negative findings is essential for the interpreter to avoid misreading a scan. here, we describe one such false positive case on fdg pet / ct, where an inflammatory lung pathology mimicked pulmonary metastases. a 45-year - old male patient with biopsy - proven adenocarcinoma of the stomach underwent a baseline fdg pet / ct scan for staging workup, which revealed metabolically active disease involving the distal esophagus, gastroesophageal junction (gej), and proximal stomach with metastases to the perigastric, mesenteric, paracaval, and pre - aortic lymph nodes. there were no pulmonary lesions or distant metastases elsewhere detected at this stage. the patient was planned for definitive therapy with neoadjuvant chemotherapy (nact) to be followed by surgery, with a curative intent. on completion of three cycles of nact with cisplatin, epirubicin, and 5-fluorouracil, the patient was referred for a repeat fdg pet / ct scan for response evaluation and re - assessment of the disease status. pet / ct scan revealed complete resolution of the nodal lesions and significant reduction in the extent as well as metabolic activity of the primary lesion in the stomach and gej. in addition, there were multiple, well - defined, fdg avid, 13 cm sized, pulmonary nodules randomly distributed over both the lungs, raising a suspicion of pulmonary metastases [figure 1 ]. the lesions revealed moderately high fdg uptake [maximum standardized uptake value (suvmax) of 6.23. ] on ct images, most of the lesions exhibited a feeding vessel sign and some of the lesions showed early cavitation within. correlation was sought with a hemogram, which revealed normal total as well as differential leucocyte count. at this stage, interpretation of the pet / ct findings was progressive disease with development of pulmonary metastases. (a - d) standard fdg - pet / ct acquisition in a follow - up case of carcinoma stomach. pet whole body 3d mip image (a) shows multiple fdg avid foci (black arrows) in the thoracic region. axial ct (b) and fused pet - ct (c, d) images of the thorax reveal multiple, peripherally located, fdg avid, pulmonary parenchymal nodules (white arrows). the larger lesion in the right lung exhibits a feeding vessel sign and shows cavitation within the patient developed fever on the second day following the pet / ct scan and gave history of chills following flushing of the indwelling central venous catheter (cvc). cvc infection was suspected and the catheter was removed. taking into account the clinical profile and blood culture results, the pet / ct findings were reviewed and a provisional diagnosis of septic pulmonary emboli (spe) was made. patient was put on intravenous antibiotics for 4 weeks, following which his fever subsided and blood culture became sterile. the tip of the removed cvc was sent for bacterial culture, which grew e. cloacae (the same organism that was found in the blood culture), confirming the source of bacteremia. chest ct done after completion of antimicrobial therapy revealed complete resolution of the lung lesions [figure 2 ]. representative axial images (in lung window), reveal complete resolution of the lung lesions that were noted in the earlier scan planned chemotherapy was continued thereafter. repeat fdg pet / ct done on completion of nact revealed absence of any fdg avid pulmonary lesions, whereas the primary disease in the stomach showed progression [figure 3 ]. whole body pet, 3d mip images, before (a) and after (b) antibiotic therapy. image (a) shows multiple fdg avid foci in the region of thorax (arrows) and minimal fdg uptake in the stomach (arrowhead) representing metabolically active local disease. image (b) reveals absence of any fdg avid focus in the thorax, where - as a large fdg avid lesion is seen in the region of stomach (arrowhead), indicating progression of the primary disease in the stomach to our knowledge, this is the first report of fdg pet / ct imaging findings of spe in a case of known malignancy with an indwelling cvc being the source of infection. although fdg - pet / ct imaging has achieved great success in the evaluation of malignant disorders, the modality is not specific for the diagnosis of cancer. uptake of fdg in living cells is determined by the cellular metabolic rate and the number of glucose transporters present in the cells. apart from tumors, activated inflammatory cells also demonstrate increased expression and upregulation of glucose transport receptors. a number of studies have reported presence of high fdg uptake in infective and inflammatory lesions, both acute and chronic. infectious diseases (mycobacterial, fungal, bacterial), sarcoidosis, radiation pneumonitis, and postoperative inflammation have been observed to show intense fdg uptake on pet scan. often, inflammatory lesions produce fdg uptake patterns that are indistinguishable from those of cancer, leading to false positive results, in patients with a known or suspected malignancy. spe is an uncommon disorder in which infection originating at an extrapulmonary site is transported to the lungs via the bloodstream. microorganisms embedded in thrombin get mobilized from the nidus of infection and lodge in pulmonary arteries leading to infarction and metastatic abscess formation. the most common sources of infection include infected heart valves, thrombophlebitis, and infected indwelling venous catheters or pacemaker wires. clinical symptoms of spe are usually nonspecific and commonly include one or more out of fever, cough, hemoptysis, shortness of breath. and chest pain. occasionally, an active extrapulmonary focus of infection may be obvious at the time of presentation. this coupled with frequent occurrence of immunosupression and neutropenia in such patients predisposes them to spe. high fdg uptake is generally accepted to be associated with malignant lesions. in a patient with known malignancy undergoing fdg pet scan, finding of multiple fdg avid, discrete, pulmonary nodules with feeding vessels stage, nearly ruling out the probability of a complete cure for the patient. as is evident from the findings in our case, spe can closely mimic metastases, with the pulmonary lesions showing high metabolic activity on fdg pet and exhibiting a morphology akin to metastatic deposits on ct. presence of high fdg uptake in spe has been documented by a number of authors and is attributed to the high glycolytic rate of activated macrophages and polymorphs that are abundantly present in most inflammatory lesions. in a patient with known malignancy undergoing fdg pet scan, if multiple fdg avid pulmonary nodules are detected as a new finding, high index of suspicion must be maintained to rule out the possibility of septic emboli and to avoid a catastrophic mistake of labeling these lesions as metastases. clinical inputs (history of fever, chills, respiratory symptoms, an obvious site of extrapulmonary infection) and hemogram results when integrated with the imaging findings can aid in providing vital clues that point towards the diagnosis of spe. clinical indicators that favor spe are history of concurrent febrile illness, neutropenia, and a positive blood culture confirming the presence of bacteremia. studied the clinical course and imaging features in 14 patients of spe and concluded that spe presented with variable, often nonspecific clinical and radiographic features and the diagnosis was usually suggested by the presence of a predisposing factor, febrile illness, and ct findings of multiple, peripheral, nodular lung infiltrates, with or without cavitation. reviewed ct scans of 18 patients with documented spe and concluded that, in an appropriate clinical setting, characteristic ct features of septic emboli can suggest the correct diagnosis. rossi. have opined that, on ct, the most characteristic features of spe include discrete pulmonary parenchymal nodules with cavitation, which often exhibit lower lobe predominance and presence of nodule - feeding vessels. in addition, subpleural heterogeneous wedge - shaped opacities may also be identified in a majority of patients with spe. although the phenomenon of mixed response to chemotherapy (where some of the cancerous lesions show regression while other lesions show progression) is well known, a patient with known cancer on chemotherapy developing new fdg avid lesions at a distant site while the pre - existing disease shows regression should alert the interpreting physician to the possibility of an unrelated pathology (e.g., infection or inflammation) as the cause of new lesions. spe is a rare condition that may mimic pulmonary metastases on fdg pet / ct imaging, especially in a patient with a known malignancy. indwelling cvc is a predisposing factor and can act as a source of infected emboli in such cases. in cancer patients with indwelling cvc, if fdg avid pulmonary nodules are detected, high index of suspicion should be maintained to rule out spe. analysis of ct morphology of the lesions is critical for arriving at the correct diagnosis. ct features most characteristic of spe include discrete, peripheral, pulmonary nodules of varying sizes, with cavitation and presence of additional subpleural wedge - shaped opacities. a positive blood culture confirms presence of bacteremia and resolution of lung lesions following antimicrobial therapy, clinches the diagnosis of spe. | inflammatory lesions may sometimes show intense tracer uptake and mimic neoplastic lesions on (18) f - fluoro - deoxyglucose (fdg) positron emission tomography / computed tomography (pet / ct). we report one such false positive case on fdg pet / ct, where septic pulmonary emboli (spe) mimicked pulmonary metastases. a 45-year - old man with stomach cancer had an indwelling central venous catheter (cvc) in situ while on neoadjuvant chemotherapy. he underwent fdg pet / ct scan for response assessment and the images revealed multiple, intensely fdg avid, peripheral, lung nodules with feeding vessels, which were suspicious for pulmonary metastases. a day later, the patient developed fever with chills and his blood culture showed bacterial growth (enterobacter cloacae). a provisional diagnosis of spe from an infected cvc was made. chemotherapy was withheld, cvc removed, and the catheter tip was sent for bacterial culture. following a 4-week course of antibiotic treatment, the patient became afebrile. culture from the cvc tip grew the same organism, as was seen earlier in the patient 's blood culture, thus pin - pointing the source of infection in our case. diagnosis of spe was clinched when follow - up ct chest done after completion of antibiotic course showed complete resolution of the lung lesions. |
selected group of patients with hearing loss at high frequency and still preserved hearing at low frequencies do not benefit from the classic hearing aid. parallelly, due to residual hearing they do not meet the qualification criteria for classic cochlear implant. nevertheless, ongoing development indicated that the hybrid system concept of electro - acoustic stimulation i.e., combination of hearing aid, that amplifies the residual hearing and cochlear implant, that stimulates cut off frequencies at the level of the basal turn of the cochlea will work best for that group of patients. preliminary observations that proceeded research and development on the hybrid system were combined with observations on conservation of residual hearing in cochlear implants recipients. results showed that 50 % of operated patients retained sufficient hearing for effective use of ipsilateral ite hearing aid. number of studies indicates that bimodal stimulation is not just a simple additive effect of both devices, but they show the synergistic action, that is especially appreciated by patients experiencing so called cocktail party effect in difficult listening environments [36 ]. new, less traumatic electrodes were introduced as well as the surgery improved from 11.2 mm cochleostomy to the round window approach. atraumaticity i.e., preservation both hearing and vestibular function become the key element in the hybrid surgery. recent advances in that field brought the hybrid - l electrode attached to nucleus freedom implant. straight, thinner and shorter (16 mm) comparing with contour advanced electrode, but with 22 active contacts hybrid - l electrode was developed to facilitate the less traumatic insertion through the round window to preserve residual hearing. new nucleus freedom hybrid - l system was introduced into the clinical practice in 2009. first experiences with new electrode were presented by lenarz. confirming the safety on both histological and clinical level [1, 9 ]. with that perspective authors would like to share their own observations on hearing preservation with hybrid - l system. study was designed as the retrospective analysis conducted at the department of otolaryngology and oncological laryngology, poznan university of medical science. twenty - one patients with high frequency hearing loss (n = 21) were included to the study. there were 12 females and 9 males, ranging in age from 16 to 77 years of age, with a mean of 49.5 years and median 52.5 years of age. nucleus freedom hybrid - l device (cochlear, australia) was implanted in every patient by the same surgeon (ws) with the standardized surgical technique described previously [8, 10 ]. patients were qualified for the surgery according to the audiometric indications (pta), minimum 5 years of stable hearing loss, insufficient gain form hearing aid (30 % of speech understanding). speech processor switch - on was done in 58 weeks, depending on acoustic component delivery. up to date, all the patients use electric as well as acoustic stimulation none of the patient use only electric stimulation. first one with classic indication for hybrid - l implant (group a, n = 13) and second with extended criteria according to lenarz. the mean value for three lower frequencies (250, 500 and 1,000 hz) were calculated. acoustic thresholds > 110 db or without recordings were considered as hearing loss and for statistic evaluation marked as 120 db. the difference between mean values before and after surgery was studied and defined as follows : hearing preservation mean value up to 10 db ; hearing impairment mean value between 10 and 30 db ; hearing loss for patients with no thresholds recorded postoperatively. in the group of 21 implanted patients in 17 cases we have observed preservation of hearing (12 patients from group a, 5 patients from group b). in 4 out of 21 patients deafness on the implanted ear was noted (group a1 patient, group b3 patients), although all these 4 patients reported significant difference in hearing after acoustic component activation. in 17 patients with preservation of hearing analysis of audiometric results revealed that within the a group (13pts.) in 9 cases average hearing thresholds were calculated for 3 evaluated frequencies. in 3 cases hearing threshold for 1,000 hz was not detectable, comparing to preoperative values : 80, 110 and 100 db. post difference of mean hearing threshold was 010 db in 6 cases, 1120 db in 3 cases, and 2130 db in 3 cases. within the b group (8pts.) in 4 cases hearing threshold for three examined frequencies was established. post difference of mean hearing threshold was 010 db in 3 cases and 2130 db in 2 cases. in one patient from the b group hearing threshold for 1,000 hz was not recorded pre- and postoperatively (table 1).table 1summary of the audiometric results in patients implanted with nucleus hybrid - l electrode recorded pre- and postoperativelypatient numbergroupimplanted earagegenderpre - op ptapost - op ptamean pre - op (db)mean post - op (db)hl (db)preservation of hearing250 hz500 hz1,000 hz250 hz500 hz1,000 hz1al16.5f30459025759055.063.38.3 + 3ar64f55606070809558.381.723.3 + 6ar18.5f20202035403020.035.015.0 + 7al77m55606012012012058.3120.061.7x9al18f6565808010012070.0100.030.0 + 10al66m45457050558553.363.310.0 + 12al53.5f305070303510050.055.05.0 + 14ar47m30508530558555.056.71.7 + 15al37f5055100558011068.381.713.3 + 16ar50f607095657010075.078.33.3 + 17ar69f25506030556545.050.05.0 + 19ar52.5f45601108510012071.7101.730.0 + 21al30.5m204090305512050.068.318.3+mean value for the group a 13.62br34.5m70708075709073.378.35.0 + 4br75.5m70909012012012083.3120.036.7x5bl49.5f45707545757563.365.01.7 + 8br54.5f55657560658065.068.33.3 + 11br75m65655590858061.785.023.3 + 13bl64m70659512012012076.7120.043.3x18br77.5m4555x7090x50.080.030.0 + 20bl20.5f80909012012012086.7120.033.3xmean value for the group b 12.7mean value for both groups (a + b) 13.1mean value for all patients in the study19.1 in all cases where threshold could not be recorded the value of 120 db was used for statistic calculation patient(s) with hearing loss excluded from the calculation summary of the audiometric results in patients implanted with nucleus hybrid - l electrode recorded pre- and postoperatively in all cases where threshold could not be recorded the value of 120 db was used for statistic calculation patient(s) with hearing loss excluded from the calculation general overviews indicated that conservation of residual hearing in ci patient can be achieved in approximately 50 % [2, 11 ]. years after first publication on the topic, carlson. interestingly pointed out that hearing preservation after standard cochlear electrode implantation remains unpredictable, but should be considered as the realistic goal. to improve the safety and increase the atraumaticity of the surgery various strategies were proposed, among which the technique called soft surgery proposed by lehnhardt, become the gold standard in all ci surgeries. other options, such as lubricants (i.e., hialuronic acid) added during the surgery are in the routine use, whereas, round window approach, as the alternative to the cochleostomy in classic ci remains under discussion. hybrid cochlear implants, unlikely the conventional ones, requires the residual hearing to give the recipient the best feedback. therefore, hearing preservation is not an option, but the key point element to be achieved. first experiences with hybrid s electrode (cochlear, australia) implanted in patients with residual hearing in low frequencies were very successful [1619 ]. although the design of the electrode (10 mm in length, six active contacts) was addressed to minimize trauma of the basilar turn of the cochlea, to reduce the risk of hearing loss, this could not be avoided. in patients with loss of residual hearing, the only electrical stimulation based on six contacts on the electrode was not sufficient, resulting in necessity of re - implantation with longer electrode. this 16 mm with 22 contacts electrode covers approximately 270 of the basal turn of the cochlea, which is an equivalent of the former nucleus 24 straight electrode. the stopper and the wing ascertains the comparable electrode position in the cochlea of the operated patients. thus, variable such as insertion depth is not an issue in cochlear nucleus freedom hybrid - l implant. that is parallel with our experience, although, the position of the round window, caused minor technical problem during the insertion (the angle) and it was not the primary goal of investigation. the overall hearing preservation rate in the current study was 80.95 % (17/21). within the classic indication group (group a) it was 92.3 % (12/13), but unfortunately, for patients with extended indications (group b) hearing preservation rate was lower62.5 % (5/8). nevertheless, it is still over 50 % above the level indicated in the literature. looking at the potential cause of hearing loss in patients from our study, surgery variations was not an issue, because all cases were operated by the same surgeon. thus, other reasons for the failure such as age of the patients (two were over 70 years), diabetes, vascular problems or inflammatory responses should be taken under consideration. our results clearly indicate that with standard procedure hearing preservation can be obtained in majority of patients. hearing preservation was not achieved in 19.05 %, but owing to design of the electrode of the cochlear nucleus hybrid - l that enables to work as ci platform alone, in patients who lost their hearing after surgery re - implantations were not required. this proves that eas is a safe and reliable method to help patients with specific type of hearing loss. | the objective of the paper is to evaluate the hearing preservation rate in patients with high frequency hearing loss, treated with cochlear nucleus freedom hybrid - l implant in the otolaryngology department, poznan university of medical sciences in poland. study was designed as the retrospective analysis. twenty - one patients were operated and implanted with nucleus freedom hybrid - l implant. pure tone thresholds were recorded prior to the surgery and at the time of speech processor switch - on. patients were subdivided into two groups with respect to their pta thresholds : group a classic indications and group b extended indications. average pta for three frequencies (250, 500, 1,000 hz) were calculated for each patient pre- and postoperatively. in the group of 21 implanted patients in 17 cases we have observed preservation of hearing (12 patients from group a, 5 patients from group b) with a mean value of 13.1 db. in 4 out of 21 patients deafness on the implanted ear was noted. our results clearly indicate that with standard procedure hearing preservation can be obtained in majority of patients. hearing preservation was not achieved in 19 %, but owing to design of the electrode of the cochlear nucleus hybrid - l that enables to work as ci platform alone, in patients who lost their hearing after surgery re - implantations were not required. this proves that eas is a safe and reliable method to help patients with specific type of hearing loss. |
induction therapy commonly refers to the administration of antibodies against specific or multiple antigenic targets of immune cells in the immediate peri - operative period. these antibodies have largely been used to provide immunosuppression at the time of antigen presentation, during the initial period after solid organ transplantation, with the purpose of reorienting the immune system by depleting potentially alloreactive immune cells. several meta - analyses have demonstrated that induction therapy improves renal graft outcome compared with conventional therapy. antilymphocyte antibodies have a beneficial effect on 2-year allograft survival, and non - depleting antibodies, such as the anti - cd25 antibodies basiliximab and daclizumab, reduce acute rejection rate [36 ]. moreover, induction drugs play a key role in the promising corticosteroid or calcineurin - inhibitor minimization strategies. the frequency of use of the different induction drugs has varied markedly over the last 15 years. while in the early 1990s the majority of us kidney transplant recipients did not receive induction therapy, in 2004 nearly 72% of recipients received some kind of antilymphocyte drug. currently, thymoglobulin is the most frequently used (37%) induction agent in the usa, and anti - cd25 antibodies are used only rarely. apart from the usa, data from the australia and new zealand dialysis and transplant (anzdata) registry showed a decline in okt3 and polyclonal antibody use and an increase in the prescription of anti - cd25 antibodies to 57.1% in 2001. non - serial studies provide data about induction use in some asian centres (18.4%), international registries (37.7%) and one south american centre (36%). one of the aims of our study was to describe the trends in induction use from 1990 to 2002 in spain and the differences in use among centres. the high rate of variation between countries and centres in induction use revealed not only differences between transplant populations but also differences in indication. other centres use lymphocyte - depleting agents for high - risk patients and non - depleting agents for low - risk patients. the lack of common indications for induction therapy would suggest that it is not well known which patients should receive it and what induction drug must be prescribed. the benefits of using induction must be weighed against the potential risk of infection and malignancy. commonly, induction is used in patients with a higher rejection risk (african - american, highly sensitized patients, patients undergoing re - transplantation), a higher delayed graft function risk (longer cold ischaemia time, expanded criteria donors, donors after cardiac death) or in patients under minimization immunosuppressive strategies. the second purpose of our study was to know in which patients in spain induction was prescribed and the differences in use between polyclonal antibodies and anti - cd25 antibodies. over the last few years, focus on kidney transplantation has shifted towards long - term graft survival due to the improvements in short - term graft survival. the effect of induction antibodies over long - term graft survival is controversial. by means of a meta - analysis of individual patient - level data, szczech. showed a benefit of induction at 2 years in all patients and even at 5 years in recipients with panel reactive antibodies (pra) 20%. a single - centre study comparing polyclonal antibody induction against no induction demonstrated that induction improved graft survival only during the first post - transplant year and did not exert its effect further. antibody induction is also associated with an elevated risk for cardiovascular death, infection - related death and malignancy - related death. data about the long - term effect of induction on patient and graft survival are scarce. we conducted a retrospective cohort study with patients receiving a kidney allograft in spain over four different years (1990, 1994, 1998 and 2002). only adult patients (18 years), receiving a single kidney and remaining alive with a functioning graft at the end of the first post - transplant year were included in the study. the following data were recorded for each patient at the time of transplantation and during hospitalization until discharge by chart review : age and gender of the donor and the recipient, source of the organ (living or deceased donor), cause of donor death, primary kidney disease, recipient body mass index, peak and current pra, number of transplants, time on renal replacement therapy, mismatches, presence of hepatitis c antibodies in the recipient, and cold ischaemia time. graft and patient survival, delayed graft function, acute rejection, first - year creatinine and first - year hypertension were also collected from the clinical charts. delayed graft function was defined as the need for dialysis within the first week after transplantation. the diagnosis of acute rejection was defined according to the criteria of each centre based on clinical and histological data. arterial hypertension was defined as blood pressure more than 140/90 mmhg or need for antihypertensive therapy. induction therapy was defined when the patient received polyclonal antibodies (alg, atg, atgam, thymoglobulin), okt3 monoclonal antibodies or anti - cd25 monoclonal antibodies (basiliximab, daclizumab). other induction drugs were used under clinical trials and were excluded from the analysis. during the study period, there were inadequate data with respect to induction therapy (data absence or duplicate), or they received different induction drugs for clinical trials. medical record review was performed by a transplant physician at each centre taking part in the study according to spanish law with reference to clinical data confidentiality (spanish official bulletin, boe no. comparison between variables was made by using student s t - test for numerical values and chi - square test for categorical data. stepwise multiple regression analysis was used to select independent risk factors for receiving induction therapy among parameters selected by univariate analysis. independent risk factors for graft loss were studied by means of cox s regression analysis. there were significant differences in the percentages of transplant patients that received induction therapy (25.7% in 1990, 40.7% in 1994, 27.1% in 1998 and 37.2% in 2002, p 20%, black race and mismatches. in our retrospective study, induction therapy was used in older recipients, with a higher pra rate, receiving a second or more transplantation, with more mismatches and diabetic. among studied variables, no donor characteristic was related with induction indication. some of these recipient variables were related with a higher immunological risk for acute rejection, such as pra, re - transplantation and mismatches, while the recipient age and diabetes are more related with the risk for delayed graft function and the intention to use steroid or calcineurin - inhibitor minimization strategies. surprisingly, the use of induction therapy in the usa was no different among groups with varying pra. by contrast, most of the us patients on steroid - avoidance regimens received induction drugs. as several meta - analyses have shown that induction therapy improves kidney transplant outcomes in a cost - effective way compared with no induction, a possible future scenario is that induction therapy will be used in all kidney transplants [36,23 ]. in this case, the doubt will be what induction we must use (polyclonal antibodies, mainly thymoglobulin, or anti - cd25 antibodies). several trials including low - immunological - risk patients have found similar rates of acute graft rejection and graft and patient survival, with higher rates of cytomegalovirus infection with polyclonal antibodies compared with anti - cd25 antibodies. in high - risk patients, the rate of acute rejection was greater in those patients treated with anti - cd25 antibodies, but graft and patient survival was the same at the first and fifth years. our study can not allow us to compare outcomes of patients receiving polyclonal antibodies vs anti - cd25 antibodies, but we analysed which kidney graft recipients were prescribed either of them. thus, spanish centres used polyclonal antibodies instead of anti - cd25 in patients with a higher acute rejection risk (high pra and re - transplants) but with a lower infection risk due to their younger age. it has been difficult to know the effect of induction therapy on long - term graft survival. in a single - centre study, 1-year graft survival was better in patients receiving polyclonal induction (93% vs 79%), but no difference was observed from the second to the 20th year in graft survival. a meta - analysis of seven randomized trials comparing lymphocyte - depleting induction with no - induction therapy showed a statistically significant improvement in 2-year graft survival in patients treated with induction drugs. another meta - analysis including 17 trials and 2786 patients found no differences in graft loss at the first and third years between those patients treated and those not treated with anti - cd25 antibodies. in a recent retrospective usrds analysis between 1997 and 2004, gill. reported no differences in 1- and 5-year graft survival in low - risk renal transplant recipients receiving polyclonal antibodies vs no - induction therapy. the benefits of using induction therapy must be weighed against the potential risk of overimmunosuppression, including the risks of infection and malignancy that may occur years after the induction use. lymphocyte - depleting induction is related with a significantly increased risk for early deaths within 6 months and after 6 months post - transplantation in relation with infection, malignancy and cardiovascular problems. however, patients with a kidney surviving more than 1 year who received induction therapy in spain showed a significantly better long - term graft survival with the same patient survival than those not treated with induction drugs. induction therapy exerts its protective role over graft survival independently of other confounding factors, such as acute rejection. this result can not be compared with previous studies due to the fact that we analysed only kidneys functioning beyond the first year. the high number of patients in our study allows us to assert that an immunosuppressive regimen including induction drugs can improve long - term graft survival, this favourable effect being different according to which groups received treatment. in our study, recipients younger than 55 years and with low pra benefited more from receiving induction therapy. in conclusion, from 1990 to 2002, the use of induction therapy in spain changed, with a progressive reduction in the use of okt3 and an increasing use of anti - cd25 antibodies. there were great differences in the rate of induction use among the different centres, although with a common trend to its use in more patients. induction therapy was mainly prescribed in patients with a higher rejection risk (higher pra titres and mismatches and re - transplants) and/or needing calcineurin - inhibitor or steroid minimization regimens to prevent delayed graft function and other secondary effects (older and diabetic recipients). | background. the use of induction drugs has increased markedly over the last 15 years in the usa, but there are few data about their use in other countries. moreover, there are not enough data about when they are indicated and their long - term effects. the aim of our study was to know the rates of use and the drugs used as induction therapy, in which patients they were prescribed and the long - term graft survival effect in spain.methods. we conducted a retrospective cohort study with adult patients (4861) receiving a kidney allograft in spain over four different years (1990, 1994, 1998 and 2002) with a functioning graft at the end of the first post - transplant year. induction therapy was defined as when the patient received polyclonal antibodies, okt3 monoclonal antibodies or anti - cd25 monoclonal antibodies.results. from 1990 to 2002, the use of induction therapy in spain changed, with a progressive reduction in the use of okt3 and an increasing use of anti - cd25 antibodies. there were great differences in the rate of induction use from one centre to another, although with a common trend to greater use at each centre. induction therapy was mainly prescribed in patients with a higher rejection risk (higher panel reactive antibody (pra) titres and mismatches and re - transplants) and in older and diabetic recipients. lastly, patients who were treated with induction therapy had significant higher allograft survival than those who did not (p value = 0.035).conclusions. the use of induction therapy in spain has changed, with an increasing use of monoclonal antibodies in recent years. induction therapy has a protective role in long - term graft survival. |
hen eggs are common food widely acceptable by people around the world, either rich or poor. not only treated as a main dish and used as an ingredient in cooking and bakery, hen eggs can be made into yolk oil (generically known as dn yu, lun yu, lun jng, dn hung yu or j z yu). yolk oil is popular in japan and korea, and among chinese people (including chinese people from the mainland china, taiwan, hong kong, malaysia, etc). in japan, yolk oil products with trademark can be purchased over the counter from pharmacies or cosmeceutical stores. in taiwan and china, most of these products can be purchased from homemade market or traditional chinese herbal shops, mostly without a brand. yolk oil is described in the well known chinese medical encyclopedia flora sinensis (bn co gng m) as a remedy for every diseases, with specific indications in skin sore and ulcer on head and mild fever with diarrhea in children (boym, 1956). nowadays, yolk oil is widely used as a complimentary medicine for treating or preventing heart diseases, especially in japan current methods for obtaining yolk oil include heating and chemical extraction, or a combination of both. it is generally believed among yolk oil users and chinese medicine practitioners that yolk oil made from heating (yoheat) method, mostly by using the traditionally wok, is more effective than yolk oil made from laboratory based chemical extraction (yoext). the yolk is dried in oven or on the wok, and then continuously heated and fried without cooking oil, until yellowish or slightly charred yolk oil effuses. the oil is then collected and stored for further use. also, it is generally thought that charred yolk oil (cyo) is more effective than the yellowish yolk oil as a medicine. however, whether yolk oil should be extracted at yellowish or charred state, remains an argument. the opponents argue that cyo may contain carcinogens and thus the threat of cancers can not be ignored, while the supporters claim that the toxicity of carcinogens is equalized by other good components of the oil, or the black materials in the oil are harmless something else. indeed, kato (1990) detected at least 7 heterocyclic amine mutagens in cyo branded samples. the worrying part of cyo is that heating method can simply be done in kitchen, and drug safety issues may not be taken into account in the heating process. an improvement of the heating method for avoiding the formation of heterocyclic amines is warranted. lipids are the main components in egg yolk, including triglycerides (65%), phospholipids (29%, out of which 86% are phosphatidylcholine and 14% phosphatidylethanolamine), cholesterol (5%), and free fatty acids (< 1%). there have been very few literatures attempting to analyze the compositions of yoheat, and thus it is unclear which components are potentially effective to human health and diseases. only a very small number of publically available modern scientific literatures have reported the effects of yoheat in human health and diseases. among these, lin reported the effects of yoheat in reducing lipid levels using rodent models (lin, 2004a ; lin, 2004b). the three experimental groups in the hamster model were fed with 2% acidic fraction oil, 4% of acidic faction oil and 2% of neutral fraction oil in their basal diets for 6 weeks, respectively, while the two experimental groups in the rat model were fed with 2% and 4% of yolk oil in their basal diets for 7 weeks, respectively. the authors showed that yoheat reduced liver total cholesterol in rats (lin, 2004a) and triglycerides in hamsters (lin, 2004b). a yoext, al721, has been reported to reverse the brain membrane hyperviscosity and, concomitantly, markedly reduce, or even completely abolish the withdrawal symptoms precipitated by naloxone in morphine - addicted mice (heron, 1982). the compound has been shown to reduce htlv - iii infectivity in human peripheral - blood lymphocytes (salin, 1985). a study design comparing the effects of yoheat and yoext in cellular and animal models may reveal essential information on the usefulness of the yolk oil. because heart diseases have become common diseases around the world, it is expected that an easy - to - make complimentary medicine like yolk oil may become even more common in the next few decades. although generic information on yoheat can be obtained from commercialized companies (e. g., http://www.tezukuri-ranyu.jp/) or books published by user groups (nomoto, 1989), there is an obvious need to verify the treatment effects, production methods and guidelines and safety of yoheat by evidence based studies. | yolk oil is common in asia. according to the flora sinensis, yolk oil is a multipurpose medicine, with specific dermatological and fever indications. nowadays, it is generally used as a complimentary medicine for heart diseases. yolk oil can be made from heating or chemical extraction method. it is generally believed that yolk oil made from heating (yoheat) method is more effective as a medicine than that from extraction (yoext). the technical details of the heating method remain an issue of argument, including the degree of char and the threat of carcinogens formed during the heating process. most yolk oil related studies used yoext as research material. nevertheless, animal studies have showed that yoheat reduced triglycerides and total cholesterol in rodent liver. it is expected an easy - to - make complimentary medicine like yoheat may become even more common and thus evidence based studies should be conducted to verify its pharmacological effects and safety. |
hereditary gingival fibromatosis (hgf) first reported by goddard and gross in 1856, is a rare, benign, nonhemorrhagic, fibrous enlargement of gingiva. it seems to be a slowly progressive keratinized gingival overgrowth with various degrees and it is genetically heterogeneous and can occur in either autosomal dominant (common) or recessive forms. hgf can potentially interfere with speech, lip competence and mastication resulting in both esthetic and functional problems. this condition is generally diagnosed alone or occasionally in association with a number of syndromes, such as zimmerman - laband syndrome, rutherford, and ramon syndromes. since exact etiology and pathogenesis of hgf has not been yet established, therefore, there is a need for further research regarding genetic inheritance of hgf. attempts should also be made to find out various preventive measures and implementation of different treatment modalities to limit or eradicate hgf and associated complications such as esthetics, speech and functional problems as much as possible. a 14-year - old male [figure 1 ], accompanied by his parents, reported with the complaint of swollen gums, presented with gradual and progressive gingival enlargement of both jaws from the age of 7 years [figure 2a c ]. the enlargement had led to incompetent lips, poor esthetics and also hindered speech and mastication. intra - oral examination revealed fibrotic enlargement of gingiva involving both upper and lower arches. his current health condition and mental state were considered normal. during the evaluation of his family history, the patient 's mother demonstrated similar intra - oral features in the form of gingival enlargement, involving to various extents, the maxilla as well as the mandible with no syndromic association [figure 3 ]. biopsy sample was taken and sent for histopathological examination showing stratified squamous epithelium with long slender rete pegs, connective tissue and dense collagen stroma [figure 4 ]. on the basis of the histopathological findings and positive family history the diagnosis of hgf was made with no syndromic association. in the treatment enlarged, gingival overgrowth was surgically removed [figure 5a and b ] (gingivectomy) and patient was referred to department of orthodontics and dento - facial orthopedics for correction of malocclusion. (c) preoperative facial view left side intraoral view of mother histopathological specimen showing stratified squamous epithelium with long slender rete pegs, connective tissue and dense collagen stroma (a) peroperative view of mandibular jaw. (b) peroperative view of maxillary jaw postoperative healing was good and desired crown lengthening was achieved. patient 's speech and masticatory problems were completely resolved with gain of adequate functional ability. esthetics was significantly improved in terms of gingival appearance after surgical excision of hypertrophied gingival tissue. patient was put in the follow - up program at 1, 3, 6 months interval followed by after 1 and 2 years. there was no recurrence of the disease even after 2 years follow - up [figure 6a c ]. syndromes that have been occasionally associated with hgf are zimmerman - laband syndrome (defects of bone, ear, nail and nose, accompanied by hepatosplenomegaly), murray - puretic - drescher syndrome (multiple dental hyaline tumors), rutherfurd syndrome (corneal dystrophy), cowden syndrome (multiple hamartomas) and cross syndrome (hypo pigmentation with athetosis). in this case, a thorough evaluation of the patient revealed no association with any of the clinical features associated with the above syndromes. the present case reported as hgf. this case is diagnosed as gingival fibromatosis due to hereditary and autosomal dominant with occurrence of enlargement in successive generation from mother to son and support with other evidence by histopathological findings. various types of treatment modalities have been employed for the excision of the enlarged gingival tissues, including of conventional surgery, electrosurgery, an apically positioned flap and lasers. in the present case, reports about recurrence rates are contradictory so the postoperative long - term benefit of periodontal surgery can not be predicted. some reports in severe cases of hgf, full - mouth - tooth clearance has been advocated, and they suggest that the condition does not recur if the teeth have been extracted. in the literature, one case has been reported that there is less chance of recurrence if the gingivectomy is delayed until the permanent dentition is in place. in our case report, there was no recurrence of the disease even after 2 years follow - up. | hereditary gingival fibromatosis (hgf) is a rare hereditary condition characterised by slow, progressive, nonhemorrhagic, fibrous enlargement of gingiva caused by increase in sub - mucosal connective tissue component. this paper presents a case report of a 14-year - old male suffering from hgf with positive family history. after through clinical examination, routine blood investigation was advised. all the parameters were within normal physiological limits. surgical excision of enlarged gingival mass was planned after meticulous scaling and root planning. patient was recalled 1-week after surgery. postoperative healing was good and desired crown lengthening was achieved with significant improvement in speech and masticatory problems. there was no recurrence of the disease even after 2 years follow - up. |
hiv is a fatal, sexually transmitted, or blood - borne disease affecting the health, cognitive abilities, and general well - being of the individuals including children and adolescents in particular. neurological and cognitive deficits have been documented in up to 80% of hiv - infected children. development delays and neurologic complications, such as encephalopathy, have been identified in children who are infected with hiv since the earliest descriptions of the disease. hiv infects a variety of cell types in the brain leading to damage of the central nervous system (cns). neuropsychological tests serve as indirect measures to assess cns functioning along with the more direct measures such as computed tomography and magnetic resonance imaging scans. studies of hiv - infected children have reported that cognitive deficits are observed in the areas of language and motor skills, verbal and memory functioning, visual - spatial integrative ability, and executive functions. the recent development of highly active antiretroviral therapy (art) has dramatically prolonged the survival of hiv - infected children. as hiv disease has moved from being a fatal to a chronic illness, cognitive, neurologic, and behavioral functioning of hiv - infected children has become a major concern. to date, not much studies of neuropsychological functioning of hiv - infected children who receive art has been done in india. the sample for the present study comprised of 20 hiv - infected children who are inmates of a home exclusively for hiv - infected children in chennai. the home which is run by a ngo is a special center, where they take care of hiv - infected children who belonged to low socioeconomic background. majority of the parents of these children are alive and 80% of them visit their children once in 3 months. after obtaining prior permission for the home authorities, all the parents and the local guardians of the inmates were contacted and the written informed consent was obtained from them after explaining the purpose of the study. all the children in the hiv - infected group were assessed individually for their intelligence using malin 's intelligence scale for indian children (ii). the inclusion criteria were (a) diagnosis of hiv infection made by the physician, which was confirmed with laboratory tests for hiv, (b) children who are receiving art, (c) children up to 12 years of age of both sexes, (d) children with hiv infection through maternal transmission, and (e) children with an intelligence quotient (iq) of 90 and above. the exclusion criteria were children with a history of severe developmental disorder (e.g., autism) or significant psychiatric disorder or comorbid medical disorder not related to hiv. out of 30, 20 hiv - infected children who fulfilled the inclusion and exclusion criteria were included in the study group. children with an iq of 90 and above were only included in the study, because hiv infection affects the brain development in young children and causing intellectual deterioration. the children with hiv infection were matched with equal number of normal controls through purposive sampling. the subjects of the control group were selected from the schools in chennai after obtaining permission from the school authorities and the written informed consent was obtained from the parents after the purpose of the study was explained to them. the children in the control group who had history of medical, psychiatric, or neurological disorders such as epilepsy that would affect the brain were excluded. those who have obtained an iq of 90 and above were included in the control group. all the children in both the groups were evaluated for their cognitive functions using a comprehensive neuropsychological battery. the battery includes nine neuropsychological tests, which measures nine neuropsychological domains namely attention, language, visual memory, verbal learning and memory, visuoperceptual functions, visuospatial functions, visuomotor functions, fine motor performance, and executive functions. these tests have been used by various authors for assessing various neuropsychological functions and found to be sensitive. the tests were administered according to standard procedure given in the manual in two sessions over a period of 2 consecutive days. the duration of each session was 2 h. the tests selected for assessing the cognitive functions are as follows : attentiondigit span (misic)language vocabulary (misic)visual memorybenton visual retention testverbal learning and memoryrey auditory verbal learning testvisuoperceptual functionspicture completion (misic)visuospatial functionsblock design (misic)visuomotor functionsobject assembly (misic)fine motor performance coding (misic)executive functionstrail making test (part - b). language vocabulary (misic) benton visual retention test verbal learning and memory rey auditory verbal learning test visuoperceptual functions picture completion (misic) visuospatial functions object assembly (misic) fine motor performance coding (misic) trail making test (part - b). the sample for the present study comprised of 20 hiv - infected children who are inmates of a home exclusively for hiv - infected children in chennai. the home which is run by a ngo is a special center, where they take care of hiv - infected children who belonged to low socioeconomic background. majority of the parents of these children are alive and 80% of them visit their children once in 3 months. after obtaining prior permission for the home authorities, all the parents and the local guardians of the inmates were contacted and the written informed consent was obtained from them after explaining the purpose of the study. all the children in the hiv - infected group were assessed individually for their intelligence using malin 's intelligence scale for indian children (ii). the inclusion criteria were (a) diagnosis of hiv infection made by the physician, which was confirmed with laboratory tests for hiv, (b) children who are receiving art, (c) children up to 12 years of age of both sexes, (d) children with hiv infection through maternal transmission, and (e) children with an intelligence quotient (iq) of 90 and above. the exclusion criteria were children with a history of severe developmental disorder (e.g., autism) or significant psychiatric disorder or comorbid medical disorder not related to hiv. out of 30, 20 hiv - infected children who fulfilled the inclusion and exclusion criteria were included in the study group. children with an iq of 90 and above were only included in the study, because hiv infection affects the brain development in young children and causing intellectual deterioration. the children with hiv infection were matched with equal number of normal controls through purposive sampling. the subjects of the control group were selected from the schools in chennai after obtaining permission from the school authorities and the written informed consent was obtained from the parents after the purpose of the study was explained to them. the children in the control group who had history of medical, psychiatric, or neurological disorders such as epilepsy that would affect the brain were excluded. those who have obtained an iq of 90 and above were included in the control group. all the children in both the groups were evaluated for their cognitive functions using a comprehensive neuropsychological battery. the battery includes nine neuropsychological tests, which measures nine neuropsychological domains namely attention, language, visual memory, verbal learning and memory, visuoperceptual functions, visuospatial functions, visuomotor functions, fine motor performance, and executive functions. these tests have been used by various authors for assessing various neuropsychological functions and found to be sensitive. the tests were administered according to standard procedure given in the manual in two sessions over a period of 2 consecutive days. the duration of each session was 2 h. the tests selected for assessing the cognitive functions are as follows : attentiondigit span (misic)language vocabulary (misic)visual memorybenton visual retention testverbal learning and memoryrey auditory verbal learning testvisuoperceptual functionspicture completion (misic)visuospatial functionsblock design (misic)visuomotor functionsobject assembly (misic)fine motor performance coding (misic)executive functionstrail making test (part - b). language vocabulary (misic) benton visual retention test verbal learning and memory rey auditory verbal learning test visuoperceptual functions picture completion (misic) visuospatial functions object assembly (misic) fine motor performance coding (misic) trail making test (part - b). a total of 45% were in the age group of 8 - 9 years, while 55% were in the age group of 10 - 12 years in both hiv - infected and hiv - unexposed groups. in terms of educational level, 90% versus 60% in primary and 10% versus 40% were studying in secondary school. regarding the status of hiv+, all the hiv - infected children were under the care of home authorities, whereas all the children in the hiv - unexposed group were under the care of their parents. parental education, age, and family income were lower in children with hiv infection than those in the control group and the details are presented in table 2. clinical status of hiv - infected children : all the children are taking art for 2 years with a good adherence and they are nutritionally adequate at the time of induction into the study. sociodemographic variables of the participants family characteristics of the two groups the neuropsychological evaluation findings of the two groups are presented in table 3. the t - test indicated significant group differences on the measures of attention, language, verbal learning and memory, visuomotor functions, fine motor performance, executive functions but not on visual memory, visuoperceptual, and visuospatial functions. comparison of scores for the two groups revealed that the hiv - infected group was more impaired on several neuropsychological measures than the control group. children with hiv infection are at high - risk for developing neurodevelopment and cognitive impairments. several studies have demonstrated neurodevelopmental impairment among hiv - infected children as early as in infancy, while other studies have demonstrated a neurocognitive deficit in hiv - infected preschool and school - aged children. our study demonstrated that hiv - infected children have a mean iq of 96 which was in the average range (the mean iq of control group was 103) and this finding is similar to other studies. however, they performed poorly on several neuropsychological measures such as attention, language, verbal learning and memory, visuomotor functions, fine motor performance, and executive functions when compared with control group. the poor neurocognitive functions in hiv - infected children might be explained by the following reasons. first, hiv infection may have a direct effect on neurodevelopment during the first few years of life, which is the time of rapid brain development occurs or it may have an indirect effect through recurrent infections or opportunitistic, leading to poorer general health. a tanzanian study of hiv - infected infants reported that infants with in utero infection had higher risk of delayed mental functioning compared to infants who were diagnosed at a later stage of life. another factor that might contribute to poor neurocognitive outcome may be due to low socioeconomic background of hiv - infected children which could lead to a variety of obstacles such as inadequate food and lack of time for cognitively stimulating activities may have negative impact on the neurocognitive development. environmental factors such as low level of maternal education, changes in care givers, and poverty which are likely to affect hiv - infected children adversely. many studies have observed that poverty and low socioeconomic status have been linked to poor outcomes on neurocognitive testing and similar findings are observed in the present study also. family structure and child rearing plays an important role in the neurodevelopment of the child. in our study, all the hiv - infected children are under the care of home authorities due to poverty in their families, while the normal controls lived with their parents. this finding is similar to a u. s. study, which reported that children living with their biological parents were less likely to manifest conduct or learning problems when compared to those living with others. in our study, the cognitive functions among hiv - infected children were impaired despite they received the art for 2 years with undetectable viral load and normal cd4 cell. this is similar to a south african study which reported that hiv - infected children with a median age of 5 years were not shown improvement in neurocognitive functions after 6 months of art. this is the first study from this part of the country to assess the neurocognitive status of hiv - infected children. the findings in this study indicate that the hiv - infected children experience difficulties with their daily living and social functioning when compared with peers of the same age without a chronic illness. there is a need for future research to develop strategies to improve neuropsychological functioning in this population. vertically transmitted hiv - infected children were only studied leaving the other subtypes of hiv transmission. this is the first study from this part of the country to assess the neurocognitive status of hiv - infected children. the findings in this study indicate that the hiv - infected children experience difficulties with their daily living and social functioning when compared with peers of the same age without a chronic illness. there is a need for future research to develop strategies to improve neuropsychological functioning in this population. regarding the limitations, children who are living in the home were only studied. vertically transmitted hiv - infected children were only studied leaving the other subtypes of hiv transmission. cognitive deficits have been observed in the domains of attention, language, verbal learning and memory, visuomotor functions, fine motor performance, and executive functions. | background and objectives : children infected with hiv are at risk for significant neurological and neuropsychological problems. this study is aimed at identifying cognitive deficits in hiv - infected children and to compare them with equal number of normal controls.materials and methods : twenty children with hiv infection who are currently on antiretroviral therapy were recruited. they were assessed for their intelligence using malin 's intelligence scale for indian children and also evaluated for their cognitive abilities with a comprehensive neuropsychological battery. they were matched with equal number of normal controls.results:hiv-infected children have shown substantial impairments in the domains of attention, language, verbal learning and memory, visuomotor functions, fine motor performance, and executive functions.conclusion:hiv-infected children have average intelligence, but they performed poorly on several neuropsychological measures. |
during the recent years, the effective treatments have been found for emotional disorders especially anxiety and depression (1). norton and hope (2) noted that this problem was related to the dissemination of effective treatments and accessibility to effective treatments. the cognitive behavioral approaches have published many therapeutic manuals and protocols for emotional disorders and their effectiveness have been confirmed by many studies (1). using these treatments face to two great obstacles including lack of access to these treatments by the clients and the lack of applying these treatments through therapists (3). lifetime prevalence of anxiety disorders and depressive disorders has been reported respectively, 29 % and 21% (4). these disorders associated with considerable burden on the societies directly and indirectly (5, 6). comorbidity between these two disorders has been reported about 40% 80% and they have high comorbidity with other disorders such as drug abuse. these chronic disorders have the risk of relapse, recurrent and low chance for improvement without treatment (7). although current cognitive therapies have been found successful in improving emotional disorders, they have some limitations such as loss of response to the current therapies among patients (8). in addition however, different therapeutic instructions have been published for disorders, training and obtaining skills in each of them need to spend money and time. moses and barlow (8) suggested that the solution for these problems was to create a unified therapeutic approach for a range of emotional disorders. unified approach or cognitive - behavioral transdiagnostic therapy is a therapy that has the same therapeutic principals for all emotional disorders without any adaptation to the especial disorder (9). remarkable attention has been devoted to the transdiagnostic therapy that has been newly practiced (1012). the transdiagnostic therapies have been shown their effectiveness in the treatment of wide variety of emotional disorders (9, 12, 13). in the area of cognitive behavioral transdiagnostic treatments, the current study was examined the effectiveness of transdiagnostic group therapy in reduction of sub - clinical symptoms of anxiety and depression and compares it with classical cognitive group therapy. this study was a comparative or relative efficacy study that compared two therapeutic models with each other based on the outcomes scales instead of comparison of the one therapy with a control group without any intervention or in the waiting list. as we know, the cognitive therapy is a common and useful intervention (14) and a standard treatment was considered for one of the control groups. forty one participants (in tow groups) were enough to gain appropriate statistical power for testing hypotheses. people with inclusion and exclusion criteria were selected and assigned randomly to experimental and control groups. this study was carried out in akhavan hospital, tehran iran during may and june 2011. the inclusion criteria included as anxiety and depressive symptoms based on the cut - off of 18 and higher in the depression subscale and score of 16 and higher in the anxiety subscale of the depression, anxiety, and stress scale (dass-42 ; 20). these cut - offs were determined based on the lovibond and lovibond s (15) method that used percentage points for determination of severity levels. according to this matter, the percentage points of 78 and higher formed low level of severity. the exclusion criteria was based on the axis i disorders, including anxiety disorders, depression, psychosis, psychosomatic and drug abuse, by using the anxiety disorders interview schedule for dsm - iv (adis - iv). participants who obtained a score equal to or greater than 18 in depression subscale and 16 in subscale of anxiety evaluated based on the anxiety disorder interview schedule for dsm - iv in order to determine inclusion and exclusion criteria. then, we explained the study, its conditions, and moral status to them and got the informed consent, after that the study instruments were administrated and readministered after treatment sessions. the study administrated on the students population of universities across tehran city. among all prepared participants, only data of 33 students analyzed after the end of invention. 16 students assigned at control group (cognitive group therapy), and 17 students in transdiagnostic group. the mean (standard deviation) age of the participants was 22.6 (sd=3.1). results showed that experimental group s age with mean (standard deviation) of 22.88 (1.62) and control group mean (standard deviation) of 23.75 (2.75) statistically not significant (m= 0.87 ; sd= 0.77 ; t= 1.12 ; p 0.001). the cognitive group therapy was considered as a control group and it caused a significant change in all variables. the results illustrated that the cognitive group therapy could reduce significantly symptoms of depression, anxiety, and stress., the cognitive group therapy provided significant changes (t=7.36, p > 0.001) and improved general performance among group. the comparison of two groups results are shown in table 4 and explained that there was not any significant difference between transdiagnostic and cognitive therapy in non of the variables expect anxiety (f=9.22, p=0.005). in other words, the transdiagnostic group therapy illustrated a significant difference in anxiety reduction not cognitive group therapy. there was not a significant difference between variables in t - test comparing the means. thus, there was not any significant difference between two groups on the symptoms of anxiety, depression, and general performance in pre test. the results of dependent t test have been displayed in table 2 that shows transdiagnostic group therapy was significant in all variables. in fact, it was significant in depressive symptoms reduction (t=13.09), anxiety (t=13.75) and stress (t=6.48) at the level of 0.001. moreover, it led to general performance improvement in the group that was measured by work and social adjustment scale (t=6.93, p>0.001). the cognitive group therapy was considered as a control group and it caused a significant change in all variables. results are showen in table 3. the results illustrated that the cognitive group therapy could reduce significantly symptoms of depression, anxiety, and stress. these results are statistically significant respectively (t=9.08, t=8.04, t=7.51 ; p>0.001). in addition, the cognitive group therapy provided significant changes (t=7.36, p > 0.001) and improved general performance among group. the comparison of two groups results are shown in table 4 and explained that there was not any significant difference between transdiagnostic and cognitive therapy in non of the variables expect anxiety (f=9.22, p=0.005). in other words, the transdiagnostic group therapy illustrated a significant difference in anxiety reduction not cognitive group therapy. the present study evaluates the effectiveness of transdiagnostic group therapy and compares it to standard cognitive group therapy in reduction of anxiety and depressive symptoms and improvement of general performance. the findings confirm study hypothesis based on the effectiveness of transdiagnostic group therapy in reduction of emotional components and improvement of general performance. in comparing post - test scores for two groups, a significant difference generally, we can conclude that transdiagnostic group therapy can reduce anxiety and depression same as cognitive therapy. however, transdiagnostic group therapy is superior to cognitive therapy in reduction of anxiety symptoms, because the transdiagnostic approach is a unified (8) and reduces the anxiety by techniques such as emotional and interoceptive exposure (25). (12) in the primary study in order to construct and evaluate the primary protocol found that transdiagnostic treatment could be effective for a range of emotional disorders. boisseau and his colleagues reported the similar results on a case study (26). indicative prevention based on cognitive approach could reduce effectively symptoms of depression and anxiety among students population (27). generally, the transdiagnostic approach have benefits in the treatment and prevention of mental disorders including the dissemination and accessibility, high capacity in applying for a group, their application for most of emotional disorders and relapse prevention. clark and taylor (28) believed that there was no need to replace transdiagnostic cognitive behavioral therapy with disorder specific therapies. although, they declared that transdiagnostic therapies could be applied as a complimentary treatment for empirical supported treatments. in other words, transdiagnostic therapies can be used before specific therapies in order to train general skills and coping with problems. clark and taylor (28) explained three major problems for specific cognitive behavioral therapies : (a) failure to demonstrate a significant additive advantage of cognitive ingredients over purely behavioral interventions, (b) difficulty in establishing cognitive mediation, and (c) neglect of common or shared features across disorders therapeutic programs based on transdiagnostic approach were effective, but it was necessary to investigate inclusion and exclusion criteria, severity of target disorders and multiple comorbidities in the therapy (29). moreover, therapeutic protocols should have less complexity. for example, selective attention to treat is most likely to be experienced by individuals with anxiety disorders but its content can be different in a variety of anxiety disorders. the transdiagnostic therapies are process oriented and the main diagnosis is not important in the therapies. although, there are some limitations for this approach, more studies should be applied to answer the questions related to this novel approach (28). there may be limitations with respect to the generality of the findings because of sample selection that was among college students. on the other hand, drop out in the sample had been occurred because of some reasons, in fact, 20% drop out took placed in the session s procedure. there were some reasons for dropping out such as : educational problems and transportation problems. applying both methods of interventions by the researcher caused individual biases that were other research limitations. recommending the future studies apply transdiagnostic interventions in the selective and universal prevention research. in addition, the study of this therapeutic model can be conducted in a variety of samples such as children, elderly and in different levels like school, family, and multi level. we believed that protective, mediator and moderator factors investigation could be done for emotional disorders. furthermore, a long - term follow up for outcomes of transdiagnostic group therapy is suggested. the study of transdiagnostic therapy mechanism and its effectiveness in the other disorders and problems is necessary for the procedure dismantling studies (12). finally, it seems that transdiagnostic interventions need to more investigations to compare specific intervention and the other disorders with this approach. ethical issues (including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc) have been completely observed by the authors. | background : the cognitive behavioral interventions based on the transdiagnostic approach for emotional disorders have received useful empirical supports in recent years. most of the researches on this area have been conducted without any control group. moreover, little information about comparative effectiveness has reported. the current study was compared transdiagnostic group therapy with classical cognitive group therapy.methods:thirty three collages students with anxiety and depressive symptoms participated in eight two - hour sessions in akhavan hospital, tehran, iran during may and june 2011. the results were analyzed by the depression, anxiety, and stress scale, and work and social adjustment scale in pre and post intervention.results:both groups showed the significant difference in research variables pre and post test. however, there was no significant difference in the results analysis using acovas except for anxiety symptoms.conclusions:the effectiveness of transdiagnostic group therapy was confirmed in reducing anxiety and depressive symptoms. implications of the study are discussed. |
the infracolic disease is resectable but the supracolic disease also involving the spleen is not and thus needle biopsy and primary chemotherapy should be considered. two cases of stage iiic ovarian cancer with calcified omental cakes (arrows) which mimic large and small bowel. | the recent rdog studies have confirmed the value of ct in the management of ovarian cancer. however we now know that metastases to the ovary may exactly mimic primary cancer. this demands a firm histological diagnosis when surgery is not planned and especially with a history of breast and gastrointestinal tract cancer. ct guided needle biopsy can provide this. |
primary sclerosing cholangitis (psc) is a chronic and progressive disease of the liver. patients with psc frequently have asymptomatic and anicteric cholestasis and many of them develop progressive biliary strictures, which lead to recurrent cholangitis, biliary cirrhosis and end - stage liver disease. these complications along with symptoms of the disease, such as chronic fatigue, pruritus, impaired memory and concentration, excessive daytime sleepiness and symptoms of frequently coexisting chronic inflammatory bowel disease (ibd) or liver cirrhosis may substantially influence patients health - related quality of life (hrqol) (1, 2). it is widely accepted that evaluation of hrqol as a multidimensional construct encompassing physical and cognitive capabilities, functional behavior, emotional status and psychosocial adjustment, plays an important role in the holistic care of patients with chronic liver diseases (3, 4). osteoporosis is a frequent comorbidity in chronic liver diseases (1), leading to fragility fractures associated with significant morbidity, mortality and further impairment of hrqol (5). in contrast to other chronic liver conditions, clinical consequences of osteoporosis in psc have been less intensively studied. particularly, to date, there have been no previous reports evaluating the fracture rate and the impact of fractures on hrqol outcomes in patients with psc. the aim of this study was to assess bone mineral density (bmd) measured by dual - energy x - ray absorptiometry (dxa), physical activity and incidence of fragility fractures in patients with psc. this study was performed on 33 patients with psc (11 females, 22 males) aged 35.3 13.38 years (ranged 19.5 to 69.4 years). psc was diagnosed using the european association for the study of the liver (easl) criteria as : 1) elevated serum markers of cholestasis ; and 2) typical bile duct changes in magnetic resonance imaging or endoscopic cholangiography (6). we included only those patients who had no other acute or chronic medical conditions that required pharmacological treatment or might influence hrqol outcomes. we excluded patients with a history of rapid weight changes within the last 12 months. among the included patients, six had an established liver cirrhosis and 20 had ibd, including 12 cases with ulcerative colitis, two with crohn s disease and six with non - classified colitis. the bowel disease was in remission in all patients. in all subjects, we measured weight and height. based on available medical reports, we collected the following data on the past fragility fractures, location of the fracture and its severity, time since diagnosis of psc, x - ray imaging and type of trauma. fragility fractures were defined as fractures of the distal forearm, neck of the femur, rib and vertebral body occurred as a result of a minimal trauma, such as a fall from a standing height or less or without identifiable trauma. blood samples were obtained to assess aminotransferases, alkaline phosphatase and bilirubin after an overnight fast from each study participant. the study protocol was approved by the ethics committee of the pomeranian medical university and conformed to the ethical guidelines of the 1975 declaration of helsinki (6 revision, 2008). bone mineral density (bmd) was measured in the lumbar spine (l1 - l4) and the hip by dual - energy x - ray absorptiometry (dxa) (ge lunar prodigy ; madison, wi ; usa ; software encore version 14.1) using automatic scan modes. osteopenia and osteoporosis were diagnosed according to the world health organization (who) criteria (7). bmd values were expressed in g / cm as well as z - scores (standard deviation from the mean bmd for normal, age - matched, gender - specific subjects) and t - scores (standard deviation from the mean bmd for normal young individuals). bmd values were compared with expected normal bmd and adjusted for gender and ethnicity using standard values for normal population provided by the manufacturer s reference database. additionally, we measured total body fat (bf) and lean mass from the total body scan using an automatic scan mode. the coefficients of variation of lumbar spine, hip and total body measurements were 0.9%, 1.5% and below 1.0%, respectively. three questionnaires were used for data collection : 1) the medical outcome study short form-36 (sf-36) ; 2) pbc-40 ; and 3) pbc-27. the medical outcomes study short form 36 (sf-36) is a widely used and validated generic questionnaire to measure hrqol in various populations with a wide variety of medical conditions (8, 9). the disease - specific instruments, pbc-40 and pbc-27 assess the symptoms of chronic cholestasis such as fatigue, excessive daytime sleepiness, pruritus and impaired memory and concentration (3, 4). sf-36 includes 36 items divided into eight domains of physical health (physical functioning, role limitation - physical, bodily pain and general health) and mental health (vitality, social functioning, role limitation - emotional and mental health). scale scores ranged from 0 (denoting the most impaired hrqol) and 100 (ideal well - being) and two summary scores ; the physical component score (pcs) and mental component score (mcs) were also calculated. pbc-40 is nowadays commonly used in the assessment of hrqol in patients with primary biliary cirrhosis (3). six domains of pbc-40 relate to fatigue, emotional, social and cognitive functions, general symptoms and itching. answers are marked on a five - point likert scale (from 1 = never to 5 = always), with higher scores denoting greater impact of symptoms and poorer hrqol. possible range of each domain was symptom domain 7 - 35, itching 3 - 15, fatigue 11 - 55, cognitive 6 - 30 and social and emotional functions 13 - 65 points (10). pbc-40 was also used in hrqol evaluation in patients with psc (11). pbc-27 is an alternative structure of the hrqol questionnaire for pbc, which appears to be simpler and equivalently sufficient in detecting the impact of pbc on patients well - being. twenty seven items are grouped into the following domains : other symptoms (possible range : 3 - 15 points), dryness (2 - 10 points), itching (3 - 15 points), fatigue (8 - 40 points), cognitive (5 - 25 points), emotional (3 - 15 points) and social (3 - 15 points) functions are evaluated on the same five - point scale as pbc-40 (4). the international physical activity questionnaire (ipaq) was used as a self - report measure of habitual physical activity, with a 7-day recall of physical activity within a month. ipaq consists of 27 questions regarding four life domains : physical activity associated with the occupation performed, work at home and around the house, moving to various places and mobility during free time devoted to recreation, playing games, sports, tourism or other muscular work and additional time spending in the sitting position. total physical activity as measured by ipaq is categorized as low (below 600 met minute / week), moderate (from 600 to 3000 met minute / week) and vigorous (above 3000 met minute / week). data are presented as means standard deviation (sd) or numbers (proportion) of patients with a condition. shapiro - wilk test was used to test for normality, while f - test (snedecor) and brown - forsythe test were used to assess the homogeneity of variances. in the case of a normal distribution and equal variances, means were compared by student s t - test ; otherwise, non - parametric methods were used. the chi - squared test for independence with a yates correction was used to analyze qualitative variables. the relationship between pairs of quantitative variables this study was performed on 33 patients with psc (11 females, 22 males) aged 35.3 13.38 years (ranged 19.5 to 69.4 years). psc was diagnosed using the european association for the study of the liver (easl) criteria as : 1) elevated serum markers of cholestasis ; and 2) typical bile duct changes in magnetic resonance imaging or endoscopic cholangiography (6). we included only those patients who had no other acute or chronic medical conditions that required pharmacological treatment or might influence hrqol outcomes. we excluded patients with a history of rapid weight changes within the last 12 months. among the included patients, six had an established liver cirrhosis and 20 had ibd, including 12 cases with ulcerative colitis, two with crohn s disease and six with non - classified colitis. the bowel disease was in remission in all patients. in all subjects, we measured weight and height. based on available medical reports, we collected the following data on the past fragility fractures, location of the fracture and its severity, time since diagnosis of psc, x - ray imaging and type of trauma. fragility fractures were defined as fractures of the distal forearm, neck of the femur, rib and vertebral body occurred as a result of a minimal trauma, such as a fall from a standing height or less or without identifiable trauma. blood samples were obtained to assess aminotransferases, alkaline phosphatase and bilirubin after an overnight fast from each study participant. the study protocol was approved by the ethics committee of the pomeranian medical university and conformed to the ethical guidelines of the 1975 declaration of helsinki (6 revision, 2008). bone mineral density (bmd) was measured in the lumbar spine (l1 - l4) and the hip by dual - energy x - ray absorptiometry (dxa) (ge lunar prodigy ; madison, wi ; usa ; software encore version 14.1) using automatic scan modes. osteopenia and osteoporosis were diagnosed according to the world health organization (who) criteria (7). bmd values were expressed in g / cm as well as z - scores (standard deviation from the mean bmd for normal, age - matched, gender - specific subjects) and t - scores (standard deviation from the mean bmd for normal young individuals). bmd values were compared with expected normal bmd and adjusted for gender and ethnicity using standard values for normal population provided by the manufacturer s reference database. additionally, we measured total body fat (bf) and lean mass from the total body scan using an automatic scan mode. the coefficients of variation of lumbar spine, hip and total body measurements were 0.9%, 1.5% and below 1.0%, respectively. three questionnaires were used for data collection : 1) the medical outcome study short form-36 (sf-36) ; 2) pbc-40 ; and 3) pbc-27. the medical outcomes study short form 36 (sf-36) is a widely used and validated generic questionnaire to measure hrqol in various populations with a wide variety of medical conditions (8, 9). the disease - specific instruments, pbc-40 and pbc-27 assess the symptoms of chronic cholestasis such as fatigue, excessive daytime sleepiness, pruritus and impaired memory and concentration (3, 4). sf-36 includes 36 items divided into eight domains of physical health (physical functioning, role limitation - physical, bodily pain and general health) and mental health (vitality, social functioning, role limitation - emotional and mental health). scale scores ranged from 0 (denoting the most impaired hrqol) and 100 (ideal well - being) and two summary scores ; the physical component score (pcs) and mental component score (mcs) were also calculated. pbc-40 is nowadays commonly used in the assessment of hrqol in patients with primary biliary cirrhosis (3). six domains of pbc-40 relate to fatigue, emotional, social and cognitive functions, general symptoms and itching. answers are marked on a five - point likert scale (from 1 = never to 5 = always), with higher scores denoting greater impact of symptoms and poorer hrqol. possible range of each domain was symptom domain 7 - 35, itching 3 - 15, fatigue 11 - 55, cognitive 6 - 30 and social and emotional functions 13 - 65 points (10). pbc-40 was also used in hrqol evaluation in patients with psc (11). pbc-27 is an alternative structure of the hrqol questionnaire for pbc, which appears to be simpler and equivalently sufficient in detecting the impact of pbc on patients well - being. twenty seven items are grouped into the following domains : other symptoms (possible range : 3 - 15 points), dryness (2 - 10 points), itching (3 - 15 points), fatigue (8 - 40 points), cognitive (5 - 25 points), emotional (3 - 15 points) and social (3 - 15 points) functions are evaluated on the same five - point scale as pbc-40 (4). the international physical activity questionnaire (ipaq) was used as a self - report measure of habitual physical activity, with a 7-day recall of physical activity within a month. ipaq consists of 27 questions regarding four life domains : physical activity associated with the occupation performed, work at home and around the house, moving to various places and mobility during free time devoted to recreation, playing games, sports, tourism or other muscular work and additional time spending in the sitting position. total physical activity as measured by ipaq is categorized as low (below 600 met minute / week), moderate (from 600 to 3000 met minute / week) and vigorous (above 3000 met minute / week). data are presented as means standard deviation (sd) or numbers (proportion) of patients with a condition. shapiro - wilk test was used to test for normality, while f - test (snedecor) and brown - forsythe test were used to assess the homogeneity of variances. in the case of a normal distribution and equal variances, means were compared by student s t - test ; otherwise, non - parametric methods were used. the chi - squared test for independence with a yates correction baseline characteristics of patients and comparisons between the subjects with and without fractures are summarized in table 1. in psc patients, mean bmd of hip was 0.97 + 0.183 g / cm and of spine was 1.11 + 0.161 g / cm. in comparison with males, females had lower hip bmd (1.02 0.015 vs. 0.88 0.21 g / cm ; p = 0.043) and comparable spine bmd (1.12 0.18 vs. 1.07 0.08 g / cm). in all subjects, mean bmd values were moderately but significantly lower in psc than expected in a matched young healthy population of the same gender and ethnicity using the reference values provided by the manufacturer s database (p = 0.011 in the lumbar spine and p = 0.044 in the hip). based on the who densitometric criteria (7), one patient (3%) had osteoporosis and 9 (27.3%) osteopenia either in the lumbar spine or hip. the incidence of a lowered bmd at any site (z - score below 1.0 sd) was not associated with presence of ibd or liver cirrhosis (p > 0.05). as many as 75% of patients declared low to moderate physical activity (below 3000 met / minute / week). in the sf-36 assessment, patients marked the lowest scoring in the general health domain and the highest in the role emotional domain. in both pbc-40 and pbc-27, patients frequently marked fatigue as well as social and emotional domains as affecting their hrqol. five of the patients who had experienced fractures also had ibd, while none of them had liver cirrhosis. three subjects with fractures had the lumbar spine and hip bmd t - scores between 1.0 and 2.5 sd ; the remaining had normal bmd.we did not find significant differences in gender distribution, age, duration of psc, bmd measurement, the incidence of ibd and liver cirrhosis and body composition between individuals with and without fractures. on the other hand, in patients who had experienced fragility fractures, mean scores in role functioning, general health, vitality and pcs domains in sf-36 were lower than subjects without fractures. a similar trend was also observed in respect of the mcs domain. in multiple regression analysis, prior fractures adjusted for gender, age and duration of psc were significantly associated with lower pcs (= -0.506 ; 95% ci : from -0.856 to -0.155 ; p = 0.006) and mcs scores (= -0.416 ; 95% ci : from -0.791 to -0.039 ; p = 0.031). mcs and pcs (except for itching) were inversely correlated with all domains tested by pbc-40 and pbc-27 (table 2). however, in multiple regression analysis, symptoms and fatigue scores and prior fractures were inversely associated with mcs (= -7.19 ; 95% ci : from -22.2 to -10.1 ; p = 0.007), but not pcs scores (p = 0.124). interestingly, prior fractures were not associated with a lower bmd below 1.0 sd in either sites (r = 0.104 ; p = 0.144). in both pbc-40 and pbc-27, patients with fractures reported higher scores in symptoms and fatigue domains than subjects without fractures. moreover in pbc-27, patients with fractures reported higher scores in dryness and emotional domains. as shown in table 3, bmd values did not correlate with age, bmi, duration of psc, fat mass and total physical activity. there was a positive correlation between lean mass (but not fat mass) and hip bmd. lean mass was also positively correlated with total physical activity (r = 0.424 ; p = 0.03). in the current study on young and middle - aged subjects with psc lasting for approximately three years, we found a high rate of fragility fractures and moderately lower bmd values in the lumbar spine and hip. in several studies performed on patients with psc, similar (13) or even higher rates of low bmd, reaching 50% of patients (14, 15) were observed. inconsistent to our results, these studies suggested that low bone mass in psc might be associated with advanced age, low bmi, severity of psc or long duration of ibd (14). however, patients participating in those studies were older and had a longer duration of psc than our population. this is consistent with other reports indicating more advanced metabolic bone disease in psc associated with end - stage liver disease evaluated for transplantation (16) or with advanced liver cirrhosis (17). additionally, advanced age and low bmi are well - known risk factors for reduced bmd in general population. our results suggested that even young patients with psc with a relatively short duration of the disease are at risk of osteoporosis. previous studies demonstrated that ibd per se is associated with an increased prevalence of bone demineralization and low energy fractures (2). however, ibd associated with psc usually follows a more quiescent course than patients without psc (18 - 20). in patients with psc, angulo. (14) found a 3.6-fold increased risk for osteoporosis in cases with duration of ibd more than 19 years. moreover, in their study the rate of bone loss per year was significantly associated with duration of ibd. hence, we speculate that in patients with a relatively short duration of disease, ibd might be a potential risk factor, but not yet a robust predictor of osteoporosis, although the relative impact of ibd - associated factors and ibd - specific inflammation on bone health is still uncertain (2). we found a positive correlation between hip bmd and lean mass. recent meta - analysis (21) and observational studies in men (22) and women (23) demonstrated that lean tissue contributes more to bone mass in the femur than fat tissue during life in healthy population, and now, as suggested by this study possibly also in psc. this finding underlines the concept that in psc, in which bmd is frequently lower, physical activity is an important component in the maintenance of bone loss and prevention of osteoporosis. because lean tissue is composed mainly of skeletal muscles, it influences bone directly via mechanical stimuli and myokines (24). therefore, a low lean mass seems to be an important risk factor of vertebral and hip fractures, ranking in importance alongside age (21). in line with this concept, we found positive associations between lean mass, hip bmd and the intensity of physical activity. in the current study, there have been no previous reports evaluating fracture rate in psc. in postmenopausal women with primary biliary cirrhosis, regardless of the small sample size, our results suggested that this prevalence may be even higher in psc. we found that patients with fractures had significantly lower sf-36 role functioning, general health, vitality and pcs scores than those without fractures ; a similar trend was also observed regarding mcs domain. moreover, in multiple regression analysis, prior fractures adjusted for gender, age and duration of psc were significantly associated with lower pcs and mcs scores. poorer mcs outcomes in patients with fractures seem to be, at least partially, associated with higher prevalence of significant subjective symptoms, like systemic symptoms and fatigue, as assessed by the pbc-40 and pbc-27. on the other hand, it has been suggested that fatigue does not seem to be a specific symptom in psc compared with the general population (26). in a study by angulo (14) comprising 237 participants, aged over 54 years, bmi lower than 24 kg / m and duration of ibd longer than 19 years, correlated with the presence of metabolic bone disease. on the other hand, campbell. (13) showed that low bone density can not be predicted by severity of liver disease in psc patients listed for liver transplantation or with hepatic decompensation. earlier reports pointed more advanced bone disease in psc patients with end - stage liver disease evaluated for liver transplantation (16) and liver cirrhosis generally was a major risk factor of bone disease in patients evaluated for liver transplantation (17). however, the natural history of psc is associated with impairment in hrqol as shown by benito de valle. (26) and more recently by our group (27). age was negatively related to all sf-36 physical domain scores and physical component summary (pcs) score and positively to sf-36 mental health, role emotional and mental component summary (mcs) scores. patients with liver cirrhosis had lower sf-36 scores of physical functioning, role functional, general health, mental health and pcs scores in comparison to non - cirrhotic individuals in benito de valle. however, neither fatigue nor psychological distress was more common in psc patients compared to general population and the disease severity was not a major determinant of hrqol in unselected patients with psc (26). recently, we also found that female gender and older age influenced hrqol in polish cohort of psc patients (27). mental component summary of sf-36 was significantly lower in females than males and men generally showed better quality of life in the study with sf-36, pbc-40 and bc-27 questionnaires (27). the results of the current study to the novel aspect of quality of life in psc patients, i.e. the impact of fragility bone fractures on hrqol. we found lower scores of role physical, general health, vitality and pcs scores in respect to sf-36 questionnaire and symptoms in both pbc-40 and pbc-27 tools in psc patients with fragility osteoporotic bone fractures. these results suggest the impairment of physical aspect of hrqol in severe osteoporosis associated with psc. fatigue is probably the most intriguing symptom affecting patients with chronic cholestatic disorders (28). it did not seem to be a specific complaint of psc in benito de vale. study (26), but al - harthy. found that fatigue was an important, but under reported symptom for patients with psc, particularly in female patients (11). ibd presence was also associated with more severe fatigue in psc patients in al - rifai. however, the result of our recent study might indicate silent bone complication as a cause of this symptom. hence, the associations presented between independent factors and outcome variables do not necessarily represent causal relationships. second, we studied a relatively modest sample of males and females from a single tertiary center, subjected to referral and selection bias. additionally, males have higher bone mass than females, especially postmenopausal females. to reduce any potential bias associated with gender - specific bmd values, we did not include women after menopause and bmd was expressed in gender - specific z - scores and t - scores. vertebral fractures are the most common form of osteoporotic fractures (30) and largely undiagnosed (31). moreover, vertebral fractures, common among corticosteroid users, are frequently asymptomatic or poorly symptomatic, but they substantially affect quality of life (32). thus, we can not exclude that undiagnosed vertebral fractures might influence hrqol outcomes in our patients. in conclusion, in young and middle - aged subjects with psc lasting for approximately three years, we found a high rate of fragility, non - vertebral fractures and moderately lower bmd in the lumbar spine and hip. fragility fractures have an impact on physical and mental aspects of hrqol, as assessed by both generic and disease - specific questionnaires. | background : osteoporosis occurs frequently in patients with chronic cholestatic liver diseases, yet data are scarce regarding the prevalence of osteoporosis and fragility fractures and their impact on health - related quality of life (hrqol) in primary sclerosing cholangitis (psc).objectives : we aimed to assess bone mineral density (bmd), physical activity and incidence of fragility fractures in patients with psc. we also sought associations between prior fractures and hrqol.patients and methods : the study was performed on 33 patients (11 females, 22 males) aged 35.3 13 years. hrqol was assessed by short form (sf)-36, primary biliary cirrhosis (pbc)-40 and pbc-27 questionnaires. bmd was measured by densitometry in the lumbar spine and hip. physical activity was assessed by questionnaire.results:in 32% of patients, bmd measured in the hip or spine was below 1.0 standard deviation. a history of fragility fractures (distal forearm and ribs) was reported in six patients (18%). in sf-36 assessment, patients with fractures had lower scores in the role functioning, general health and vitality domains and physical component summary (pcs) than those without fractures. prior fractures adjusted for gender and psc duration were associated with lower pcs and mental component summary (mcs) scores. symptoms and fatigue (assessed by pbc) and prior fractures were inversely associated with mcs (p = 0.007).conclusions : in middle - aged subjects with psc, we found a high rate of non - vertebral fractures and a moderately decreased bmd in lumbar spine and hip. fragility fractures had an impact on physical and mental aspects of hrqol. |
primary pancreatic lymphoma (ppl) is a rare disease constituting less than 0.5% of all pancreatic malignancies, and ppl is difficult to diagnose preoperatively even by various modalities. regarding the treatment for ppl, chemotherapy including r - chop is considered as standard. on the other hand, for the patient who had a symptom caused by obstruction of biliary tract, surgical treatment was effective ; furthermore, a response rate of 100% was obtained by successful resection, and the long - term survival rate with combination of surgical treatment and adjuvant chemotherapy was superior to current chemotherapy. herein we present a case of pancreas head tumor managed as tumor - forming pancreatitis, who proved to have ppl after pylorus - preserving pancreaticoduodenectomy, and chemotherapy was performed. after 4 years of combined treatment, the patient has no signs of recurrence. for the patient with ppl, surgical treatment combined with chemotherapy can be an effective treatment contributing to long - term survival. a 16-year - old male with an unremarkable past medical history presented with abdominal pain and jaundice. the patient 's laboratory findings on admission included a white blood cell count of 4.5 10/l, hemoglobin of 15.8 g / dl, a platelet count of 19.0 10/l, creatinine of 1.0 mg / dl, total protein of 7.6 g / dl, total bilirubin of 8.1 mg / dl, and liver enzymes including a sgot of 157 iu / l, sgpt of 312 iu / l, amylase of 2,605 iu / l, c - reactive protein of = 0.3 mg / dl, and dupan-2 of 970 u / ml. abdominal computed tomography (ct) showed a 3.0 4.5 cm homogeneously enhancing mass (fig. the common pancreatic duct was dilated and the common bile duct was stenosed (fig. magnetic resonance imaging (mri) findings showed a 4.5 cm tumor localized between the inferior vena cava and pancreatic head with low signal intensity on t1w images and high intensity on t2w images (fig. endoscopic retrograde cholangiopancreatography (ercp) findings were compatible with smooth stenosis of the common bile duct and main pancreatic duct without signs of infiltration (fig. the patient was diagnosed with tumor - forming pancreatitis and pylorus - preserving pancreaticoduodenectomy was performed. 4a), and immunochemical staining was positive for b cell markers cd20 and cd79a and negative for t cell marker cd10 (fig. the patient 's postoperative course was uneventful and he was discharged from the hospital on the 28th postoperative day. ppl is a rare disease constituting less than 0.5% of all pancreatic malignancies, and less than 2% of extranodal lymphomas. most cases are intermediate or high grade non - hogdkin 's lymphoma of diffuse large b cell type. ppl demonstrates a male preponderance (male to female ratio of 7:1) and the mean age at presentation is 55.0 years. saif reported that the major presenting symptoms are abdominal pain in 83% of patients and abdominal mass in 58% of cases. other symptoms include jaundice, reflux, weight loss, bowel obstruction and diarrhea. in our case, a survey of the literature reveals that our patient is the youngest case of resected ppl in our country. the diagnostic criteria for ppl, as indicated by dawson., include : (a) neither superficial lymphadenopathy nor enlargement of mediastinal lymph nodes on chest radiography ; (b) a normal leukocyte count on peripheral blood smear ; (c) the main tumor mass confined to the pancreas with lymph node involvement confined to the peripancreatic region ; (d) no hepatic or splenic involvement. ct and ultrasound are the most common imaging techniques used for the detection of ppl. ultrasound usually shows a homogeneous, hypoechoic mass and ct commonly demonstrates a homogeneous lesion with attenuation that is less than that of muscle. merkle. found ct especially helpful in the differential diagnosis of a tumor localized to the pancreatic head. they reported that the combination of a bulky localized tumor in the pancreatic head without significant dilatation of the main pancreatic duct suggests a diagnosis of pancreatic lymphoma over adenocarcinoma. enlarged lymph nodes below the level of the renal veins with invasive tumor growth not respecting anatomic boundaries and infiltrating either retroperitoneal or upper abdominal organs and the gastrointestinal tract are additional reliable findings of ppl. concerning ercp findings, normal duct, ductal displacement, mild ductal stenosis and stricture of the main pancreatic duct were found in 30, 10, 50, and 10% of patients, respectively. regarding mri, the mass usually shows low signal intensity on t1-weighted images and homogeneously high signal intensity on t2-weighted images, similar to our findings. for a definitive diagnosis, report that ct - guided biopsy was performed for 4 cases of ppl, and in all cases, definitive diagnosis was obtained. however, tuchek. reported that it was difficult to perform ct - guided biopsy safely and, therefore, this method should be performed only by trained physicians in tertiary centers. regarding treatment, anthracycline - based chemotherapy is the standard treatment for non - hodgkin 's lymphoma, and six to eight cycles of r - chop are usually performed for patients of all ages. on the other hand, kevin. reported that it is difficult to obtain long - term survival by chemotherapy alone, and battula. reported that the 5-year survival rate of ppl treated with current chemotherapy was less than 50%, and its rate was inferior to combination of surgical procedure and chemotherapy, therefore they concluded that pancreaticoduodenectomy may have a therapeutic role in association with chemotherapy. as another adaptation of choice surgical procedure, lin. reported that patients with biliary tract or gastrointestinal obstruction should receive biliary or gastric bypass to alleviate symptoms. in our case, the patient was not diagnosed with ppl preoperatively, but since there was obstruction of the biliary tract, pylorus - preserving pancreaticoduodenectomy was performed. his postoperative course was uneventful and 8 cycles of r - chop were performed with no signs of recurrence after 4 years. it is, therefore, important to choose the appropriate treatment depending on disease progression and the patient 's condition. | primary pancreatic lymphoma (ppl) is a rare disease that is difficult to diagnose preoperatively. we describe the youngest case of ppl treated by surgical excision and chemotherapy. a 16-year - old male presented with abdominal pain and jaundice. abdominal computed tomography showed a 3.0 4.5 cm homogeneously enhanced mass localized between the inferior vena cava and pancreatic head ; the common pancreatic duct was dilated and the common bile duct was stenosed. magnetic resonance imaging findings showed a 4.5 cm tumor localized between the inferior vena cava and pancreatic head with low signal intensity on t1w images and high intensity on t2w images, which enhanced inhomogeneously. endoscopic retrograde cholangiopancreatography findings were compatible with smooth stenosis of the common bile duct. he was diagnosed as pancreatitis secondary to pancreatic tumor and pylorus - preserving pancreaticoduodenectomy was performed. postoperative diagnosis was ppl and chemotherapy was performed. after 4 years of treatment he has no signs of recurrence. |
the recent study by burri and colleagues examined the usefulness of arterial blood gases (abg) in the diagnosis and prognosis of 530 dyspneic patients with abg drawn upon presentation to the emergency department. the study was a retrospective analysis of prospectively collected data performed at a single center. they concluded that abg can not be used to distinguish between pulmonary and other causes of dyspnea. prior to the introduction of automated blood gas analyzers, blood po2 and pco2 could be obtained only by laborious and often inaccurate laboratory methods, such as gas tonometry and van slyke manometric extraction of plasma total carbon dioxide. improvements in electrode technology came with the development of the astrup ph electrode, the stow / severinghaus - type pco2 electrode and the polarographic oxygen clark electrode. although pulse oximetry has largely superseded the use of arterial po2 (pao2), the ease by which abg can now be measured has made this test de rigueur in the workup of dyspneic patients. we agree with burri and colleagues in that pao2 values serve mainly to triage and to guide treatment, rather than to differentiate among the causes of dyspnea. except for anxiety hyperventilation, it is difficult to rely solely on abg to identify a specific clinical syndrome. the poor predictive value of pao2 in diagnosing patients with pulmonary embolism is well known. in the pioped i study of patients with angiographically proven pulmonary embolism and no prior cardiopulmonary disease, 26% had pao2 > 80 mmhg. the probability of diagnosing an acute pulmonary embolism based on changes in pao2 did not achieve statistical significance. burri and colleagues also report that arterial ph was a significant predictor of short - term and long - term outcome. multiple physiological buffers act to preserve the concentration of hydrogen ions in blood within a relatively small range. decreases in arterial ph are likely to reflect severe impairments or even exhaustion of systemic compensatory mechanisms in patients with acute dyspnea. the predictive value of the arterial ph in dyspneic patients noted by burri and colleagues supports this hypothesis. burri and colleagues propose the use of venous blood ph in the initial evaluation of acute dyspnea, based on data from several single - center studies reporting relatively close limits of agreement (-0.11 to + 0.04) between arterial ph and venous blood ph. one must keep in mind, however, that muscular activity or regional microcirculatory alterations could result in misleadingly low regional ph values in septic or agitated patients. substituting venous blood ph for arterial ph in the evaluation of dyspneic patients is an appealing notion, but one that requires testing in multicenter, prospective clinical studies. until these studies corroborate the equivalence between arterial and peripheral venous blood gases, or until non - invasive techniques to monitor arterial pco2 and ph become available, we shall continue to support the use of abg in the initial evaluation and treatment of patients with acute dyspnea. abg : arterial blood gases ; icu : intensive care unit ; paco2 : arterial partial pressure of carbon dioxide ; pco2 : partial pressure of carbon dioxide ; po2 : partial pressure of oxygen. | arterial blood gases (abg) are obtained commonly in dyspneic persons presenting to emergency departments. the study by burri and colleagues found that the information contained in abg fails to distinguish between pulmonary and other causes of dyspnea. on the other hand, arterial ph was highly predictive of icu admission and outcome. until large clinical studies show equivalence between peripheral venous and abg, we will continue to advocate the use of abg in the evaluation of acute dyspnea. |
glaucoma is the second leading cause of blindness, affecting approximately 60.5 million people worldwide or about 2.6% of the population over the age of 40. although the most prevalent form of glaucoma in western countries and in many other parts of the world is open - angle glaucoma (oag), angle - closure glaucoma (acg) is the most common type of glaucoma among the han chinese. factors associated with the development of glaucoma include increased intraocular pressure (iop), higher cup - to - disc ratio, aging, thinner central corneal thickness, family history of glaucoma, myopia, and chronic and systemic diseases such as diabetes [3, 4 ]. most ophthalmologists treat glaucoma by lowering the iop using one of three modalities topical antiglaucoma drugs, laser treatment, or glaucoma surgery. types of topical medication include -blockers, -agonists, prostaglandin analogs (pags), carbonic anhydrase inhibitors (cais), cholinergic agonists, and adrenergic agonists. because some patients require multiple daily dosing, fixed combination eye drops have been developed to enhance and reinforce patient compliance. recent studies have shown that glaucoma treatment in some developed countries causes a significant financial burden on the health care system [79 ]. in this study, we used the national longitudinal health insurance database 2000 (lhid2000) to examine trends in glaucoma medication expenditure in taiwan from 1997 to 2010. the taiwan national health insurance (nhi) program is a mandatory single - payer health insurance system under which all residents are covered. the nhi program has been in existence since 1995 and by the end of 2010 the coverage rate was 99% of taiwan 's population of 23.1 million. medical care in taiwan is well known for its low cost, convenience, high efficiency, high quality, and excellent medical accessibility [1012 ]. the nationwide population - based dataset provides an opportunity to explore the trends in glaucoma medication expenditure. this study was designed as a population - wide retrospective review using the taiwan national health insurance research database (nhird). the nhird contains a large number of computerized records including registration files, medication and treatment regimens, and information on surgery. claim reimbursement data from patients since 1999 are provided to researchers in an electronically encrypted form. data contained in the lhid2000 are randomly selected from one million subjects from the nhird and are made available for research purposes. data from the lhid2000 used in this study included patient identification numbers, gender and age, monthly salary, occupation type, diagnostic data, antiglaucoma drug codes, and surgery codes. diseases are defined in accordance with a codes (a230) before 2000 and international classification of diseases, 9th revision, clinical modification (icd-9-cm365), after 2000. the study received approval from the ethics committee of the institutional review board of the changhua christian hospital and was conducted in accordance with the tenants of the declaration of helsinki. topical glaucoma medications included pgas, -blockers, -agonists, cais, cholinergic agonist, -blockers / cais fixed combinations, -blockers/-agonists fixed combination, -blockers / pgas fixed combinations, and adrenergic agonists the trend test for expenditure was analyzed with linear regression weighted with the inverse of squares residual. we also assessed the effect of demographic factors on glaucoma expenditure using a generalized estimating equation regression model (gee). a p value < 0.05 was considered to indicate statistical significance ; all tests were two - tailed. all statistical analyses were conducted using the statistical package sas for windows (version 9.2). the number of beneficiaries included in the lhid2000 sample declined from 916,626 persons in 1997 to 859,913 persons in 2010. the number of patients receiving antiglaucoma drugs increased from 3105 in 1997 to 7033 in 2010 (table 1). after adjusting for inflation, the total annual medical expenditures increased from $ 0.21 million in 1997 to $ 0.63 million in 2010 (slope = $ 37,618/year, p < 0.001) (table 1). the increase in total expenditures was remarkable in both genders, every age group, every income group, and every occupation group (p < 0.05). the annual glaucoma medication expenditures were most prominent in the age group 65 years, followed by the age group 4064 years and the age group < 40 years (figure 1). the white - collar occupation group spent more money on medications than the other occupation groups. the mean medication expenditure per person increased from $ 67.3 in 1997 to $ 90 in 2010 after adjusting for inflation (slope = $ 2.8/year, p < 0.001) (table 1). the mean cost per capita increased year on year from 1997 to 2006 and then decreased gradually after 2007. the trend in mean medication expenditures per person was similar in each category (gender, age, income, and occupation group). estimates from the gee conducted to determine demographic variables associated with the increase in glaucoma medication expenditure indicate that patients 40 years incurred higher costs for medication than patients under the age of 40 years (p < 0.001). in the same analysis, men incurred higher costs than women (p < 0.05), populations with higher income incurred higher costs for medications than populations with lower income (p < 0.05), glaucoma medication expenditures increased year on year (p < 0.001), and blue - collar workers had lower expenditures than the other types of workers (p < 0.001) (table 2). the annual expenditures for most classes of glaucoma medications increased during the study period, except for -blockers (slope = $10,152/year, p < 0.001) and cholinergic agonists (slope = $432/year, p < 0.001) (table 1). the decrease in expenditure for -blockers was associated with the administration of pgas (p = 0.002), and -agonists (p < 0.001). a significant increase in expenditures on pgas (slope = $ 23,779/year, medications containing pgas accounted for 46% of the total glaucoma medication expenditure in 2010 (table 1) (figure 2). we also found a decreasing trend in per capita medication expenditures for -blockers (slope = $0.5/year, p < 0.001) and cholinergic agonists (slope = $0.5/year, p < 0.001). however, the trends for other glaucoma medications were not significant (table 1). the numbers of trabeculectomies performed during the study period are shown in table 1. during the period 19972010, we found that the frequency of trabeculectomy had two break points, one in the year 1999 and the other in the year 2000 based on the chow test. during the study period, the total expenditures for glaucoma medications significantly increased by 3.03-fold because of an increase in patient numbers and an increase in mean medication expenditures per person. the increase in patient numbers may be related to the early diagnosis due to advanced diagnostic modalities (e.g., optical coherence tomography), more accurate diagnosis, overdiagnosis, aging population, or good medical accessibility in taiwan [10, 11, 15 ]. the increase in mean medication expenditure per person may be associated with the administration of pgas and more aggressive glaucoma treatment. the rising cost of glaucoma drugs after pgas launching also occurred in ireland, scotland, australia, denmark, and france. as seen in table 2, mean glaucoma medication expenditures increased markedly after the nhi system began covering treatment with pgas in 2000. the reduction of expenditure per capita after 2007 and the mean medication cost in taiwan was much lower than in denmark and the united states [8, 9 ] reflecting the success of bargaining medication cost and prescription policy (beta - blocker should be first - line drug) by taiwan nhi. however, the increasing total medication expenditure imaged that the cost down policy could not offset the growing medical demand. in this study, expenditures for glaucoma medications were significantly higher for men than for women, whereas in the usa the opposite is true. the possible reasons are differences in knowledge of health issues between genders in different societies and the higher prevalence of acg in taiwan [2, 17 ]. women are at higher risk of acg that can be treated with laser iridotomy or cataract surgery rather than glaucoma medications. cataract surgery is readily available in taiwan and the procedure reduces the likelihood of developing the disease [13, 19 ]. it is not surprising that glaucoma medical expenditures were highest among patients 65 years because aging is one of the risk factors for developing glaucoma. in this study, the glaucoma medical expenditures for the age group over 65 years significantly increased and accounted for more than half the amount of medical expenditures ; a reflection of taiwan 's aging population has great financial burden in glaucoma care. furthermore, we also found that expenditures for glaucoma medications increased significantly for patients in the age group < 40 years, possibly because of advanced diagnostic tools, more aggressive glaucoma treatment, and the high prevalence of myopia among younger people in taiwan [5, 15, 20 ]. the study results show that blue - collar workers have lower glaucoma medical expenditures, while higher income families incur higher glaucoma medical expenditures, indicating inequality in health care services under universal health coverage in taiwan. such inequality may be the result of differences in general knowledge of health care, out - of - pocket payment policy, and other socioeconomic disparities [10, 21, 22 ]. the total expenditure for -blockers decreased because of the reduction in cost by the bureau of nhi and the decrease in usage of -blockers, mainly due to the increased availability of pgas and -agonists. however, considering the drug price, the taiwan nhi stipulates that -blockers are first - line medications and pgas, cais, and fixed combinations are second - line medications. therefore, -blockers still have a considerable market share in taiwan, despite the fact that pgas are more effective, are associated with fewer adverse effects, require only once - a - day dosing, and are associated with greater patient compliance. the medical expenditures in the usa have also undergone similar changes relative to insurance coverage. even though most people in taiwan are chinese in origin and have a higher prevalence of acg [2, 17 ], there has been a decreasing trend in the application of cholinergic agonists, which may be offset by the increased availability of other drugs like pgas and the increase in frequency of cataract surgery in taiwan [13, 23 ]. the trend in increasing expenditures for fixed combinations of medications during the period of study can be attributed to patients ' preference, because the fixed combinations improve medical adherence and reduce eye discomfort. our study reveals that the number of trabeculectomies decreased significantly in 1999 and 2000, at the time when pgas were launched and the taiwan nhi began to reimburse expenses for pgas. a similar situation of decreasing the number of trabeculectomies after pgas listing was also reported in scotland, france, and australia. however, the side effects of trabeculectomy are greater than those associated with pgas. according to cutler and mcclellan, technological change affects treatment in two ways treatment substitution and treatment expansion or both. treatment expansion takes place when treatments become safer and easier, and patients pay more attention to their conditions when therapy is more effective or less side effective. based on the results of this study, we presume that pgas may have the effect of treatment expansion for trabeculectomy under universal health coverage. poor patient adherence may reduce medication expenditures at first but eventually will increase medical and surgical expenditures due to symptom complications. most people are employees and pay the insurance fee according to their salary. however, the health insurance fee is lower for those living on their investments. thirdly, we did not characterize which types of glaucoma and which education levels contributed to medical expenditures. this nationwide population - based study demonstrated an increasing trend in glaucoma medical expenditures from 1997 to 2010 in taiwan. the main factors contributing to these trends include administration of brand - name drug products such as pgas, the increasing glaucoma population, good medical accessibility, and possibly more aggressive treatment. expenditures were highest among men, patients over 40 years of age, and patients with higher incomes and were lowest among blue - collar workers. | medical care in taiwan is well known for its low cost, high efficiency, high quality, excellent medical accessibility, and high equity. we investigate the trends in medication expenditures for glaucoma from 1997 to 2010. the results show that higher medical expenditures were incurred by patients who were aged 40 years, male patients, and patients in the highest salary population whereas lower medical expenditures were incurred by blue - collar workers. the medications with the most significant increases in expenditure were prostaglandin analogs (pgas), -agonists, and fixed combinations, whereas the medications with the most significant decreases in expenditure were -blockers and cholinergic agonists. the number of trabeculectomies shows two downward break points in 1999 and 2000 when pgas were listed and were reimbursed. these results suggest socioeconomic disparities in glaucoma care, as well as understanding of the changes in the expenditure of glaucoma medications under such universal health insurance coverage system. |
a 64-year - old female was diagnosed with thyroid cancer in 2004 and was treated by thyroidectomy. during follow - up, blood free t4 levels were increased and an i-131 whole body scan was performed. 1). additional chest computed tomography (ct) showed a 1.2-cm - long nodule at the left lower lobe, which matched the location in the i-131 scan results (fig. 2). a thoracoscopic approach was first attempted, but failed due to adhesion of left upper lobe. thus, a thoracotomy was performed. a 1-cm - long nodule surrounded by pulmonary tissue was observed next to the inferior pulmonary vein. a blood vessel branching off from the pulmonary vein and going through the nodule was also observed. the blood vessel was ligated, excised, and isolated from the inferior pulmonary vein. endogia (covidien, mansfield, usa) was then used for wedge resection of the nodule (fig. it was identified as ectopic thyroid tissue, but it was not observed to be malignant (fig. 5). the patient 's chest tube was removed on day 5 after surgery, and the patient was discharged on day 10 without other complications. the thyroid migrates from endodermal protrusion of the first and second pharyngeal arch at 3 weeks to 7 weeks of gestation. a thyroglossal duct forms along the descending path of the thyroid and undergoes atrophy during the embryonic stage. during this development, abnormal migration of the thyroid causes it to be misplaced, which is known as ectopic thyroid. the prevalence of ectopic thyroid is 1 in every 100,000 to 300,000 in the general population and 1 in 4,000 to 8,000 in patients with thyroid disease. ectopic thyroid is mostly symptomless, but can have various symptoms based on the size and location. in particular, when located inside or around the trachea, the patient may experience aphagia, hoarseness, dyspnea, or a foreign body sensation as the size increases. however, most cases of ectopic intrapulmonary thyroid are symptomless, as in this case study. it is observed through radiation screening and ct scans and is reported to cause dry cough, dyspnea, hemoptysis aphagia, and superior vena cava syndrome. in addition, it is reported that orthotropic thyroid coexists in all cases of intrapulmonary thyroid. radiologic examinations for diagnosis include ct, magnetic resonance imaging, tc-99 m, i-131, and i-123. blind bronchoscope aspiration and real - time ultrasound aspiration have been used to obtain samples for histological diagnosis. a differential diagnosis of ectopic intrapulmonary thyroid includes thyroid cancer metastasis, thymoma, neuroma, and germ cell tumor. among ectopic thyroid cases, prior to ectopic intrapulmonary thyroid treatment, the patient 's age, presence of orthotropic thyroid, symptoms due to nodules and their severity, thyroid hormone level, and malignancy potential need to be considered to perform surgical or medical treatment. depending on the position of the tissue, sternotomy or thoracotomy can be performed for surgical treatment. also, thoracoscopic treatment can also be considered. in conclusion, ectopic thyroid occurs due to early developmental differentiation problems and can be positioned in different locations based on thyroid development. it is usually symptomless, but can cause symptoms depending on its location, size, and malignance. this case reports a patient with a history of thyroid cancer and abnormal thyroid function. surgery was performed in order to determine malignancy, and to diagnose and treat the ectopic thyroid. | an ectopic thyroid is caused by abnormalities in migration of the thyroid during development and rarely occurs in the thoracic cavity or the abdominal cavity. we report the case of a 64-year - old female who had abnormal findings from a thyroid hormone test during follow - up after thyroid cancer surgery. based on the radioisotope diagnostic test, an ectopic thyroid inside the thoracic cavity was suspected. through surgical treatment, the patient was diagnosed with ectopic intrapulmonary thyroid. ectopic intrapulmonary thyroid is reported to be very rare and the case is described along with a literature review. |
peroxisome proliferator activated receptor- (ppar) regulates metabolic homeostasis and adipocyte differentiation, and it is activated by oxidized and nitrated fatty acids. here we report the crystal structure of the ppar ligand binding domain bound to nitrated linoleic acid, a potent endogenous ligand of ppar. structural and functional studies of receptor - ligand interactions reveal the molecular basis of ppar discrimination of various naturally occurring fatty acid derivatives. |
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bipolar disorder i (bdi) is an episodic illness characterized by recurrent manic, mixed, and depressive episodes, with an estimated global lifetime prevalence of 1%5%.1 the illness is associated with high levels of mortality and morbidity2 and functional impairment,3 and high rates of suicide.4 despite the phenomenon of switching from depression to mania or from mania to depression being a core aspect of the clinical presentation of bdi, the neurobiology of the switching process is still poorly understood. recently, we reported that 3-methoxy-4-hydroxyphenylglycol (mhpg), more so than homovanillic acid (hva) or brain - derived neurotrophic factor (bdnf), is a biomarker for the switch from the manic to the remission state in bdi.5 however, the usefulness of mhpg levels as a biomarker for the switch from depression to remission or from depression to mania in bdi is not yet clear. most antidepressants have been associated with an increased risk for treatment - emergent affective switch. consequently, antidepressants targeting the serotonergic, noradrenergic, dopaminergic, bdnf, and other systems may affect the switching process in bdi.6 in fact, mhpg and hva levels are higher in bipolar manic patients than in both bipolar depressed and control patients.7 furthermore, a meta - analysis showed that bdnf levels are decreased in both bipolar mania and bipolar depression when compared with those in both control groups.8 thus, the association of bdi with peripheral biomarkers is not uniform. there is currently no biomarker that could serve as an objective index for evaluating the severity or switch progression of bdi. moreover, the long - term clinical consequences of the switch process are still poorly understood. the main purpose of this study was to determine whether plasma levels of catecholamine metabolites and bdnf are correlated with mood states or severity of manic or depressive symptoms. our report describes the first long - term, naturalistic, retrospective case study examining mhpg (a noradrenaline metabolite that is associated with noradrenaline levels in the brain), hva (a dopamine metabolite that is associated with dopamine levels in the brain), and bdnf levels in both the depressed and manic states of bdi. the subjects included two japanese bdi patients admitted to in- or outpatient clinics at sato hospital, koutokukai : a 50-year - old man (case 1) and a 44-year - old woman (case 2). case 1 had 20 incidences of hospitalization and release between x9 and x+4 years (x is the study start year). he has been divorced twice and went into voluntary bankruptcy because of debts due to excessive spending and gambling while in the manic state. case 2 had 14 incidences of hospitalization and release between x2 and x+5 years (x is the study start year). the severity of depressive and manic states were assessed every 2 weeks by independent experienced raters. asberg depression rating scale (madrs) was used to measure depression, and the young mania rating scale (ymrs) to measure mania. blood was withdrawn by venipuncture from each subject, between 10 am and 5 pm on the assessment day, into a blood collection tube containing edta (ethylenediaminetetraacetic acid) as an anticoagulant. the tubes were immediately cooled to 4c and then centrifuged at 2,000 g for 20 minutes. plasma mhpg and hva levels were analyzed by high - performance liquid chromatography with electrochemical detection (hplc - ecd)9,10 using both the internal standard (5-hydroxyindolecarboxylic acid) method and standard addition methods. plasma bdnf levels were analyzed by sandwich elisa (enzyme - linked immunosorbent assay) according to methods described previously.11 written informed consent was obtained from all patients participating in the study. the study protocols were approved by the ethics committee of sato hospital, koutokukai, and the ethics committee of graduate school of pharmaceutical sciences, tohoku university. the spearman s rank correlation coefficient () was calculated to investigate the relationship between the plasma levels of mhpg, hva, and bdnf and the rating scales of madrs and ymrs for each individual patient. standardized coefficient beta () was calculated for the factors (biomarkers) that appeared to be linked to the symptoms (madrs and ymrs), using the multiple regression analysis. thus, multivariate analysis was applied to the rating scales as the independent variables and the biomarkers as the dependent variables, to determine an influence factor that influences the dependent variable. analyses were performed using the spss software (ibm, tokyo, japan) version 19.0 for macintosh. a description of the mood states of the patient (determined by rating madrs and ymrs) throughout the 2 years and 4 months that he participated in the study is shown in figure 1a. for most of the study period a total of 72 data points were obtained, and all data points were analyzed. the mean scores of the ymrs and the madrs over the study period were 8.312.0 and 11.815.5 points, respectively. the mean plasma mhpg, hva, and bdnf levels over the study period were 8.02.3 ng / ml, 10.44.0 ng / ml, and 6,7043,164 pg / ml, respectively. we investigated the relationship between the plasma levels of mhpg, hva, and bdnf and the rating scales of madrs and ymrs. the homoscedasticity of all variables was rejected by the shapiro wilk test (p<0.05). we found a significant positive correlation between ymrs scores and plasma mhpg levels (=0.429, p<0.001). we also found a significant negative correlation between madrs scores and plasma mhpg (=0.542, p<0.001) and hva (=0.429, p<0.001) levels (figure 2a). in contrast, no significant correlation was found between ymrs scores and the plasma hva level. furthermore, no significant correlation was found between the plasma bdnf levels and the mood state (both ymrs and madrs scores). in the multiple regression analysis, the standardized coefficient beta () for madrs was 0.466 (mhpg). in this analysis, a description of the mood states of the patient (determined by rating madrs and ymrs) throughout the 5 years and 11 months that she participated in the study is shown in figure 1b. for most of the study period for case 2 a total of 183 data points were obtained, and all data points were analyzed. the mean scores of the ymrs and the madrs over the study period were 7.110.8 and 12.812.9 points, respectively. the mean plasma mhpg, hva, and bdnf levels over the study period were 8.12.7 ng / ml, 11.94.0 ng / ml, and 7,7813,743 pg / ml, respectively. we investigated the relationship between the plasma levels of mhpg, hva, and bdnf and the rating scales of madrs and ymrs. we found a significant positive correlation between ymrs scores and plasma mhpg (=0.488, p<0.001) and hva (=0.319, p<0.001) levels. we also found a significant negative correlation between madrs scores and plasma mhpg (=0.465, p<0.001) and hva (=0.381, p<0.001) levels (figure 2a). in contrast, no significant correlation was found between the plasma bdnf level and the mood state (both ymrs and madrs scores). the standardized coefficient beta () for ymrs was 0.266 (mhpg), 0.235 (bdnf), and 0.233 (hva). the standardized coefficient beta () for madrs was 0.292 (mhpg), 0.270 (hva), and 0.136 (bdnf). a description of the mood states of the patient (determined by rating madrs and ymrs) throughout the 2 years and 4 months that he participated in the study is shown in figure 1a. for most of the study period a total of 72 data points were obtained, and all data points were analyzed. the mean scores of the ymrs and the madrs over the study period were 8.312.0 and 11.815.5 points, respectively. the mean plasma mhpg, hva, and bdnf levels over the study period were 8.02.3 ng / ml, 10.44.0 ng / ml, and 6,7043,164 pg / ml, respectively. we investigated the relationship between the plasma levels of mhpg, hva, and bdnf and the rating scales of madrs and ymrs. the homoscedasticity of all variables was rejected by the shapiro wilk test (p<0.05). we found a significant positive correlation between ymrs scores and plasma mhpg levels (=0.429, p<0.001). we also found a significant negative correlation between madrs scores and plasma mhpg (=0.542, p<0.001) and hva (=0.429, p<0.001) levels (figure 2a). in contrast, no significant correlation was found between ymrs scores and the plasma hva level. furthermore, no significant correlation was found between the plasma bdnf levels and the mood state (both ymrs and madrs scores). in the multiple regression analysis, the standardized coefficient beta () for madrs was 0.466 (mhpg). in this analysis, a description of the mood states of the patient (determined by rating madrs and ymrs) throughout the 5 years and 11 months that she participated in the study is shown in figure 1b. for most of the study period a total of 183 data points were obtained, and all data points were analyzed. the mean scores of the ymrs and the madrs over the study period were 7.110.8 and 12.812.9 points, respectively. the mean plasma mhpg, hva, and bdnf levels over the study period were 8.12.7 ng / ml, 11.94.0 ng / ml, and 7,7813,743 pg / ml, respectively. we investigated the relationship between the plasma levels of mhpg, hva, and bdnf and the rating scales of madrs and ymrs. the homoscedasticity of all variables was rejected by the shapiro wilk test (p<0.05). we found a significant positive correlation between ymrs scores and plasma mhpg (=0.488, p<0.001) and hva (=0.319, p<0.001) levels. we also found a significant negative correlation between madrs scores and plasma mhpg (=0.465, p<0.001) and hva (=0.381, p<0.001) levels (figure 2a). in contrast, no significant correlation was found between the plasma bdnf level and the mood state (both ymrs and madrs scores). the standardized coefficient beta () for ymrs was 0.266 (mhpg), 0.235 (bdnf), and 0.233 (hva). the standardized coefficient beta () for madrs was 0.292 (mhpg), 0.270 (hva), and 0.136 (bdnf). antipsychotics have good efficacy in controlling manic symptoms in bdi patients.12 however, these can induce depression or extrapyramidal symptoms in bipolar manic patients.1315 hence, antipsychotics are a temporary treatment in severe manic cases, and clinicians know that there is an increased risk of depression or extrapyramidal symptoms if high doses of antipsychotics are continued to be prescribed. conversely, although antidepressants have good efficacy in controlling depressive symptoms in bdi,16 most antidepressants have been associated with increased risk for treatment - emergent mania / hypomania.17 a study by altshuler has detected an increased likelihood of switching in bdi patients treated with antidepressants.18 for these reasons, antidepressant monotherapy is not recommended for patients with bipolar depression.19 many reports suggest that regulating monoaminergic transmission or the bdnf system might increase individual susceptibility for bdi switching. this is the first report in which two cases of bdi have been continuously evaluated for plasma mhpg, hva, and bdnf levels in a long - term retrospective study. these results are supported by previous findings that the switch to mania is associated with increased functional brain noradrenaline and dopamine.6,20 interestingly, in both cases, we found a significant positive correlation between ymrs scores and plasma mhpg levels, and a significant negative correlation between madrs scores and plasma mhpg levels (more so than for hva or bdnf). that is, plasma mhpg acted as a peripheral biomarker, consistently indicating the switch of symptoms from manic to depressive state in both bdi cases. mhpg is the major noradrenaline metabolite in the brain.21 although hva was significantly correlated with ymrs score in case 2, no significant correlation was found between ymrs score and hva levels in case 1. multiple regression analysis also showed that mhpg level was the most effective predictor of mood state in both cases. the results of the present study suggest that changes in the noradrenergic system are more likely to induce switching in bdi than are changes in the dopaminergic and bdnf systems. given the role of noradrenaline in treatment - emergent affective switch, we suggest that noradrenaline reuptake changes and dysfunction of noradrenaline autoreceptors may be involved in switching in bdi. some antidepressants are noradrenaline reuptake inhibitors, which inhibit the action of the noradrenaline transporter. conversely, depressive states may involve diminished presynaptic noradrenaline release or increased presynaptic noradrenaline reuptake. interestingly, two naturalistic studies found a significantly lower rate of treatment - emergent affective switch when patients were treated with selective serotonin reuptake inhibitors, compared with tricyclic antidepressants.22,23 the levels of biomarkers were markedly different from individual to individual.5 therefore, it is important that the biomarker level of an individual patient is known, because it may change over time. also, the difference in correction coefficient between cases 1 and 2 may be due to the difference in effect size. more large - scale design follow - up studies are needed to understand the pathologic state for the treatment of bdi. in summary, the results show that plasma levels of mhpg may reflect the pathophysiology of bdi from manic to depressive states better than do the plasma hva or bdnf levels. these data suggest that peripheral mhpg (which is associated with noradrenaline levels in the brain) could be used as a biomarker of both the manic and depressive states for bdi. the mhpg level is likely to reflect the clinical characteristics of the switch process in bdi, and has prognostic significance for the treatment of manic and depressive states. this study is registered in the umin clinical trials registry (umin - ctr) : analysis of genome and blood components for elucidation and treatment of mood disorders (https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr.cgi?function=brows&action=brows&recptno=r000007415&type=summary&language=e;umin000006264). | backgroundtreatment of the depressive and manic states in bipolar disorder i (bdi) is a challenge for psychiatrists. despite the recognized importance of the switch phenomenon, the precise mechanisms underlying this process are yet to be shown. we conducted a naturalistic study in two bdi patients to determine whether biological markers (monoamine metabolites and brain - derived neurotrophic factor [bdnf ]) are associated with the switch between depressive and manic states.case presentation and methodsblood sampling and mood assessments were performed at 2-week intervals over a period of 2 (case 1, n=72) and 6 (case 2, n=183) years. plasma concentrations of 3-methoxy-4-hydroxyphenylglycol (mhpg) and homovanillic acid (hva) were analyzed by high - performance liquid chromatography with electrochemical detection. plasma bdnf was assayed by sandwich elisa (enzyme - linked immunosorbent assay).resultsmhpg had the highest standardized coefficient () in the multiple regression analysis. we found a significant positive correlation between young mania rating scale scores and plasma mhpg levels (case 1 : =0.429 ; case 2 : =0.488), and a significant negative correlation between montgomery asberg depression rating scale scores and mhpg levels (case 1 : =0.542 ; case 2 : =0.465). conversely, no significant correlation was found between the level of bdnf and the presence of a manic or depressive state, and although hva had a slightly stronger correlation than mhpg, the levels of neither of these were found to significantly correlate with the symptoms.conclusionthese data suggest that peripheral mhpg levels (which is related to noradrenaline levels in the brain) could be used as a biomarker of mood states in bdi. the noradrenaline level in the brain is likely to reflect the clinical characteristics of the switch process in bdi, and has prognostic significance for the treatment of both manic and depressive states. |
posttranslational modifications play a key role in the regulation of protein activity, localization, and stability, as well as in the control of protein - protein interactions. among them, ubiquitylation counts as one of the most prominent and best - characterized modifications. ubiquitin is a small protein that can be covalently attached to lysine residues of other proteins through the interplay of different ubiquitin - activating (e1) and -conjugating (e2) enzymes and ubiquitin ligases (e3) (hershko and ciechanover, 1998). the analysis of mice lacking various components of the ubiquitylation machinery highlighted the importance of ubiquitylation in several cellular and physiological processes, spanning from autophagy, transcription factor regulation, and dna repair to the control of cell cycle and cell death (bergink and jentsch, 2009, bernassola., 2010, hershko and ciechanover, 1998, mukhopadhyay and riezman, 2007), which in turn can affect complex processes such as the immune response (bhoj and chen, 2009, corn and vucic, 2014), the maintenance of tissue homeostasis (kumari., 2014), and tumorigenesis (hoeller and dikic, 2009, paolino., 2014). life or death cell fate decisions are critical for development, against infectious diseases, or cancer formation. apoptosis was considered the sole form of programmed cell death during development or disease (elmore, 2007). however, besides apoptosis, alternative programmed necrosis or necroptosis can be induced by death receptors (vandenabeele., survival, apoptosis, and necroptosis can all be triggered by the same cell - surface receptors. there has been intense interest in how these fate decisions are made by identifying the gatekeepers of apoptotic versus necroptotic cell fate and the signaling cascades involved (christofferson and yuan, 2010). one of the most paradigmatic signaling pathways that regulates cellular fate is triggered by the tumor necrosis factor receptor 1 (tnfr1). tnfr1 engagement elicits cell survival pathways via nf-b induction (beg and baltimore, 1996, van antwerp. when nf-b activation is inhibited, tnf stimulation triggers caspase-8-mediated apoptosis via a defined death effector complex (micheau and tschopp, 2003, wang., 1996). in specific conditions, blockade of both nf-b and apoptosis may result in programmed necrotic cell death (cho., 2009, he., 2009, necroptosis is mediated via the rip1/rip3/mlkl kinase pathway and recently has been implicated in normal development and in pathogenesis of diseases, including ischemic injury, neurodegeneration, and intestinal inflammation (duprez., 2011, kaiser., 2011, kang., 2013, 2011). hect domain and ankyrin repeat - containing e3 ligase 1 (hace1) is an e3 ligase initially identified in the context of sporadic wilms tumor (anglesio., 2004). a number of reports showed that hace1 is downregulated in various types of tumors (anglesio., 2004, hibi., 2008, kk., 2013, zhang., 2007), and a previous study from our group highlighted the important function of hace1 as a tumor suppressor : mice lacking hace1 showed an increased propensity to develop spontaneous tumors as well as a reduced survival in different cancer models (zhang., 2007). in addition to this function, hace1 has been implicated in golgi membrane dynamics (tang., 2011), 2014), and heart failure following cardiac injury (zhang., 2014). however, the complexity of the molecular and cellular processes potentially regulated by hace1 is still largely unexplored. here genetic inactivation of hace1 impairs nf-b activation and apoptosis downstream of tnfr1, whereas rip3-regulated necroptotic cell fate is not affected. importantly, we found that hace1 mutant mice exhibit enhanced intestinal inflammation and colon cancer following epithelial injury, which can be rescued by simultaneous genetic inactivation of either rip3 or tnfr1. to identify pathways relevant to hace1 function, we scanned the signaling pathways downstream of multiple ligand / receptor pairs, using hace1 mutant (hace1) mouse embryonic fibroblasts (mefs). intriguingly, hace1 mefs stimulated with tnf showed compromised phosphorylation and incomplete degradation of the nf-b inhibitor ib. as a consequence, tnf - induced phosphorylation and activation of the nf-b subunit p65 was strongly reduced in hace1 mefs (figure 1a). impaired nf-b activation was confirmed using an electrophoretic mobility shift assay (emsa) filter plate assay (figure 1b). re - expression of wild - type (wt) hace1 in hace1 mefs, but not that of the e3 ligase inactive hace1 c876s mutant (anglesio., 2004), restored tnf - induced p65 nf-b activation (figure 1b ; figure s1a). moreover, tnf - induced nf-b - dependent expression of interleukin-6 (il-6) (bollrath and greten, 2009) also was markedly inhibited in hace1-deficient mefs (figure 1c). in addition to defective nf-b induction, tnfr1-induced jnk and consequent c - jun phosphorylation were markedly reduced in hace1 mefs, a phenomenon that was accompanied by increased and prolonged erk1/2 and p38-mapk phosphorylation (figure 1a). tnfr1 and nf-b are essential for the in vivo clearance of the intracellular pathogen listeria monocytogenes (pfeffer., 1993, 1995). considering the observed in vitro defects in tnfr1/nf-b activation, we tested whether hace1 also controls the response to l. monocytogenes in vivo. importantly, we observed impaired clearance of l. monocytogenes (figure 1d), accompanied by enhanced lethality of the hace1 mice as compared to their wt littermates (figure 1e), a phenotype that is compatible with and might be dependent on defective tnfr1/nf-b activation. thus, hace1 e3 ligase activity is essential for tnf - induced activation of both nf-b and jnk downstream signaling in vitro, and hace1 controls in vivo immunity to l. monocytogenes. when nf-b activation is blocked, tnf stimulation results in apoptotic cell death (beg and baltimore, 1996, van antwerp., 1996, wang., 1996). given that tnf - mediated nf-b activation is impaired in hace1 cells, we reasoned that loss of hace1 might sensitize cells to tnfr1-induced apoptosis. surprisingly, tnf stimulation alone did not induce significant cell death in hace1 cells (figures 2a and 2b), even when we monitored these cells for 72 hr (figure s1b). moreover, whereas, as expected, control wt mefs underwent apoptosis in response to treatment with tnf and actinomycin - d for 8 hr (actd ; used to block nf-b anti - apoptotic transcriptional activity ; lasek., 1996), hace1 mefs remained resistant to apoptotic death (figures 2a and 2b) and cells remained viable, albeit only partially, even at later time points (figure s1c). treatment with actd alone did not induce cell death in control nor hace1 mefs (figure s1d). consistent with a critical role for hace1 in mediating tnf / actd - induced apoptosis, activation of caspase-8 and its downstream death effector caspase-3 as well as cleavage of rip1 kinase (rip1) were markedly impaired in hace1 mefs (figure 2c). we also failed to detect caspase-3 activation in hace1 mefs using a devdase activity assay (figure s1e). similarly, apoptotic death and caspase-3 activation were absent in hace1 mefs challenged with tnf plus cycloheximide (chx) (figure 2d ; figure s1f), an inhibitor of the nf-b - mediated survival pathway (tsuchida., 1995). importantly, re - expression of wt hace1, but not e3 ligase - dead c876s hace1, restored tnf / actd- as well as tnf / chx - induced cell death in hace1 mefs (figures 2e and 2f). smac mimetic drugs such as bv6 induce intrinsic apoptosis by blocking the inhibitor of apoptosis (iap) proteins downstream of tnfr1 (vercammen. we therefore used this model as an additional trigger of tnf - induced apoptotic cell death. baseline ciap1 and xiap expression levels before and after tnf stimulation were comparable in control and hace1 mefs (figure s1 as expected, we observed tnf / bv6-induced cell death in control mefs ; by contrast, hace1 mefs again were resistant to apoptosis induced by tnf and bv6 (figure s1h). these results show that tnfr1-induced apoptosis depends on hace1. to test whether hace1 also regulates tnfr1-mediated apoptosis in vivo, we first challenged control and hace1 mice with recombinant murine tnf. hace1 mice were completely resistant to tnf - induced death and systemic inflammation, as determined by the liver injury markers alt and ast (figures 3a3c). to extend these data to additional in vivo models, we next challenged mice with lipopolysaccharide (lps)/d - gal, which induces liver damage and death in wt mice, but not in tnfr1 mutant mice (pfeffer., 1993). hace1 knockout mice were, to a large extent albeit not completely, resistant to lps / d - gal - induced liver damage and apoptotic death (figures 3d3f). these in vivo data indicate that hace1 deficiency renders mice resistant to apoptotic liver damage and death downstream of tnfr1. in the condition where both nf-b and caspase activation are either genetically or pharmacologically blocked, tnf stimulation may result in a non - apoptotic form of cell death termed necroptosis (cho., 2009, christofferson and yuan, 2010, declercq., 2009, to determine whether loss of hace1 also interferes with tnf - induced necroptosis, wt and hace1 mefs were pre - treated with the pan - caspase inhibitor z - val - ala - asp - fluoromethyl ketone (z - vad) and then stimulated with tnf / actd to induce cell death. we observed that both wt and hace1 mefs are comparably sensitive to necroptosis triggered by tnf / actd / z - vad (figure 4a). these findings also were confirmed using tnf / chx / z - vad as an alternative necroptotic drug combination (figure 4a). of note, treatment with z - vad alone or in combination with either actd or tnf did not induce cell death in control or hace1 mefs (figures s1d and s2a). two kinases, rip1 and rip3, mediate necroptotic cell fate downstream of tnfr1 (christofferson and yuan, 2010, declercq., 2009). pre - treatment with the specific rip1 kinase inhibitor necrostatin 1 (nec-1) (degterev., 2008) reverted necroptotic cell death in both control and hace1 mefs (figure 4a). we next generated hace1 ripk3 double - knockout (hace1 ripk3) mefs to genetically determine the contribution of rip3 on cell death in hace1 mefs. these double - mutant cells exhibited growth curves comparable to control mefs (figure s2b). importantly, whereas hace1 mefs were sensitive to necroptosis, hace1 ripk3 mefs were resistant to necroptotic death (figure 4b). thus, hace1 is required for tnf - induced nf-b activation and apoptosis induction, but it is dispensable for the necroptotic response following tnfr1 stimulation. to identify molecular targets of hace1 that could explain impaired nf-b activation and defective apoptosis downstream of tnfr1, we established a hace1-dependent in vitro ubiquitylation assay using hemagglutinin (ha)-ubiquitin, the ubiquitin - activating e1 enzyme, and multiple e2 enzymes. the most efficient e2 for hace1 e3 ligase activity was found to be ubc13 (figure s3a). using recombinant hace1, ubc13, the e1 enzyme, and ha - ubiquitin, we then performed in vitro ubiquitylation assays on microchips spotted with > 9,000 natively folded human proteins. intriguingly, our microchip array data revealed multiple hace1 ubiquitylation targets, many of which had been annotated previously to the tnfr1/nf-b - signaling pathway (figures s3b and s3c). using in vitro ubiquitylation assays previous reports indicated that traf2 plays a pivotal role in driving nf-b and jnk activation and inhibiting necroptosis after stimulation with tnf. this function is modulated by k63-linked ubiquitylation of traf2 (karl., 2014, li., 2009, petersen., 2015, yeh., 1997). to identify the nature of traf2 ubiquitylation mediated by hace1, we performed an in vitro ubiquitylation assay. we found that hace1 directly catalyzes k63-linked ubiquitylation of traf2 (figure s4a). importantly, hace1 is essential for this particular modification : lack of hace1 led to the abrogation of tnf - driven k63-linked ubiquitylation of traf2 (figure s4b). we failed to detect tnf - induced k48-specific ubiquitylation of traf2 in control and hace1 mefs (figure s4c) as well as in in vitro ubiquitylation assays (not shown). defective ubiquitylation was restored in hace1 mefs reconstituted with wt hace1, but not in the e3 ligase - defective hace1 c876s mutant (figure s4d). similar to hace1 mefs, rnai - mediated downregulation of hace1 in wt mefs impaired ubiquitylation of traf2 following tnf stimulation (figure s4e). activation of tnfr1 induces rapid formation of a traf2-containing plasma membrane - bound signaling complex (complex i) (micheau and tschopp, 2003). within complex i, k63 ubiquitylation of traf2 has been previously associated with the ability to activate nf-b and to induce jnk signaling (habelhah., 2004, li., 2009, liang., 2010, xia and chen, 2005). endogenous hace1 associated with traf2, an interaction that was markedly enhanced upon tnf stimulation (figures s5a and s5b). moreover, endogenous hace1 co - immunoprecipitated with tnfr1 in wt mefs, an interaction that appears to be constitutive (figures s5c and s5d). cell surface expression of tnfr1 in hace1 mefs was comparable to that in control cells (figure s5e). immunoprecipitation of complex i using flag - tagged human tnf (micheau and tschopp, 2003) showed that all the essential components of complex i, such as ikk, ikk, traf2, as well as rip1, were recruited to the activated tnfr1 in both control and hace1 mefs (figure 5a ; figure s5c). rip1 kinase appeared to be normally phosphorylated and ubiquitylated within complex i (figure 5a ; figures s5f and s5 g). serial immunoprecipitation experiments, carried out to pull down traf2 from the tnf - activated complex i (lu., 2013), showed a marked defect in k63 ubiquitylation of traf2 in hace1 versus control mefs (figure 5b ; figure s5h). thus, loss of hace1 has no apparent effect on the assembly of complex i ; however, hace1 is essential for the ubiquitylation of complex i - associated traf2. following initial formation of complex i, tnfr1 stimulation results in the assembly of a secondary cytoplasmic complex, termed complex ii (micheau and tschopp, 2003), which drives caspase-8-dependent apoptotic cell death (declercq., 2009, kaiser., 2011, 2011). to investigate whether the observed resistance of hace1 mefs to tnf - induced apoptosis originates from defective complex ii formation, we analyzed the assembly of the apoptotic complex ii following tnf / actd stimulation. in hace1 mefs, the association of traf2 with fadd (figure 5c), as well as its tnf stimulation - dependent association with rip1 kinase (figure 5d), was severely impaired. further in vitro assays using recombinant proteins showed that traf2 and fadd interaction depends on ubiquitylation of traf2 (figure s6a). moreover, the recruitment of rip1 kinase and caspase-8 to fadd also was severely affected (figure 5e), demonstrating that loss of hace1 expression results in defective assembly of the caspase-8-activating complex. loss of hace1 does not impair necroptosis downstream of tnfr1 stimulation (figure 4a). necroptosis is mediated via the formation of complex ii containing, among other proteins, fadd, caspase-8, and both rip1 and rip3 kinases. when caspase-8 is inactivated, rip1 and rip3 kinases together induce programmed necrosis (cho., 2009, christofferson and yuan, 2010, declercq., 2009, galluzzi and kroemer, 2008, zhang., 2011). as reported previously (wang., 2008), z - vad inhibition of caspase-8 in tnf / actd - treated wt mefs was associated with enhanced stability of the fadd / caspase-8/rip1 kinase complex (figure 5e ; figure s6b). intriguingly, the association of rip1 kinase and caspase-8 with fadd was found to be virtually absent in hace1 mefs undergoing tnf / actd / z - vad - triggered necroptosis (figure 5e ; figure s6b). in both control and hace1 mefs, this interaction is mirrored by the phosphorylation of the necroptotic downstream mediator mlkl (figure 5 g) (vanden berghe., 2014), indicating that induction of rip1/rip3-dependent necroptosis is not affected by the absence of hace1. interestingly, rip1/rip3 interactions can be detected, albeit at reduced levels, in hace1 mefs even under apoptotic conditions (figure 5f). however, we failed to detect any association between the rip1/rip3 kinases and caspase-8 under necroptotic conditions in hace1 mefs treated with either tnf / actd / z - vad or with tnf / bv6/z - vad (figures s6b and s6c) (he., 2009). importantly, we still observed rip1 phosphorylation, a marker of rip1 kinase activity and necroptotic pre - disposition (declercq., 2009), in both control and hace1 mefs treated with tnf / actd / z - vad (figure 5e ; figure s6b) or tnf / bv6/z - vad (figure s6c). thus, loss of hace1 expression impairs the assembly of the canonical complex ii without any apparent effect on the formation and activity of the necrosome formed by rip1 and rip3 kinases and phosphorylation of mlkl. our data so far showed that loss of hace1 protects cells from tnf - induced apoptosis in vitro while leaving tnf - mediated rip1/rip3 kinase - dependent necroptosis intact. since tnfr1, nf-b, and rip3 kinase recently have been implicated in intestinal homeostasis (gnther., 2011, pasparakis, 2009, welz., 2011), we tested whether hace1 might play a role in gut inflammation. naive hace1 mice showed normal body weight (figure s7a), and morphological and flow cytometric analyses of the intestine of naive hace1 mice did not reveal any overt alteration compared to wt littermates (figures s7b and s7c). however, when we challenged mice with dextran sodium sulfate (dss), a chemical irritant that disrupts the intestinal epithelial barrier and induces colitis (de robertis., 2011), we observed markedly increased intestinal inflammation in hace1 versus control hace1 mice. hace1 mice suffered a more pronounced weight loss than wt mice when treated with dss (figure 6a), and histopathological analysis revealed markedly more severe inflammation, tissue destruction, and epithelial cell death in hace1 mice compared to wt littermate controls (figure 6b ; figure s7d). in addition, hace1 mice showed more severe diarrhea and increased intestinal bleeding (hemoccult test) compared to wt littermates (figures 6c and 6d). flow cytometric analysis of the immune system in the lamina propria revealed markedly increased infiltration of cd11b gr-1 ly6 g inflammatory monocytes in the intestines of hace1-deficient mice after treatment with dss (figure 6e), confirming the increased severity of intestinal inflammation in hace1 compared to control wt mice. nf-b acts downstream of surface receptors recognizing pro - inflammatory cytokines, including tnf itself, as well as pathogen - associated pattern receptors, which in turn leads to the expression of further inflammatory mediators (bonizzi and karin, 2004). on the other hand, nf-b expression in epithelia such as the intestine plays a fundamental role in ensuring epithelial cell survival and in maintaining the structural integrity of mucosal surfaces (pasparakis, 2009). hace1 mrna is expressed at comparable levels in a variety of immune cells as well as in intestinal epithelial cells (figure s7f). to discern in which cells hace1 is acting to prevent severe colitis, we generated bone marrow chimeric mice lacking hace1 exclusively in the immune system by transferring hace1-deficient bone marrow cells in lethally irradiated hace1-sufficient host mice. as t cells in the intestine are largely radioresistant, we used lymphopenic rag2 mice as hosts (korag2) to prevent host - derived wt t cells to persist in the intestine, thus affecting the validity of the system. as a control group, we reconstituted irradiated rag2 mice with wt bone marrow (wtrag2). treatment of these chimeric mice with dss revealed that the absence or presence of hace1 in the immune system had no apparent effect on the development of colitis : both groups showed comparable weight loss (figure s7 g) and similar recruitment of inflammatory cells to the intestinal lamina propria (figure s7h). these results indicate that the loss of hace1 in radiosensitive immune cells is not responsible for the increased susceptibility of hace1 mice to colitis. importantly, genetic inactivation of rip3 kinase in hace1 ripk3 double - knockout mice and inactivation of tnfr1 in hace1 tnfr1 double - knockout mice completely rescued all parameters of the severe inflammatory phenotype in hace1 mice to levels observed in wt littermates, including weight loss, tissue damage, death of epithelial cells, recruitment of inflammatory cells, as well as diarrhea and intestinal bleeding (figures 6a6e ; figure s7d). also, the expression of several pro - inflammatory mediators was upregulated in the colon of hace1 mice ; in hace1 tnfr1 and hace1 ripk3 mice the levels of these pro - inflammatory factors were again restored to levels detected in wt controls (figure s7e). the increased death of intestinal epithelial cells in dss - treated hace1 mice is probably the consequence of exaggerated immune activation and inflammation resulting from the preferential induction of tnf - driven necroptosis instead of apoptosis in the absence of hace1 (pasparakis and vandenabeele, 2015). of note, while others reported increased severity of colitis in ripk3 (moriwaki., 2014) and, to a lesser extent, in tnfr1 mice (stillie and stadnyk, 2009, wang., 2012), in our hands single - knockout mice showed no change in weight loss compared to wt littermates (figure 6f). the difference in ripk3 mice might be due to the time point of analysis : moriwaki. (2014) reported a defect of ripk3 animals in the recovery from dss, while we focused on the earlier phase of tissue destruction during which enhanced necroptosis is known to be detrimental (gnther., 2011). together, our data show that the increased susceptibility of hace1-deficient mice to dss - induced colitis depends on deregulated tnfr1 signaling and rip3 kinase. we initially identified hace1 as a tumor suppressor gene downregulated in multiple cancers (anglesio. since we observed increased intestinal inflammation, we next explored whether hace1 mice are prone to develop colon cancer upon challenge with dss and the genotoxic drug azoxymethane (aom) (tanaka., 2003). since hace1 mice are particularly sensitive to dss, we reduced the dss treatment regimen to a level not sufficient to induce colon cancer in wt mice (de robertis. as expected using this low dss dose, we failed to detect any tumors in control hace1-expressing mice (figure 7a). however, in hace1 mice, we observed a markedly increased incidence of colon cancer as determined by colonoscopy or histopathological examination (figures 7a and 7b). colonic tumors in hace1 mice show features consistent with adenomatous polyps as well as nuclear atypia, multiple mitotic figures, areas of carcinoma in situ, and focal invasion (figure 7b). most importantly, hace1 ripk3 as well as hace1 tnfr1 double - knockout mice were similar to wt mice and did not show aom / dss - induced colonic tumors (figures 7a and 7b), indicating that simultaneous genetic inactivation of tnfr1 and rip3 kinase considerably reduced colon cancer formation secondary to hace1 deficiency. in conclusion, hace1 deficiency predisposes to colonic inflammation and carcinogenesis, which is markedly alleviated by genetic inactivation of tnfr1 and rip3 kinase. loss of hace1 impairs tnfr1-mediated activation of nf-b signaling and apoptosis in vitro and in vivo, but it does not inhibit tnfr1-dependent rip1/rip3-induced necroptosis. moreover, our data show that enhanced inflammation and colon cancer in hace1 mutant mice can be reverted by genetic inactivation of rip3 kinase and tnfr1. loss of hace1 does not impair formation of complex i at tnfr1 ; however, nf-b and jnk activation are markedly impaired in hace1 mefs, a function dependent on its e3 ligase activity. our results indicate that hace1 expression is essential for k63 ubiquitylation of traf2 within complex i upon tnfr1 stimulation, which previously has been associated with the ability to activate the canonical nf-b pathway and to induce jnk signaling (habelhah., 2004, liang., 2010, xia and chen, 2005). although nf-b signaling is impaired, hace1 mefs do not undergo apoptosis in response to tnfr1 stimulation, which can be biochemically explained by the fact that hace1 cells fail to form pro - apoptotic complex ii. moreover, our results show that traf2 ubiquitylation is required for traf2 to associate with the key death molecule fadd, which in turn nucleates the caspase-8 apoptosis effector complex. however, besides traf2, we found multiple additional hace1 ubiquitylation targets that previously have been linked to cell death and signaling pathways downstream of tnfr1, suggesting that hace1 could regulate tnfr1-regulated cell fate at various interception points. our in vitro and in vivo data indicate that necroptosis in hace1 mutant cells is mediated via the rip1/rip3/mlkl pathway. since we failed to detect formation of an intact necrosome, such death signals might progress independently of the canonical molecular necroptotic complex. necroptosis in the context of hace1 deficiency appears to occur without direct caspase-8 interaction with rip1/rip3 kinases, raising the possibility that alternative rip1/rip3 kinase complexes might exist when hace1 is inactivated. of note, in our experimental system, induction of necroptosis in hace1 knockout mefs still requires caspase inhibition via z - vad. whether regulation of cflip (oberst., 2011) could explain this remaining caspase dependency requires further exploration. moreover, a feedback inhibition loop via rip3 kinase activity has been reported recently in the control of caspase-8-mediated apoptosis (newton., 2014), suggesting complex interplays between the apoptotic and necroptotic death effector machineries. we initially identified hace1 as a candidate tumor suppressor gene in a child with wilms tumor (anglesio. in accord with its putative tumor suppressor function, we found hace1 to be downregulated in multiple cancers (hibi., 2008, sakata., 2009). that hace1 can indeed suppress cancer was shown in hace1 knockout mice that develop tumors of multiple tissues (zhang., 2007). since the tnfr1 and rip1/rip3 kinase pathway has been reported to induce oxidative stress and possibly alter cellular metabolism (christofferson and yuan, 2010, declercq., 2009, galluzzi and kroemer, 2008), our results suggest that this activation pathway downstream of tnfr1 might not only trigger necroptosis but also could be involved in oncogenesis. moreover, rip1/rip3 kinases downstream of tnfr1 have been implicated in a pro - inflammatory cascade in the intestine unleashed by smac mimetics or caspase-8 deficiency (kang., 2013, vince., 2012), driven by the release of necroptosis - mediated damage - associated molecular pattern molecules (damps) (duprez. mutant mice indeed develop increased intestinal inflammation following epithelial injury and increased incidence of colon cancer. importantly, genetic deletion of either rip3 kinase or tnfr1 not only reversed the enhanced inflammation but also strongly reduced the development of colon cancer in hace1 single - mutant mice. these data demonstrate that deregulation of the tnfr1-mediated rip3 kinase signaling is a key driving principle of inflammation and subsequent colon cancer development controlled by hace1. the mice, hace1, hace1, hace1 tnfr1, and hace1 ripk3 on a c57bl/6 background, were bred and maintained under specific pathogen - free conditions at the institute of molecular biotechnology of the austrian academy of sciences (imba) animal facility. for experiments, age- and sex - matched mice (812 weeks of age) all animal experiments were approved by austrian authorities, and mice were kept following the guidelines of austrian animal laboratory law. mefs derived from hace1 and hace1 mice were prepared as described previously (zhang., 2007). mefs were seeded at 2 10 cells per 60-cm petri dish and cultured in dmem supplemented with 5% fetal calf serum (fcs) and 1% pen - strep (sigma) in a 5% co2 incubator. the next day, cells were treated with 10 ng / ml mouse tnf (3014, immunotools), 1 g / ml actd - mannitol (a-5156, sigma), 5 g / ml z - vad (bachem), and 50 nm nec-1 (n-9037, sigma). in addition, cells were treated with 10 ng / ml mouse tnf (3014, immunotools) and 10 g / ml chx (c1988, sigma). following the treatments, the cells were lysed in 1 ml ice - cold np-40 buffer (10 mm tris [ph 8.0 ], 150 mm nacl, and 1% nonidet p-40) supplemented with protease and phosphatase inhibitor tablets (11873580001 and 04906837001, roche). the lysates were then centrifuged at 14,000 rpm for 10 min at 4c. immunoprecipitations were performed according to the manufacturer s protocol. for high - dose lethal tnf challenge, animals were injected via the tail vein with 450 g / kg of purified endotoxin - free tnf (immunotools) in 200 l pbs (ph 6.8). for induction of hyper - acute shock, tnf was used at a dose of 5 g / kg. lps (l2880, sigma) was administrated intraperitoneally (i.p.) at a concentration of 10 g / kg. d - gal (g0500, sigma) was given to mice together with lps at a concentration of 1 g / kg. liver enzymes were assayed using the cobas c111 system (roche). for histology, livers were embedded in paraffin, sectioned, and stained with h&e. listeria monocytogenes (strain lo28) was prepared for infection as described previously (stockinger., 2009). the bacterial suspension (500 l) was injected into the peritoneum of 8- to 10-week - old male hace1 and hace1 littermate mice. survival of infected mice was monitored for 10 days. for determination of the bacterial load, mice were euthanized 96 hr after infection and spleens were homogenized in pbs. serial dilutions of homogenates were plated on oxford agar plates and colonies were counted after growth at 37c for 24 hr. if not otherwise stated, all data are expressed as the means sd. significance was assessed by student s two - sided t test unless otherwise stated in the figure legends. for multiple comparisons, we used a two - way anova test with a bonferroni post - test. differences with p values < 0.05 were considered as significant (p < 0.05, p < 0.01, and p < 0.001). g.s.- f., f.i., g.k., p.v., h.k., a.p., and | summarythe hect domain e3 ligase hace1 has been identified as a tumor suppressor in multiple cancers. here, we report that hace1 is a central gatekeeper of tnfr1-induced cell fate. genetic inactivation of hace1 inhibits tnf - stimulated nf-b activation and tnfr1-nf-b - dependent pathogen clearance in vivo. moreover, tnf - induced apoptosis was impaired in hace1 mutant cells and knockout mice in vivo. mechanistically, hace1 is essential for the ubiquitylation of the adaptor protein traf2 and formation of the apoptotic caspase-8 effector complex. intriguingly, loss of hace1 does not impair tnfr1-mediated necroptotic cell fate via rip1 and rip3 kinases. loss of hace1 predisposes animals to colonic inflammation and carcinogenesis in vivo, which is markedly alleviated by genetic inactivation of rip3 kinase and tnfr1. thus, hace1 controls tnf - elicited cell fate decisions and exerts tumor suppressor and anti - inflammatory activities via a tnfr1-rip3 kinase - necroptosis pathway. |
a 75-year - old asian female without systemic disease initially presented with nasal congestion, mucous rhinorrhea and progressively decreasing sense of smell. she was treated as allergic rhinitis with oral antihistamine and nasal steroid spray, but no obvious improvement. one month after the symptoms, paranasal sinus computed tomography (ct) showed heterogeneous soft tissue densities in the left maxillary, frontal, bilateral ethmoid, and sphenoid sinuses, with extensive bony destruction [fig. 1 ]. paranasal sinus ct (a : coronal view, b : axial view) shows sinusitis with bony erosion over left maxillary, frontal, bilateral ethmoid and sphenoid however, 6 months later, the patient was referred to our clinic with a 2 weeks history of declining vision in one eye (oculus sinister [os ]). examination disclosed no light perception and revealed a relative afferent pupillary defect, but normal fundus appearance [os ; fig. 2a ]. visual field testing (oculus dexter) showed a temporal defect with moderate constriction [fig. the diagnosis was not confirmed with suspicion of central retinal artery occlusion or optic neuropathy. orbital magnetic resonance imaging (mri) was suggested and indicated multiple sinusitis with bone erosion and intracranial extension. furthermore, encasement of the right optic nerve and infiltration of the left optic nerve near the optic chiasm were noted without abnormal optic nerve enhancement after contrast [fig. 4 ]. due to the finding of the intracranial lesion, surgical debridement for sinusitis with suspected optic neuropathy was suggested by the ophthalmologist but not performed immediately. fundus appearance reveals no obvious abnormality, including disc, macula and vessels initially 2 weeks after left visual loss (a). six months later with bilateral visual loss, disc does not reveal pale appearance (b) (poor quality due to loss of fixation) visual field (vf). vf (oculus dexter) shows temporal defect with moderate constriction while visual loss optic nerve in orbital magnetic resonance imaging. encasement of the right optic nerve (marked as r) by the fluid - filled onodi cell () and the fluid - filled anterior clinoid process (+) is suspected on t2-weighted image (a) ; left optic nerve (l) is infiltrated by the anterior cranial fossa lesion (#) (b, t1 with enhancement and fat suppression), which are indicated in coronal view (c, t1 with enhancement and fat suppression, right optic nerve marked as, left optic nerve marked as) moreover, 3 weeks after the first episode of vision loss (os), vision loss suddenly occurred in the other eye. under the impression of optic neuropathy associated with presumed sinusitis (oculus uterque [ou ]), the patient received mega - dose steroids (methylprednisolone 250 mg q6 h for 5 days) and emergent bilateral sinusectomy. because of unusual clinical course, the findings were highly suggestive of sinonasal undifferentiated carcinoma (snuc)-related retrobulbar infiltrative optic neuropathy (ou) ; therefore, the patient underwent concurrent radiochemotherapy as malignancy therapy. three months after the first episode of visual loss, ocular examination still showed no light perception (ou). the pattern visual evoked potential (vep) failed to perform due to poor vision and flash vep [fig. 6 ]. at the final visit (6 months after the first visual loss episode), no light perception (ou) persisted even though the disc appeared pinkish [fig. two months after visual loss (oculus dexter [od ]) and 3 months after visual loss (oculus sinister [os ]), pattern vep fails due to tracing inability with no light perception ; flash vep reveals delayed latency (od : 120 ms, os : 140 ms) and decreased amplitude (od : 11 uv, os : 7.5 uv) electroretinography (erg), erg reveals no obvious abnormal amplitude both in rod and cone function rhinogenic optic neuropathy is characterized by optic nerve damage secondary to the sinonasal lesion, and includes various sinonasal diseases such as sinusitis, mucocele, and sinus surgery, with the main mechanism being compression and inflammatory changes. in addition to optic neuropathy, orbital invasion is a common manifestation with ocular findings such as lid edema, blepharoptosis, conjunctival chemosis, conjunctival injection, diplopia, limitation of ocular motility, globe displacement, epiphora, anisocoria, eye pain, and photophobia. the efficacy of steroid therapy for rhinogenic optic neuropathy has not been elucidated, possibly due to the limited number of cases. surgical intervention in the form of orbital or optic nerve decompression accompanied by systemic steroids resulted in favorable visual outcome ; however, poor prognosis was still mentioned in case reports of sphenoid sinus mucocele and eosinophilic chronic rhinosinusitis. preoperative visual acuity tended to correlate with prognosis ; therefore, patients with severely impaired vision should be given more attention and treated aggressively. snuc, a rare and aggressive malignancy, was first described in 1986 as a highly aggressive neoplasm arising from the nasal cavity and paranasal sinuses. the most common locations of snuc are the nasal cavity and ethmoid sinus ; in addition, orbital and intracranial invasion and distant metastasis are frequent findings. bone destruction and invasion of adjacent structures such as the orbit, cranial vault, and skull base is frequently seen. in addition to retrobulbar optic neuropathy, other ophthalmic symptoms of the oculomotor nerve palsy, optic atrophy, and proptosis are possible manifestations. due to the frequent cranial invasion, there are several possible mechanisms to explain the sudden visual loss with intracranial mass lesions, including ischemia due to vascular compression of the visual pathways or demyelination secondary to compression. in all previous cases of snuc related optic neuropathy, one eye is affected more than the other eye, or just only one eye affected, while this case is totally blindness in both eyes. the cause of bilateral visual loss was possibly ischemia, originating from a compromised visual pathway vascular system caused by the snuc, which could not be excluded even though no optic disc pale was noted 3 months after first visual loss (os). distinguishing mucosal inflammatory and neoplastic lesions of the paranasal sinuses by medical imaging modalities may be appropriate but difficult, as seen in our patient. snucs have typical but nonspecific imaging characters, such as a large, expansile, and aggressive lesion with bone destruction and invasion of adjacent structures, including the anterior cranial fossa, adjacent paranasal sinuses, and orbits. on ct scans, the lesions are typically isointense to muscle on t1-weighted scans, iso - to hyperintense to muscle on t2-weighted scans, and heterogeneous on enhancement with gadolinium [fig. these imaging findings could not provide much advantage in differentiating snuc from other pathological entities in this region. sinonasal undifferentiated carcinoma (snuc) in different magnetic resonance imaging modalities. to differentiate snuc by different modalities, the lesion reveals isointense to muscle on t1-weighted image (a, marked as) and heterogeneous appearance after enhancement (c, marked as) overall survival of patients with snuc is poor in most reported series, especially for those presenting with brain parenchymal invasion. the visual outcome is not available in the literature owing to the limited number of cases ; however, surgical decompression with steroid therapy did not results in any improvement in our patient. no clear consensus exists regarding treatment for snuc, but a combination of operation, chemotherapy, and radiotherapy may be suggested. to the best of our knowledge, this is the first report of bilateral sequential total blindness induced by snuc - related rhinogenic optic neuropathy. because of aggressive optic neuropathy and poor survivor in snuc, it is important to differentiate between chronic sinusitis and snuc, or other sinonasal malignancies, based on clinical presentation and image study of mri. atypical clinical presentation includes poor response to medication, such as antihistamine and antibiotics, ocular and visual symptoms ; furthermore, imaging suggests sinus wall bony erosion, multiple invasion, and obvious intralesional necrosis. more attention and aggressive surveillance, such as early biopsy, should be given to the patients mentioned above. however, in our experience, surgical decompression by sinusectomy with systemic steroids and antibiotics did not improve the visual outcome, which emphasizes the rapid disease progression and importance of early diagnosis and treatment. | sinonasal undifferentiated carcinoma - related rhinogenic optic neuropathy is rare and may lead to visual loss. to the best of our knowledge, this is the first report of bilateral sequential visual loss induced by this etiology. it is important to differentiate between chronic sinusitis and malignancy on the basis of specific findings on magnetic resonance images. surgical decompression with multidisciplinary therapy, including steroids, chemotherapy, and radiotherapy, is indicated. however, no visual improvement was noted in this case, emphasizing the rapid disease progression and importance of early diagnosis and treatment. |
chronic obstructive pulmonary disease (copd) is characterized by chronic airflow limitation that is not fully reversible and is highly prevalent worldwide. although cigarette smoking is a major risk factor for copd, the mechanism by which inhaled smoke contributes to airflow obstruction is not fully understood. current theories for this include persistent airway inflammation that is modified by oxidative stress, excess proteinases, autoimmunity, and apoptosis of alveolar cells. gene expression studies of diseased lungs can generate high - throughput results to shed light on the molecular processes underlying copd pathogenesis. whole - genome expression of copd in humans has been studied using airway epithelium and resected lung tissue in several groups [39 ]. background and cross - hybridization are not issues in rna - seq and the technology can quantify both lowly and highly abundant transcripts. in addition, this method can provide information about splice variants, allele - specific expression, and fusion transcripts. rna - seq data of airways was recently published [13, 14 ] ; however, the number of subjects in these studies was relatively small. we performed gene expression profiling using rna - seq of lung tissues resected from large numbers of subjects with copd or with control subjects to better understand the molecular mechanisms responsible for the pathogenesis of copd. subjects were patients who required resection for lung cancer and who were registered in the asan biobank from january 2008 to november 2011. the inclusion criteria were a postbronchodilator fev1/fvc ratio (ratio of forced expiratory volume in the first second to forced vital capacity) of less than 0.7 for the copd group and normal spirometry for the control group in accordance with american thoracic society / european respiratory society criteria. this study was approved by the institutional review board of asan medical center (2011 - 0711) and written informed consent was obtained from all patients. total rna was isolated from apparently normal fresh frozen lung tissue that was remote from the lung cancer. rna integrity was assessed using an agilent bioanalyzer and rna purity was assessed using a nanodrop spectrophotometer. one g of total rna was used to generate cdna libraries using the truseq rna library kit. the protocol consisted of poly a - selected rna extraction, rna fragmentation, reverse transcription using random hexamer primers, and 100 bp paired - end sequencing using the illumina hiseq 2000 system. all data have been deposited in the ncbi gene expression omnibus (geo) public repository and can be accessed through the accession number gse57148. for quality control, differential gene expression (deg) analysis was performed using tophat and cufflinks software. to estimate expression levels, the rna - seq reads were mapped to the human genome using tophat (version 1.4.1) and quantified using cufflinks software 2.0.0. the numbers of isoform and gene transcripts were calculated and the relative abundance of transcripts was measured in fragments per kilobase of exon per million fragments mapped (fpkm). expression levels were extracted as a fpkm value for each gene of each sample using cufflinks software. statistical significance was determined using student 's t - test. the false discovery rate (fdr) the analysis steps used are summarized in figure 1. to investigate whether degs are related to clinical phenotypes, we performed a linear regression analysis for 5 clinical phenotypes with respect to its gene expression. we considered each clinical phenotype as the responder for regression and each gene expression as the predictor. several genes whose expression level was found to be related to copd status by rna - seq were validated using taqman real - time pcr. functional enrichment analysis was performed using gene set enrichment analysis (version 2.0.8), which combines information from previously defined gene sets obtained from the molecular signature database (version 3.1). biological gene functional annotation analysis was performed using david (version 6.7) with a list of degs. biolattice (version 1.1) was used to annotate coexpressed gene groups to go biological process terms and visualize their relations. to detect differential alternative splicing between the two groups, subjects from each group were evaluated using a multivariate bayesian algorithm called multivariate analysis of transcript splicing. differential alternative splicing, including exon skipping, mutually exclusive exons, alternative 5 or 3 splice site usage, and intron retention, was investigated. a connectivity map was used to identify potential drugs that might reverse the gene expression pattern associated with the pathogenesis of early copd. the connectivity map is a collection of genome - wide transcriptional expression data from cultured human cells treated with bioactive small molecules. the basic assumption of the connectivity map is that transcriptional perturbation can occur or be treated by certain drugs that intrigue similar changes. the demographic characteristics of 98 subjects with copd and 91 control subjects are shown in table 1. all subjects were male and the mean age and the mean pack years of cigarette smoking history were higher in the copd group than in the control group. as expected, pulmonary function was significantly lower in the copd group than in the control subjects. in copd group, 28 subjects took inhaled corticosteroid, 40 subjects took tiotropium, and 22 subjects took short - acting beta - agonist. the total number of reads produced from each sample was 38,742,474 7,332,014 reads (mean standard deviation). the difference in the number of reads between copd samples and control samples was not statistically significant. after read alignment with tophat and read quantification with cufflinks using ucsc hg19 transcriptome reference, a total of 189 samples and 23,146 genes were analyzed. a total of 248 genes had zero fpkm values in all samples. after filtering for genes with zero counts in whole samples, noncoding genes, and low variance genes, 16,676 genes were analyzed. out of these genes, 2,312 genes were differentially expressed between the two groups (fdr corrected q < 0.01) (table 3). there were many overlaps between t - test and edger (see supplementary table 1 in the supplementary material available online at http://dx.doi.org/10.1155/2015/206937). regression analysis of the degs with clinical phenotypes revealed that genes were more associated with fev1 (forced expiratory volume in the first second) and fev1/fvc ratio than with age and smoking history (figure 2). to validate the rna - seq results, we performed qrt - pcr of the fgg, mcl1, pde4a, s100a6, serpine1, sftpc, and tmsb4x genes using the same rna samples that were used for rna - seq. the rna - seq and qrt - pcr results were in good agreement (figure 3). a heat map shows the genes that were differentially expressed between the two groups (figure 4). david revealed that the expression of genes involved in protein catabolism, oxidative phosphorylation, and chromatin modification differed most significantly between the two groups (table 4). expression of genes encoding proteasome components including psma2, psmb1, psmc5, and psmd4 was lower in the copd group than in the control group. gene set enrichment analysis revealed that the most significantly downregulated pathways in the copd group were oxidative phosphorylation and biosynthetic process (fdr q < 0.25). we used k - means clustering to construct 30 groups of coexpressed genes for biolattice. after matching with hypergeometric distributions to annotate those gene sets to go terms, concept lattice for coexpressed gene clusters with go biological process terms was visualized showing that genes related to transcription and oxidative phosphorylation are enriched in the major clusters (figure 5). in the copd group, the heat map shows hierarchical clustering of two subgroups using euclidean distance (figure 6). four hundred and four genes that were differentially expressed between these two subgroups in copd subjects (q < 0.05) are enriched in the mitochondrial and steroid biosynthesis pathway. group 1 showed tendency for lower fev1 and lower dlco (carbon monoxide diffusing capacity). when comparing each group with the control, degs between group 1 and the controls only consisted of 18 genes, and their p values were negligible. meanwhile, degs between group 2 and the controls consisted of 4072 genes and their p values were even higher than those of degs between the copd group and the controls. isoforms that were differentially expressed between the copd and control groups were evaluated by cufflinks. however, among the deis, 310 were not in the deg list, which are enriched in genes encoding proteins that function in cell junctions and focal adhesions. the multivariate analysis of transcript splicing program (mats) revealed that specific alternative splicing events were significantly more common in copd subjects than in control subjects. five categories of differentially expressed isoforms were identified by mats : skipped exon, alternative 5 splice site, alternative 3 splice site, mutually exclusive exons, and retained introns. significantly different events between the copd group and the control group are shown in the supplementary table 2 (fdr q < 0.01). intron retention of hnrnph1 and vim occurred significantly more in the copd group than in the control group. mutually exclusive exons occurred more frequently in the copd group than in the control group in 78 genes. a connectivity map was used to identify potential drugs that might reverse the gene expression pattern associated with the pathogenesis of early copd. gene expression changes arising from treatment with several drugs were negatively correlated with the expression patterns that differ in the copd group compared to the control group. gene expression changes arising from treatment with mg-262 (p = 0.00004) and puromycin (p < in this study, we used rna - seq to identify genes whose expression differs between copd and control subjects. in total, 2312 genes were identified with fdr q < 0.01. we validated a subset of rna - seq data with qrt - pcr, and the results were in good agreement. previous studies have investigated the gene expression profiles of copd patients using microarray, serial analysis of gene expression, and rna - seq. microarray data indicates that mical2 and notch2 are upregulated in the resected lung tissue of copd patients ; the expression of these genes was higher in the copd group than in the control group in the present study. the expression of genes encoding ribosomal proteins and s100a6 was lower in the copd group than in the control group, and rna - seq previously indicated that the expression of these genes is reduced in the small airway epithelium of smokers. however, there is little overlap between studies in terms of the genes that are differentially expressed between people with reduced lung function and those with normal lung function. this may be because different methods were used or because different phenotypes were examined. of interest, previous studies of the copd transcriptome reported a high degree of overlap in the biological processes affected. in the current study, the expression of mitochondrial genes was previously shown to be reduced in the lung tissues of copd subjects using serial analysis of gene expression. a recent report showed that expression of the mitochondrial membrane protein phb1 is downregulated in lung tissue of copd patients, suggesting that mitochondrial stability is reduced. another report revealed that mitochondrial mass is reduced in airway epithelial cells exposed to particulate matter, and this defect is also observed in the daughter cells. one possible explanation may be related to the fact that an increase in the co2 level can reduce oxidative phosphorylation. however, considering that our copd subjects were in relatively early stages of the disease, they are not expected to have a clinically meaningful increase in co2 levels. in the current study, many genes related to the 20s proteasome including psma2, psmb1, psmc5, psmd4, and psmd13, as well as ubiquitin ligase complex genes including stub1, sels, and derl2, were downregulated in copd subjects. the ubiquitination - proteasome pathway is dysregulated in copd, but the mechanism by which this occurs is not fully understood. the expression of 26s proteasome - associated genes is lower in lung tissues of moderate copd subjects than in those of the smoker control subjects with normal lung function. the expression and activity of the proteasome are reduced in lung tissues of copd subjects due to dysregulation of nrf2. interestingly, the proteasome inhibitor mg-262 was on top of a list of drugs that reverse the gene expression pattern of copd. in recent experiments in mice, a proteasome inhibitor was suggested to be a therapeutic agent for pulmonary arterial hypertension via inhibition of pulmonary vascular smooth muscle cell proliferation and correction of endothelial dysfunction. inhibition of proteasome inhibitors can reverse diaphragmatic function in a copd mouse model. in the current study chromatin modification is an important mechanism in epigenetics. in the current study, the expression of mll, which plays an important role in h3k4 methylation, and chd, which is important for chromatin modification and opening of chromatin to allow transcription, the expression of hdac10 was decreased in the lung tissue of copd subjects, while the expression of hdac2 was previously reported to be decreased in copd. several studies report the mechanism by which the expression of hdac2 is reduced in copd, but further studies are required to explain how the expression of several genes related to chromatin modification is increased in copd. in a recent study investigating genes associated with the severity of emphysema, the major pathways affected were inflammation and tissue repair. however, the inflammation pathway was not majorly affected in copd subjects in the current study, probably because the subjects were at relatively early stages of the disease. pathway analysis results of the deis were similar to those of the degs ; however, genes encoding proteins that function in cell junctions and cell migration were found specifically in the deis and not in the degs. alternative splicing of genes is tissue - specific and has important roles in development, physiological responses, and the pathogenesis of diseases. interestingly, the gene encoding vimentin, which is a structural protein, had more retained introns in the copd group than in the control group, which may alter the sequence of the protein. copd subjects were older and had more pack years of smoking than the control group. however, regression analysis results showed that most of degs had stronger correlation with lung function than age or smoking amount. the results of this study may not be applicable to nonsmoking or female copd subjects. microdissection of lung tissue or single cell sequencing would be required to determine whether the differential expression is altered in all lung cells or only in a specific subset of cells. finally, it is difficult to determine whether the dysregulated pathways identified in this study are a cause or a consequence of the pathogenesis of copd. experiments in which the increase / decrease of the degs is reversed and shown to slow disease progression are needed to confirm that these pathways are causally involved in the pathogenesis of copd. in conclusion, reduced oxidative phosphorylation, modulation of protein catabolism, and dysregulation of transcription are important molecular features of early stages of copd. genes and splicing variants were identified that were differentially expressed between copd subjects and control subjects. | background and objectives. chronic obstructive pulmonary disease (copd) is a complex disease characterized by airflow limitation. although airway inflammation and oxidative stress are known to be important in the pathogenesis of copd, the mechanism underlying airflow obstruction is not fully understood. gene expression profiling of lung tissue was performed to define the molecular pathways that are dysregulated in copd. methods. rna was isolated from lung tissues obtained from 98 subjects with copd and 91 control subjects with normal spirometry. the rna samples were processed with rna - seq using the hiseq 2000 system. genes expressed differentially between the two groups were identified using student 's t - test. results. after filtering for genes with zero counts and noncoding genes, 16,676 genes were evaluated. a total of 2312 genes were differentially expressed between the lung tissues of copd and control subjects (false discovery rate corrected q < 0.01). the expression of genes related to oxidative phosphorylation and protein catabolism was reduced and genes related to chromatin modification were dysregulated in lung tissues of copd subjects. conclusions. oxidative phosphorylation, protein degradation, and chromatin modification were the most dysregulated pathways in the lung tissues of copd subjects. these findings may have clinical and mechanistic implications in copd. |
success of osseointegrated implants is predominantly based upon patient selection, conservation of hard and soft - tissues along with following proper surgical techniques and prosthetic protocols. the presence of healthy peri - implant soft - tissue to provide an optimal seal between the oral environment and the implant with its associated super structure plays a crucial role in long - term success of dental implants. the protective barriers for an osseointegrated implant are compromised due to the presence of parallel arrangement of gingival connective tissue fibers resulting in a weak peri - implant mucosal seal. controversies exist in the scientific literature regarding the relationship between the adequacy of the keratinized mucosa and the health of peri - implant tissues. lang and loe established that a minimum of 2 mm of keratinized gingiva (kg) is required to maintain gingival health regardless of the patient 's oral hygiene. however, according to de trey and bernimoulin, the width of attached gingiva is not the only determining factor for implant survival. factors like the patient 's age, oral hygiene maintenance, esthetic considerations and patient 's expectations, should also be taken into consideration. several experimental studies (wennstrm., mericske - stern., adell., lekholm., schou.) have suggested that there is no correlation between implant success rate and the presence of peri - implant keratinized soft - tissue as long as plaque control is maintained. on the other hand, studies by buser, schroeder., and kirsch and ackermann have reported that keratinized mucosa around the abutments is an important requisite for peri - implant health, presence of an adequate amount of kg around to the implant reduces gingival inflammation and hyperplasia and minimizes marginal peri - implant gingival tissues retraction. though implant survival rate is not merely dependent on the width of keratinized tissues, in areas of esthetic concern and difficulties in plaque control the presence or augmentation of keratinized tissue around implants would be desirable for routine oral hygiene maintenance without causing discomfort. hence, soft - tissue augmentation should be considered to provide a keratinized mucous zone that is as wide as possible embracing the implant prosthesis. soft - tissue management in areas of implant rehabilitation may be performed before the surgical phase or after the surgical phase and before the prosthetic phase or after the completion of the prosthetic phase. various periodontal plastic surgical procedures have been proposed for obtaining adequate amounts of keratinized tissue, which include free gingival auto grafts, lateral pedicle rotating flap, apically positioned flap (apf), coronally positioned flap, sub - epithelial connective tissue grafts and cellular dermal grafts. the apf technique has shown to predictably increase the width of keratinized tissue around natural teeth. the increasing height of attached gingiva occurs because of an apical alteration of the mucogingival junction, which includes apical displacement of the muscular insertions. the advantages of apf technique are minimal post - operative bone loss, controlled post - operative position of the gingival margin and it does not require a second surgical site and hence better patient acceptance and comfort. disadvantages being that it can not be advocated for a thin gingival biotype and occasionally formation of a white scar may occur. though a few studies have shown the effectiveness of apf in improving kg around natural teeth, except a case report by park., no studies have been done so far to evaluate the gain of keratinized tissue around dental implants using apf ; hence, the current study aims at evaluating the clinical improvement in kg and esthetic outcome by applying apf technique around dental implants. the study enrolled 12 patients with partially edentulous dentition requiring the replacement of missing teeth either in the anterior or posterior region of maxilla and mandible who had reported to the department of periodontogy and oral implantology of srm dental college, chennai, india. signed informed consent was obtained from the subjects before entering into the study and the study protocol was reviewed and approved by the institutional ethical committee. a total of 12 patients including 6 non - periodontitis and 6 with mild periodontitis were included in this study. baseline evaluation of the full mouth plaque score, probing pocket depth, clinical attachment level, gingival biotype and the width of kg was recorded [table 1 ]. patients with moderate to severe periodontitis, pregnant women, lactating mothers, smokers and those with systemic diseases were excluded from the study. all patients had full mouth scaling and root surface debridement done and if any periodontal surgical procedures were to be performed was completed. reevaluation of base line parameters was carried out after 8 weeks during which four patients who had poor oral hygiene maintenance and persisting active periodontal disease sites were excluded from the study [table 1 ]. finally, a total of 10 endosseous dental implants (self - threaded ez - hi tech life care implants, israel) were placed in selected sites in 8 patients [table 2 ]. apf surgery was performed at the implant site on the buccal aspect either at the time of implant placement (one stage surgical protocol) or during the implant recovery stage (two stage surgical protocols) for increasing the width of kg. a para - crestal incision was placed and a full thickness mucoperiosteal flap was raised 2 mm apical to the cemento enamel junction of the adjacent natural teeth followed by a split thickness flap. two vertical releasing incisions were placed on either side on the buccal gingiva at the mesial and distal line angles of the adjacent teeth. the vertical releasing incisions were sutured with simple interrupted sutures and a periosteal anchoring suture was placed at the base of the flap [figures 1 - 5 ]. periodontal dressing was placed if necessary and routine post - operative instructions and medications were given. patients were followed at 1 week, 4 weeks and at 12 weeks post - operatively. the width of kg was evaluated at baseline and at the end of 12 weeks after performing apically displaced flap surgery [figures 6 and 7 ]. in addition, soft - tissue esthetic outcome was assessed by an independent examiner using visual analog scale (vas) at 12 weeks following the surgery. baseline and 8 weeks re - evaluation values before implant placement implant sites, baseline and 12 week post.operative width of kg and vas score pre - operative edentulous site para crestal and papilla preservation incision given, flap reflection and implant osteotomy performed split thickness flap raised with vertical releasing incisions flap apically displaced and sutured 12 weeks post - operative implant with prosthesis the statistical package for social sciences program spss v19.0 (spss inc, chicago, il, usa) was used to perform paired t - test to evaluate the changes in the width of kg at baseline and at 12 weeks post - operatively. the statistical package for social sciences program spss v19.0 (spss inc, chicago, il, usa) was used to perform paired t - test to evaluate the changes in the width of kg at baseline and at 12 weeks post - operatively. table 2 shows the baseline and 12 weeks post - operative width of kg and vas score around 10 dental implants placed in eight patients. the mean width of kg at baseline was 1.47 mm and 5.42 mm at 12 weeks post - operatively as seen in table 3. paired samples statistics the gain in kg from baseline was 3.95 mm with the p value of 0.000, which was highly statistically significant as seen in table 4a and b. paired samples correlations the assessment of esthetic outcome using vas gave an average score of 7.1 indicating good esthetics. figures 8 and 9 show the diagrammatic representation of the changes in the width of kg at baseline and at 12 weeks post - operatively and the vas score respectively. the plaque accumulation around implants leads to marginal tissue inflammation, which may result in peri - implantitis. block and kent had reported that the presence of keratinized tissue was significantly associated with good mucosal health and crestal bone loss of 2 mm or greater was seen in areas with lack of kg. warrer., in their animal experimental study in monkeys concluded that implants without keratinized mucosa resulted in more recession and attachment loss when compared with implants with adequate keratinized tissue. chung., suggested that plaque accumulation and gingival inflammation were higher around dental implants with keratinized mucosa < 2 mm., kim., schrott., reported that increased width of keratinized mucosa around dental implants is associated with lesser soft - tissue recession and greater hard tissue stability resulting in an advantage for long - term maintenance of implants. the fact that mucosal thickness and the width of keratinized mucosa are of significance particularly in the esthetic zone, where narrow and thin gingival biotype may result in greater gingival recession, led to the introduction of many surgical procedures for soft - tissue augmentation around dental implants. numerous studies have shown that increase in the amount of kg can be obtained by several different types of procedures, either before, simultaneously or after implant placement. though subepithelial connective tissue graft is the gold standard for gingival augmentation, use of such soft - tissue grafts have the intrinsic drawback of second surgical site morbidity, increased surgical time and cost and additional patient discomfort due to prolonged healing time needed at the donor site, which can overcome by apf technique. park., 2010 reported two cases, where the pre - existing narrow band of keratinized mucosa was displaced bucco - apically by apf with a partial thickness flap and stabilized by using a pre - fabricated implant - retained stent clipped over provisional abutments. in the present study, the technique of apf around implants showed statistically significant improvement with a mean gain of 3.95 mm in the width of kg at the end of 12 weeks (p < 0.000), the results of which correlate with case report by park., who showed a gain in kg following apf and to the best of our knowledge this is the first study to assess the esthetic outcome of apically displaced flap around implants. the esthetic outcome evaluated by an independent examiner using vas gave an average score of 7.1 indicating good esthetics. the technique of apf resulted in significant improvement in keratinized tissue, which is both functionally and esthetically acceptable. further longitudinal studies with a control group and adding clinical parameters such as plaque index, gingival index and evaluation of crestal bone loss should be done to assess long - term stability of the gain in kg and its influential role in maintaining peri - implant health. | purpose : dental implants though a successful treatment modality there exists controversies regarding the relationship between the adequacy of the keratinized gingiva (kg) and peri - implant health. the presence of an adequate amount of peri - implant kg reduces gingival inflammation and hence soft - tissue augmentation should be frequently considered. among the various periodontal plastic surgical procedures, the apically displaced flap increases the width of keratinized tissue with reduced patient morbidity. the current study aims at evaluating the esthetic improvement in kg around dental implants applying apically positioned flap (apf) technique.materials and methods : a total of 10 endosseous dental implants were placed in eight systemically healthy patients. apf surgery was performed at the implant site on the buccal aspect either at the time of implant placement (one stage surgical protocol) or during the implant recovery stage (two stage surgical protocols) for increasing the width of kg and reviewed until 12 weeks post - operatively. the width of kg was evaluated at baseline and at the end of 12 weeks after surgery. paired t - test was performed to evaluate the changes in the width of kg at baseline and at 12 weeks post - operatively. in addition, soft - tissue esthetic outcome was assessed by using visual analog scale (vas).results : the mean width of kg at baseline was 1.47 mm and 12 weeks post - operatively was 5.42 mm. the gain in kg from baseline was 3.95 mm with the p value of 0.000, which was highly statistically significant. the assessment of esthetic outcome using vas gave an average score of 7.1 indicating good esthetics.conclusion:the technique of apf yielded a significant improvement in keratinized tissue, which is both functionally and esthetically acceptable. |
aplastic anemia is a form of anemia in which the bone marrow fails to produce adequate numbers of peripheral blood elements. the incidences of atherosclerosis and myocardial infarction in patients with congenital coagulation disorders and chronic thrombocytopenia are very low. however in cases requiring invasive procedures (e.g., dental procedure and endoscopic procedure), patients at times need platelet transfusion. in this paper, a case of late stent thrombosis was reported within 4 hours after transfusion of platelets for anticipated bleeding in an aplastic anemia patient. a 52-year - old man who had been diagnosed with aplastic anemia and stable angina visited the emergency room of the authors ' hospital with a complaint of chest pain. the chest pain had developed 1 hour before the patient 's arrival at the hospital. the initial electrocardiogram showed a hyperacute tall t wave in the precordial leads (fig. the cardiac markers were in the normal range, but thrombocytopenia was noted (initial white blood cell count : 3350, hemoglobin : 12.5 g / dl, platelet : 45000/l). his past history revealed that he had been admitted to a hospital a year ago for acute coronary syndrome and percutaneous coronary intervention (pci) of the left anterior descending artery (lad), for which a cypher select stent (3.528 mm, johnson & johnson - cordis, miami, fl, usa) procedure was performed (fig. he had been taking 100 mg of aspirin, 75 mg of clopidogrel and 100 mg of oxymetholone (anabolic steroids) for aplastic anemia daily since the last one year. however, he had stopped taking aspirin and clopidogrel for 3 days before presenting himself at the hospital since he was scheduled to be treated for a decayed tooth. his laboratory findings showed that he had severe thrombocytopenia (platelet count : 12000/l). therefore, transfusion of 6 pints of platelet concentrates was performed on the same day of the patient 's visit to the hospital. a dose of 7,000 units of unfractionated heparin was injected during the cag with another loading dose of 300 mg of clopidogrel. after the pci, the chest pain subsided. on the second day, blood started oozing from the puncture site, hence a manual compression dressing was applied for 1 hour, after which there was no more complications. on the 12th day, the patient 's dental caries was treated with tooth extraction without bleeding complications, and the patient was discharged. he was also asked to continue oxymetholone (anabolic steroids) therapy for aplastic anemia. aplastic anemia is a form of anemia in which the bone marrow fails to produce adequate numbers of peripheral blood elements. the incidences of atherosclerosis and myocardial infarction in patients with congenital coagulation disorders and chronic thrombocytopenia are very low. there are a few reports of cases of aplastic anemia associated with myocardial infarction during treatment with anabolic steroids.1) steroids have been commonly used to treat aplastic anemia, are a possible risk factor for coronary thrombosis, because steroids have an accelerating effect on metabolic changes and can induce a hypercoagulable state, each of which may promote atherosclerosis. therefore, steroids may have helped to precipitate the clinical events in the patient.2) andrade.3) reported a case of acute stent thrombosis in a patient with giant cell arteritis, but there is no case report of long - term oral steroid therapy associated with very late stent thrombosis. even if other risk factors for stent thrombosis (e.g., incomplete stent expansion or clopidogrel resistance), according to time sequence, could not be completely excluded, platelet concentrate transfusion might have played an important role in coronary stent thrombosis in this study. sabovic and simona4) suggested that a substantial number of platelets and whole blood transfusions might be risk factors for coronary thrombosis. that is, plasminogen activator inhibitor - ii, a strong prothrombotic factor, which accumulates in stored blood products in a time - dependent manner, could extensively increase in the plasma after transfusions. this prediction, which may be clinically relevant, must be further clarified.5)6) unfortunately, this marker or other coagulation factors have not been examined. cornet.7) reported three case studies of coronary stent occlusion after platelet transfusion. actually, since peri- or intra - procedural massive bleeding was noted in all three cases, platelet transfusion was done inevitably. in our case, one could argue that platelet transfusion was not necessary in a patient who was scheduled for a minor dental procedure. the use of intravenous immunoglobulin (ivig) concomitant with platelet transfusion is indicated in patients with life - threatening severe thrombocytopenia or hemorrhage, or when therapeutic procedures with some risk of bleeding are performed.8) ivig should be administered only after careful consideration of the risks and benefits, and should be reserved for patients at risk of severe hemorrhage, and the use of a bare - metal stent over a drug - eluting stent should always be considered as the first option.9 - 11) in conclusion, the authors ' observations emphasize the risks of platelet transfusion and support withholding such a treatment unless vitally indicated, in patients with coronary artery stent implantation and even in those on dual antiplatelet therapy. | aplastic anemia is a condition in which the bone marrow fails to produce adequate numbers of peripheral blood elements. the incidences of atherosclerosis and myocardial infarction in patients with congenital coagulation disorders and chronic thrombocytopenia are very low. in this paper, a case of late stent thrombosis within a drug - eluting stent occurring after platelet transfusion in a patient with aplastic anemia is presented. the authors ' observations emphasize the risks of platelet transfusion and the authors ' support withholding such a treatment unless vitally indicated, in patients with coronary artery stent implantation and even in those on dual antiplatelet therapy. |
complete transposition of the penis and scrotum is an uncommon congenital malformation of the external genitalia in which the scrotum is located cephalic to the penis. there have been described fewer than 20 cases of complete penoscrotal transposition (cpst) with an intact scrotum.[36 ] most of the reported cases of cpst are sporadic. cpst is often characterized by major and sometimes life - threatening associated malformations involving the urogenital, cardiovascular, intestinal, and skeletal systems. a 3.6 kg newborn boy was noted at birth to have a complete transposition of the external genitalia. a 3.5-cm - long, hypospadic and hypoplasic penis arose from the perineum, just above the anus and beneath a normal scrotum with both normal testis inside [figure 1 ]. external genitalia before surgery at 18 months, we performed the first surgical procedure achieving penis advancement to the level of the scrotum [figure 2 ]. external genitalia after the first surgical procedure when he was 30 months old, we performed the second procedure advancing the penis without the urethra, achieving a satisfactory position of the penis above the scrotum and a scrotal hypospadias [figures 3 and 4 ]. in a third surgical procedure external genitalia before the second surgical procedure external genitalia after the second surgical procedure after this three - stage procedure, we achieved a satisfactory penis position and excellent cosmetic results [figure 5 ]. fewer than 20 cases have been reported in the literature.[137 ] the embryological sequence responsible for this malformation remains unclear ; however, it has been suggested that an abnormal positioning of the genital tubercle in relation to the scrotal swellings during the critical fourth to fifth week of gestation could affect the migration of the scrotal swellings. during normal development scrotal swellings migrate inferomedially during the 911 week, and fuse in the midline caudal to the penis by the 12 week of gestation. as chadha. suggested, the phallic tubercle is intrinsically abnormal and affects the corporal bodies development explaining the flaccid and hypoplasic penis. others suggested that a failure of labioscrotal migration due to a unilateral or bilateral gubernaculum defect leads to anomalies such as incomplete, complete transposition or ectopic scrotum. it has been suggested that many cases reported as penile agenesis may actually represent cases of concealed cpst. some reviews report a high incidence of associated urogenital malformations such as flaccid penis, hypospadias, urethral atresia, or bifid scrotum. renal abnormalities such as renal agenesis, ectopic pelvic kidney, or dysplastic kidneys are the most frequent extragenital malformations associated. reported other less frequent abnormalities : mental retardation (60%), imperforate anus (33%), central nervous system abnormalities (29%), preaxial limb defects (24%), and congenital heart disease (19%). the presence of a multicystic pelvic kidney in the case that we report advocates that review. there is not any description of the technique for correction and results of these so severe cases of pst., we tried to advance the penis to the level of scrotum without disconnecting the urethra from the corpora cavernosa. in the second procedure, it was necessary to disconnect the urethra from the corpora cavernosa to achieve the penis position above the scrotum. extreme penoscrotal transposition with severe hypospadias and chordee is difficult to differentiate from penile agenesis with a midline skin tag anterior to the anus. in both cases, the penile reconstruction and repositioning are often unsatisfactory and female sex reassignment, unlike ethically controversial, may be a prudent therapeutic in selected cases. we think that surgical correction is possible in selected cases by achieving a satisfactory penis position and excellent cosmetic results. | complete penoscrotal transposition (cpst) with an intact scrotum is a rare anomaly in which the scrotum is located cephalic to the penis. it is the most severe degree of malformation of a spectrum of abnormalities in scrotal development. there are few cases reported in the literature, and there are few descriptions of the technique for correction and results. we describe a new case of cpst and its sequential correction. |
the term of preterm birth used to define the premature neonates considering pregnancies age of less than 34 weeks and corticosteroids are commonly prescribed to promote embryos lung maturity. the amniotic fluid index (afi) has been an integral component of fetal assessment during antepartum ultrasound examination for > 20 years. decreased amniotic fluid or oligohydramnios, is typically defined as an afi below 5-cm, which represents the value below the first percentile. in term and near - term gestations, this 5-cm threshold has been associated with increased rates of complications, including small for gestational age neonate, nonreassuring fetal heart rate (fhr), stillbirth, and neonatal death. the amniotic fluid volume is most abundant in the early third trimester, subsequently decreasing until term. before 34 weeks, the value of 8-cm is below the fifth percentile for gestational age. potential risks associated with borderline amniotic fluid in the preterm period are not fully understood. dexamethasone and betamethasone cause to produce surfactant in fetus lung and thereby it reduces the resistant between layers of airways and sacs to simply slide on each other and eventually easily breathing of neonate after birth preventing respiratory distress syndrome in neonate. the preferred gestational age for administration of dexamethasone and betamethasone is usually 28 - 34 weeks. the usage dose of betamethasone is 0.17 mg / kg daily for 2 doses to reduce the risk of intraventricular hemorrhage, chronic lung diseases, necrotizing enterocolitis, and retinopathy of prematurity, sepsis, and neonatal intensive care unit administration. considering the side effects of each drug, some studies have been done to evaluate the side effects of dexamethasone and betamethasone. normal range of afi in fetus is > 8 - 23 cm and its average is 12.8-cm in jackson study that measured the effect of corticosteroids on afi which in about 72% of cases the afi was decreased. biophysical profile parameters (bpp) consists of five parameters including : fetal tone, fetal gross movement, fetal breathe, afi, nonstress test (nst) changes, each of these parameters is assigned number 0 - 2. fetal gross movement, nonstress test (nst) changes, each of these parameters is assigned number 0 - 2. the normal score of bpp is 8 - 10 and 6 - 8 is unclear and below 6 considers as abnormal. bpp for the most of the fetus (95.95%) is normal. in the jackson study that measured bpp after administration of corticosteroids, fetal gross movement and afi score were decreased in 44% and 87% of cases, respectively. to evaluate the effect of corticosteroids on nst, the parameters that change in nst are a short and long beat to beat which are decreased generally, and it can be neglect the corticosteroids effect on acceleration. the difference between dexamethasone and betamethasone on nst, afi, and bpp, dexamethasone has no clear effect on nst, afi, and bpp but betamethasone usually decreases afi in 63% of cases, short beat to beat in nst in all cases and fetal movement in 80% cases but other parameters have no changes. thus because of the importance of nst, afi, and bpp on decision for the fetus, the changes after administration of dexamethasone and betamethasone on bpp, nst, and afi and differences between their effects on the bpp, afi, nst are important for us. knowing of these changes prevents rash decision for patients. in this study, we compared the effect of betamethasone versus dexamethasone on the afi in the women at risk of preterm labor to the best decision may be made for each patient. this study is a double - blind clinical trial study registered in www.irct.ir with code of irct201212307513n2 and conducted on 70 patients for 28 - 34 weeks. women having at least once preterm labor in their previous pregnancy have been submitted in al zahra hospital and shahid beheshti clinic of obstetrics and gynecology in 2012 - 2013. in this study, neither the person who collected the data nor the mothers who received the drugs were aware of the prescribed medications. inclusion criteria in our study were pregnancy, having at least once preterm labor or being at risk of preterm labor, no consumption of any drugs only ferrous sulfate or folic acid, having no background illness. the women with intrauterine growth retardation fetus, vaginal leak, gestational diabete mellitus, preeclampsia, consumption of any drugs, vaginal bleeding or if the neonate had anomaly after birth were excluded from the study. in our study, all cases with decrement of afi, bpp fewer than 8 and nonreactive nst were hospitalized. sample size was calculated with confidence level of (z = 1 = 1/96) and the power of (2 = 1b = 0.84). the first group were received 8 mg each 12 h for 4 doses dexamethasone when the patient at risk of preterm labor admitted in clinic or hospitalized and the second group were received 12 mg betamethasone each 24 h similarly. nevertheless, the measurement bias of that sonographer in different time of measurement could be about 5-cm. in order to avoid such bias, in this study, only more than 5-cm decrement in afi and afi under 8-cm is considered as a meaningful decrease. afi sonography performed and repeated 3 days after administration of corticosteroids in each group and data were collected. nst has three parameters of variability in fhr : long beat to beat.short beat to beat.acceleration. one observer performs nst after and before of administration of corticosteroids in each group and short beat to beat and a long beat to beat, and acceleration were evaluated. by the observer one sonographer done sonography bpp before and after corticosteroids (3 days after taking corticosteroids) and afi lower than 8-cm or any decrement more than 5-cm was registered. the bpp that consists of five components that each of them has a score 0 or 2. bpp score was nl (8 - 10), (6 - 8) bpp would be repeated, and (4 - 8) were hospitalized. bpp data combine two sources 1-ultrasound imaging and nst which has been previously explained above. if the nst is reactive, its score is 2 otherwise its score is 0. afi which has been explained above is 0 if afi 0.05). the average of afi before and after giving the medication has been presented in table 2. according to student 's t - test, no statistically difference between the two groups was seen in the before and after intervention (p > 0.05). nevertheless, about 20% of cases had afi decrement after betamethasone administration. there was no significant difference in afi between these two groups before and after administration of corticosteroids but according to repeated measures anova, time had statistical effect on afi (p = 0.034) [figure 1 ]. furthermore, no statistically difference between nst acceleration tests was detected before and after corticosteroids administration in both groups. distribution of basic and general variables distributions of nst and bpp variables amniotic fluid index changes in the before and after intervention between the two groups the only statistically significant difference was found between the results of long beat to beat and short beat to beat test between the two groups. the results of bpp sonography have revealed no significant difference in fetal thoracic movement and fetal tone between the two groups but remarkably a great variance in fetal movement. in the group who has received dexamethasone and in betamethasone receiver group there was normal fetal movement in 90% and 60% of the cases, respectively (p = 0.001). furthermore, the average of bpp index between betamethasone and dexamethasone recipients was associated with a meaningful difference. this study was performed to check, analyze, and compare the effect of betamethasone and dexamethasone on fetal index including afi, bpp, nst in pregnant women with the pregnancy age of 28 - 34 weeks experiencing prematurity (premature delivery). mothers were divided into two groups having no significant difference in their demographic information or history of past pregnancy. thus, the changes in the results (alternance) could be due to the difference of the corticosteroids they were given at the last week of their pregnancy. according to our study results, although in some cases afi was decreased, no significant influence on afi was observed owning to corticosteroids injection, such that regarding to afi, no remarkable variance was found between the two groups. only it was observed that 3 days after corticosteroids injection, there was a 2.11 0.1 cm decrease in afi of betamethasone group and 0.9-cm in the dexamethasone group but as mentioned before there was no great variance. however, some other studies in contrary to ours have been related the afi changes to corticosteroids injection. in jackson study, the afi has been decreased exactly after corticosteroids injection in about 72% of their sample size. in kazardoost study which has been done in yasooj medical school in 2010, betamethasone injection was also considered as a reason for afi decrease. the nst results showed no great variance in acceleration index between the recipients of dexamethasone and betamethasone with a significant difference between short beat to beat and a long beat to beat index in the mentioned groups. short beat to beat index was normal in 98% of women in the dexamethasone group and 66% of betamethasone group. long beat to beat in all of the dexamethasone group and 80% of betamethasone recipients was normal. moreover, the results of bpp sonography have introduced a noticeable difference in fetal movement and thereby a biophysical profile showed difference between these two groups. furthermore, 90% of dexamethasone recipients and the same results were reported with bpp indicating that the dexamethasone group had greatly higher bpp. in kazardoost study, which was mentioned previously, all fetal movement had decreased after injection of betamethasone. according to mulder. study, only short and long beat to beat index were decreased due to corticosteroids injection, with no effect on acceleration. doyle. in a study concluded that the benefits of late dexamethasone may not outweigh actual or potential adverse effects but considering the evidence of both benefits and harms of treatment, and the limitations of the evidence at present, it appears prudent to reserve the use of late dexamethasone to infants who can not be weaned from mechanical ventilation, and to minimize the dose and duration of any course of treatment. all studies taking into account corticosteroids influence on fetal indexes including afi, bpp, and nst, reported no great changes in these indexes due to corticosteroids injection. in rotmensch study on two groups of women exposed to (experiencing) preterm delivery prescribed with dexamethasone and betamethasone and the results were observed 48 h after injection. fetal thoracic movement in 83% of betamethasone group and 90% of the dexamethasone group had been decreased. there was also a 48.6% decrease in fetal movement for betamethasone group, and there was a meaningful variance between these two groups. in another study, the bpp status was assessed after corticosteroids injection. according to the study, for 44% of samples, gross fetal movement was decreased, and afi and bpp index decrement has been reported for 87% of samples. in our study, there were no differences in fetal tone and fetal thoracic movement between the two groups before and after of corticosteroids administration. based on the results of this study and other similar works in this area, it can be concluded that dexamethasone has lower effect on afi, bpp, and nst indexes respect to betamethasone and for this reason it is more preferred rather than betamethasone. according to this study, afi has no significant change before and after corticosteroids administration. zk and hgt have collected data and conducted the statistical analysis and prepared the first draft of manuscript. | background : the aim of this study was to compare the biophysical profile parameters (bpp), especially amniotic fluid index (afi), before and after administration of corticosteroids (dexamethasone and betamethasone) and these two groups with each other.materials and methods : this double - blind clinical trial study was done on 70 patients for 28 - 34 weeks women having at least one preterm labor in their previous pregnancy has been submitted in al zahra and shahid beheshti clinic in 2012 - 2013. 70 women were randomly allocated in two groups. the first group were received 8 mg each 12 h for 4 doses dexamethasone when the patient at risk of preterm labor admitted in clinic or hospitalized and the second group were received 12 mg betamethasone each 24 h similarly. nonstress test (nst) and bpp and sonography afi were done in all cases, and then nst was done in the before and 3 days after intervention. data were analyzed by spss software version 20 using t - test, chi - square, and fisher 's test.results:there was no significant difference between the range of afi before and after the drug intervention (p > 0.05). two groups had no significant difference in afi before and after administration of corticosteroids. no significant difference was seen in nst acceleration tests between the two groups before and after corticosteroids administration.conclusion:biophysical profile had a significant difference in dexamethasone group respect to that of betamethasone group. furthermore, the biophysical profile had a significant difference before and after the administration of corticosteroids in two groups. it is noted that afi had no role in this matter. |
prophylactic measures are primarily focused on lifestyle changes and primary and secondary interventions with drugs such as antihypertensive agents, statins, and platelet inhibitors. socioeconomic position has been associated with the risk for stroke. a larger burden of cardiovascular risk factors in lower income groups appears to explain at least part of their higher stroke risk. the relative impact of individual risk factors on total stroke risk in different socioeconomic groups is, however, unclear. more knowledge in this area could help focus the preventive initiatives that would reduce social inequality. the impact of socioeconomic position on stroke incidence has so far been studied only in subpopulations and not at the national level. socioeconomic position is often geographically determined ; therefore, a study based on an entire nation might provide a more balanced image of social inequality in society. the aim of the study reported here was to investigate the impact of socioeconomic position on the burden of stroke in denmark. we studied the association between the socioeconomic position of patients hospitalized for stroke after the age of 40 years in denmark during the years of 20032012 and that of the general danish population. furthermore, we studied the cardiovascular risk factor profile associated with stroke in different socioeconomic groups. all citizens of denmark over the age of 40 and who had lived in denmark during some or all of the period of 20032012 were included. information on stroke outcome was obtained from the danish stroke register (formerly the stroke section, danish national indicator project) as described in detail elsewhere. danish hospitals are obliged to report a predefined set of data to the register for all patients admitted to hospital for acute stroke, including age, sex, stroke severity on admission measured on the scandinavian stroke scale, stroke subtype, and a predefined cardiovascular profile. the scandinavian stroke scale is a validated neurological scale of stroke severity from 0 to 58, lower scores indicating more severe strokes. the cardiovascular profile included information on alcohol consumption (14/21 > 14/21 drinks per week for women and men, respectively [under / over the limit set by the by the danish national board of health ]), current daily smoking, body mass index (weight / height), diabetes mellitus, atrial fibrillation (af) (chronic or paroxystic), arterial hypertension, previous myocardial infarction, previous stroke, and intermittent arterial claudication. by march 2012 the recommended alcohol intake limit was lowered to 7/14 drinks per week for women and men, respectively. diabetes, af, arterial hypertension, previous myocardial infarction, previous stroke, and intermittent arterial claudication were diagnosed on current danish standards and were either known before the onset of stroke or diagnosed during hospitalization. for all people in the study population, we obtained information on level of education and disposable income, as described in. here, education was grouped into 3 categories, from basic to high : (1) basic / high school, defined as 7 to 12 years of primary, secondary, and grammarschool education ; (2) vocational, defined as 10 to 12 years of education including vocational training ; and (3) higher, defined as 13 or more years of education. disposable income was defined as household income after taxation and interest per person, adjusted for the number of people in the household and deflated according to the 2000 value of the danish crown (dkk). people with a high negative income due to debt and interest on debt (> 50 000 dkk per year) were excluded from all analyses. for the analyses, disposable income was categorized into the first to fifth quintile of income distribution. we included incident hospital admissions for first ischemic stroke between january 1, 2003, when the danish stroke register was fully established, and july 28, 2012. for patients with multiple hospital admissions, information on previous stroke was obtained from the patient, his / her care persons or from hospital records available on the acute admission or from the danish stroke register. patients with recorded history of previous stroke (either ischemic or hemorrhagic) were excluded ; however, patients with previous transient ischemic attacks (tia) were not excluded. tias are not registered in the danish stroke register (ie, not an outcome of the study). hence, we studied only incidence of hospitalization for firstever ischemic stroke in the danish population between 2003 and 2012. patients 30 ; information available for only 73% of stroke patients. the risk ratios of hospitalization for stroke associated with disposable income are shown in table 2 (both unadjusted and adjusted for age, sex, calendar year, and length of education). a highly significant stepwise relation was found between income and risk for hospitalization for stroke, the risk being almost twice as high in the lowest income group than in the highest. analyses for people over and under 65 years gave similar results (not shown). incidence rates per 1000 personyears and relative risks of hospitalization for ischemic stroke in the danish population over 40 years by length of education and disposable income, 20032012 for age, sex, calendar year, and income / education. the risks for hospitalization for stroke associated with length of education are also shown in table 2 (both unadjusted and adjusted for age, sex, calendar year, and income). in the adjusted analysis, the risk of stroke hospitalization of the higher education groups was lower than in the vocational and short education group (p 97%, and that for alcohol consumption, smoking, and intermittent arterial claudication it was > 85%. moreover, treatment would have been uniform, as 96% of patients were treated in a stroke unit. missing values were associated with increasing age, although we do not find patterns in missingness conditional on age. therefore, we consider our study population to be representative of the danish population hospitalized for stroke, and have conducted complete case analysis. although our observations therefore apply to the majority of stroke patients, they apply only to those who are hospitalized. a limitation of the study is thus that hospitalization rates are not reflecting true incidence of stroke. we do not have data to analyze whether this affects the estimated risk ratios, ie, whether missing stroke registration is associated with socioeconomic status. this study presents novel information about social inequality in the risk of stroke based on a nationwide data. although denmark has a highly developed social security system, with free, equal access to health care services, there is considerable social inequality in relation to the risk for stroke. in comparison with people with the highest income, those with the lowest had a 2fold higher risk of being hospitalized for stroke. among people of working age, those with the shortest education had a 36% higher risk for stroke than those with the longest education. among patients with stroke obesity, smoking, and high alcohol consumption lifestyle, therefore appears to play an essential role for inequality in the risk for stroke and should remain a main target of preventive measures. our study provides new insights into the role of medically treatable risk factors for social inequality in risk of stroke. diabetes was almost two times more common among stroke patients in the lowest income group indicating that prevention of diabetes is crucial to social inequality in risk of stroke. medically amenable classical stroke risk factors such as hypertension and af appeared to be less important suggesting that consequences of an unhealthy lifestyle in the strict sense can not be eliminated by medical intervention. our study indicates that a focus on lifestyle and prevention of diabetes is a precondition for reducing social inequality in stroke in denmark. | backgrounda greater burden of stroke risk factors in general is associated with a higher risk for stroke among people of lower than those of higher socioeconomic position. the relative impact of individual stroke risk factors is still unclear.methods and resultswe studied the relations between socioeconomic position, measured as household income and length of education, and all hospital admissions for a first ischemic stroke among 54 048 people over the age of 40 years in denmark in 20032012 in comparison with the general danish population (23.5 million personyears). we also studied the cardiovascular risk factor profile associated with socioeconomic position in stroke patients. relative risks for stroke were estimated in loglinear poisson regression models. the risk for hospitalization for a first ischemic stroke was almost doubled for people in the lowest income group, and the risk of those of working age (< 65 years) was increased by 36% among people with the shortest education. diabetes, obesity, smoking, and high alcohol consumption in particular and, to a lesser extent, previous myocardial infarction or intermittent arterial claudication were significantly overrepresented among stroke patients with lower socioeconomic position. atrial fibrillation and hypertension were not.conclusionsin denmark, there is a strong relation between low socioeconomic position and risk for hospitalization for stroke. lifestyle, as indicated by smoking, obesity, and alcohol consumption, and diabetes appears to increase the risk for stroke in people with lower socioeconomic position. |
statistical significance, most often defined as a p value of < 0.05, simply means that an observed quantitative difference would occur by chance < 5% of time and does not necessarily imply biological significance. in the nonparametric analysis of survival data, the order of the events rather than the timing of the events is the basis for assessing differences between treatment groups. for example, if all mice in group 1 die before the mice in group 2, the results will be statistically significant regardless of whether the group 2 mice die 1 h later or 1 month later or not at all. thus, in an experiment in which all deaths occur within a day or two but animals are monitored to determine precise survival times, group differences could be statistically significant but not biologically relevant. with this in mind, it has become apparent that in many recent publications in various highly respected journals, animals were monitored for survival multiple times a day or even hourly to obtain statistically significant results. these experimental settings allowed statistically significant results to be obtained even when the differences in survival time were clearly of limited biological significance. for example, in many studies, deletion of a particular gene led to a 1-day decrease or increase in the median time to death with a p value as low as < 0.001. this could happen if all mice in a treatment group, for example, were kept in the same cage or were monitored as a group. to elaborate on this systematic problem, we generated three sets of hypothetical data that were designed to compare the efficacies of vaccination for protection against viral challenge. each data set consisted of two experimental groups (mock - treated mice and vaccinated mice), and each group consisted of five mice (fig. 1). for the first set of data, both groups of mice were monitored once a day and all the mice died 10 days after viral challenge. this yielded a p value of 1, hence not rejecting the null hypothesis and not indicating any efficacy of vaccination (fig. for the second data set, the same experiment was conducted except that the mice were monitored on an hourly basis. in this case, a difference of 1 h median time to death was observed between the two groups (fig. in this example, all the mice died around the same time, but in the mock - treated group, all mice died before any of the mice in the vaccinated group died. alternatively, it could be that all the mice in the mock - treated group were assessed before the vaccinated mice, so that there was a temporal delay in data gathering. in either case, the null hypothesis in this instance was clearly rejected (p = 0.0027) and could be used to argue that indeed the vaccination was effective in providing significant protection. given the clustering of deaths observed, however, the results may have been due to nothing more than a cage effect, with no biological significance. 1c had a difference of 100% in survival rates between the mock - treated group and the vaccinated group, with at least 10 more days of protection than the results shown in fig. this is because the log rank test considers only the order in which the animals die. thus, from these hypothetical data sets, it is apparent that the use of statistical significance in survival analyses could be extremely deceptive. the biological effect, i.e., duration of protection, clearly needs to be assessed along with the statistical significance of the data. a mismatch between statistical significance and biological significance could be a red flag for a poorly designed study that measures something other than treatment efficacy. two groups of mice (5 mice per group) were either mock treated or immunized with vaccine x and were challenged with a lethal dose of virus. survival of the infected mice was monitored either daily (a) or hourly (b). p values were derived by the kaplan - meier log rank test using graphpad prism 4 software. statistical analyses gave identical p values for panels b and c ; however, it is clear that biologically significant protection was seen only in panel c. pbs, phosphate - buffered saline. the intent of this article is to serve as a reminder that statistical and biological significance should never be used interchangeably in survival studies that attempt to predict protective efficacy. in our opinion, for acute infections that cause death in 7 to 10 days, a 3-day difference in survival is the minimum value that would warrant further development. on the other hand, a 3-day difference in survival would be biologically irrelevant for chronic infections such as tuberculosis, in which desired differences would be weeks, months, or even years. nevertheless, we along with others (1, 2) appreciate that defining particular quantitative changes as biologically or clinically significant is subjective, context dependent, and sometimes obscure. we suggest that the biological outcome from the experiment be considered first and then statistics applied to determine if the results are likely to be due to chance. in this process, it should be remembered that a cutoff p value of 0.05 is relative ; a p value of 0.1 indicates that a particular result would occur by chance 10% of the time. it is hoped that these considerations will assist investigators in focusing more on the most promising outcomes in preclinical disease models and increase the impact of experimental results on development of effective cures or vaccines in humans. | abstractin most preclinical disease models, survival analyses are the gold standard for measuring the efficacy of medical interventions such as therapeutics or vaccines. in these analyses, treatment regimens that promote the survival and/or reduce the morbidity of experimental subjects (e.g., mice) are tested for efficacy. although these analyses appear to be relatively straightforward, there are associated caveats regarding interpretation of the results that we wish to discuss in this editorial. of particular concern is overinterpretation of the biological significance of survival data based on statistical significance rather than durability of protection. |
as a service to our authors and readers, this journal provides supporting information supplied by the authors. such materials are peer reviewed and may be re - organized for online delivery, but are not copy - edited or typeset. technical support issues arising from supporting information (other than missing files) should be addressed to the authors | unique -hemiaminal ether gold carbene intermediates were accessed by a gold - catalysed 1,1-carboalkoxylation strategy and evolved through a highly selective 1,2-n - migration. this skeletal rearrangement gave functionalised indenes, and isotopic labelling confirmed the rare c n bond cleavage of the ynamide moiety. the effect of substituents on the migration has been explored, and a model is proposed to rationalise the observed selectivity. |
venous thromboembolism (vte) affects one to two in 1,000 adults, causing severe mortality and morbidity.1 vte comprises deep venous thromboembolism and pulmonary embolism. the risk factors for vte include advanced age, obesity, hypertension, diabetes, dyslipidemia, pregnancy, major surgery, and hormone replacement therapy.13 in addition, particular psychiatric patients under physical restraints, with catatonia, or with neuroleptic malignant syndrome are at high risk of vte.46 in addition, antipsychotic drug use is known as a risk factor of vte. there have been several vte case reports in psychiatric patients receiving antipsychotics between the 1950s and 1980s.7 this relationship was also discovered among patients receiving clozapine in the 1990s.8 however, the association between the use of antipsychotics and vte remains unclear. almost all studies have suggested a positive relationship between antipsychotic use and vte, and these studies have estimated a wide range of risk from 1.1 to 29.3,918 with only one study indicating negative findings.17 because the prolactin response to antipsychotics is related to dopamine d2-receptor blockade,1921 hyperprolactinemia is a frequent consequence of certain antipsychotic treatments. the major effects of hyperprolactinemia among women include amenorrhea, galactorrhea, the cessation of normal cyclic ovarian function, loss of libido, occasional hirsutis,22,23 and long - term osteoporosis risk.24 the effects among men include impotence, loss of libido, hypospermatogenesis,22,23 and the long - term risk of low bone density.24 several studies have suggested that increased levels of prolactin are associated with platelet aggregation.25,26 erem reported that the levels of fibrinogen and antithrombin iii among patients with prolactinoma were significantly increased compared with control participants. similarly, anaforoglu found that adenosine diphosphate (adp)-stimulated expression was significantly greater in the hyperprolactinemia group than in the control group ; this adp - stimulated expression provides evidence of platelet activation. based on these findings, prolactin is regarded as a hormone - associated coagulant, and patients with hyperprolactinemia are considered at risk for vte. recently, skokou and gourzis29 reported a case of pulmonary embolism in a woman with bipolar disorder who was treated with amisulpride, aripiprazole, and paroxetine. although the contribution of aripiprazole and paroxetine can not be excluded completely, the most likely factor underlying the thromboembolic event was hyperprolactinemia (92 ng / ml) caused by the amisulpride. therefore, we examined the effect of elevated prolactin levels on enhanced coagulation in psychiatric patients treated with antipsychotics. in addition, sex - specific effects were evaluated because prolactin responses to antipsychotics differ between men and women. this study was conducted at hirosaki - aiseikai hospital (hirosaki, japan), kuroishi - akebono hospital (kuroishi, japan), and hirosaki university hospital (hirosaki, japan). the sample consisted of patients registered between january 1, 2012 and september 1, 2013. all patients (58 inpatients and 124 outpatients) had schizophrenia and were aged between 18 and 90 years. we conducted the present study after obtaining approval from the ethics committee at the hirosaki university school of medicine. participants provided written informed consent after receiving a full description of the study. to be included in this study, the participants could not have received hormone replacement therapy or any anticoagulant or oral contraceptive medications. patients who were pregnant, had malignant tumors, had a history of requiring physical restraint over the last 3 months, or had difficulty walking were excluded. the patients must have received the same antipsychotic treatment for more than 3 consecutive months. research psychiatrists conducted the psychiatric diagnoses using the structured clinical interview for the diagnostic and statistical manual of mental disorders, fourth edition, of the american psychiatric association. participants were coded as having schizophrenia, mood disorders, or dementia and were classified as either positive or negative for hypertension, diabetes, or dyslipidemia. we did not control when the meal and medication were taken prior to sampling blood. a cubital vein blood sample was taken and divided into two aliquots after the anthropometric assessments. body weight and body mass index (bmi) (weight in kg / height squared) were assessed. the blood levels of d - dimer, fibrinogen degradation products (fdp), and thrombin antithrombin complex (tat) were also assessed. blood samples were drawn into vacuum tubes containing sodium citrate and ethylenediaminetetraacetic acid as anticoagulants. serum prolactin concentrations were measured using an automated electrochemiluminescence immunoassay (roche diagnostics, tokyo, japan). the coefficients of variation (cvs) of the intra- and interassays were less than 10%. d - dimer and fdp measurements were performed by the latex flocculation method using commercial kits for d - dimer (kainos laboratories, inc., tokyo, japan) and fdp (sekisui medical co., ltd, tokyo, japan). a tat assay was performed with an enzyme - linked immunosorbent assay (tfb, inc., the normal range is <3 ng / ml. we compared descriptive statistics (age, height, body weight, bmi, duration of illness, and chlorpromazine equivalent) between men and women using student s t - test. unless otherwise stated, continuous variables are expressed as the mean standard deviation (sd). logarithmic conversions were performed for nonnormally distributed variables, including d - dimer (log d - dimer), fdp (log fdp), and tat (log tat). for multiple statistical comparisons between the three activated coagulation factors, we applied bonferroni s corrections. to investigate the relationship between prolactin levels and individual markers of activated coagulation the individual markers of activated coagulation were calculated in the controls for different levels of prolactin (g / l). we compared levels across the 0th25th (first quantile), 25th50th (second quantile), 50th75th (third quantile), and 75th100th (fourth quantile) percentiles. multiple regression analysis was performed to determine the correlations between the markers of activated coagulation (log d - dimer, log fdp, and log tat) and the clinical variables for each group and for all participants. in the between - group analyses, the markers of activated coagulation were set as the dependent variables, and age, sex, bmi, hypertension, diabetes, dyslipidemia, smoking status, chlorpromazine equivalent, and prolactin level were set as the independent variables. statistical analyses were performed using spss 21.0 (ibm corporation, armonk, ny). this study was conducted at hirosaki - aiseikai hospital (hirosaki, japan), kuroishi - akebono hospital (kuroishi, japan), and hirosaki university hospital (hirosaki, japan). the sample consisted of patients registered between january 1, 2012 and september 1, 2013. all patients (58 inpatients and 124 outpatients) had schizophrenia and were aged between 18 and 90 years. we conducted the present study after obtaining approval from the ethics committee at the hirosaki university school of medicine. participants provided written informed consent after receiving a full description of the study. to be included in this study, the participants could not have received hormone replacement therapy or any anticoagulant or oral contraceptive medications. patients who were pregnant, had malignant tumors, had a history of requiring physical restraint over the last 3 months, or had difficulty walking were excluded. the patients must have received the same antipsychotic treatment for more than 3 consecutive months. research psychiatrists conducted the psychiatric diagnoses using the structured clinical interview for the diagnostic and statistical manual of mental disorders, fourth edition, of the american psychiatric association. participants were coded as having schizophrenia, mood disorders, or dementia and were classified as either positive or negative for hypertension, diabetes, or dyslipidemia. we did not control when the meal and medication were taken prior to sampling blood. a cubital vein blood sample was taken and divided into two aliquots after the anthropometric assessments. body weight and body mass index (bmi) (weight in kg / height squared) were assessed. the blood levels of d - dimer, fibrinogen degradation products (fdp), and thrombin antithrombin complex (tat) were also assessed. blood samples were drawn into vacuum tubes containing sodium citrate and ethylenediaminetetraacetic acid as anticoagulants. serum prolactin concentrations were measured using an automated electrochemiluminescence immunoassay (roche diagnostics, tokyo, japan). the coefficients of variation (cvs) of the intra- and interassays were less than 10%. d - dimer and fdp measurements were performed by the latex flocculation method using commercial kits for d - dimer (kainos laboratories, inc., tokyo, japan) and fdp (sekisui medical co., ltd, tokyo, japan). a tat assay was performed with an enzyme - linked immunosorbent assay (tfb, inc., blood samples were drawn into vacuum tubes containing sodium citrate and ethylenediaminetetraacetic acid as anticoagulants. serum prolactin concentrations were measured using an automated electrochemiluminescence immunoassay (roche diagnostics, tokyo, japan). the coefficients of variation (cvs) of the intra- and interassays were less than 10%. d - dimer and fdp measurements were performed by the latex flocculation method using commercial kits for d - dimer (kainos laboratories, inc., tokyo, japan) and fdp (sekisui medical co., ltd, tokyo, japan). a tat assay was performed with an enzyme - linked immunosorbent assay (tfb, inc., we compared descriptive statistics (age, height, body weight, bmi, duration of illness, and chlorpromazine equivalent) between men and women using student s t - test. unless otherwise stated, continuous variables are expressed as the mean standard deviation (sd). logarithmic conversions were performed for nonnormally distributed variables, including d - dimer (log d - dimer), fdp (log fdp), and tat (log tat). for multiple statistical comparisons between the three activated coagulation factors, we applied bonferroni s corrections. to investigate the relationship between prolactin levels and individual markers of activated coagulation, correlations among these variables were determined using pearson s correlation analysis. moreover, the individual markers of activated coagulation were calculated in the controls for different levels of prolactin (g / l). we compared levels across the 0th25th (first quantile), 25th50th (second quantile), 50th75th (third quantile), and 75th100th (fourth quantile) percentiles. multiple regression analysis was performed to determine the correlations between the markers of activated coagulation (log d - dimer, log fdp, and log tat) and the clinical variables for each group and for all participants. in the between - group analyses, the markers of activated coagulation were set as the dependent variables, and age, sex, bmi, hypertension, diabetes, dyslipidemia, smoking status, chlorpromazine equivalent, and prolactin level were set as the independent variables. statistical analyses were performed using spss 21.0 (ibm corporation, armonk, ny). table 1 summarizes the clinical characteristics and laboratory parameters for all participants, divided by sex. the median age and this difference was significant (p=0.008). the median sd prolactin level was 25.621.1 g / l for men and 52.757.5 g / l for women ; this difference was significant (p<0.001 ; table 1). the body weight for men was significantly higher than that for women (p=0.024, table 1). however, the bmi of women was significantly higher than that of men (p=0.020 ; table 1). the dose of antipsychotic administered to men was higher than that administered to women (p=0.048 ; table 1). among all the participants, prolactin levels were not significantly correlated with the markers of activated coagulation (table 2). however, a positive correlation was observed between prolactin and markers of activated coagulation such as log d - dimer (p=0.002) and log fdp (p=0.026) among the men. however, after applying bonferroni s correction, the significance of the correlation between prolactin and fdp disappeared. conversely, no correlation was observed between the prolactin level and markers of activated coagulation among the women (figure 1). table 3 shows the means of the markers of activated coagulation in the four prolactin groups. these results indicate a significant difference in the d - dimer level among men (p=0.048 ; figure 1) but not among women. table 4 shows the multiple regression results for the markers of activated coagulation. among men, age, hypertension, diabetes, and prolactin level log tat was not associated with any of the dependent variables among men and women. the dose of antipsychotics was not associated with any marker of activated coagulation in men or women., no correlations were observed between the prolactin levels and the markers of activated coagulation in any subgroup of patients receiving a given antipsychotic medication. the results of this study indicated that the prolactin level was significantly correlated with certain markers of activated coagulation (log d - dimer and log fdp) in men receiving antipsychotic treatments. this study is the first to show an association between the prolactin level and vte risk among psychiatric patients. our data showed that men with higher levels of prolactin had enhanced markers of activated coagulation (log d - dimer and log fdp) but that women did not show this tendency. raaz suggested that a high density of prolactin enhances the adp - stimulated aggregation of platelets. however, prolactin alone does not influence the process of aggregation ; rather, the combination of prolactin and adrenalin does. prolactin affects aggregation through the gq protein, which induces the shape change of activated platelets. prolactin receptors are not directly related to gq proteins ; rather, they bypass g - protein - related pathways and directly interact with protein kinase c. protein kinase c, coupled to protein gq, is a key enzyme in the signal transduction pathway. several case control studies have suggested that the risk of vte gradually rises with increasing levels of prolactin. these studies have found an association between prolactin levels and vte,19,30 although the evidence for the link between prolactin and the occurrence of vte in psychiatric patients remains inconclusive. therefore, the strength of the current study is its large sample size of approximately 100 men and women with hyperprolactinemia, which is nearly three times that of any previous study.27,28 no significant differences in any marker of activated coagulation were observed between men and women in this study. this result may be explained by the opposing effects of age and bmi on each sex. in this study, the age of the men was significantly higher than that of the women (p=0.008). alternatively, the bmi of the women was significantly higher than that of the men (p=0.020). as a result, the effects of aging and high bmi, which are known to be risk factors for vte, on the markers of activated coagulation might have offset each other in men and women. in addition, although the daily dose of antipsychotics administered to men was significantly higher than that administered to women (p=0.048), no difference in the ratio of chlorpromazine equivalent (mg / day) to body weight (kg) was detected between men and women (p=0.500). therefore, it is assumed that the blood concentration of this antipsychotic may not be different between men and women. several previous studies have shown that aripiprazole exerts a negligible effect on prolactin concentration31 and alleviates the increases in the prolactin concentrations induced by other antipsychotics32 but that other antipsychotics often increase the prolactin concentrations.33 therefore, this mixed patient cohort might not display any association between the markers of activated coagulation and the dose of antipsychotic. nevertheless, prolactin was correlated with the markers of activated coagulation levels only in men. the median sd illness duration among men was 24.814.3 years, whereas that among women was 17.111.3 years, representing a significant difference (p=0.001 ; table 1). therefore, men might be exposed to higher levels of prolactin over a longer period. the mechanisms that underlie the sex differences in vte remain unknown. at the molecular level, sex differences affect platelets and markers of activated coagulation activity, and sex hormones such as estrogen regulate markers of activated coagulation.30,34,35 for example, hyperprolactinemia results in low levels of estrogen, and this hormone is associated with coagulation activity.35,36 therefore, certain markers of activated coagulation might offset the elevated prolactin concentrations in women.37,38 however, because we did not measure the levels of sex hormones, whether sex hormones affected our results remains unknown. the levels of d - dimer and fdp (but not tat) were positively associated with prolactin levels in men. in general, obtaining a blood sample from a small and dehydrated vein can falsely elevate plasma tat levels. omote indicated that several plasma markers of activated coagulation, including d - dimer and tat, changed under various difficult sampling conditions, with the exception of d - dimer. therefore, we hypothesize that the plasma tat levels of this study might have been increased by difficult sampling, and that the relationship between plasma tat and prolactin levels might not be significant. the reasonable hypothesis concerning the formation of vte must be judged in the context of three basic factors : damage to the vessel wall, venous stasis, and the abnormal process of coagulation.40 these three factors are known as virchow s triad. additional studies are required to clarify the mechanisms responsible for vte in psychiatric patients. because damage to the vessel wall is primarily associated with operations or bone fractures we assumed that the mechanisms of both the abnormal process of coagulation and venous stasis influenced the markers of activated coagulation in the patient group. antipsychotics, which are often used in combination with psychotropic medications, might cause sedation and reduce daily activity, thereby contributing to venous stasis. the lifestyles of patients on long - term antipsychotics are characterized by a lack of exercise, weight gain, and unhealthy diets. furthermore, metabolic symptoms caused by antipsychotics, such as weight gain, hyperglycemia, and dyslipidemia, increase the risk for vte.41,42 because many articles have reported that aging increases the risk for vte,43 the influence of age might be stronger than the level of prolactin on the markers of activated coagulation in this study. in fact, the effect sizes (partial correlation coefficients) of age were greater than those of prolactin in both men (0.379 vs 0.221 for log d - dimer and 0.336 vs 0.132 for log fdp) and women (0.438 vs 0.021 for log d - dimer and 0.479 vs 0.009 for log fdp) (table 4). the stratification of patients according to age is required for examining the influence of prolactin on the risk of vte. first, we did not employ a control group ; thus, we were unable to compare our results or determine the relationship between vte and the level of prolactin in healthy individuals. second, we studied patients with several psychiatric diseases ; thus, the direct influence that each disease has on the markers of activated coagulation remains unknown. furthermore, measuring the influence that illness severity has on markers of activated coagulation is difficult because we did not evaluate the diseases using that scale. third, we measured several markers of activated coagulation but did not investigate vte itself using a multislice computed tomography scanner. therefore, whether increases in these markers of activated coagulation cause vte is unknown and should be investigated in the future. fourth, although many risk factors for vte were examined as potential confounders in the current study, other risk factors such as family history and thrombophilia were not recorded. this lack of data might underestimate the risk of vte, but because thrombophilia is rare, our findings are unlikely to change substantially. hyperprolactinemia is associated with an increase in markers of activated coagulation among men receiving antipsychotics. additional studies are required to prospectively evaluate the relationship between the level of prolactin and vte among patients receiving antipsychotics. | objectivethe strong association between psychiatric patients who receive antipsychotics and the incidence of venous thromboembolism (vte) is known. although previous reports suggest that hyperprolactinemia often increases markers of activated coagulation, few studies have examined the direct relationship between the prolactin level elevated by antipsychotics and activated markers of activated coagulation.methodthe participants included 182 patients with schizophrenia (male = 89, female = 93) who received antipsychotic treatments for at least 3 months. markers of vte (d - dimer, fibrin / fibrinogen degradation products, and thrombin antithrombin complex) and serum prolactin concentrations were measured.resultsprolactin levels were significantly correlated with the logarithmic transformation of the d - dimer (r=0.320, p=0.002) and fibrin / fibrinogen degradation product levels (r=0.236, p=0.026) but not of the thrombin antithrombin complex level (r=0.117, ns) among men. however, no correlations were found between the vte markers and prolactin levels among women. these results were confirmed using multiple regression analyses that included demographic factors and antipsychotic dosages.conclusionthe current study indicates that hyperprolactinemia is associated with an increase in markers of activated coagulation among men receiving antipsychotics. this finding clinically implies that monitoring and modulating prolactin levels among men are important to decrease the risk of vte. |
we assessed 2 cohorts. cohort 1 comprised 1,032 children (617 boys and 415 girls) with acute lrtis, hospitalized in beijing children s hospital (bch), from whom nasopharyngeal aspirates were collected from may 2007 through march 2009. the patients ranged in age from 2 weeks to 16 years (mean age 31.3 months, median 9 months). cohort 2 comprised 506 bch outpatient children (277 boys and 229 girls) with acute urtis, from whom throat swabs were collected from may through august 2009. these patients ranged in age from 4 months to 16 years (mean age 46.7 months, median 37 months). virus nucleic acids in clinical samples were extracted by using the nuclisens easymag system (biomrieux, marcy - letoile, france) according to the manufacturer s instructions. pcr (rt - pcr) by using primers selective for the 5 untranslated region (utr) (3). the viral protein (vp) 1 gene was amplified by using 3 pairs of primers as previously described (3,5,6). the 5 and 3 utr sequences were determined by using the race system (invitrogen, carlsbad, ca, usa) according to the manufacturer s protocol. after being cloned into the pgem - t easy vector (promega, madison, wi, usa), all pcr products were verified by sequencing. in addition, all screened specimens were tested for known respiratory viruses as previously described (8,9). mycoplasma pneumoniae was detected by using the gelatin particle agglutination test kit (serodia - myco ii, fujirebio, japan). for phylogenetic analysis, we constructed neighbor - joining trees based on the distances of safv nucleotide or amino acid sequences by using mega 4.0 (10). we used simplot (version 3.5.1) to analyze possible recombination between viral genome sequences (11). llc - mk2 cells were used to isolate safv as previously described (12). cells were collected either when cytopathic effects were observed or after 12 days postinoculation, and then they were tested for safv by rt - pcr. we detected safv rna in 4 (0.4%) of the 1,032 nasopharyngeal aspirates from patients with lrtis and in 3 (0.6%) of the 506 throat swab specimens from outpatients with urtis. the safv - positive patients (4 girls and 3 boys) were 5 months to 9 years of age (table). safv infection did not appear to have a predominant time for occurrence : cases were detected in a range of months for the periods covered (august and december 2007, october and november 2008, and june 2009). all safv - positive patients exhibited symptoms of respiratory tract infection, such as coughing, gasping, sneezing, or fever. of the 4 safv - positive patients with lrtis, 2 had underlying illnesses, i.e., tuberculosis, hepatic dysfunction, or respiratory failure (table). no major differences were found in disease duration between patients who had underlying diseases and those who did not. co - infections with additional respiratory pathogens were detected for 3 of 7 safv - positive patients, all in the first cohort. these pathogens were respiratory syncytial virus (1 patient), enterovirus (1 patient), and m. pneumoniae (1 patient) (table). virus was not isolated from other safv - positive samples (data not shown). we identified 3 genetic lineages, safv-1, -2 and -3, on the basis of phylogenetic analysis of vp1, the most diverse protein of picornaviruses (6) (figure 1). multiple - alignment analysis, based on reference sequences of each lineage available in genbank, showed that the identity of the vp1 amino acid sequences among the strains in the same lineage was 92.6%100% and among strains belonging to different lineages, 63.3%100% (table a1). we found no obvious differences in amino acid sequences, nor any new motifs in these sequences, between strains detected in respiratory samples and those in fecal samples within the same lineage. phylogenetic analysis of saffold cardiovirus (safv) strains obtained in beijing, china, 20072009, based on viral protein (vp) 1 (a), p1 capsid proteins (b), and full - length genomes (c). the trees, with 500 bootstrap replicates, were generated by using the neighbor - joining algorithm in mega 4.0 (10). strains identified in this study are indicated by a specific identification code (bch or bchu), followed by the patient number and labeled with dark triangles (bch133, bch350, bch895, bch1031, bchu79, bchu115, and bchu353). are bch1031 gu943513, bchu115 gu943514, and bchu79 gu943518 ; of the p1 gene, bch350 gu943515, bch895 gu943516, and bchu353 gu943517 ; and of the vp1 gene of bch133, gu126461. safv-1 (prototype), uc1-uc7, can112051 - 06, d / vi2229/2004, br/118/2006, d / vi2273/2004, d / vi2223/2004, lz50419, gl317, pak12, pak971, afg1449, pak1141, pak2578, pak5003, pak5152, pak6572, and nl2007, temv - gdvii, theiler - like virus ngs910, and vilyuisk virus were used as reference sequences (genbank accession nos. nc009448, nc010810, eu604745-eu604750, am922293, eu681176-eu681179, fj586240, fj464767, fj463600-fj463602, fj463604, fj463605, fj463615-fj463617, fm207487, x56019, ab090161, and eu723237). scale bars indicate nucleotide substitutions per site. to further characterize the variation of safvs, we amplified the p1 region sequences of 6 strains identified in this study. the identity of all available nucleotide sequences of the p1 region among safvs was 68.5%97.2%, whereas that of amino acid sequences was 74.9%99.4%. the vp1 cd and vp2 ef loop structures, which display the greatest amino acid divergence among different safvs and are associated with tropism and virulence (6,7), were analyzed by amino acid alignment. similar to previously reported findings (6,7), we found that the sequences in both loops among safvs were highly diverse among safvs (figure 2). the amino acid identities among all known safvs were 53.1%100% in cd loops and 40.3%100% in ef loops ; amino acid identities of safvs versus animal cardioviruses were 29.1%41.1% in cd loops and 19.2%32.6% in ef loops. we did not find any major differences among the available amino acid sequences of cd loops (91.1%100.0%) or ef loops (97.9%100.0%) of safvs within the same lineage for samples collected from either the gastrointestinal or respiratory tracts. alignment of saffold cardiovirus (safv) viral protein (vp) 1 cd (a) and vp2 ef (b) loop sequences from strains isolated in beijing, china, 20072009. columns highlighted in black show absolute amino acid conservation ; those highlighted in gray show amino acids with highly similar properties. strains identified in this study are labeled with dark triangles (bch133, bch1031, bchu115, bch350, bch895, bchu353 and bchu79) (genbank accession nos. gu126461, gu943513gu943518), safv-1 (prototype), safv-1 gl317, safv-2 can112051 - 06, safv-2 br/118/2006, safv-2 d / vi/2229/2004, safv-2 pak971, safv-2 uc1, safv-3 pak2578, safv-3 d / vi/2223/2004, safv-3 d / vi/2273/2004, safv-3 nl2007, safv-4 pak12, safv-5 pak5152, safv-6 pak 6572, safv-7 afg1449, safv-8 pak1141, theiler - like virus ngs910, tmev gdvii, and emcv were used as reference sequences (genbank accession nos. nc009448, fj464767, am922293, eu681177, eu681176, fj463601, nc010810, fj463605, eu681179, eu681178, fm207487, fj463600, fj463616, fj463617, fj463602, fj463604, ab090161, x56019, x87335). the complete genome sequence of bch1031 (safv-3) was obtained, as were nearly complete genome sequences of bchu115 (safv-3) and bchu79 (safv-2), which covered full - length coding sequences as well as the 3 utr and a partial 5 utr (figure 1). simplot analysis showed relatively high (> 86%) identity between strains within the same lineage. no clear evidence of genetic recombination between the safv strains was found (figure a1). although 8 lineages of safv have been detected in fecal samples worldwide, only safv-2 has been detected in respiratory samples (2,4). in addition, only safv-1 had been reported in china (5,13). in this study, we found that safv lineages 1, 2, and 3 co - circulated in patients with respiratory infections in beijing. although safv is known to be transmitted by the fecal oral route (1,37), as are other picornaviruses (14), our detection of safv in respiratory samples suggests that various safv lineages may also be transmitted through the respiratory tract and may be associated with disease. however, other respiratory viruses and m. pneumoniae were co - detected in 3 of the 7 safv - positive patients. given that we did not conduct assays for common respiratory bacteria (and the number of safv - positive cases was limited), whether safv actually caused the observed symptoms in the patients can not be definitively determined. the genetic diversity of safvs in respiratory samples can complicate the relationship between safvs and disease because different genotypes of the same picornavirus species may cause different clinical signs and symptoms (6,14). further investigations are needed to clarify any possible link between the pathogenicity and genetic diversity of safv. | to clarify the potential for respiratory transmission of saffold cardiovirus (safv) and characterize the pathogen, we analyzed respiratory specimens from 1,558 pediatric patients in beijing. we detected safv in 7 (0.5%) patients and identified lineages 13. however, because 3 patients had co - infections, we could not definitively say safv caused disease. |
a 66-year - old male patient with a history of hypertension and heavy alcoholism was referred to asan medical center for further evaluation of an abdominal distension that had developed 15 days prior. on admission, the patient had experienced 6 kg of weight loss during the previous 2 months. a physical examination revealed fever (38.2c) and abdominal tenderness. laboratory investigations revealed an elevated white cell count of 10.610/l and a c - reactive protein level of 6.69 mg / dl (the reference value is 10 l) of the whole surgical field. additionally, we found the inflamed distal esophagus to be suggestive of an infected pseudocyst and planned to perform distal esophageal resection ; however, the patient s family declined further esophageal procedures in consideration of the additional surgical risks. postoperatively, vital signs were stable and the patient fasted for 2 weeks until the absence of an esophageal fistula or perforation on egd was confirmed. in addition, the patient underwent anticoagulation with iv heparin, which was switched to oral warfarin to prevent potential thromboembolism. the patient was able to ingest full meals, and a follow - up ct confirmed significant improvement of the lesions in the distal esophagus, which had been thickened diffusely with fat necrosis (fig. two years after surgery, the patient was doing well clinically, without any complications. this was an extremely rare case of a patient with multiple aortic mural thrombi and necrotizing mediastinitis caused by acute pancreatitis. although most cases of acute pancreatitis are self - limited, one - fourth of patients have been reported to develop vascular complications, such as hemorrhage into a pseudocyst, erosion of the peripancreatic arteries, splanchnic venous thrombosis, and formation of pseudoaneurysms. arterial thrombus formation, however, is very uncommon, and aortic involvement is extremely rare. in a review of the literature, we were only able to find 3 cases of aortic mural thrombi formation secondary to acute pancreatitis, all of which involved the retropancreatic aorta. to our knowledge, this is the first report in the literature describing thrombi in the ascending aorta. we initially thought that the intramural aortic mass on the chest ct was a pseudolesion due to motion artifacts that turned out to be multiple floating thrombi in the ascending aorta according to subsequent ct evaluations and tee. primary aortic thrombosis is defined as the development of thrombosis without underlying atheromatous lesions in the aorta. the etiologies of primary aortic thrombosis are diverse, ranging from chemotherapy to treat cancer to heparin - induced thrombocytopenia and inflammatory bowel disease. in addition, primary aortic thrombosis has been reported to occur in extremely rare cases of acute pancreatitis. although the pathophysiology of acute pancreatitis leading to aortic thrombosis is not clear, several hypotheses have been suggested. first, acute severe pancreatitis emits proteolytic enzymes that injure the vascular wall directly, which may result in its acting as a nidus for intraluminal thrombus formation. second, the activation of thrombogenic factors from pancreatitis, such as fibrinogens and platelets, can also be triggered by the release of trypsin into the blood stream. lastly, acute pancreatitis possibly can lead to hypovolemia, which can increase the risk of vascular stasis and vasospasm. after the second set of imaging evaluations in the present case, we assumed that the acute pancreatitis had progressed and was complicated by a paraesophageal pseudocyst, which subsequently developed into phlegmonous esophagitis and resultant acute mediastinitis. these inflammatory processes in the mediastinum might have been facilitated by the thrombogenic environment in the proximal aorta. in conclusion, we experienced a rare case of acute pancreatitis resulting in necrotizing mediastinitis, paraesophageal pseudocyst, and multiple aortic thrombi. it was successfully treated by medical therapies combined with an urgent operation in which mediastinal debridement and complete removal of the aortic thrombi were performed. | the formation of aortic thrombi is an extremely rare complication of acute pancreatitis. here we report a case of acute pancreatitis complicated by a paraesophageal pseudocyst, necrotizing mediastinitis, and the formation of multiple thrombi in the ascending aorta. the patient was successfully treated by surgical therapy, which included extensive debridement of the mediastinum and removal of the aortic thrombi under cardiopulmonary bypass. although esophageal resection was not carried out concomitantly, the lesions were resolved and the patient remained free of complications over 2 years of follow - up care. |
the most common site is legs and melanomas in men are most common on the back. melanoma of the clivus is an extremely rare case presentation with only a few cases reported in the literature. conventional imaging techniques like computed tomography (ct) and magnetic resonance imaging (mri) may be suboptimal in evaluating such tumor, and may lead to inaccurate staging. a multimodality whole body imaging technique, 2-deoxy-2-[18f ] fluoro - d - glucose positron emission tomography / ct (18f - fdg pet / ct) is being increasingly used in oncology for staging of multiple malignancies to know the spread of the tumor in the body. this rare case is important because it highlights the extensive disease that can be caused by a clival tumor and the role of noninvasive imaging, that is, 18f - fdg pet / ct in correct staging and hence, guiding further management of the disease. a 55-year - old woman, presented to the hospital with chief complaints of headache, decreased vision in the left eye, and occasional episodes of vomiting since 3 months. mri brain revealed altered signal intensity lesion with solid, hemorrhagic, and few cystic components in basiocciput, basisphenoid, clivus, sella, and right petrous apex ; displacing optic chiasma superiorly. there was associated soft tissue component extending into cavernous sinus with partial encasement of cavernous segment of right internal carotid artery. cemr study revealed a large moderately enhancing mass lesion involving the clivus with sellar - suprasellar extension with encasement of bilateral internal carotid arteries suggestive of plasmacytoma / chordoma or metastasis [figure 1 ]. she underwent endonasal transsphenoidal excision of clival tumor and the black colored, relatively avascular tumor was confirmed to be melanocytic melanoma of clivus on histopathological examination. the patient was thoroughly examined to rule out any lesion on the skin and mucosa with other investigations including chest x - ray. a week after the surgery, this patient was referred to our department for a whole body 18f - fdg pet / ct scan for restaging. whole body pet - ct scan was performed after intravenous (iv) administration of 10 mci of 18f - fdg. pet and contrast - enhanced ct images were acquired and reconstructed to obtain transaxial, coronal, and sagittal views. the study revealed residual hypermetabolic well - defined lobulated soft tissue lesion in the basisphenoid and sella turcica region extending into the extraaxial space of right middle cranial fossa causing destruction of the sella turcica, sphenoid sinus, dorsal sella, and clivus ; suggestive of residual disease. also multiple metabolically active skeletal lesions were noted suggestive of skeletal metastasis [figure 2 ]. (a and b) magnetic resonance images - t1 and t2 weighted axial sections of the brain (preoperative) showing altered signal intensity lesion with solid, hemorrhagic, and few cystic components in basiocciput, basisphenoid, clivus, sella, and right petrous apex ; displacing optic chiasma superiorly associated with soft tissue component extending into cavernous sinus with partial encasement of cavernous segment of right internal carotid artery (a) maximal intensity projection image of the patient from base of skull to mid - thigh showing focal areas of hypermetabolism throughout the body corresponding to multiple metastatic skeletal lesions. physiological uptake noted in heart, liver, bowel, kidneys, and urinary bladder, (b) sagittal positron emission tomography and fused pet - computed tomography images reveal abnormal fluoro-2-deoxy - d - glucose uptake in spinal column corresponding to lytic lesions on ct, (c) metabolically active well - defined lobulated soft tissue lesion in basisphenoid and sella turcica region, extending into the extraaxial space of right middle cranial fossa and indenting the medial temporal lobe causing destruction of the sella turcica, sphenoid sinus, dorsal sella, and clivus, (d) hypermetabolic lytic intradiploic lesions noted in left anterior frontal, high frontal, and parietal region eighty five percent of the patients diagnosed in early stages can be cured with surgery. primary intracranial malignant melanoma is a rare entity with incidence estimated to be 0.005 cases per 100,000 population. the age of the patients usually range from 15 - 71 years, with a peak incidence in the 5 decade. symptoms at presentation include headache ; vomiting due to intracranial hypertension ; hydrocephalus ; focal neurological deficits due to compression of the brain, spinal cord, or cauda equina ; subarachnoid hemorrhage ; and seizures. to our knowledge, very few cases of primary melanoma of the clivus have been cited in the literature previously. metastases involving this area have been previously described as a single case report or included in series with other skull base tumors. in 2009, a literature review was performed by pallini., which reveals that out of 46 patients who underwent surgery for clival bone tumor, seven proved to be metastatic, representing 0.18 and 0.42%, respectively of intracranial and skull base tumors which were treated in their institution in the study period between january 1995 and december 2007. the primary tumors associated were lung adenocarcinoma (n = 2), prostate carcinoma (n = 2), skin melanoma (n = 1), hepatocarcinoma (n = 1), and lung squamous cell carcinoma (n = 1). in 2010, chaudhary., presented a case of an atypical clival meningeal melanoma treated with a multidisciplinary staged transcrural and transsphenoidal endoscopic surgical approach. no other metastases was evident for 2 years after initial symptoms and with no evidence of a cutaneous source, diagnosis of a primary meningeal lesion of the clivus was made. bone metastases occur in a significant proportion of patients with metastatic melanoma. in such patients survival, in 108 patients in 2008, which revealed median survival following diagnosis of bone metastases in malignant melanoma to be 3.2 months (range 0.3 - 47.4 months). bone metastases most commonly occurred in patients with the primary melanoma originating on the back and lower limbs and spine was the commonest site of bone involvement, followed by ribs, pelvis, long bones, and skull. fdg pet is a sensitive and specific technique for patients with melanoma but has limitations with small (less than 1 cm), pulmonary, and brain metastases. it is felt to be superior to ct alone in detecting abdominal, nodal, subcutaneous, and skin sites. it is useful in assessing extent of disease in patients with surgically resectable disease by conventional methods as it may render them unresectable in a considerable population. in our patient, surgical removal of the tumor was done from an outside institution and was referred to us for further management. pet - ct scan was performed for the patient in view of histological diagnosis of melanocytic melanoma. also, no primary site could be localized after thorough general examination of the skin. the findings on pet - ct scan suggest that the clival mass represents the primary site of malignancy. this case adds to the literature on the occurrence of intracranial malignant melanoma in patients with extensive skeletal metastasis, and supports the finding that such tumors need to be followed carefully. | malignant melanoma of the clivus is a rare entity, for which there is little evidence - based literature for guiding clinicians to understand the importance of disease staging via noninvasive imaging strategy. this report highlights the case of a 55-year - old lady with histopathologically confirmed melanocytic melanoma of the clivus postoperative status, with multiple skeletal metastasis, demonstrated on 2-deoxy-2-[18f ] fluoro - d - glucose positron emission tomography / computed tomography (18f - fdg pet / ct scan). the experience gained with this patient demonstrates the feasibility and usefulness of this noninvasive application in accurate staging and hence, correct decision making regarding further treatment. |
in the 1990s two independent discoveries opened up the previously unsuspected world of noncoding rnas (ncrnas). the phenomenon of rna interference (rnai) was being uncovered as cosuppression in plants [1, 2 ], quelling in fungi [3, 4 ], and rnai in nematodes through the 1990s and at least the broad strokes of the mechanism were elucidated by the turn of the 21st century. at the same time, another curious phenomenon was being observed by victor ambros, gary ruvkun, and colleagues in nematodes [7, 8 ]. like rnai, this phenomenon, initially called short temporary rna (strna), was at first regarded as a one - off curiosity but, again like rnai, persistence paid off with the explosive validation of the microrna (mirna) [912 ]. the two worlds of rnai and mirnas merged when it was observed that both rnai and mirnas employed the same mechanism to carry out their mission of regulating eukaryotic gene expression. over the past several years rnai has become a powerful tool for understanding the role played by dozens of plant and animal genes in a wide range of cellular processes, both normal and pathogenic. moreover, rnai is proving to be a potentially powerful tool in attacking pathogenic cellular processes. similarly, the world of mirnas has grown from the two original nematode genes to now number more than one thousand loci in plants and animals and their role in regulating cellular processes has expanded to a point where virtually all normal and pathogenic cellular processes are affected at some point by one or more of these tiny entities. hence, the discovery of mirnas represents a hallmark in rna science for understanding rna - dependent regulation of many complex biological processes such as development, function of metabolic pathways, cell fate and death. in addition, the universe of small rnas has expanded to include not only mirnas but new classes including endogenous small interfering rnas (sirnas), 21u rnas, and piwi - interacting rnas (pirnas). of these small rna classes, that stable hairpin makes mirna prediction in sequenced genomes a relatively tractable exercise. on the other hand, de novo finding of mirnas in species whose genomes have yet to be sequenced and discovering new classes of small rnas must still rely upon in vitro cloning from purified cellular rnas. thus, reliable and reproducible methods for cloning small rna species are of paramount importance and will remain so into the foreseeable future. here, we present a compilation of extant small rna cloning methods, options for sequencing, and some of the small rna results that we have obtained in the still unsequenced allotetraploid cotton genome. there are a number of strategies that have been proposed for cloning small rnas. before discussing these, however, there is one factor common to all of them that is essential to be aware of. small rnas, whether from plant cells, animal cells, or other sources, represent a small fraction of the total rna mass present. agilent technologies quantifies the quality of cellular rna in the form of their rna integrity number (rin). very high quality intact rna has a rin of 10.0 and the lower the rin, the more degraded the rna. rin values between 6.5 and 10.0 represent a continuum of acceptable to excellent rnas. using rin as the point of departure, agilent assessed the relative fraction of total rna that is within the small rna size range in forty tissues from human, mouse, and rat. first, for all but five tissues, the relative mass of small rnas is below 3% and, second, there is a significant negative correlation (r = 0.58 ; p <.01, df = 38) between overall rna quality as assessed by rin value and relative small rna mass. clearly, increasing amounts of rna degradation will introduce a greater mass of small fragments that lie in the true small rna zone. this will result in a greater mass of competing rna that will make it more and more difficult to see the real small rnas that are the targets of interest even if the majority of the degraded rnas are themselves unclonable by some of the methods discussed below. while there will be variation from rna source to rna source, it is clear that larger rna components like mrnas, rrnas, and trnas, comprise by far the bulk of the total rna and that the relative mass of the true small rna fraction should and will be the smallest in very high quality rna. as can be seen, the true mirna region is indeed a very small part of the total mass. given this, it is essential to the small rna cloning process that rna quality, as assessed by measures like rin, be as high as possible and that as much of the competing rna mass as possible be removed so that a target - rich small rna component can be purified prior to starting the cloning process. one of the simplest ways is to simply run a sample of total rna on a denaturing polyacrylamide gel (dpage) and excise the area of the gel containing the small rna fraction (see the appendix). the problem with this method is that the enriched small rnas must be removed from the gel and purified for further manipulations and this routinely results in a substantial loss of what is already a small amount of mass to begin with. there are ways to minimize this loss of material and we will discuss one of these in the next section. other methods for enriching the small rna fraction have been developed including column capture and release methods like the mirvana protocol from ambion and the timed size exclusion method, represented by the flashpage fractionator system, also from ambion. the point is that, whatever method is employed, the small rna fraction of total cellular rna must be enriched to increase the likelihood of successfully cloning small rnas. once the small rna fraction is enriched and purified, there are several ways to proceed to clone the individual small rnas contained in the fraction. reviewed the basic small rna cloning methods. in all cases the target species for direct cloning is an rna varying in size between 18 and 25 nucleotides (nt) having a free 3 hydroxyl group and a free 5 phosphate group. although some variation exists, the universal initial step in the cloning process is first to ligate a 3 adaptor sequence through the free 3 hydroxyl. the 3 adaptor will serve as the site for later annealing of an oligonucleotide primer for reverse transcription. as seen in figure 3, there are several possible ways to accomplish this adaptor joining. in one option however, as many small rna species in plants have been shown to contain 2-o - methyl modifications on their 3 ends, this method may be of only limited utility since such modifications block polya polymerase extension. both of the other 3 adaptor joining options are designed to prevent later circularization of the linkered rnas. in one variation, the rnas are dephosphorylated prior to adaptor ligation and then rephosphorylated for subsequent processing [23, 24 ]. in the other variation, the 5 end of the adaptor is preadenylated and the 3 end blocked by a nonstandard group such as a dideoxynucleotide [10, 25 ]. preadenylation of the adaptor obviates the need to dephosphorylate the target rnas because the adaptor joining via t4 rna ligase can be carried out in the absence of atp. given the obvious advantage that this method confers by reducing the number of operations required to process target rnas, new england biolabs (neb) has introduced a truncated t4 rna ligase that specifically reacts with preadenylated 3 linkers [2527 ]. regardless of the method chosen, however, producing a stable and reactive 3 linkered small rna population is the goal of the first step in cloning. the next phase of cloning is to join a second adaptor to the small rna population. as shown in figure 3, there are now but two ways to do this and the choice is dictated by the methods chosen for 3 adaptor joining. if the method chosen is the polyadenylation route, then the 5 adaptor joining method is to carry out a template switch. this method relies on the property of a number of reverse transcriptases to add a small number of nontemplated nucleotides to the 3 ends of cdnas. since the nontemplated nucleotides tend to be mostly deoxycytidines, an adaptor containing a poly - g 3 run can be used to switch the template from the mirna to the adaptor. the other path is to use a 5 adaptor with a 3 hydroxyl group that will ligate to the 5 phosphate of the target rnas. this is carried out with a t4 rna ligase in the presence of atp and is followed by a reverse transcription using a primer complementary to the 3 linker. in both cases, the resulting cdna population is pcr amplified in preparation for cloning and/or sequencing. pcr amplicons can be directly cloned using any one of several pcr cloning vectors or the amplicons can be processed to form concatamers which are then cloned. concatamer formation from amplicons is a direct descendant of the serial analysis of gene expression (sage) methodology developed in the 1990s by velculscu and colleagues [24, 28 ]. the obvious advantage of concatamer cloning is that individual clones will contain more small rnas than the ones that will be present if the pcr amplicons are simply shot - gun cloned. this is a consideration for conventional sanger dye - terminator sequencing but, as will be discussed later, new generation deep sequencing methods have circumvented the need for concatamers and, indeed, for cloning at all. one aspect of the cloning methods shown in figure 3 is that small rnas will all contain a 5 phosphate group following 3 adaptor joining. this constant feature that allows for subsequent 5 adaptor joining was believed to represent the universal state of small rnas in vivo. in 2007, attempts to clone a specific small rna in c. elegans called cel-1 repeatedly failed even though there was ample evidence that it existed. their persistence in uncovering the reason for cel-1 being refractory to conventional small rna cloning methods paid off in their discovery that cel-1, and, now, other small interfering rnas, was tri - phosphorylated on its 5 end. they developed an alternative method for cloning troublesome rnas featuring the use of two 3 ligations with the reverse transcription step in between the two ligations. this alternative method, named by them 5 ligation independent cloning, is completely indifferent to the state of the 5 end of the target rnas. the reverse transcription step following the initial 3 adaptor ligation makes the initial 5 end the new 3 end with a hydroxyl group ready for a second 3 ligation step regardless of what may or may not have been present on that initial 5 end. the 5 ligation independent cloning option revealed that a secondary pool of small rnas was being produced in c. elegans via a completely different pathway from conventional mirnas. while each small rna cloning strategy has its own strengths and weaknesses, the method employing a preactivated, adenylated 3 linker sequence, pioneered by david bartel, has proved to be a readily accessible and flexible method. the adenylation of the 5 end of a dna oligonucleotide provides a preactivated linker that will specifically ligate to the 3 hydroxyl group of rna in the presence of the enzyme t4 rna ligase. this reaction proceeds in the absence of atp, which is known to promote circularization of the target rnas in solution. the 3 end of the preactivated linker is blocked with a nonstandard base, such as dideoxycytidine (ddc), to prevent circularization of the linker. the synthesis reaction begins with an deoxyoligonucleotide synthesized with a 3 block, such as ddc, and a 5 phosphate. adenylation at the 5-end of the oligonucleotide is achieved through the introduction of adenosine 5-phosphorimidazolide in the presence of magnesium chloride as the catalyst. once purified, the linker, with the form rapp-(dntp)n - ddc, will react with the free 3 hydroxyl of an rna in the presence of t4 rna ligase and the absence of atp to create a 3-linkered rna plus amp. this reaction is quite efficient so long as a relatively small mass of t4 rna ligase is used. aravin and tuschl showed that the enzyme itself in commercial preparations of t4 rna ligase is adenylated and that this can cause circularization of the target rna species and other unwanted side reactions that severely reduce production of the desired ligation product. a truncated t4 rna ligase called t4 rnl-2 truncated, that specifically and efficiently ligates adenylated linkers to rnas in the absence of atp without producing side reactions is available from new england biolabs [2527 ]. new england biolabs offers one with a 3 amino block and integrated dna technologies (idt) offers three linkers, each with a 3 ddc block. once the target small rnas are 3 ligated, any unligated linkers are removed by a denaturing polyacrylamide gel electrophoresis (dpage) purification of the ligated material. as with initial small rna enrichment, one way to significantly reduce this loss is to process the acrylamide gel slice containing the rnas using a column originally developed by edge biosystems for cleaning up sanger dye terminator cycle sequencing reactions. called performa columns, these spin columns will retain the acrylamide gel, salts, and urea while passing as much as 95% of the rna into the collection tube (see the appendix). the 3-linkered rnas so recovered will have a 3 end block courtesy of the linker but will retain their 5 phosphate groups. this provides a coupling group for ligation of an oligonucleotide composed of a few 5 dna bases and a run of 3 rna bases that will ligate to the target rnas in the presence of t4 rna ligase and atp. again, a commercial 5 linker, called 5 mrs, is available from idt that is compatible with each of their 3 linkers as well as the neb 3 linker. doubly - ligated rnas are converted into an all dna substrate by reverse transcription using an rt primer complementary to the 3 linker. these cdnas are then amplified in a pcr reaction that uses the rt primer as the reverse pcr primer and a forward pcr primer compatible with the 5 linker. thus, all target rnas can be amplified for subsequent cloning using a universal pcr primer pair. following pcr amplification the target - containing amplicons can be cloned with any one the vector systems designed for pcr cloning. the generally accepted criteria for adding a new mirna to the ever growing catalog being ably curated in mirbase [30, 31 ] are that the sequence of the mature 21 to 23 nt candidate is not already present among extant mirnas, that the sequence is expressed, and that there is flanking sequence ranging in size from 60 to more than 100 nt that, with the mature sequence inside, forms a thermodynamically stable hairpin secondary structure [19, 32 ]. direct cloning and sequencing from an enriched pool of small rnas satisfies the first two of these three criteria at the same time. for this reason, sequencing is obviously a crucial part of mirna cloning and, given that there are usually hundreds of small rnas being expressed at various levels in tissues of interest, the more efficiently that clones can be sequenced, the better the chances of discovering new candidates. in the world of sanger - type, this solution makes use of the simultaneous sequencing capabilities of multi - capillary platforms like the ge healthcare megabace or the abi 3730xl 96-capillary machines. on these platforms small rnas can be sequenced either as single insert shot - gun clones (e.g.,) or as concatamers as shown in figure 3. this is clearly an improvement over any previously available method but one of the most important technological advances of the post - genome era is the development of several massively parallel signatures sequencing (mpss) systems that not only produce several orders of magnitude with more quality sequences per run but also allow researchers to skip the actual cloning steps in figure 3 altogether. the first of the massively parallel sequencing systems to arrive on the scene was the roche pyrosequencing platform originally developed at 454 life sciences. this platform utilizes the phenomenon of pyrophosphate release that accompanies nucleotide incorporation to initiate a light detection reporting system based on the cleavage of oxyluciferin by luciferase. reactors following ligation of 5 and 3 adaptors that serve as the universal templates for clonal amplification inside the reactors. universal adaptor ligation and subsequent clonal amplification provide an ideal opportunity to feed 5 and 3 ligated small rnas directly into the sequencing flow by making fusion primers that incorporate both the rna linker and roche (454) adaptor sequences. these fusion primers would be 40-mers composed of the roche (454) 5 adaptor plus the 5 linker sequences on one end and the 3 linker plus the roche (454) 3 adaptor sequences on the other end (table 1). in addition, these primers can be barcoded so that mixed rna populations could be simultaneously sequenced and the sequences deconvoluted later based upon the barcodes (table 1). the performance obtained by the roche 454 life science commercial system genome sequencer (gs - flx) platform of 99.5% accuracy and average read lengths of over 250 bp resulting in outputs exceeding 200 000 reads with acceptable phred values (a dna sequence quality score) is ideal for searching genomes for new small rnas and, indeed, such studies have already resulted in the discovery of the curious 21u rna class of small rna in c. elegans. according to the latest updates, current 454 flx platform is capable of sequencing 400600 million high - quality bases in ten hours with an average of ~400 bp long reads and a raw base accuracy of 99% (http://www.454.com/products-solutions/system-features.asp ;). this makes the 454 flx platform with several hundred times higher throughput compared to the current state - of - art sanger - based capillary sequencing system. however, current limitations of this platform compared to sanger system are relatively shorter read length as well as challenges with sequencing of homopolymer regions. the latter limitation is due to nonterminating chemistry during pyrosequencing that introduces nucleotide substitution errors. another of the next generation sequencing platforms, based on a four - color dna sequencing - by - synthesis (sbs), introduced by illumina / solexa (http://www.solexa.com/), also incorporates the use of oligonucleotide adaptor ligations to produce millions of short, ligated nucleic acid fragments that are then covalently bound to a solid surface and ultimately interrogated by reversible fluorescent terminator synthesis reactions [36, 41, 42 ]. in comparison with the current 454 flx platform, illumina / solexa platform has a higher throughput sequencing capability that equals to 11.5 billions of 35 bp reads per run. the read length is well suited to the 21 to 31 nt size range of the so - far known small rna classes. although 454 flx and illumina / solexa platforms utilize the same ssb sequencing principle, the sequencing chemistries (pyrosequencing versus fluorescent - based solid phase) and consequently the limitations of two systems are substantially different. the major limitation of the illumina / solexa platform with regard to small rna applications is also the potential for nucleotide substitution errors though the use of fluorescent - based solid phase dye terminators makes homopolymeric runs less problematic. also in the small rna size range of read lengths is the applied biosystems ' sequencing by oligo ligation and detection (solid) platform. solid is the combination of mssp and polymerase colony (polony) sequencing [41, 42, 44, 45 ] that creates emulsion pcr generated clonal amplicons on 1 m magnetic bead from genomic fragments. sequencing - by - ligation is carried out on enriched beads through the repeated cycles of ligation of mixture of sequencing and 8-mer fluorescently labeled oligonucleotide probes to the amplicons and detecting the color [36, 42, 45 ]. the solid system delivers 13 billion bases read per run or 200300 million bp sequence data per day with 25 to 35 bp lengths and a raw base accuracy of 99% [41, 42 ]. this comparatively higher throughput level of solid system is achieved by using smaller beads and random array format compared to 454flx system (26 m and ordered format). however, similar to the illumina / solexa system, there is a potential for incorporating substitution errors and with the shorter read lengths these can be misleading when sequencing small rnas. although yet - unavailable for many small scale molecular biology laboratories with limited funding constraints, these new generation sequencing platforms are already being widely used by plant researchers to characterize plant small rnas. a pioneer mpss effort has revealed more than 2 million small rnas from flower and seedling tissues of model plant arabidopsis thaliana, yielding over 75 thousand distinct sequence signatures. the small rnas in various arabidopsis [47, 48 ] and maize mutant backgrounds were deep sequenced and characterized. recently, small rna / mirna pools in rice were characterized using these next generation sequencing platforms [50, 51 ]. chellappan and jin published an excellent review of small rna cloning and discovery methodology in plants and have compared the deep parallel sequencing of small rna libraries using aforementioned 454, illumina / solexa, and solid technologies. in general, all of the next generation sequencing technologies offer unprecedented sequencing depth in a very short time. the power of these platforms is that they are only capable of finding all or nearly all of the small rnas expressed in a particular tissue but they can do so in a quasiquantitative manner due to the enormous number of sequence reads generated, dramatically reducing the cost. however, since next generation sequencing platforms are still under development and most likely will be improved for higher throughput and accuracy at reduced cost, at present, the suitability of any particular platform for small rna sequencing comes down to study objectives and the availability of the platforms. there are many excellent methods available that utilize known microrna sequences for the purpose of determining both absolute and relative expression levels in various tissues and under various conditions. these methods primarily focus upon either quantitative, or real - time, pcr or microarray hybridizations. however, as noted above, the primary objective of small rna cloning is different, it is discovery of both new mirnas and new classes of small rna. in this final section, we will briefly present results that we have obtained using an adenylated cloning linker strategy (refer to [33, 53 ] for detailed protocol) to investigate the pool of small rna signatures and discover plant small rnas in root tip and developing ovule tissues of a widely grown upland cotton g. hirsutum l. these results are initial surveys, but the first effort of wet - bench works toward studying the small rna world for a complex still unsequenced the genus gossypium l. includes approximately 45 diploid a - g to k genomic groups and 5 allotetraploid (ad1ad5 lineages formed by a- and d - genome hybridization about 1 - 2 million years ago) species. the genomes of allotetraploid cottons have a chromosome complement of 2n = 4x = 52, a haploid genome size of 22003000 mb dna, and a total recombination length of approximately 5200 cm (an average of 400 kb per cm). accordingly, allopolyploid cotton genomes are one of the largest plant genomes with its complex nature, and are an important model system to study fundamental biological studies in plants. furthermore, cotton fiber is regarded as a unique single - celled model system to study cell growth initiation, elongation, differentiation and cellulose biosynthesis in plants [5759 ]. as of february 2009, a search of the genbank nucleotide database for gossypium revealed a total of 452, 634 nucleotide sequences, corresponding to an 8, 239 core subset of nucleotide, 375, 447 expressed sequence tag (est), and 68948 genome survey sequence (gss) records (http://www.ncbi.nlm.nih.gov ; searched on february 16, 2009). efforts toward sequencing entire cotton genome(s) are in progress and the smallest genome, g. raimondii (d5), will soon be completely sequenced and available for researchers. nevertheless, one of the major present sources of cotton genomic sequences, available through genbank, only corresponds to an 11.4 mb of cotton genome. this is a serious obstacle for systematically searching the cotton genome for small rna / microrna signatures although several investigators have reported initial efforts to identify these tiny elements in cotton using in silico bioinformatics analysis [6163 ]. this underlies the necessity for wet laboratory cloning of cotton small rna sequences for de novo discovery of unique small rnas and micrornas from various tissues in cotton, which then subsequently will be validated with availability of a complete dna sequence of cotton genome(s). using the adenylated cloning linker strategy outlined above, we have conducted an initial survey of small rna content in the 35 days old root tip tissue of texas - marker-1 (g. hirsutum standard line) and sequenced ~ 300 individual colonies with the 3 and 5 specific linker ligated small rna inserts. our sequencing efforts have confirmed 20 microrna signatures from 8 families including mir-156 (7), mir-156 (1), mir-166 (4), mir-167 (1), mir-168 (1), mir-169 (2), mir-171 (2), mir-396 (1), and mir-457 (1), suggesting their involvement during early root development of cotton seed germination process (figure 5). these very abundant micro - rnas have known targets including transcription factor and stress response genes in other plants, and mir-156 and mir-166 are considered two of the largest and oldest mirna families in plants. in addition, we found several unidentified 21-mer small rnas that possibly have a potential to be cotton - specific micrornas. we also have several 24-mers that match dcl3 processed small rnas in arabidopsis and many unidentified 24-mers that might also be dcl3 processed small rnas in cotton. two (+ /) gene hits that are notable are the ashbya gossypii opt1 gene and a hit on myb2. thus, the results of our initial attempts using size - directed small rna cloning strategy demonstrated that the cloning method does work for finding small rnas / micrornas in cotton. they also confirmed the difficulty of finding plant micrornas since we only have 20 micrornas, representing only 8 loci, in more than 300 sequenced clones from cotton root tissue small rna library. recently, using the same size - directed small rna cloning strategy with adenylated linkers, we have characterized the small rna sequence signatures in eleven postanthesis (dpa) periods of fiber development (010 dpa) (figure 6). sequencing more than 6500 individual colonies from 11 ovule small rna libraries, we identified nearly 2500 candidate small rnas comprising of 583 unique sequence signatures of 2124 nt size range. as reported by abdurakhmonov., results showed (1) the presence of only a few mirbase - confirmed plant micrornas (mir172, mir390 and ath - mir853-like), and these were differentially represented in specific dpa periods of ovule development. (2) the vast majority of sequence signatures were expressed in only specific dpa period and this included nearly all of the 24 nt sequences, further, they showed (3) the existence of specific pattern of sequence diversity and abundance between 02 to 310 dpa periods, possibly corresponding to the transition of fiber initiation to elongation phase of fiber development. further, target predictions in silico using ovule - derived small rna sequences putatively indicated their involvement in numerous important biological processes including processes involving previously reported fiber - associated proteins (figure 7). results collectively demonstrate that the initiation and elongation stages of cotton fiber development are at least partially regulated by specific sets of small / micrornas. however, to get a better picture of cellular mechanisms of small rna network during fiber development process, there is urgent need for so - called deep sequencing efforts of small rna pools using next generation sequencing platforms [36, 49 ] that will undoubtedly increase multi - dpa representation of small rnas. the discovery of the world of small, regulatory rnas has provided geneticists with a phenomenal array of opportunities as well as questions. this discovery has also led to the development of a powerful set of new molecular tools that can be used to answer those questions and take full advantage of those opportunities. the techniques built around rna interference, real - time pcr, and microarrays allow an unprecedented level of precision in unraveling the mechanisms of gene expression and regulation. so, too, have the developments in small rna cloning and next generation dna sequencing discussed here opened previously barred windows on genome organization that will continue to feed into the functional genomics pipeline. the size - directed small rna cloning strategy using adenylated linkers, highlighted with its application for the yet - unsequenced cotton genome small rna characterization, is an efficient methodology for studying these tiny molecules in various plant genomes, especially suitable for the small - scale plant genome laboratories worldwide, that lack access to the still - expensive next generation sequencing platforms. run total rna spiked with 10 pmoles of the mispike (integrated dna technologies) 21-mer control rna on a 12% to 15% denaturing page (7 m urea) for 90 minutes at 275 v (be sure to monitor the gel so that the small fragments do not run off). stain the gel with gelstar nucleic acid stain (lonza cat. select rna fragment(s) to be purified and cut it (them) from the gel as shown in figure 8. place the gel slice in a 1.5 ml tube and crush with a glass rod. (note : we have had very good results using the 1.5 ml tubes and disposable pestles from kontes glass company). add 200 l idt sterile, nuclease - free water and continue to crush the gel into a fine slurry. place the tube at 70c for 10 minutes. following manufacturer 's recommendations, prepare a performa dtr column for each gel slice. vortex the gel slurry, transfer the entire volume onto the column and spin at 3000 rpm for 3 minutes. discard the dtr column. add 3 l 10 mg / ml glycogen, 25 l of 3 m naoac (ph5.2), and 900 l ice cold 100% etoh to the eluent. spin tubes at full speed (10 000 rpm) for 10 minutes to pellet the rna. this protocol successfully removes the urea and other salts with substantially less loss of rna than is seen with conventional crush and soak methods followed by nap-5 column desalting or by dialysis methods. detail list of small rna cloning products and protocol for mircat can be found from idt product manual at (http://www.idtdna.com/support/technical/technicalbulletinpdf/mircat_user_guide.pdf). | the rna revolution that started at the end of the 20th century with the discovery of post - transcriptional gene silencing and its mechanism via rna interference (rnai) placed tiny 21 - 24 nucleotide long noncoding rnas (ncrnas) in the forefront of biology as one of the most important regulatory elements in a host of physiologic processes. the discovery of new classes of ncrnas including endogenous small interfering rnas, micrornas, and piwi - interacting rnas is a hallmark in the understanding of rna - dependent gene regulation. new generation high - throughput sequencing technologies further accelerated the studies of this tiny world and provided their global characterization and validation in many biological systems with sequenced genomes. nevertheless, for the many yet - unsequenced plant genomes, the discovery of small rna world requires in vitro cloning from purified cellular rnas. thus, reproducible methods for in vitro small rna cloning are of paramount importance and will remain so into the foreseeable future. in this paper, we present a description of existing small rna cloning methods as well as next - generation sequencing methods that have accelerated this research along with a description of the application of one in vitro cloning method in an initial small rna survey in the still unsequenced allotetraploid cotton genome. |
the human body has simple and compound organs that obtain their nourishment through four humors. one of them is bile (yellow bile). according to iranian traditional medicine (itm), there are various kinds of natural medicines with their specific mechanisms of action affecting on bile in the human body. hakim aghili shirazi (18th century), one of the great scholars in itm field, introduced all types of natural medicines influencing bile in his valuable book written in persian, makhzan - ul - adwiah, about single herbal medicines (mofradat). the aim of this review article was to introduce all types of natural medicines influencing bile in the human body. the classification of natural medicines influencing bile was studied in this article as viewed by hakim aghili shirazi in makhzan - ul - adwiah. reviewing makhzan - ul - adwiah, this natural influencing bile is defined in ten categories. these are haabes - e safra (obstructive of bile), daafe - e safra (expellant of bile), raafe - e safra (resolver of bile), ghaate - e safra (stopper of bile), ghaame - e safra (suppressant of bile), kaasere - e safra (fractionating of bile), mohregh - e safra (burner of bile), moder - e safra (bile diuretic), mosaken - e safra (bile reliever), and moshel - e safra (bile laxative). recognition of the precise mechanisms of these natural medicines is necessary to prescribe a suitable remedy for bilious diseases by traditional medicine specialists. | background : the human body has simple and compound organs that obtain their nourishment through four humors. one of them is bile (yellow bile). according to iranian traditional medicine (itm), there are various kinds of natural medicines with their specific mechanisms of action affecting on bile in the human body. hakim aghili shirazi (18th century), one of the great scholars in itm field, introduced all types of natural medicines influencing bile in his valuable book written in persian, makhzan - ul - adwiah, about single herbal medicines (mofradat). the aim of this review article was to introduce all types of natural medicines influencing bile in the human body.methods:the classification of natural medicines influencing bile was studied in this article as viewed by hakim aghili shirazi in makhzan-ul-adwiah.results:reviewing makhzan - ul - adwiah, this natural influencing bile is defined in ten categories. these are haabes - e safra (obstructive of bile), daafe - e safra (expellant of bile), raafe - e safra (resolver of bile), ghaate - e safra (stopper of bile), ghaame - e safra (suppressant of bile), kaasere - e safra (fractionating of bile), mohregh - e safra (burner of bile), moder - e safra (bile diuretic), mosaken - e safra (bile reliever), and moshel - e safra (bile laxative).conclusion : each group has a specific function and mechanism on bile. recognition of the precise mechanisms of these natural medicines is necessary to prescribe a suitable remedy for bilious diseases by traditional medicine specialists. |
constitutive autophagy is required for cellular housekeeping (e.g., elimination of damaged or long - lived organelles). it is a highly sensitive process that cells are induced in response to a wide range of stressful conditions (physical, chemical, or metabolic) in order to maintain cellular homeostasis. while the inflammatory responses are generally beneficial for host protection, this process needs to be spatially and temporally tightly regulated to avoid a state of excessive and/or sustained inflammation that is potentially detrimental. indeed, prolonged exposure of tissues and organs to high concentration of inflammatory mediators represents a stressful environment for cells and can result in severe damage. since an abnormal inflammation could disrupt cellular homeostasis, autophagy acts by at least two means to protect cells from excessive long lasting inflammation : (i) indirectly by allowing efficient clearance of damaged organelles (mitochondria, e.g.,) or intracellular pathogenic microorganisms that both constitute potent inflammatory stimuli and (ii) directly by suppressing proinflammatory complexes. naturally, regulatory networks that control autophagy activity are able to sense output signals from various inflammatory mediators - associated signaling, allowing a proper modulation of the process according to inflammation state. in this review, following a brief introduction on molecular mechanisms controlling autophagy, we will make an overview of interrelations existing between inflammation and autophagy. facing tremendous number of studies describing relationships between inflammatory mediators and autophagy, it is nearly impossible to be completely exhaustive, but we will highlight some of the best - characterized interactions between these two processes. in the last part of the review, we will discuss in more detail the crosstalk between autophagy and inflammation during pathophysiological situations, especially inflammatory bowel diseases. macroautophagy corresponds to the sequestration of cytoplasmic structures into double- or multimembrane vesicles termed autophagosomes. complete autophagosomes then transit along microtubules to deliver their content to degradative compartments, lysosomes, forming autolysosomes. the term microautophagy refers to the direct engulfment of the cytosolic material by invagination of the lysosomal membrane. the third form of autophagy is chaperone - mediated autophagy (cma), during which, proteins containing a pentapeptide motif (kferq - like sequence), are recognized by the cytosolic chaperone hsc70 (heat shock cognate protein of 70 kda) and its cochaperones that deliver them to the surface of lysosomes. the substrate - chaperone complex binds to the lysosomal protein lamp-2a (lysosome - associated membrane protein type 2a) and the substrate is unfolded., we will focus only on macroautophagy (hereafter referred to as autophagy) and its interrelations with inflammatory processes. autophagy was first described as a nonselective bulk degradation process, sequestering a portion of the cytosol and used by the cell during nutrient deprivation period. in light of studies during last decade, it turns out that autophagy can also be selective, allowing, under certain conditions, the sequestration of specific substrates such as mitochondria (mitophagy), endoplasmic reticulum (er- or reticulophagy), lipid droplets (lipophagy), peroxisomes (pexophagy), endosomes, lysosomes, secretory granules, ribosomes (ribophagy), cytoplasmic aggregates (aggregaphagy), inflammatory proteins, and invading pathogens (xenophagy). structures targeted for destruction by autophagy are often ubiquitinated. a series of autophagy receptors, termed slrs, for sequestosome 1- (sqstm1-) like receptors contain ubiquitin - binding domain (ubd) associated with a lir (lc3-interacting region) motif and act as adaptors between k48- or k63-linked polyubiquitin chains on a targeted - substrate and atg8 paralogs (lc3, gabarap), bridging autophagic cargoes to nascent autophagosomes. substrates can also be delivered to autophagosome in ubiquitin - independent manner, as exemplified by mitophagy. in some cases, autophagy - mediated degradation of mitochondria relies on polyubiquitylation of proteins at the outer mitochondrial membrane and is dependent on pink1 (pten - induced putative kinase protein 1) and the e3 ligase parkin. in other cases, however, mitophagy is dependent on mitochondrial outer - membrane proteins (e.g., nix) that can directly link mitochondria to autophagosomal membranes via their own lir domain. finally, an alternative way has been observed in neuronal cells and involves externalization of an inner mitochondrial membrane phospholipid, named cardiolipin, to the outer mitochondrial membrane and its direct recognition by lc3 [7, 8 ]. autophagy can be divided into 6 main steps : initiation, vesicle nucleation, elongation, membrane elongation, closure, maturation, and degradation. initiation step leads to the formation of an isolation membrane, called phagophore, most often in close vicinity with the endoplasmic reticulum (er). various organelles, including the er, the golgi apparatus, mitochondria, the plasma membrane, and endosomes, have been proposed to serve as membrane reservoir for phagophore generation and growth. initiation of autophagy requires two protein kinases complexes : (i) the ulk1/2-atg13-fip200 complex, which is coupled with the autophagy suppressor tor complex 1 (mtorc1), and (ii) the beclin1-vps34-vps15-atg14 complex. this last complex is usually inhibited by interactions with proteins from the golgi apparatus, antiapoptotic bcl2 proteins, and other signals transducers. given the fact that autophagy is a highly dynamic process, its activation is largely dependent on a set of posttranslational modifications such as phosphorylation, acetylation, and ubiquitylation. the mtorc1 complex consists of the mtor kinase, which is a master cell - growth regulator integrating numerous intracellular and extracellular signals (growth factor, nutrients, and cellular energy status), gl, pras40, and raptor. under basal condition, the mtorc1 complex associates with the ulk1/2-atg13-fip200 complex and phosphorylates ulk1/2 and atg13, resulting in the inhibition of ulk1/2 kinase activity. under stressful conditions (e.g., nutrient deprivation), ampk activates ulk1/2 (in complex with atg13 and fip200) directly by site - specific phosphorylation and indirectly by inhibiting mtorc1. dephosphorylation of mtor - dependent inhibitory sites on the ulk1/2-atg13-fip200 complex releases ulk1/2 activity allowing autophosphorylation of this complex and its interaction to the atg101 protein in an atg13-dependent manner. these events lead to the subsequent recruitment and activation of the beclin1-atg14-vps34-vps15 complex at the membrane, inducing nascent phagophore formation. this vesicle nucleation step relies on dynamic assembly of both autophagy initiation complexes (ulk1/2-atg13-atg101-fip200 and beclin1-atg14-vps34-vps15) on the exocyst, a scaffolding protein complex involving the ras - like small g - protein ralb and its effector exo84, which acts as an activation platform for core autophagy machinery. the beclin1-associated phosphatidylinositol 3-kinase class iii, vps34, marks the site where the phagophore emerges from the er, by generating a pi3p - enriched structure, called omegasome. this event leads to the recruitment of pi3p - binding proteins such as dfcp1 (double fyve - containing protein 1), alfy, and wipi (wd - repeat domain phosphoinositide interacting) family proteins. members of the wipi family, wipi1, wipi2, and wipi4, recognize pi3p accumulation at the nascent autophagosome and are necessary for the recruitment of the autophagosome elongation complex atg12atg5atg16l1 complex. elongation of the phagophore membrane involves two ubiquitin - like proteins : atg12 and atg8. similarly to ubiquitination, atg12 is conjugated to atg5 (substrate) by atg7 (e1-like enzyme) protein and atg10 (e2-like enzyme). then the second ubiquitin - like reaction involves atg7 (e1-like enzyme) and atg3 (e2-like enzyme) enzymes and is required for conjugation of ubiquitin - like molecules of the atg8 family (lc3, gabarap, and gate-16) to the lipid phosphatidylethanolamine. whereas atg5-atg12-atg16l1 complex will dissociate from closed autophagosomes, lc3-ii, the lipidated form of lc3, remains associated with the autophagosome until fusion with the lysosome. it has been observed that atg5-atg12-atg16l1-positive / lc3-negative pre - autophagosomal structures coalesce through snare - mediated homotypic fusions, thereby increasing the size of the membrane constituting the phagophore, a prerequisite for optimal acquisition of lc3 and progression from autophagosome precursor to phagophore. although its function is not still fully understood, the transmembrane protein atg9 is also proposed to orchestrate membrane delivery to the phagophore assembly site. however, the lack of mutant cells defective for this step does not allow for precise molecular insights. once edges of the phagophore are sealed, sequestering substrates to be degraded, the autophagosome migrates along microtubules to a perinuclear location and interacts with endosomes and lysosomes. fusion of these organelles is orchestrated by snare proteins (vamp3 and vamp7) and rab gtpases (at least rab7 and rab11) [2022 ]. the beclin1 protein, which is essential for autophagy initiation, also plays a role in autophagosomal maturation by indirectly modulating rab7 protein activity. the last stage of autophagy is the efflux into the cytosol of metabolites (amino acids, sugars, and lipids) that are generated by autolysosomal degradation. recycling of amino acids from autophagosomes involves three vacuolar amino acid permeases atg22, avt3, and avt4. interestingly, mtor, which is the master suppressor of autophagy, is reactivated upon autophagy termination by amino acids release and stimulates extrusion of lysosome - derived membranes from autolysosomes to form new functional lysosomes. at the cellular level, presence of pathogens is detected by pattern recognition receptors (prrs) located at the plasma membrane (toll - like receptors (tlrs) 1, 2, 4, 5, and 6), at endosomal membranes (tlr3, tlr7, tlr8, and tlr9) or in the cytosol (nod - like receptors (nlrs), retinoic acid - inducible gene - i- (rig - i-) like receptors (rlrs), c - type lectin like receptors (clrs)) [2528 ]. these innate immune receptors recognize highly conserved structural motifs present on microbes termed pathogen - associated molecular patterns (pamps), such as the bacterial cell wall components lipopolysaccharide (lps), flagellin, and lipoproteins ; bacterial and viral nucleic acids ; and the fungal cell wall components zymosan and mannan. they also detect danger - associated molecular patterns (damps) that signalize host cellular damage. connections exist between autophagy and innate immune receptors, where prrs serve as sensor for microbial presence and autophagy ensures their intracellular elimination through lysosomal degradation. it is important to notice that prrs repertoire varies substantially from one cell type to another ; thus by contrast to starvation - induced autophagy, prrs - mediated autophagy will be more cell type dependent. for example, in the colon, tlr5 is only exposed on the basolateral side of enterocytes and is absent from apical side. screening of pamps library for their effects on autophagy showed that prototype ligands of various tlrs, including tlr1, tlr3, tlr4, tlr5, tlr6, and tlr7, induce autophagy in mouse and human macrophages [30, 31 ]. tlr9-dependent induction of autophagy has also been reported in human colonic epithelial cells stimulated by bacterial cpg motifs. invading pathogens, such as listeria monocytogenes (l. monocytogenes) and staphylococcus aureus, have also been shown to activate autophagy in macrophages in a tlr2-dependent manner and thereby triggered their own elimination [33, 34 ]. interestingly, activation of autophagy through tlr stimulation by agonists enables also the elimination of noncognate intracellular pathogens. connections between tlr and autophagy are complex to study since various downstream signaling effectors are engaged, according to the innate immune receptor activated. data discrepancies exist regarding the adaptor (myd88 versus trif) involved in autophagy induction by tlr4 stimulation in macrophages [31, 35 ]. delgado. showed that activation of autophagy via tlr7 upon macrophages stimulation with ssrna is dependent on myd88. mechanistically, connection between tlr - signaling and autophagy is supposed to be mediated by the adaptor proteins trif or myd88 that are found to coimmunoprecipitate with beclin1 and reduce the binding of beclin1 to the inhibitory protein bcl2, leading to autophagy activation. physical association of myd88 with mtor has also been reported allowing activation of master transcription factors (interferon - regulatory factor- (irf-) 5 and irf-7) for proinflammatory cytokine- and type i ifn - genes. in addition, mycobacterial infection of human macrophages and zebrafish embryos induced dram1 (dna damage - regulated autophagy modulator 1) mediated selective autophagy in a myd88 and nf-b - dependent manner. nod receptors stimulation by bacterial peptidoglycan components induces autophagy and results in enhanced bacterial killing and antigen presentation [38, 39 ]. the molecular interaction between the cytoplasmic nod1 and nod2 receptors and autophagy they showed that atg16l1 interacts with nod receptor that enables recruitment of autophagy machinery at the bacterial entry site and the subsequent delivery of bacteria into autophagosomes for degradation. nodophagy entails other autophagy - related proteins such as atg5, atg7, and receptor - interacting serine - threonine kinase-2 (ripk-2). autophagy regulation by nod proteins is evolutionarily conserved, since the drosophila homologue pgrp - le, recognizing also peptidoglycan fragments, is able to induce autophagy upon l. monocytogenes challenge. modulation of autophagy by other nlr family members, involved in inflammasomes assembly, has been reported [4143 ]. activation of aim2- or nlrp3-mediated inflammasomes triggers the activation of the small g protein ralb and autophagosome formation. through binding to exo84 in addition, nlrp4 physically interacts with the c - vps complex (vps11, vps16, vps18, and rab7) that controls membrane tethering and fusion of vacuolar membranes, thereby blocking maturation of autophagosomes to autolysosomes. to summarize, prrs, by sensing various microbial products from bacteria, virus and fungi are able to modulate autophagy at multiple steps (initiation and maturation). by binding to their specific receptors located at the cytoplasmic membrane, cytokines modulate autophagy. in a general way, th1 cytokines (il-2, tnf-, and ifn-) are considered as autophagy inducers whereas th2 cytokines (il-4, il-5, il-6, il-10, and il-13) and anti - inflammatory cytokines are regarded as autophagy repressors. cytokines that have been reported to induce autophagy encompass il-1, il-2, il-6, tnf-, tgf-, and ifn-. autophagy induction by cytokines may constitute an important mechanism in the elimination of invasive pathogens. ifn- illustrates this mechanism since it promotes degradation of intracellular mycobacterium tuberculosis and chlamydia trachomatis by inducing autophagy [46, 47 ]. induction of autophagy by ifn- involves immunity - related gtpase such as murine irgm1, human irgm, irga6, and members of the 65-kda guanylate binding protein family [4649 ]. of note, unlike the irgm1 gene in mice, the human irgm1 ortholog, irgm, is not responsive to ifn-, but interestingly it remains able to induce autophagy upon infection in epithelial cells and macrophages [4951 ]. a single nucleotide polymorphism (snp) in irgm (261 t) is associated with resistance to m. tuberculosis infection in people carrying this protective variant. in irgm1/ macrophages, an alternative activation pathway of autophagy by ifn-,, ifn- induces autophagy through irf-1 signaling pathway and this autophagy activation is associated with cell death. induction of autophagy by type i ifns has also been reported in several cancer cell lines and involved jak / stat and pi3k - mtor pathways. type i ifn proteins include the ifn- subtypes, ifn- and ifn-, and play pleiotropic functions, such as antiviral, antiproliferative, and immunomodulatory activities. the proinflammatory cytokine and death ligand, tnf-, represents also an autophagy inducer in various cell types, including t - lymphoblastic leukaemic cells, osteoclasts, or ewing sarcoma cells [5658 ]. tnf- regulation of autophagy has been shown to be dependent on various signaling pathways including jnk and erk signaling and via the production of reactive oxygen species (ros) [5962 ]. the role of tnf- in autophagy is also supported by studies on m. tuberculosis, since reactivation of tuberculosis associated with anti - tnf- treatments (infliximab, adalimumab, certolizumab pegol, and etanercept) is suspected to, at least partially, depend on suppression of the antibacterial autophagic process [63, 64 ]. il-6 enhanced autophagic activity in myeloid cells and mouse pancreatic tumor cells [65, 66 ]. in contrast, il-6 overexpression blocks autophagy in human bronchial epithelial cells by supporting interaction between beclin1 and the antiapoptotic bcl2 protein mcl-1. other cytokines that have been described to induce autophagy include il-1, which is suspected to be an endogenous inhibitor of the mtor pathway and tgf-, which stimulates autophagy through various signaling pathways such as jnk, smad, and tak1 pathways [69, 70 ]. conversely, some th2 and anti - inflammatory cytokines exert autophagy - suppressive functions. regarding il-4 and il-13 cytokines, they inhibit starvation - induced autophagy through stimulation of pi3k / akt signaling pathway and are able to counteract ifn--induced autophagy in a signal transducer and activator of transcription 6 (stat6) dependent manner. the anti - inflammatory cytokine il-10 has also been described to inhibit starvation - induced autophagy by stimulating the pi3k / akt signaling pathway. several lines of evidence indicate that reactive oxygen species (ros) are early autophagy inducers upon nutrient deprivation. this is supported by data showing that treatment with antioxidants partially or completely reverses the process. under nutrient deprivation, it has been reported as the expulsion of reduced glutathiones (gsh), which are powerful antioxidants, by the plasma membrane translocator abcc1mrp1 (multidrug resistance protein 1) in the extracellular milieu. this results in a shift of the intracellular redox state toward oxidizing conditions and may favor oxidation of redox - sensitive proteins, inducing consequently the early autophagy. it is postulated that superoxides and hydrogen peroxide, the main ros produced by mitochondria upon nutrient deprivation, could activate autophagy by at least two ways. one molecular mechanism that has been described is the activation of the ampk (5-adenosine monophosphate - activated protein kinase), an energy sensor of the cell, through s - glutathionylation of its cysteines. s - glutathionylation is a posttranslational modification of cysteines frequently induced in cells as a response to ros production. ampk stimulates autophagy by phosphorylating tsc2 (tuberous sclerosis complex 2), an inhibitor of mtorc1, raptor, a component of the mtorc1 complex, and by phospho - activating ulk1 [11, 75, 76 ]. another mechanism by which ros induce autophagy is by oxidizing a cysteine residue near the catalytic site of the cysteine protease atg4, thereby stimulating its proteolytic activity and enhancing autophagy [77, 78 ]. until recently, transcription control of autophagy was clearly underappreciated, but compelling evidences during last years demonstrate that a network of transcription factors is involved in fine - tuning of autophagy. some transcription factors (tfs), which orchestrate inflammatory responses, have been also described as transcriptional regulators of the core autophagy genes. a prototypical example of these tfs is the nuclear factor-b (nf-b) p65/rela family member, which has been shown to upregulate becn1 and sqstm1 transcription [79, 80 ]. other inflammation - related tfs that are able to modulate autophagy genes transcription include hypoxia inducible factor-1 (hif-1), jun, signal transducers, and activators of transcription (stat) 1 and stat3. hif-1 stimulates autophagy by enhancing transcription of bnip3 (bcl2/adenovirus e1b 19 kda protein - interacting protein 3) and bnip3l (bnip3-like) encoding genes. these two proteins, which belong to the bh3-only bcl2 family proteins, are known to activate autophagy. c - jun is recruited to the beclin1 and atg8 genes promoter regions in response to ceramide treatment and thereby enhances transcription of these genes [83, 84 ]. a role as negative autophagy regulators has been described for stat1 and stat3, since an impaired expression of those tfs correlates with an increased autophagy activity and stimulates transcription of some atg genes including atg12 and beclin1 [85, 86 ]. tfs belonging to the nuclear receptors family, such as peroxisome proliferator - activated receptor (ppar), ppar, and farnesoid x - activated receptor (fxr), which function as ligand - activated tfs and modulate inflammatory response, can also modulate autophagy [87, 88 ]. ppar and fxr have opposite effect on autophagic genes transcription, since pharmacological activation of ppar leads to an upregulation of the expression of autophagy genes, whereas fxr activation leads to a repression. copetti and colleagues demonstrate that these two nuclear receptors compete for binding to the same site on autophagy genes promoters. inflammatory state has also been described to modulate the activity of transcription factor eb (tfeb), a master regulator that orchestrates the expression of autophagy and lysosomal genes. hence, tfs represent an additional complex layer of regulation ; thereby inflammation deeply affects autophagy - associated transcriptional program. inflammasomes are intracellular signaling platforms that detect a set of substances emerging during infections, tissue damage, or metabolic imbalances and that proteolytically activate the highly proinflammatory cytokines il-1 and il-18. these multimeric protein complexes usually consist of three partners : an inflammasome sensor protein, which can be a pamp- or damp - detecting module in the form of a nlr, such as nlrp3 and nlrc4, or an endogenous dna (released from mitochondria) detecting module such as aim2 (absent in melanoma 2), the adaptor protein asc and caspase-1 that enzymatically processes pro - il-1 and pro - il-18 for their activation. activation of inflammasomes also causes pyroptosis, which corresponds to a rapid and proinflammatory form of cell death. mice those are deficient in atg16l1, which is essential for autophagy, present higher il-1 and il-18 levels in sera in response to lps stimulation or during colitis. these results have been confirmed by pharmacological (3-methyladenine treatment) or genetical (lc3b and beclin1) inhibitions of autophagy that all result in higher il1- production upon pamps stimulation [43, 92 ]. at the opposite, tlr- or however, the molecular mechanisms by which autophagy regulates inflammasome activation are not yet fully understood. autophagy could act directly by sequestering and degrading ubiquitinated - inflammasomes and pro - il-1 molecules or indirectly by lowering endogenous sources inducing inflammasome formation, such as mitochondrial ros and dna [43, 9294 ]. in addition to its role in clearance of polyubiquitinated proteins and bacteria by autophagy [95, 96 ] and in the regulation of the nutrient - sensing pathway, p62/sqtm1 is also involved in the regulation of inflammatory mediators production. the p62/sqtm1 protein can induce the upregulation of oxidative stress response genes, particularly those controlled by nrf2, an antioxidant transcription factor. under basal condition, nfr2 is sequestered in the cytoplasm by keap1. upon oxidative stress, p62/sqstm1 interacts and competes with the nrf2-binding site on keap1, leading to the stabilization and the nuclear translocation of nrf2 and transcription of target genes, including key ros scavengers encoding genes. thus, the nrf2 activation is reinforced by degradation of p62/sqstm1-keap1 complex by autophagy and through the activation of p62/sqstm1 transcription by nrf2. besides the conventional secretion process by which proteins endowed with a leader peptide undergo modification in the er, transit through the golgi apparatus, and are secreted upon fusion of post - golgi carriers with the plasma membrane, an unconventional secretion process based on autophagy has been described. this secretion process first described in yeast is involved in the secretion of proteins devoid of leader peptide. the principal features defining secretory autophagy are the involvement of atg proteins and autophagy process, and dependence on golgi reassembly and stacking protein, grasp55 and grasp65. the secretory autophagy is a rising investigation area and a number of points remain to be elucidated to understand how autophagy orients peptide to secretion or simple degradation. it has been reported in mammalian cells that secretory autophagy contributes to the secretion of proinflammatory cytokines (il-1 and il-18) and alarmins (high mobility group box (hmgb)-1), under transient and specific circumstances. mononuclear phagocytes and among them macrophages play a central role in the orchestration and expression of innate and adaptive immune responses. one of the hallmarks of these cells is their high diversity and plasticity. in tissues, in response to a large variety of environmental cues (e.g., growth factors, cytokines, microbial products, or glucocorticoids), mononuclear phagocytes undergo transcriptional reprograming that shape their phenotype and functions, m1 (classical) and m2 (alternative) phenotypes, being the extremities of a continuum of activation states. then, depending on the macrophage microenvironment, m1 macrophage polarization is mediated by stat1 and irf-5, whereas m2 polarization is supported by stat6, irf-4, and ppar. multiple other signaling molecules, transcription factors, epigenetic mechanisms, and posttranscriptional regulators involved in fine - tuning of macrophage polarization have been characterized. of note schematically, m1 macrophages stimulate a proinflammatory response against intracellular microorganisms and tumors cells, whereas m2 macrophages are immunosuppressive cells and participate in cancer progression by supporting key processes such as angiogenesis, tissue repair, and remodeling. in addition to playing role in macrophages migration and differentiation, which will not be discussed in this review, evidences showed that autophagy is involved in the control of macrophage polarization. binding of hepatoma - derived factors to tlr2 stimulate m2 macrophage polarization, a phenotype expressed by most of tumor - associated macrophages (tam) from established tumors, by regulating cytoplasmic nf-b level through selective autophagy [106, 107 ]. pharmacological treatment with bafilomycin a1, a lysosomal inhibitor, or genetic defects (knockdown of atg5) that impaired autophagy prevents degradation of nf-b (p65) and forces the m2 macrophages to secrete high levels of inflammatory cytokines which characterized m1 phenotype. in addition, the mtor pathway has been described as a critical element of the monocyte differentiation to tam. inhibition of mtor through rapamycin treatment caused the monocytes to differentiate toward a m1 phenotype, whereas activation of mtor by rna interference - mediated knockdown of the mtor repressor tsc2 induced the differentiation of monocytes toward a m2 macrophage phenotype. a defect in cats, a cathepsin whose expression is upregulated in several tumors, is associated with accumulation of autophagosomes and attenuation of the autophagic flux in macrophages. it has been recently demonstrated that cat s - mediated autophagic flux is required to maintain polarization of the m2 tams phenotype. given the multiple roles played by autophagy in immune homeostasis, malfunctions of this process have been associated with the pathogenesis of a variety of diseases. hence, autophagy may impact on the onset or progression of various human diseases associated with a chronic inflammatory state, including inflammatory bowel diseases (ibds), paget 's disease, infectious diseases (tuberculosis), lysosomal storage disorders, and autoimmune disorders (systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, and type 1 diabetes). among them, ibds are the most studied inflammatory diseases for their link with autophagy alteration and especially crohn 's disease (cd). cd and ulcerative colitis (uc) represent the two major forms of idiopathic ibd. it is now widely accepted that the etiology of ibd involves environmental and genetic factors that lead to dysfunction of the epithelial barrier with consequent deregulation of the mucosal immune system and responses to gut microbiota. human genetic studies over the last decade and experimental studies have revealed the keystone role played by autophagy in the disorders associated with cd. the initial identification of the link between autophagy and cd arose from genome - wide association studies (gwas), which revealed the association between a coding single nucleotide polymorphism (snp) in the atg16l1 gene of the autophagy core machinery and an increase risk of cd onset. two others genes associated with autophagy have been confirmed as cd susceptibility genes : irgm (immunity - related gtpase family m) and lrrk2 (leucine - rich repeat kinase 2) [113, 114 ]. more recently, whole exome sequencing of cd patients has identified a cd - associated missense variant (v248a) in the autophagy receptor ndp52. cd - associated polymorphisms in atg16l1 and irgm encoding genes have been reliably found in many patient cohorts as reported by a meta - analysis [116, 117 ]. of note, irgm polymorphisms are also associated with an increased risk in developing uc, what is not true for other autophagy - related genes associated with cd - susceptibility. regarding irgm, a synonymous variant within the coding region (rs10065172, ctg > ttg, leucine) was initially linked to cd, and this silent polymorphism was found to be in perfect linkage disequilibrium with a 20 kb deletion upstream of irgm. results showed that the 20 kb deletion does not impair, in a reliable manner, expression of the irgm mrna, but that silent polymorphism alters the recognition of the irgm mrna by a family of microrna (mir), mir-196a and mir-196b [51, 118 ]. in a specific study to identify rare snps in autophagy - related genes, a cd - associated snp in the ulk1 gene locus has been identified, but the functional relevance of this genetic variation in the disease is still unknown. association between granulomas, one of the microscopic hallmarks of cd, and variants in autophagy atg4a, atg2a, fnbp1l, and atg4d genes has also been reported. in addition, three mutations in nod2, including a frameshift mutation (l1007fsinsc) that results in a truncated nod2 protein and two amino acid substitutions (r702w and g908r), have been reported to be strongly associated with cd onset [121, 122 ]. mutations in nod2 encoding gene impaired ability of this innate immune receptor to interact with atg16l1 and recruit the autophagy machinery for degradation of invasive bacteria. atg16l1, independently of its role in autophagy, acts as a negative regulator of the nod1 and nod2-mediated proinflammatory signaling pathways, by negatively regulating the activation of the rip2 kinase. these studies strengthen the connection existing between nod2, the first gene historically associated with cd etiology, and autophagy - related - genes associated recently with cd by gwas, highlighting the importance of autophagy in cd onset. er stress is caused by the accumulation of unfolded or misfolded proteins in the er. cells secreting large amounts of protein, such as paneth cells and goblet cells in the intestine, are highly susceptible to er stress for survival and to executing their secretory functions. the unfolded protein response (upr) enables the cell to resolve the er stress by facilitating the folding, export, and degradation of proteins accumulating in the er during stressed conditions. three er membrane resident proteins, ire1 (inositol requiring transmembrane kinase endonuclease 1), perk (pancreatic er kinase), and atf6 (activated transcription factor 6) sense the presence of unfolded protein in the er lumen and induce a transcriptional program necessary for the upr. in the absence of misfolded proteins in the er lumen, ire1, perk, and atf6 are maintained as inactive complexes through association with grp78. complex connections exist between er stress and inflammation. in mice, genetic deletion of molecules involved in the upr, such as ire1, the transactivator of upr 's xbp1 (x - box binding protein 1) and agr2 (anterior gradient 2) target genes, which is a member of the er protein disulfide isomerase (pdi) gene family are associated with either spontaneous intestinal inflammation and/or increased sensitivity to the experimental induction of colitis [125127 ]. in human, genetic studies have identified the upr - related genes xbp1 and ormdl3 (orosomucoid - like 3), which is involved in er calcium homeostasis, as risk loci associated with crohn 's disease. evidences for increased er stress response in patients with cd include an increased expression of grp78 and an increased xbp1 splicing in the small intestine and colon of cd patients comparatively to controls [125, 128, 129 ]. intestinal epithelial cell - specific deletion of xpb1 (xbp1) and most notably in paneth cells leads to autophagy induction linked to er stress related perk - eif2 signaling pathway. interestingly, spontaneous ileitis observed in xbp1 mice is converted into severe cd - like transmural ileitis when both mechanisms, er stress and autophagy, are compromised (atg7/xbp1 and atg16l1/xbp1 double transgenic mice). thus, autophagy may function as a buffer mechanism in intestinal epithelial cells protecting cells from inflammation generated upon sustained er stress. defective autophagy promotes er stress in hepatocytes and increased xbp1 splicing and grp78 expression have been observed in atg16l1 mice crypt compartment compared to wild - type. in addition, patients with cd carrying the atg16l1 risk variant frequently exhibit er stress in their paneth cells, in contrast to those harboring the normal variant. invasive bacteria that escape into the cytosol such as shigella flexneri or listeria monocytogenes or those that reside in intracellular vacuoles such as salmonella typhimurium or mycobacterium tuberculosis can be entrapped into autophagosomes and delivered to lysosomes for degradation. key molecules that target bacteria for xenophagic degradation had been identified and include nlrs (nod1 and nod2), ubiquitin, galectins, and diacylglycerol (dag). recent evidences show that cell infection with pathogens, such as s. flexneri or s. typhimurium, triggers an amino acid starvation response, an inhibition of the mtor signaling, and thereby induces autophagy. this suggests that xenophagy response against invasive pathogens might derive from primordial metabolic stress response, reinforcing the link existing between cell metabolism and immune cellular defense. dysbiosis of the fecal-, lumenal-, and mucosal - associated microbiota have been described in cd patients and substantial evidence supports an abnormal colonization of the mucosa and lesions of patients with ileal form of cd by escherichia coli belonging to the pathovar designated adherent - invasive e. coli (aiec) [135, 136 ]. one putative consequence of impaired autophagy is an uncontrolled pathobionts overgrowth due to defective intracellular bacterial clearance. pathobiont term refers to microorganisms that are usually recognized as harmless but that can become pathogens under certain environmental conditions (e.g., host detrimental diet or host genetic susceptibility). such a hypothesis is supported by data demonstrating that inflamed tissues of the terminal ileum of cd patients with the risk allele of atg16l1 harbor a higher abundance of three pathosymbiont groups, enterobacteriaceae (mostly e. coli), bacteroidaceae (mostly bacteroides fragilis group), and fusobacteriaceae, in comparison with those with the protective allele. it has been shown that autophagy protects against dissemination of pathobionts and true invasive intestinal pathogens, since intestinal epithelial cell - specific deletion of atg5 exhibits increased dissemination of these bacteria to extraintestinal sites. silencing expression of atg16l1 by sirna in human macrophages and epithelial cells and expression of the risk variant of atg16l1 by epithelial cells impair aiec handling by autophagy and favor their persistence within host cells [139, 140 ]. monocytes from cd patients homozygous for the atg16l1 risk allele showed impaired killing of aiec under inflammatory conditions compared with those homozygous for the atg16l1 protective allele. similarly impaired xenophagy against aiec bacteria has been described in dendritic cells isolated from donors with cd - associated nod2 variants and in peritoneal macrophages isolated from nod2 knockout mice [38, 139 ]. similarly to atg16l1 and nod2, decreased expression level of irgm totally impairs autophagy initiation and leads to uncontrolled replication of aiec bacteria. an unexpected result was that overexpression of irgm, as observed in the intestinal mucosa of cd patients (compared to controls), also altered the autophagosome maturation step, supporting intracellular aiec persistence. risk polymorphisms associated to cd in the autophagy - related nod2, atg16l1, and irgm genes are associated with aberrant inflammatory responses. defects in autophagy caused by alteration of the expression of autophagy - related genes lead to an exacerbated inflammatory response in human thp-1 macrophages. surprisingly, in a mouse model of atg16l1 deficiency, this abnormal higher inflammatory response has been shown to confer protection against uropathogenic e. coli (upec) infection, by allowing an accelerated clearance of these bacteria in an il-1-dependent manner [141, 142 ]. at the opposite, forced induction of autophagy decreased proinflammatory cytokine release. recently, it has been shown that aiec infection upregulated levels of micrornas like mir-30c and mir-130a in intestinal epithelial cells by activating nf-b. upregulation of these micrornas inhibited autophagy by reducing the levels of atg5 and atg16l1 and led to increased numbers of intracellular aiec and exacerbated inflammatory response. in vitro, blocking of mir-30c and mir-130a in aiec - infected cells restored functional autophagy. interestingly, ileal samples from patients with cd have increased levels of these same micrornas and reduced levels of atg5 and atg16l1. as illustrated by studies detailed above in this review, autophagy and autophagy - related proteins are essential components modulating inflammatory response either directly by acting on stability or secretion of inflammatory mediators or indirectly by suppressing intracellular stressors (e.g., damaged organelles, intracellular pathogenic microorganisms, and er stress). autophagy regulatory network integrates a wide range of signals from innate immune receptors sensing pamps (tlrs and nlrs), cytokines, and ros and thereby is able to respond appropriately according to the degree of inflammatory state (figure 1). autophagy has a significant impact on health, since it has been shown that specific induction of autophagy can increase lifespan in multiple animal species. we could assume that the ability of autophagy to restrain detrimental side effects of inflammation might contribute to its positive role on health. an interesting parallel, supporting this hypothesis, is the fact that, during aging, there is a decrease in autophagy efficiency, concomitantly to an increase in the basal inflammation level. novel therapies designed to enhance autophagy might represent an attractive strategy to overcome autophagy insufficiencies associated with some human inflammatory diseases, such as cd. beyond the well - known autophagy inducer, rapamycin, already used in clinic as an immunosuppressive and antitumor drug, high - throughput screening studies have identified additional molecules, already fda approved, that are able to modulate the autophagic process [148, 149 ]. as a proof of concept, in vivo delivery in mice of a specific autophagy inducer peptide, tat - beclin1, has demonstrated positive results in the treatment of neurodegenerative disease and viral infections. however, considering the multiple roles of autophagy, further investigations are needed to examine in which situations stimulated autophagy is beneficial and does not generate detrimental side effects. | autophagy is an intracellular catabolic pathway essential for the recycling of proteins and larger substrates such as aggregates, apoptotic corpses, or long - lived and superfluous organelles whose accumulation could be toxic for cells. because of its unique feature to engulf part of cytoplasm in double - membrane cup - shaped structures, which further fuses with lysosomes, autophagy is also involved in the elimination of host cell invaders and takes an active part of the innate and adaptive immune response. its pivotal role in maintenance of the inflammatory balance makes dysfunctions of the autophagy process having important pathological consequences. indeed, defects in autophagy are associated with a wide range of human diseases including metabolic disorders (diabetes and obesity), inflammatory bowel disease (ibd), and cancer. in this review, we will focus on interrelations that exist between inflammation and autophagy. we will discuss in particular how mediators of inflammation can regulate autophagy activity and, conversely, how autophagy shapes the inflammatory response. impact of genetic polymorphisms in autophagy - related gene on inflammatory bowel disease will be also discussed. |
polycystic ovary syndrome (pcos) is a multifactorial, complex genetic, endocrine and metabolic disorder. characteristics are chronic anovulation, polycystic ovaries and hyperandrogenism (1). according to different criteria, 4% to 8% of women exhibit pcos in the world (2). the prevalence of pcos in iran was 14.6% using rott s definition (3). pcos has a tremendous negative impact on the physiology and metabolism of the body such as metabolic syndrome, insulin resistance (ir), dyslipidemia, hypertension, hyperinsulinemia, abdominal obesity, type 2 diabetes mellitus (4), endometrial hyperplasia, cardiovascular disease (5), eating and mood disorders (6). it is beneficial for health and has protective effects on cardiovascular disease (8) and metabolic syndrome (9). in recent years, intake of omega-3 in diets is less than omega-6, and this imbalance in omega-6 to omega-3 ratio can be related to chronic diseases (10). symptoms of pcos differ among women with pcos and vary over time in each woman in the presence of particular precipitating factors, the most significant of which is an alteration in body weight (11, 12). there is evidence that obese pcos women have more severe hyperandrogenism and a significantly higher incidence of anovulatory cycles, oligomenorrhea and/or hirsutism compared to normal weight ones (12) and weight has an effect on the levels of androgens (13), adiponectin, and visfatin (14). demonstrated the highest dose of omega-3 causes the more reduction in fat mass (16). visfatin is an adipokine that mimics the effect of insulin and is secreted by visceral fat tissue (18) and it is related to insulin resistance (19). visfatin is related to body mass index (bmi) (20) and waist to hip ratio (whr) (21). some studies showed that visfatin increased in subjects with abdominal obesity and pcos (19, 22, 23). visfatin similarity to insulin may stimulate the androgen synthesis and be the pathogenesis of pcos (24). the regulatory effect of omega-3 on the level of some adipokines like leptin was evaluated (25), but its effects on visfatin levels was limited (26). adiponectin, as another adipokine, improves insulin sensitivity and has an anti - inflammatory effect (27). some studies demonstrated that the low level of adiponectin can be related to pathogenesis of pcos and its metabolic complications (29). showed the effect of omega-3 on elevating the level of adiponectin in pcos women (30). release of lh in pcos women is more irregular than other women (31). elevation in serum lh and lh : fsh ratio in pcos women is common (32, 33). also, prolactin level is higher in these women despite fsh level (34). others showed no effect on lh and fsh after supplementation by polyunsaturated fatty acid (pufa) (36, 37). there were many studies about the effect of omega-3 on metabolic statues of many diseases (3840), but studies on pcos patients were limited (41). the objective of this study was evaluating the effect of omega-3 supplementation on these hormone profiles, some adipokines, and some anthropometric indices in pcos women. the study was a randomized double blind placebo - controlled clinical trial and was executed from november 2011 to may 2012. it was approved by shahid sadoughi university of medical sciences ethics committee (irb no 50338), yazd, iran. this study was a part of the large trial registered in iranian registry of clinical trial (registry no irct2012071410281n1). it was conducted on 84 women with polycystic ovary syndrome (26.92 5.05 years, bmi=31.69 kg / m), referred to the fertility and infertility research center and shahid sadoughi hospital in yazd. after the examination, evaluation and para - medical assessment by obstetrician, they were recruited. inclusion criteria were : pcos diagnostic criteria, age between 2040 years, and bmi of 2540. exclusion criteria included metabolic disease, thyroid disease, hyperprolactinemia, hypercortisolemia, congenital adrenal hyperplasia, cushing s syndrome, pituitary disorder, neoplasm, renal and liver diseases, history about intake of any drug during the last 3 months that may affect the insulin sensitivity or hormonal profile such as : oral contraceptives, glucocorticoids, ovulation induction agents, anti - diabetic and anti - obesity drugs, estrogenic, anti - androgenic or anti - hypertensive medication, being in any diet in the last 6 months, any addiction to tobacco and alcohol, taking omega-3 supplement in the last 3 months, menopause, allergy to sea foods, and the use of anti - coagulant medicine. and the participants were excluded if they needed ocp for treatment, were not willing to be in the study, did not obey the protocols of the study, or could not digest more than 20% of supplements. the rotterdam eshre / asrm criteria for confirming the diagnosis of pcos were : oligo - anovulation, clinical / or biochemical evidence of hyperandrogenism, and polycystic ovaries on ultrasound examination (11). thorough clinical examination with special focus on clinical hyperandrogenism (i.e. hirsutism and or hypergonadism) was done. after explaining the objectives of the study to the patients the 24 hr recall was completed for each patient before and after the intervention to eliminate the effect of change in dietary intake. recall s data were analyzed using nutritionist 4 software (n - squared inc, san bruno, ca, usa). subjects were randomly divided in to two groups of a and b to reduce the bias. researchers did nt know which groups take the omega-3 supplement or placebo until the end of the study. both groups were given three capsules of omega-3 or placebos every day for 8 weeks (42). each capsule of omega-3 contained 180 mg epa and 120 mg dha, and each placebo included 1 g paraffin (zahravi, iran). all subjects were followed weekly by phone call, and they referred to the office bimonthly to take the supplements to reduce the drop out. all participants were asked to maintain their usual diet and lifestyle habits. for biochemical analysis, serum levels of visfatin, adiponectin, lh, fsh, and prolactin were measured. serum levels of visfatin (cusabio, china), adiponectin (medignost, germany), lh (diametra, germany), fsh (diametra, germany), and prolactin (diametra, germany) were measured by elisa reader (statfax2600, usa). weight, height, and bmi (weight in kg divided by height in m) were measured before the intervention for checking the inclusion criteria. weight (kg) was measured twice to the nearest 0.1 kg, with no shoes. height (m) statistical analysis included mean and standard deviation, percentage, ratio, chi - square, pearson correlation coefficient test, paired t - test, student t - test, anova, wilcoxon, and mann - whitney. spss 12 software was used (spss inc, chicago il) for analyzing the data and a p - value less than 0.05 was considered statistically significant. the remaining 78 participants completed the study. finally, 78 people (92.8%) among the patients, 39 patients in the group receiving omega-3 and 39 patients in the group receiving placebo, finished the eight week trial. mean of bmi was 31.465.74 kg / m in omega-3 and 31.883.86 kg / m in placebo group. there were not any significant differences between groups in terms of age, weight, and other characters before the study. there were no significant differences between and within groups in energy, macronutrients, omega-3, omega-6, and omega-6/mega-3 daily intake in the beginning of the study and after the intervention (table 1). mean and standard deviation of intake of omega-3, omega-6, omega-6/omega-3, energy, and macronutrients between both groups before and after the intervention paired sample t - test, comparison of quantitative variables between both groups before and after intervention paired sample t - test, independent sample t - test, data after the study- first data, the results of our study showed that omega-3 treatment was associated with significant improvement with lh, adiponectin concentration, whr, and lh / fsh ratio in pcos. according to this study, no changes were seen in energy and macronutrient intakes and bmi in both groups after the intervention. the study of mohammadi. showed no changes in whr after 8 weeks of supplementation (30). however, some studies showed a reduction in whr, bmi after 8 weeks of omega-3 supplementation in women with type 2 diabetes (26) and reduction of weight in mice (44, 45). obesity and its comorbidities (diabetes, cancer, and heart disease) are linked to inflammation. omega-3 had anti - inflammatory properties and has some effects on mediating receptors (46). our result about adiponectin level was compatible to previous studies in human (30, 47, 48) and animals (49, 50). the stimulation of adipoq (adiponectin gene) in adipose tissue as a ligand of ppar- (peroxisome proliferator - activated receptor) is one of the effects of epa and dha (44). evidence about the effect of omega-3 on visfatin level is limited. according to the study of hajianfar., visfatin concentration increased after 8 weeks of the omega-3 supplementation (2 capsule / day, each capsule contain epa 360 mg, dha 240 mg) (26). in an in vitro study, it was suggested that ampk activation has a role in upregulation of visfatin gene after omega-3 intervention (51). the duration of the study and dose of epa / dha may not be enough to detect visfatin changes. the improvement of lh concentration and lh / fsh ratio can cause the improvement of reproductive system. this trend can be explained by the mechanism related to arachidonic acids (52). high arachidonic acid can make 2-series prostaglandins (pgs), thromboxanes (txs), and 4-series leukoterienes (lts) and they induce star. it is the first and main step for steriodogenic pathway which results in production of androstenedione and testosterone (52). our data showed that omega-3 supplementation in 8 weeks has no effect on fsh concentration in pcos women. they examined 3 months of n-3 pufa application on hormone status in 12 pcos samples, but they did not see any changes on fsh concentration (53). karakas.s study showed similar results (36). in our study, lh concentration and lh / fsh ratio significantly decreased after the intervention. some studies showed no changes in lh concentration (52) and lh / fsh ratio after the intervention (37) like karakas.s study in using rich - pufa diet for 3 months (36). the limitation of our study was a shortness of intervention period and the clinical impacts were not collected. it is suggested that more clinical trials be conducted in this field with longer duration of intervention and in women with similar pattern of ovulations. it has been suggested to measure the concentration of fatty acid on rbc membrane to check the protocol adherence and effect of omega-3 on other adipokines. omega-3 supplementation in pcos women can show a benefit effect on adiponectin, lh concentration, whr, and lh / fsh ratio. this effect was not associated with bmi, visfatin, fsh, and prolactin levels. | background : polycystic ovary syndrome (pcos) is a multifactorial, metabolic disorder. characteristics are chronic anovulation, polycystic ovaries and hyperandrogenism. the aim of this study was to determine the effect of omega-3 supplementation on visfatin, adiponectin, and anthropometric indices in pcos women.methods:the study was a randomized double blind placebo - controlled clinical trial. it was conducted on 84 women with polycystic ovary syndrome (26.925.05 years, bmi=31.69 kg / m2) who referred to the fertility and infertility research center and shahid sadoughi hospital in yazd. after the examination, evaluation and para - medical assessment by obstetrician, they were recruited. they took 3 capsules of omega-3 (each one contained 180 mg epa and 120 mg dha) or placebo (each contained 1 g paraffin) daily for 8 weeks. statistical analysis was paired t - test and student t - test, and a p<0.05 was considered statistically significant.results:after the intervention, visfatin concentration did not change in neither groups. but, at the end of the study, the mean of adiponectin concentration increased (p<0.001) in omega-3 group. moreover, the mean of changes in this factor was significantly different between groups (p<0.005). fsh did not change in two groups of the study. however, the mean of lh decreased about 1.74 mlu / ml in omega-3 group (p<0.005). the mean of change of lh / fsh ratio between groups was significant (p<0.05). after the intervention, prolactin did not meaningfully change in both groups.conclusion:our results showed that 8 weeks of supplementation of omega-3 may have some beneficial effects on pcos biochemical characteristics such as lh, lh / fsh, and adiponectin. |
during the past year, the health care financing administration (hcfa) has conducted a number of meetings to discuss quality of care measurement, data development and release, and research priorities. in june 1987, hcfa convened a quality of care research symposium for the purpose of detailed discussion of the current status of quality of care research and identification of research needs. eighteen experts in various aspects of quality of care measurement and analysis gave presentations, followed by four smaller work groups in which speakers and attendees contributed to the discussions. the purpose of this article is to provide an overview of some of the issues addressed at the symposium, the general themes of the discussions, and the research priorities identified. since the mid-1970 's there has been a plethora of literature regarding many aspects of quality assurance and utilization review. despite the marked surge in quality assessment literature, the caliber has been varied and the status of the conceptualization and measurement instrumentation of health care quality has not progressed much beyond that of the early 1970 's. through his lectures and his numerous writings over the past 20 years, avedis donabedian has developed an integrating conceptual framework, which has come to be utilized by most researchers in the field. the framework is often verbally reduced to the major components : structure, process, and outcome. donabedian has noted that these are not attributes of quality, but are approaches to the acquisition of information about the presence or absence of the attributes that constitute or define quality. donabedian proposed an integration of the dimensions of quality and their analysis through an emphasis on : the need to adequately conceptualize the components of health (physical - physiological function, psychological function, and social function). the levels of aggregation and organization of the providers of care (such as individual physician, team, department, hospital, etc.). the levels of aggregation of the actual or potential recipients of care (such as individual or groups of patients and entire populations or subpopulations). by emphasizing the various possible levels of aggregation of patients, populations, and providers, donabedian has begun to enumerate the many aspects of medical care quality assessment (donabedian, 1980). the speakers at the symposium reaffirmed the multifaceted nature of quality and reiterated donabedian 's point that there is no single definition of quality for medical care and, therefore, there is no single measure of it. no one composite index of quality of health care has been, or probably ever can be, developed. the symposium discussions made clear that there is a need for several levels of analysis and monitoring. there was strong support expressed for the use of epidemiologic techniques for monitoring health care at one level of aggregation and diagnosis - specific criteria development for chart review at another level. there was an expressed need for : epidemiologic monitoring of patient outcomes such as mortality, morbidity, and disability. local area analyses to identify problems in both patient outcomes and health care processes. analysis and monitoring of both the outcomes and processes of care at the institution or medical care plan level. although historically there have been heated debates about the relative merits of the use of structural measures, procedural criteria, or patient outcome measures, the speakers at the symposium supported the appropriateness of the use of both process and outcome measures of quality. there was a strong expression of overriding need for determinations of the interrelationships among the structural, the procedural, and the outcome measures of quality of care. during the past 20 years, progress in the assessment of the quality of health care has been impeded by lack of agreement about the appropriate indicators of good health care as well as lack of detailed data bases on the condition of and the care provided to patients. although these necessary data can be made available in clinical trials or other types of special studies, in the day - to - day world of practicing medicine, the detail of available data is often insufficient to analyze and understand complex interrelationships. to date, uniformly collected data have not been sufficiently detailed to permit process studies of health care quality to be conducted. in addition, the data are neither uniform nor easily accessed, and errors in recording and abstraction are other long - recognized problems. in particular, there is need for uniform reporting of patient characteristics, particularly a uniform clinical data base. development in the following area was considered a high priority : a global measure of patient physiologic status and physiologic reserve that can provide a reliable determination of patient prognosis (likelihood of responding to treatment). such a measure does not represent quality, but is important for interpretation of other process and outcome measures. robert brook (the rand corporation) opened the symposium with the provocative question : will imperfect information about health care quality lead to better health or will it lead to increased social divisiveness ? as with all research, the readily available health care data do not always adequately measure the desired theoretical concepts. therefore, quality of care analyses often have been limited to those aspects for which data exist or that are relatively inexpensive to obtain. although hospital discharge abstract data bases and administrative data bases such as that of the medicare program have facilitated analyses of patient length of stay and patient - based mortality and readmission, these are admittedly either poor proxies for quality or they are vulnerable to ambiguous interpretation. the symposium participants recognized the concerns of health care providers regarding the use of imperfect measures of quality of care ; however, several speakers urged that research and the dissemination of data and research results should not await the perfect data base. it was pointed out that a number of existing surveys contain data that, if linked with medicare utilization data, could provide a more thorough analysis. it was emphasized that sampling is an appropriate means of estimating phenomena in a population and that surveys can be used to periodically collect specific, necessary data. linkages among extant data bases, such as tumor registries, state - maintained death certificates, national center for health statistics surveys of disease incidence and prevalence, surveys of functional status, etc., are feasible, particularly the linkage of these clinically oriented data bases with the utilization data from the medicare statistical system. this, however, was not felt to be a thoroughly satisfactory solution to the need for improved and expanded routine data bases for the types of priority data previously described. there was also felt to be a need for information on all sectors of health care, including ambulatory care in physician offices, home health care, and nursing home care, as well as the frequently studied inpatient care. in particular, given the increasing interest in health maintenance organizations and other forms of capitated health care, there is a strong demand for information on both the capitated and fee - for - service sectors. mark blumberg (kaiser foundation health plan, inc.) emphasized the need for measures and data collection that permit analysis of comparability across provider types and across larger health care systems. the participants also expressed a need for more analysis of the quality of care provided to specific subpopulations. in particular, concern was raised that cost - saving efforts might motivate providers to discriminate in the types of patients accepted and/or in the types of care provided. although there is need for analysis of the appropriateness of clinical decisionmaking, there is also clearly a role for population - based analyses of health status, mortality, morbidity, disability, and health care utilization that is independent of the adquacy of the medical processes employed by health care practitioners. currently there are no quality of care data bases on regional, state, or federal levels. but as the pressure for comparable information on health care quality measures increases so that consumers may make enlightened decisions regarding selection of individual providers, health care plans, and insurance packages, there will likely be increased pressure for better, comparable data bases. it appears that researchers, practitioners, and purchasers of care are beginning to agree that it is desirable, even imperative, to develop information on attributes felt to reflect aspects of quality in health care. this heightened awareness, verging on impatience, is likely to further the development and refinement of measures, data bases, routine monitoring mechanisms, and the analytic skills of all concerned parties. it is generally believed that improvements in the measurement and monitoring of health care will ultimately improve both the quality of health care and the utilization of resources. in summary, the thoughtful presentations and discussions at the symposium acknowledged that there is reason for concern about the effects of incomplete data on quality of care, but that there is also reason for moving ahead with carefully designed measurement, analysis, and feedback., codman research group, hanover, new hampshire ; mark blumberg, m.d., kaiser foundation health plan, inc.,, associate professor and chairman, division of geriatric medicine, medical college of virginia ; william munier, m.d., practicing physician in wellesley, massachusetts ; robert keller, m.d., practicing physician in belfast, maine ; kathleen lohr, ph.d., assistant director, pennsylvania health care cost containment council ; anne flood, ph.d., college of medicine, university of illinois ; duncan neuhauser, ph.d., professor of epidemiology and community health, case western reserve university ; christopher r. blagg, m.d.,, d.sc, senior staff health services researcher, the rand corporation, santa monica, california ; douglas wagner, ph.d., intensive care research unit, george washington university, washington, d.c. ; kathleen m. griffin, ph.d., cae, executive vice president, american college of health care administrators, bethesda, maryland ; gary gaumer, ph.d., vice - president, abt associates inc., cambridge, massachusetts ; kenneth manton, ph.d., director, center for demographic studies, duke university ; james prevost, m.d., director of research, joint commission on accreditation of healthcare organizations ; joseph d. restuccia, ph.d., associate professor of health care and operations management, boston university. | the research issues in the assessment of quality of health care have not changed significantly during the past 20 years. what has changed is the increased interest among providers, consumers, and policymakers for ways to measure and compare quality among providers. clearly there is an increased sense of urgency in the need for research in such areas as the development of improved measures of physiologic status, physiologic reserve, studies of all health care providers, studies of various subpopulations, more use of existing population and sample survey data bases through linkages, and implementation of uniform clinical reporting. |
periodontal diseases are ubiquitous, affecting all dentate animals. among various periodontal disease affecting humans, the most prevalent is gingivitis, affecting more than 90% of the population, regardless of age, sex, or race. dental plaque is a sticky film of invisible layer of bacteria that hangs around the gums, tongue, teeth, and other dental restorations. plaque if not removed regularly, leads to tooth and periodontal disease which eventually leads to tooth loss. it is defined as inflammation of the gingiva in which the junctional epithelium remains attached to the tooth at its origin level. the prevention of gingivitis by daily and effective supragingival plaque control is necessary to arrest its progression into periodontitis. although mechanical plaque control methods have the potential to maintain adequate levels of oral hygiene, clinical experience and population - based studies have shown that such methods are not being employed as accurately as they should be by a large number of people. therefore, several chemotherapeutic agents have been developed to control bacterial plaque, aiming at improving the efficacy of daily hygiene control measures. the interest in plants with antibacterial and anti - inflammatory activities has increased to overcome the consequence of current problems associated with the wide - scale misuse of chemotherapeutic agents that induce microbial drug resistance. various natural products like astronium urundeuva, calendula, aloe vera, curcuma zedoaria, and other herbs that had been used effectively since ages in ayurveda are revisited and are been tested for their effectiveness in treating oral diseases with appreciable results. punica granatum (family punicaceae), generally known as pomegranate, is a shrub or small tree native to asia where several of its parts have been used as an astringent, and for hemostatic as well as diabetic control. the fruit of this tree is used for the treatment of throat infections, coughs, and fever due to its anti - inflammatory properties. in a study evaluating the effects of pomegranate on gingivitis, pereira and sampaio showed a significant reduction in gingival bleeding after using a dentifrice containing the pomegranate extract. yet in another similar study with a control group by salgado., the effect of a gel with a pomegranate extract was tested on a group with experimental gingivitis which hardly mimics the naturally occurring gingivitis. therefore, the purpose of the present study was to compare and evaluate the efficacy of a gel containing the pomegranate extract in plaque formation in comparison to a control formulation and to evaluate the effect of the pomegranate gel on clinical parameters of naturally developed gingivitis. fresh pomegranates were obtained and their seeds were separated and ground into fine juice in an electric grinder. the concentrated extract was obtained through direct percolation by filtering the juice in a buckner funnel through a filter paper. at this stage, a control gel was prepared by dissolving 5 g of carboxymethyl cellulose in 100 ml of distilled water and stirring it gently for 15 min until a gel of consistency (0.05%) convenient for usage, as the orabase gel, is obtained. similarly, the test gel was prepared by dissolving 5 g of carboxymethyl cellulose in 100 ml of the concentrated extract of pomegranate juice. a very small amount of methyl paraben (2 mg) was added as a preservative to both test and control gels. a densitometric high - performance thin layer chromatography analysis was performed for the development of the characteristic finger printing profile. the extract of the fruit was mixed with high - performance liquid chromatography grade methanol. the solution was centrifuged for 5 min and used for high - performance thin layer chromatography analysis. then, 2 l of the samples were loaded in the form of bands of width 7 mm in the 10 10 silica gel 60f254 thin layer chromatography plate using the hamilton syringe and camag linomat 5 instrument. the sample - loaded plate was kept in the thin layer chromatography twin trough developing chamber (after saturation with solvent vapor) with the respective mobile phase (toluene acetone formic acid 4.5:4.5:1) up to 90 mm. hptlc analysis : baseline display showing peak (rf values) control and experimental gels were formulated and packed into small plastic containers for delivery to patients and it was made sure that volunteers were unaware about the content of the gel. all volunteers were delivered fresh samples of gels sufficient for the 7-day usage on their periodic recall visits. the volunteers were instructed to massage the provided gel onto their gums twice daily, morning and night, for 35 minutes. no other oral hygiene instructions were given to the volunteers and they were asked to follow their routine oral hygiene method. gels were delivered during first two visits only to check the sustainability of the product on consecutive visits. a total of 40 patients (17 males and 23 females aged 2045 years with chronic generalized gingivitis from the outpatient department of periodontics, sri hasanamba dental college and hospital, hassan, karnataka, india) were enrolled for this study based on inclusion and exclusion criteria. all subjects had at least 24 natural teeth, were apparently healthy, and had clinical signs of gingivitis. all patients were informed about the nature of the study and signed the informed consent form in compliance with the study guidelines. patients with a probing depth of 4 mm in any tooth, history of antibiotic coverage in last 6 months preceding the study or on long - term exposure to anti - inflammatory drugs, and history of periodontal therapy including oral prophylaxis in the last 6 months, and also smokers, pregnant women, and those who were allergic to the pomegranate fruit were excluded from the study. this study was a randomized, and intragroup comparisons of four groups of patients in four experimental phases of 7 day each for 21 days were done. the patients were randomly divided into 4 groups of 10 in each : group a : patients received scaling and root planning (srp) and were delivered a gel containing the pomegranate extract.group b : patients received srp and were delivered the placebo gel.group c : patients did not receive any basic therapy but were delivered a pomegranate gelgroup d : patients did not receive any therapy and were delivered a placebo gel for application. group a : patients received scaling and root planning (srp) and were delivered a gel containing the pomegranate extract. group c : patients did not receive any basic therapy but were delivered a pomegranate gel group d : patients did not receive any therapy and were delivered a placebo gel for application. during each visit of the 21-day experimental period, that is on baseline, day 7, day 14, and day 21, the plaque index (pi), gingival index (gi), and papillary bleeding index (pbi) were recorded and plaque samples were collected using sterile probes on one particular surface for each patient ; fresh samples of the gel sufficient for usage for next 7 days were delivered. collected plaque samples were transferred and spread onto two clean, sterile microscopic slides, and were stained with gram 's stain. stained slides were used to make a reliable semiquantitative assessment of morphologically different types of bacteria. for the recorded pi (silness and loe), gi (loe and silness) and pbi (mulhemann), intragroup comparisons were done using paired t - test or wilcoxon 's signed rank test between baseline and day 21, and intergroup comparisons were done by one - way anova followed by duncan 's range test. for groupwise comparisons, for all the tests, a p value of 0.05 or less was considered for statistical significance. for microbiological analysis results, the comparison of the microbial status between different groups was analyzed using the chi - square test. fresh pomegranates were obtained and their seeds were separated and ground into fine juice in an electric grinder. the concentrated extract was obtained through direct percolation by filtering the juice in a buckner funnel through a filter paper. at this stage, a control gel was prepared by dissolving 5 g of carboxymethyl cellulose in 100 ml of distilled water and stirring it gently for 15 min until a gel of consistency (0.05%) convenient for usage, as the orabase gel, is obtained. similarly, the test gel was prepared by dissolving 5 g of carboxymethyl cellulose in 100 ml of the concentrated extract of pomegranate juice. a very small amount of methyl paraben (2 mg) was added as a preservative to both test and control gels. a densitometric high - performance thin layer chromatography analysis was performed for the development of the characteristic finger printing profile. the extract of the fruit was mixed with high - performance liquid chromatography grade methanol. the solution was centrifuged for 5 min and used for high - performance thin layer chromatography analysis. then, 2 l of the samples were loaded in the form of bands of width 7 mm in the 10 10 silica gel 60f254 thin layer chromatography plate using the hamilton syringe and camag linomat 5 instrument. the sample - loaded plate was kept in the thin layer chromatography twin trough developing chamber (after saturation with solvent vapor) with the respective mobile phase (toluene acetone formic acid 4.5:4.5:1) up to 90 mm. control and experimental gels were formulated and packed into small plastic containers for delivery to patients and it was made sure that volunteers were unaware about the content of the gel. all volunteers were delivered fresh samples of gels sufficient for the 7-day usage on their periodic recall visits. the volunteers were instructed to massage the provided gel onto their gums twice daily, morning and night, for 35 minutes. no other oral hygiene instructions were given to the volunteers and they were asked to follow their routine oral hygiene method. gels were delivered during first two visits only to check the sustainability of the product on consecutive visits. a total of 40 patients (17 males and 23 females aged 2045 years with chronic generalized gingivitis from the outpatient department of periodontics, sri hasanamba dental college and hospital, hassan, karnataka, india) were enrolled for this study based on inclusion and exclusion criteria. all subjects had at least 24 natural teeth, were apparently healthy, and had clinical signs of gingivitis. all patients were informed about the nature of the study and signed the informed consent form in compliance with the study guidelines. patients with a probing depth of 4 mm in any tooth, history of antibiotic coverage in last 6 months preceding the study or on long - term exposure to anti - inflammatory drugs, and history of periodontal therapy including oral prophylaxis in the last 6 months, and also smokers, pregnant women, and those who were allergic to the pomegranate fruit were excluded from the study. this study was a randomized, and intragroup comparisons of four groups of patients in four experimental phases of 7 day each for 21 days were done. the patients were randomly divided into 4 groups of 10 in each : group a : patients received scaling and root planning (srp) and were delivered a gel containing the pomegranate extract.group b : patients received srp and were delivered the placebo gel.group c : patients did not receive any basic therapy but were delivered a pomegranate gelgroup d : patients did not receive any therapy and were delivered a placebo gel for application. group a : patients received scaling and root planning (srp) and were delivered a gel containing the pomegranate extract. group c : patients did not receive any basic therapy but were delivered a pomegranate gel group d : patients did not receive any therapy and were delivered a placebo gel for application. during each visit of the 21-day experimental period, that is on baseline, day 7, day 14, and day 21, the plaque index (pi), gingival index (gi), and papillary bleeding index (pbi) were recorded and plaque samples were collected using sterile probes on one particular surface for each patient ; fresh samples of the gel sufficient for usage for next 7 days were delivered. collected plaque samples were transferred and spread onto two clean, sterile microscopic slides, and were stained with gram 's stain. stained slides were used to make a reliable semiquantitative assessment of morphologically different types of bacteria. the microbiological status was coded in grades as below : < 5 organisms + 510 organisms for the recorded pi (silness and loe), gi (loe and silness) and pbi (mulhemann), intragroup comparisons were done using paired t - test or wilcoxon 's signed rank test between baseline and day 21, and intergroup comparisons were done by one - way anova followed by duncan 's range test. for groupwise comparisons, for all the tests, a p value of 0.05 or less was considered for statistical significance. for microbiological analysis results, the comparison of the microbial status between different groups was analyzed using the chi - square test. the experimental gel had good acceptance and did not show adverse effects, such as ulceration or allergic reactions. control and experimental groups a and b showed statistically significant differences with respect to pi, gi, and pbi [table 1 ] from the baseline to day 21 whereas the groups which were treated with gels without srp (groups c and d) did not show this difference. plaque index, gingival index, and papillary bleeding index but the difference between these respective groups at the end of the experimental period was statistically significant only with respect to the pbi. with respect to the microbiological results [table 2 ], there was a statistically significant difference in the values of gram - negative cocci and bacilli in the groups which used the pomegranate extract both with and without srp whereas with respect to gram - positive cocci and bacilli, this difference was not seen between the groups. plaque is the main agent responsible for the breakdown of periodontal tissues leading to periodontal disease. the removal of this plaque regularly is of paramount importance in the prevention of periodontal disease. the inability of the adult population to perform adequate mechanical tooth cleaning has stimulated the search for chemotherapeutic agents added to dentifrices to improve plaque control and prevent gingivitis. so various means have been established and search is going on to reduce the bacterial load. herbal products are one group of agents which has been used extensively in reducing the bacterial population. phytotherapeutic products have been investigated with these purposes and have shown satisfactory results.[57 ] this made us to evaluate the efficacy of p. granatum as an antiplaque agent. the antibacterial activity of p. granatum has been evaluated in previous studies with good results. trivedi and kazmi, using extracts of fruit barks, have observed an antibacterial activity against bacillus anthracis and vibrio cholerae while machado. showed a similar effect against staphylococcus aureus, in agreement with prashant and asha. all the above - mentioned studies had used either the bark or the leaves of the p. granatum tree or were tried and tested on only one particular group of organism. this made us to take up this study which finds the efficacy of the fruit extract in preventing plaque and also its effect on morphotypes of organisms which gives an overall view of its effect on plaque organisms as established in goodson 's work where he has distinguished the presence of morphotypes of organisms under different conditions. on the basis of this work, analysis of the p. granatum fruit extract in morphotypes of plaque bacteria was also made. considering that flavoring agents can promote a moderate antiplaque affect and contents of test and control gels were different only with respect to the presence of the pomegranate extract, results show that this agent did not have an additional effect in reducing plaque formation and signs of gingivitis unless it was supplemented with basic therapy. these data are not in agreement with those of a previous in vitro study by periera. which showed that a bacterial strain present in supragingival plaque, namely, streptococcus sanguis, was sensitive to different concentrations of the pomegranate extract, which demonstrated an inhibitory action similar to that of chlorhexidine. it should be highlighted, however, that in vitro studies do not reproduce exactly the oral conditions. in the present study, the pomegranate extract gel did not avoid plaque formation during the trial, as suggested by kakiuchi. and pereira. but there was a significant reduction in the plaque score compared to the group which used the placebo gel. hydrolysable tannins that form complexes of a high molecular weight with soluble proteins, increase bacterial lysis, and moreover interfere with bacterial adherence mechanisms on tooth surfaces. these results are consistent with those reported by pereira and sampanio, who showed a significant reduction in gingivitis using the dentifrice containing the pomegranate extract. nevertheless, it is noteworthy that a control group was not included in that study, which had not allowed the assessment of the actual gingivitis reduction rate related exclusively to mechanical plaque control. according to ross. the anti - inflammatory effect of pomegranate may be attributed to its considerable immunoregulatory activity over macrophages and t- and b - lymphocyte subsets. the score used in this study only evaluates the presence or absence of bleeding ; it does not allow the assessment of other characteristics of the inflammatory process, such as edema, changes in gingival contour, and loss of tissue attachment. therefore, the possible effect of the p. granatum extract on controlling the severity of gingivitis is not very clear. microbiological data showing unaltered values of gram - positive cocci and bacilli suggest that the pomegranate extract might have had no effect on the natural habitats of the oral cavity as it 's a natural product unlike chemotherapeutic agents which alongside being bactericidal to pathogenic flora also disrupt the normal flora of the oral cavity. but the reduction in the counts of gram - negative bacilli and cocci in the groups which used the pomegranate extract in comparison to the group using the control gel suggests that it might have had some bactericidal activity on these pathogenic species. the use of carboxymethyl cellulose as the carrier for the pomegranate extract has proved to be efficient in improving the gingival condition as carboxymethyl which is a commonly used orabase gel has significant sustainability on the oral tissues. though the difference between the groups with the pomegranate extract with respect to the gi and pi is not statistically significant, clinical conditions showed considerable difference from the groups which used the control gel. the group which was treated without srp did not show any statistically significant difference between the groups which used pomegranate and control gel showing that the pomegranate extract on itself would not be so very effective in treating gingival disease without the basic therapy. so the basic therapy remains the gold standard for the treatment of the periodontal therapy. due to the lack of clinical trials with a similar design investigating the effect of pomegranate on gingivitis, the data of this study were evaluated by parallel inferences, taken off from nonspecialized articles. therefore, more clinical trials with a similar design using different concentrations of pomegranate are necessary to verify its action upon plaque microflora in vivo and severity of gingivitis. further research will be needed to identify the real benefits of pomegranate as a therapeutic and preventive agent for gingivitis, in addition to its common use in popular medicine. within the limits of this clinical study, it can be concluded that the gel containing pomegranate extract was efficient in treating gingivitis when used along with mechanical cleaning in controlling plaque and gingivitis. | background : chronic gingivitis is the most prevalent in all dentate animals. regular methods for controlling it have been found to be ineffective, which have paved the way for the use of herbal products as an adjunctive to mechanical therapy as they are free to untoward effects and hence can be used for a long period of time.objective:the antigingivitis effect of a gel containing pomegranate extract was evaluated using a 21-day trial in patients with chronic gingivitis.materials and methods : forty patients participated in this randomized clinical study, carried out in four phases of 7 days each over 21 days. the patients were randomly assigned to four groups : first group was treated with mechanical debridement and an experimental gel and the second group with mechanical debridement and a control gel ; the third group was nt subjected to mechanical debridement and only experimental gel was used. the fourth group was treated with control gel only. all the groups were subjected to various clinical and microbiological parameters to evaluate the antiplaque and antigingivitis effect of the pomegranate extract.results:the group which used the pomegranate gel along with mechanical debridement showed significant improvement in all the clinical and microbiological parameters included in the study when compared with the other three groups.conclusion:hence it can be concluded that the pomegranate gel when used as an adjunct with mechanical debridement was efficient in treating gingivitis. |
there are certain prognostic factors to determine the neurological outcome of the patients in spinal cord injury (sci). etiology, severity of trauma, and preoperative neurological condition are the most important factors influencing the prognosis. patients with a complete sci have a 20 in all of the patients with asia a, b, c, and d (lesion). however, si was 40% in all of the patients with asia a, b, c, and d (lesion). however, cc was 50% in all of the patients with asia a, b, c, and d (lesion) and 15. in our series, the patients with si > 20 had a higher rate of neurological deficit (p 40% had a higher rate of neurological deficit (p 50% had a higher rate of neurological deficit (p 20, the patients with cc > 40%, and the patients with lvbh > 50% are likely to have a more severe neurological injury. | aim : our aim was to determine whether a combination of sagittal index (si), canal compromise (cc), and loss of vertebral body height (lvbh) is associated with the severity of neurological injury in patients with thoracolumbar burst fractures.materials and methods : seventy - four patients with thoracolumbar burst fracture undergoing instrumentation between 2010 and 2015 were analyzed retrospectively. the degree of neurological injury was determined using the american spinal injury association (asia) scoring system. the association between the morphology of the fracture and the severity of neurological injury was analyzed.results:there was a strong association between fracture morphology and the severity of neurological injury. of the patients, 77.5% with si 20, 81.6% with cc 40%, and 100% with lvbh 50% had lesion according to asia. all of 7 patients with asia a had si 20, cc 40%, and lvbh 50%. on the other hand, 79% of the patients with asia e had si 25, the patients with cc > 40%, and the patients with lvbh > 50% are likely to have a more severe neurological injury. |
approximately 50100 million infected cases were reported annually. among those infected, 500000 patients had severe infection and required hospital admission ; most were children. the cost of care was as high as $ us 2.1 billion per year in the united states of america. no specific treatments are available except for symptomatic, which are effective in early detection. in patients with severe infection, early detection or correct prognostication may avoid such severe complications [4, 6 ]. clinical risks and various laboratory results were studied to explore their roles in the prediction of dengue severity. among many of them were girls, children above 5 years of age, persistent abdominal pain, lethargy, cold hand and feet, hepatomegaly, abnormal bleeding, overweight, malnourished children, ascites, plural effusion, leucopenia (white blood count 6 years (or = 1.46, 95% ci = 1.121.91, p = 0.005), hepatomegaly (or = 12.31, 95% ci = 8.8417.15, p 5000/l (or = 1.40, 95% ci = 1.131.75, p = 0.002), and platelet 50000/l (or = 3.95, 95% ci = 3.144.96, p 11.5 (dss). the score 11.5 predicted dss correctly in 39 out of 90 patients, with 1-level overestimation in 1 out of 11 patients (1.4%), with no 2-level overestimation (0%), or a total of 1.4% overestimation (table 3). dengue infection is an urgent condition requiring prompt diagnosis and treatment before patients enter into bleeding or shock states. previous scoring systems trying to evaluate or forecast disease severity included the dengue fever scoring system based on epidemiological information and clinical signs or symptoms, which might be useful in detecting df very early prior to laboratory results. other scoring systems were the pediatric logistic organ dysfunction (pelod) score and the pediatric risk of mortality iii (prism iii), used to evaluate the mortality rates, and the disseminate intravascular coagulation (dic) score to diagnose dic and to discriminate df and dhf from other febrile illnesses. decision tree algorithms were also applied to classify dengue infections into df, dhf grade i, dhf grade ii, and dhf grade iii. they may also predict patients with low risk who may be discharged safely and select patients with high risk who should be admitted for close monitoring. some studies included only adult patients [25, 29 ] which may or may not be relevant for children. in studies which included children, some studies used information on the first few days of admission in pediatric intensive care unit (picu). in some studies, included dhf patients did not experience shock and may not be inferred to patients with dss. risk prediction in many studies was based on nonroutine laboratory investigations [19, 20, 26, 28, 29 ]. despite their high predictive powers, an investment on such facilities may not be feasible and may not be cost effective. our study developed a scoring system based on clinical risk and routine laboratory parameters and categorized patients with dengue infection into df, dhf, or dss.patients scoring 11.5 were classified as a risk group for dss. patients may safely be handled as outpatients with symptomatic treatments and may be advised to observe any abnormal signs or symptoms with a follow - up appointment. these patients should be admitted to be closely monitored for early signs of plasma leakage, hemoconcentration, coagulopathy, thrombocytopenia. our scoring system predicted dss correctly with a positive predictive value (ppv) of 88%, similar to other studies which reported a ppv of 8295% [20, 26 ]. however, the fact that the derived scores in df and dhf were more or less overlapping made it less likely to be used to differentiate dhf for df. other nonroutine predictors specifically for dhf may be required. in case of continuous routine laboratory investigations, our score may also be used to monitor the patients risk for dss as the disease progresses from day to day. in case of outpatients, this will help clinicians make decision when to admit the patients to hospital. application of this scoring system into routine patient care may help reduce unnecessary admission and also reduce case facility in severe cases that are admitted based on high risk scores. the derived dengue infection severity score classified patients into df, dhf, or dss, correctly into their original severity levels. | objectives. to develop a simple scoring system to predict dengue infection severity based on patient characteristics and routine clinical profiles. methods. retrospective data of children with dengue infection from 3 general hospitals in thailand were reviewed. dengue infection was categorized into 3 severity levels : dengue infection (df), dengue hemorrhagic fever (dhf), and dengue shock syndrome (dss). coefficients of significant predictors of disease severity under ordinal regression analysis were transformed into item scores. total scores were used to classify patients into 3 severity levels. results. significant clinical predictors of dengue infection severity were age > 6 years, hepatomegaly, hematocrit 40%, systolic pressure 5000 /l, and platelet 50000 /l. the derived total scores, which ranged from 0 to 18, classified patients into 3 severity levels : df (scores 11.5, n = 50, 6.4%). the derived score correctly classified patients into their original severity levels in 60.7%. an under - estimation of 25.7% and an over - estimation of 13.5% were clinically acceptable. conclusions. the derived dengue infection severity score classified patients into df, dhf, or dss, correctly into their original severity levels. validation of the score should be reconfirmed before application of routine practice. |
tumors undergo sustained growth in a dynamic environment where oxygen and nutrients are often scarce (denko, 2008 ; hanahan and weinberg, 2011). to cope with the energetic requirements of rapid proliferation in these challenging conditions (fritz and fajas, 2010), tumor cells profoundly reorganize their core metabolism (cairns., 2011 ; glucose utilization, which provides atp, essential anabolic intermediates, and antioxidative defenses (hsu and sabatini, 2008 ; vander heiden., 2009), is boosted and dissociated from oxygen availability (the warburg effect ; warburg, 1956 ; warburg., 1927). key to the warburg effect is the decrease of mitochondrial respiration (frezza and gottlieb, 2009), which allows cancer cells to grow in the hypoxic conditions found in the interior of the tumor mass (hsu and sabatini, 2008). the molecular mechanisms that inhibit oxidative phosphorylation (oxphos) in tumors are understood only partially. the transcription factor hif1 (hypoxia - inducible factor 1) decreases the flux of pyruvate into the krebs cycle and, hence, the flow of reducing equivalents needed to power the electron transport chain (etc) and stimulates glycolysis by inducing glucose transporters and glycolytic enzymes (denko, 2008 ; semenza, 2010b). hif is activated by hypoxia as well as by the accumulation of the krebs cycle metabolites succinate and fumarate that inhibit the prolyl hydroxylases (phds) responsible for proteasomal degradation of the hif1 subunit (selak., 2005). succinate accumulation can originate from loss - of - function mutations in any of the genes encoding for succinate dehydrogenase (sdh) subunits (or their assembly factor sdhaf2), which cause hereditary paraganglioma - pheochromocytoma syndrome and are associated to a number of other neoplasms (bardella., 2011). within this conceptual framework, we have analyzed the activity of trap1, an evolutionarily conserved chaperone of the hsp90 family mainly located in the mitochondrial matrix and overexpressed in a variety of tumor cell types, where it exerts antiapoptotic functions through mechanisms that are only partially understood (altieri., 2012 ; kang. our results indicate that trap1 supports tumor progression by downmodulating mitochondrial respiration through a decrease in the activity of sdh, which leads to hif1 stabilization even in the absence of hypoxic conditions, by increasing succinate levels. we found that trap1 is localized in mitochondria of cancer cell models (figures s1a and s1b available online), as expected (altieri., 2012), and that downregulation of trap1 expression by rnai abrogated any transforming potential. in fact, knockdown of trap1 expression made saos-2 osteosarcoma cells, hct116 colon carcinoma cells, and hela cervix carcinoma cells (dubbed shtrap1 cells ; figures s1c s1e) unable to both form foci (figure 1a) and grow in soft agar (figure 1b) without affecting the rate of cell growth (figure 1c). notably, shtrap1 tumor cells lost the ability to develop tumor masses when injected into nude mice (figure 1d). conversely, when the trap1 complementary dna (cdna) was expressed in either rwpe-1 prostate epithelial cells or fibroblasts, these nontransformed cells acquired the capacity to form colonies in soft agar (figures 2a and 2d), and downregulation of trap1 expression in rwpe-2 prostate cells, which are transformed by expression of v - ki - ras in rwpe-1 cells (rasola., 2010a), abolished their tumorigenic features (figure 2b). moreover, stable transfection of a murine trap1 cdna, which is insensitive to human - directed small hairpin rna (shrna) constructs, reinstalled the tumorigenic capability of shtrap1 cells (figure 2c). mitochondrial localization of trap1 was essential for its proneoplastic activity, as expression of a trap1 cdna devoid of its mitochondrial targeting sequence was not tumorigenic in either cancer or nontransformed cells (figures 2d and 2e). we then asked whether trap1 promotes transformation by acting on mitochondrial metabolism, thus contributing to the warburg phenotype. we used a blue native (bn)-page approach (figure 3a), which allows the separation and characterization of protein complexes under nondenaturing conditions (wittig and schgger, 2008), to investigate a possible interaction between trap1 and etc complexes. by cutting bn - page bands and running them on an sds - page, we could observe the association between trap1 and both complex iv (cytochrome oxidase, cox) and complex ii (succinate dehydrogenase, sdh) (figure 3a). moreover, by performing an immunoblot directly on the bn - page, we found trap1 to be in correspondence with both complex iv and complex ii bands ; notably, these bands were diffused, and trap1 colocalized with their upper portion, suggesting that trap1 contributes to form a multimeric complex of higher molecular weight than the etc complex per se (figure 3b). we confirmed the interaction between trap1 and complex ii / sdh through further approaches, including (1) immunoprecipitation, finding coimmunoprecipitation (coip) of trap1 with sdh and vice versa (figure 3c), and (2) mitochondrial protein crosslinking with dimethyl 3,3-dithiobis - propionimidate (dtbp), a homobifunctional compound that reacts with the primary amines of two interacting proteins at an average distance of about 8 (giorgio., 2009), followed by trap1 immunoprecipitation in order to determine whether trap1 and sdh are closely associated. we found that two trap1/sdh complexes are formed in mitochondria (figure 3d). complex ii couples the krebs cycle to oxphos by oxidizing succinate to fumarate and then transferring electrons to coenzyme q ; hence, the enzyme is called either sdh or succinate : coenzyme q reductase (sqr ; cecchini, 2003 ; lemarie and grimm, 2011). sqr activity can be assessed spectrophotometrically in permeabilized mitochondria after inhibition of the other etc complexes by recording the reduction of 2,6-dichlorophenolindophenol (dcpip) in the presence of succinate as an electron donor and coenzyme q1 as an intermediate electron acceptor. the slope of the absorbance decline of dcpip is directly proportional to sqr activity (see figure s2a). we found that sqr enzymatic activity was increased in mitochondria from shtrap1 cells relative to those derived from control cells (figures 4a, s2a, and s2b). trap1 did not affect either the cytochrome oxidase enzymatic activity of complex iv (figure s2c) or complex ii protein levels (figure s2d) or mitochondrial mass (figure s2e). specificity of trap1 inhibition on sdh was assessed by (1) expression of the mouse trap1 (in shtrap1 cells) that is insensitive to human - directed shtrap1 constructs, which resulted in inhibition of sqr activity to values similar to those of mock cells (figure 4a), and (2) using an inhibitor of trap1/hsp90 atpase activity (felts., 2000), 17-allylamino-17-demethoxygeldanamycin (17-aag), whose availability to mitochondria was recently shown in situ (xie., 2011) ; 17-aag specifically increased sqr activity in control mitochondria, whereas shtrap1 mitochondria were insensitive to the drug (figures 4a, 4b, and s2b). notably, the effect of 17-aag was unrelated to hsp90, as hsp90 protein levels were the same in mock and shtrap1 cells (figure s2f). the sqr activity of etc complex ii was further inhibited in mitochondria from control cells that progressed through the focus - forming assay compared to mitochondria from the same cells kept in standard culture conditions, whereas no change in sqr activity could be appreciated in mitochondria from shtrap1 cells during the focus - forming experiments (figure 4b). 17-aag could still reactivate the sdh enzyme in mitochondria of trap1-expressing cells undergoing the focus - forming process (figure 4b), indicating that even the enhanced inhibition of sqr activity occurring during the in vitro transformation progression is mediated by trap1 and remains reversible. in further accord with an inhibitory function of trap1 on etc complex ii, mitochondria from mef cells stably expressing trap1 showed a diminished sqr activity compared to controls, and this inhibition was increased during the focus - forming assay ; 17-aag reactivated sdh selectively in mitochondria from trap1-expressing mefs (figure 4c). trap1 expression was shown to be increased in a variety of tumor types (kang. we analyzed the sqr activity of etc complex ii in a set of human colorectal cancer samples and compared it with that measured in the surrounding nontransformed mucosa for each patient. in all colorectal cancer samples at stage iv, characterized by metastases to lymph nodes and to distant sites, and in the majority of samples of stage i iii, characterized by the absence of distant metastases, trap1 was upregulated relative to normal mucosa (costantino. when we measured sqr activity in extracts from these samples, we found that trap1 upregulation was always paralleled by a decrease in sqr activity, and that this decrease could be partially rescued by adding 17-aag before starting recordings (figure 4d). in a small subset of stage i iii colorectal cancers, trap1 expression was not induced relative to surrounding nontumor tissues. in these samples, we could not detect any difference in sqr activity between samples from tumor and normal mucosa (figure 4e), strengthening the link between trap1 and the regulation of complex ii activity. the sqr assays described so far measure the maximal enzymatic activity of complex ii, as the complex is made accessible in permeabilized mitochondria and exposed to an excess of substrates. we next analyzed whether trap1 also affects the oxygen consumption rate (ocr) of living cells. downregulation of trap1 markedly increased mitochondrial - dependent respiration in all cancer cell models we tested (figures 5a, s3a, and s3b) ; in full accord with the effect of the drug on sqr activity (see figure 4a), the trap1 inhibitor 17-aag increased ocr only in trap1-expressing cells (figure s4a). in shtrap1 cells, the extra ocr was used to make atp, as it was inhibited by the atp synthase blocker oligomycin ; moreover, addition of the uncoupler fccp increased respiration well above the basal level, indicating an increased respiratory capacity that remained fully sensitive to etc inhibition by rotenone (figure 5a). the comparison with control cells is striking because, unlike shtrap1 cells, they already utilize their maximal respiratory capacity under basal conditions, as shown by the lack of ocr increase with fccp (figure 5a), an arrangement implying that any additional atp requirement must be provided by glycolysis. consistently, we found that oxphos marginally contributes to atp synthesis in mock cells, whereas a high proportion of the intracellular atp content is provided by glycolysis, with a marked increase of glycolytic atp during the in vitro tumorigenic process ; instead, in shtrap1 cells, most of the atp comes from oxphos (figure 5b). moreover, expression of the trap1 cdna in nontransformed fibroblasts markedly inhibited basal ocr and abolished any respiratory reserve (figure 5c), mimicking the respiratory pattern of trap1-expressing tumor cells. expression in shtrap1 cells of the murine trap1 insensitive to human - directed shtrap1 constructs determined an ocr pattern indistinguishable from that of mock cells (figure 5d). a low concentration of the etc complex ii inhibitors 3-nitropropionic acid (3-np), which inactivates sdh after covalent binding with an arg residue in the catalytic core of sdha (huang., 2006), or thenoyltrifluoroacetone (ttfa), which blocks electron transfer from succinate to coenzyme q at the quinone - binding site in subunits b and d (huang., 2006), inhibited ocr in shtrap1 cells but were inactive in the presence of trap1 (figures s4b and s4c), paralleling the downmodulation of the sqr activity induced by 3-np only in trap1-expressing mitochondria (figure 5e). we also found that 3-np inhibits death in a dose - dependent fashion in shtrap1, but not in mock saos-2 cells placed in conditions of long - term starvation that mimic the paucity of nutrients found in the inner tumor mass during the phases of its rapid accrual (figure 5f). moreover, treatment with 3-np partially restored the ability of shtrap1 cells to form colonies in soft agar, whereas it was ineffective on the colonies formed by control cells (figure 5 g). it was shown that succinate induces hif1 by inhibiting phds, the enzymes that hydroxylate hif1, allowing its subsequent ubiquitin - dependent degradation (selak., 2005). we observed that during the focus - forming assay, the intracellular level of succinate increased only in trap1-expressing cells (figure 6a), matching the downmodulation of their sdh enzymatic activity (figure 4b). in keeping with these results, hif1 was detectable exclusively in trap1-expressing cells during the focus - forming process (figure 6b), whereas it was hydroxylated on pro residues (i.e., primed for proteasomal degradation) both in culture conditions, independently of the presence of trap1, and in shtrap1 cells exposed to focus - forming conditions (figure 6c). hif2, which shares some redundant functions with hif1 and whose expression is increased in a broad spectrum of cancer cell types (keith., 2012), was not stabilized in our experimental conditions (figure 6d). we then used pimonidazole, a compound that is reductively activated under hypoxic conditions and forms protein adducts by reacting with cys residues (arteel., 1998), to understand whether hif1 stabilization could at least partially depend on hypoxic conditions occurring during the formation of foci. remarkably, we could not detect any induction of pimonidazole - protein adducts in trap1-expressing cells during the process of in vitro tumorigenesis, even when hif1 had already been stabilized (figure 6e), which demonstrates that pseudohypoxic conditions elicited by the presence of trap1 are sufficient to promote hif1 stabilization. tumor samples obtained from nude mice xenografted with trap1-expressing saos-2 cells (see figure 1d) were characterized by densely packed cells, amidst which fibrotic and necrotic areas could be observed (figure 6f, marked as f and n, respectively) ; hif1 was clearly detected in the majority of cells, the signal being particularly strong in the nuclei of cells where proliferation markers were also evident (compare the mib / ki67 and the hif1 staining in figure 6f). in these samples, cells displayed a punctate trap1 signal that fits well with its mitochondrial localization (see the high - magnification trap1 staining in figure 6f). the addition of dimethyl succinate, a membrane - permeable succinate analog, to the focus - forming culture medium both stabilized hif1 (figure 7a) and rescued the capability to form colonies (figure 7b) in shtrap1 cells, while it did not further increase the tumorigenicity of trap1-expressing cells (figure 7b). moreover, hif1 inhibition either with a cell - permeable esterified form of -ketoglutarate (1-trifluoromethylbenzyl--ketoglutarate, takg), which reverses hif1 stabilization by restoring phd enzymatic activity (mackenzie., 2007 ; tennant., 2009), or with rnai on hif1 or hif1, the latter being the stable subunit of the heterodimeric hif transcription factors (keith., 2012), fully abolished formation of foci in trap1-expressing tumor cells and in mefs transfected with a trap1 cdna (figures 7c7f). taken together, these data indicate that trap1 prompts neoplastic growth by inducing a succinate - dependent stabilization of hif1. tumor cells tend to increase their glycolytic activity without a matching increase of oxidative phosphorylation (warburg, 1956 ; warburg., 1927). inhibition of the tumor suppressor p53 or activation of the transcription factor hif1 curtails oxphos by inducing the autophagic degradation of respiratory complexes and by abrogating the synthesis of some of their subunits (such as sdhb) or assembly factors (denko, 2008 ; semenza, 2010a ; vousden and ryan, 2009). conversely, oxphos inhibition can play a causal role in tumorigenesis. inactivating mutations in mitochondrial dna (mtdna) genes encoding for subunits of etc complexes i and iii were found associated with renal oncocytomas (gasparre., 2008) as well as thyroid and prostate cancers (abu - amero., 2005 ; however, these mutations are confined to a small set of neoplasms, and the lack of clear - cut molecular mechanisms hampers the definition of whether oxphos inhibition as such can play a general tumorigenic role. key findings of the present work demonstrate that the mitochondrial chaperone trap1, which is widely expressed in most tumors, but not in highly proliferating, nontransformed cells (http://www.proteinatlas.org/), is a component of the molecular machinery that decreases mitochondrial respiration and that this event is crucial for neoplastic progression. indeed, we find that trap1 behaves as an oncogene since (i) without trap1, tumorigenesis is blunted both in vitro and in vivo, and (ii) trap1 expression confers tumorigenic potential to nontransformed cells. we observe that trap1-mediated inhibition of sdh limits the maximal rate of respiration and leads to succinate accumulation followed by hif1, but not hif2, stabilization. remarkably, the membrane - permeable succinate analog dimethyl succinate could both elicit hif1 stabilization and rescue the tumorigenic phenotype of shtrap1 cells, highlighting the mechanistic connection between trap1-dependent succinate accumulation and hif1-dependent tumor formation. once activated by inhibition of the proteasomal degradation of its subunit (either hif1 or hif2), and by the ensuing association with the stable subunit (hif1), hif1 can boost evolution of neoplasms by promoting angiogenesis, epithelial - mesenchymal transition, and the glycolytic switch (brahimi - horn., 2011 ; semenza, 2010a). in multiple tumor models, both hif1 and hif2 promote neoplastic progression by regulating sets of genes that are only partially shared, and independent roles for hif1 and hif2 can depend on the cancer type or on its growth and progression stages (keith., 2012). however, both subunits can play a tumor suppressor function in specific tumor types, such as renal cell carcinoma (hif1) or lung adenocarcinoma (hif2), through poorly defined mechanisms. in our model, hif1 stabilization, and the ensuing hif activation, have a crucial proneoplastic role, as promoting hif1 degradation or knocking down either hif1 or hif1 abolishes the neoplastic potential of trap1-expressing cells. instead, we could not observe any hif2 stabilization, ruling out its role in the trap1-dependent tumorigenic process. it is interesting that hif2 accumulates at higher o2 concentrations than hif1 (keith., 2012), and the possibility exists that, at variance with what we observe for hif1, pseudohypoxic conditions are insufficient or unable to stabilize hif2. hif1 stabilization emerges after several days of in vitro transformation. this is not at all surprising because excess succinate can be utilized in multiple pathways, including increased heme synthesis (frezza., 2011), and because sdh becomes more strongly inhibited by trap1 during the focus - forming assay. the latter observation also suggests that a threshold sdh inhibition must be reached to allow for succinate accumulation. despite a partial respiratory inhibition, trap1-expressing cells fully utilize their residual respiratory capacity to produce atp, as shown by ocr experiments, but reorient their metabolism toward glycolysis to meet any energy demand that exceeds respiratory capacity, in complete accord with warburg s observations. trap1 is likely to begin a feedforward loop, as it inhibits sdh and respiration (hence oxphos) and induces hif1, which in turn further inhibits oxphos (denko, 2008 ; semenza, 2010b) and directly downregulates sdh by induction of mir-210 (puissgur., 2011). notably, we have determined that trap1-dependent stabilization of hif1 occurs in a pseudohypoxic way (i.e., in the absence of any hypoxic stress). this observation has potential implications for the kinetics of tumor development, as trap1 could induce hif1 transcriptional activity even before the dysregulated accrual of the tumor mass creates hypoxic areas in its inner core. at variance with the complete block of the sdh enzyme, which is an extreme case caused by loss - of - function mutations only seen in specific subsets of tumors (bardella., 2011), the partial and reversible sdh inhibition caused by trap1 and its increased expression levels would mediate a more general proneoplastic function, which fits with trap1 identification as a bona fide inducible target of the proto - oncogene c - myc (coller., 2000). tumor cells could be endowed with a multichaperone mitochondrial complex, as trap1 interacts with cyclophilin d, hsp90, and hsp60 (ghosh., 2010 ; kang and altieri, 2009). given the multiplicity of chaperone client proteins, we can not exclude further interactions of trap1 with other mitochondrial components. for instance, trap1 is involved in the inhibition of the mitochondrial permeability transition pore (kang., 2007), whose opening irreversibly commits cells to death (rasola and bernardi, 2007 ; rasola., 2010b), and we have observed that keeping the pore locked can be used by tumor cells to evade apoptosis (rasola., 2010a). thus, trap1 could take part in several mitochondrial changes that crucially contribute to the neoplastic phenotype. targeting its chaperone activity and molecular interactors could dismantle the metabolic and survival adaptations of neoplastic cells, paving the way to the development of highly selective, mitochondriotropic antineoplastic drugs. experiments were performed on different cell models : human saos-2 osteosarcoma cells, human hct116 colorectal carcinoma cells, rwpe1/rwpe2 prostate epithelial cells, hela cervix carcinoma cells, and mefs. trap1, hif1, and hif1 expression was knocked down by stable transfections with shrnas. stable transfection of a trap1 cdna was performed in mef cells that show negligible levels of the endogenous protein. tissue samples from both tumor and normal, noninfiltrated peritumoral mucosa were obtained from patients with colorectal carcinoma during surgical cancer removal after express written informed consent was obtained from all patients. three different tumorigenesis assays were performed : (1) focus - forming assays, in which cells were grown to confluence and kept in culture for the subsequent 25 days ; tumor cells lose contact inhibition and overgrow to form foci ; (2) soft agar assays, in which cells are embedded in an agar matrix ; only transformed cells, which escape anoikis death signals, can grow to form colonies ; (3) cell injection in nude mice, in order to follow the growth of primary tumors. samples obtained from these assays were also exploited for investigating changes in complex ii enzymatic activity in succinate levels and in hif1 stabilization and distribution. cytofluorimetric analyses were utilized to analyze cell death induction with the use of annexin v - fitc and propidium iodide probes as well as mitochondrial mass with the use of n - acridine orange. mitochondria were isolated through sequential centrifugations after mechanical cell disruption. in order to establish submitochondrial protein localization, bn - page experiments were carried out on isolated mitochondria in order to identify etc complexes. after a first electrophoresis in nondenaturing conditions, bands were visualized with coomassie blue staining, cut, and run on sds - page for the identification of protein components by western immunoblot. crosslinking assays were performed on isolated mitochondria incubated with the membrane - permeable, homobifunctional reagent dtbp prior to trap1 immunoprecipitation. complex ii enzymatic activity was investigated, measuring the sqr activity with classical spectrophotometric approaches on cell or tumor lysates. complex iv enzymatic activity was investigated, measuring the oxidation of reduced cytochrome c. each measurement of the respiratory chain (rc) complex activity was normalized for protein amount and for citrate synthase activity. in vivo respiration was followed in a kinetic mode by measuring the oxygen consumption rate (ocr) of cell monolayers with an extracellular flux analyzer. immunohistochemical inspections were performed on serial sections of paraffin - embedded tumor samples obtained from xenografted nude mice following standard procedures. immunoelectron microscopy (immuno - em) inspections were performed on antibody - labeled fixed cells using the gold - enhanced protocol. intracellular succinate level was analyzed on lysates obtained by scraping cells placed in a cold methanol / acetonitrile solution. after spinning down the insoluble material, the supernatant was collected, and metabolites were separated using a liquid chromatography system coupled online to an ltq orbitrap mass spectrometer equipped with an electrospray ionization source. student s t test was used to compare pairs of data groups. in all figures, bar graphs report mean sd values (n 3) ; p < 0.05. | summarywe report that the mitochondrial chaperone trap1, which is induced in most tumor types, is required for neoplastic growth and confers transforming potential to noncancerous cells. trap1 binds to and inhibits succinate dehydrogenase (sdh), the complex ii of the respiratory chain. the respiratory downregulation elicited by trap1 interaction with sdh promotes tumorigenesis by priming the succinate - dependent stabilization of the proneoplastic transcription factor hif1 independently of hypoxic conditions. these findings provide a mechanistic clue to explain the switch to aerobic glycolysis of tumors and identify trap1 as a promising antineoplastic target. |
fear of hypoglycaemia and gain in body weight are barriers for initiation of insulin therapy. modern insulin analogues are a convenient new approach or tool to glycaemic control, associated with low number of hypoglycaemia and favourable weight change. a1chieve, a multinational, 24-week, non - interventional study, assessed the safety and effectiveness of insulin analogues in people with t2 dm (n = 66,726) in routine clinical care. please refer to editorial titled : the a1chieve study : mapping the ibn battuta trail. the patient characteristics for the entire cohort divided as insulin - nave and insulin users is shown in the table 1. the majority of patients (48.8%) started on or were switched to biphasic insulin aspart. other groups were insulin detemir (n = 54), insulin aspart (n = 8), basal insulin plus insulin aspart (n = 8) and other insulin combinations (n = 33). overall demographic data after 24 weeks of treatment, overall hypoglycaemia reduced from 20.8 events / patient - year to 4.3 events / patient - year in insulin user group whereas hypoglycaemic events increased from 0.3 events / patient - year to 2.0 events / patient - year in insulin nave group. however, this hypoglycaemia incidence in insulin naive group at 24 weeks was still lower than that observed in insulin users at baseline. blood pressure and lipid profile improved in the total cohort, but the findings were limited by number of observations. quality of life also improved after 24 weeks of treatment [tables 2 and 3 ]. all parameters of glycaemic control improved from baseline to study end in the total cohort [table 4 ]. overall efficacy data of the total cohort, 98 patients started on biphasic insulin aspart ogld, of which 69 (70.4%) were insulin nave and 29 (29.6%) were insulin users. after 24 weeks of treatment, hypoglycaemic events reduced from 21.5 events / patient - year to 1.2 events / patient - year in insulin user group whereas hypoglycaemia increased from 0.4 events / patient - year to 2.4 events / patient - year in insulin nave group. quality of life also improved at the end of the study [tables 5 and 6 ]. biphasic insulin aspartoral glucose - lowering drug safety data all parameters of glycaemic control improved from baseline to study end in those who started on or were switched to biphasic insulin aspart for both insulin nave and insulin user groups [table 7 ]. biphasic insulin aspartoral glucose - lowering drug efficacy data of the total cohort, 8 patients started on basal + insulin aspart ogld, of which 1 (12.5%) was insulin nave and 7 (87.5%) were insulin users. after 24 weeks, hypoglycaemic events reduced from 26.0 events / patient - year to 9.3 events / patient - year in insulin user group [tables 8 and 9 ]. basal+insulin aspartoral glucose - lowering drug safety data all parameters of glycaemic control improved from baseline to study end in those who started on or were basal + insulin aspart oglds for insulin - user group [table 10 ]. basal+insulin aspartoral glucose - lowering drug efficacy data of the total cohort, 54 patients started on insulin detemir ogld, of which 46 (85.2%) were insulin nave and 8 (14.8%) were insulin users. after 24 weeks of starting or switching to insulin detemir, hypoglycaemia reduced from 11.4 events / patient - year to 0.0 events / patient - year in insulin user group whereas hypoglycaemic events increased from 0.0 to 1.1 events / patient - year in insulin nave group. an improvement in quality of life was also observed at the end of the study [tables 11 and 12 ]. insulin detemiroral glucose - lowering drug safety data all parameters of glycaemic control improved from baseline to study end in those who started on or were switched to insulin detemir oglds for insulin - nave group whereas mean hba1c and fpg values improved for insulin user group [table 13 ]. insulin detemiroral glucose - lowering drug efficacy data of the total cohort, 8 patients started on insulin aspart ogld, of which 5 (62.5%) were insulin nave and 3 (37.5%) were insulin users. after 24 weeks of treatment, hypoglycaemic events reduced from 65.0 events / patient - year to 6.5 events / patient - year in insulin user group [table 14 ]. insulin aspartoral glucose - lowering drug safety data mean fpg values improved from baseline to study end in those who started on or were switched to insulin aspart oglds for insulin - nave group [table 16 ]. of the total cohort, 98 patients started on biphasic insulin aspart ogld, of which 69 (70.4%) were insulin nave and 29 (29.6%) were insulin users. after 24 weeks of treatment, hypoglycaemic events reduced from 21.5 events / patient - year to 1.2 events / patient - year in insulin user group whereas hypoglycaemia increased from 0.4 events / patient - year to 2.4 events / patient - year in insulin nave group. quality of life also improved at the end of the study [tables 5 and 6 ]. biphasic insulin aspartoral glucose - lowering drug safety data all parameters of glycaemic control improved from baseline to study end in those who started on or were switched to biphasic insulin aspart for both insulin nave and insulin user groups [table 7 ]. of the total cohort, 8 patients started on basal + insulin aspart ogld, of which 1 (12.5%) was insulin nave and 7 (87.5%) were insulin users. after 24 weeks, hypoglycaemic events reduced from 26.0 events / patient - year to 9.3 events / patient - year in insulin user group [tables 8 and 9 ]. basal+insulin aspartoral glucose - lowering drug safety data all parameters of glycaemic control improved from baseline to study end in those who started on or were basal + insulin aspart oglds for insulin - user group [table 10 ]. of the total cohort, 54 patients started on insulin detemir ogld, of which 46 (85.2%) were insulin nave and 8 (14.8%) were insulin users. after 24 weeks of starting or switching to insulin detemir, hypoglycaemia reduced from 11.4 events / patient - year to 0.0 events / patient - year in insulin user group whereas hypoglycaemic events increased from 0.0 to 1.1 events / patient - year in insulin nave group. an improvement in quality of life was also observed at the end of the study [tables 11 and 12 ]. insulin detemiroral glucose - lowering drug safety data all parameters of glycaemic control improved from baseline to study end in those who started on or were switched to insulin detemir oglds for insulin - nave group whereas mean hba1c and fpg values improved for insulin user group [table 13 ]. of the total cohort, 8 patients started on insulin aspart ogld, of which 5 (62.5%) were insulin nave and 3 (37.5%) were insulin users. after 24 weeks of treatment, hypoglycaemic events reduced from 65.0 events / patient - year to 6.5 events / patient - year in insulin user group [table 14 ]. insulin aspartoral glucose - lowering drug safety data mean fpg values improved from baseline to study end in those who started on or were switched to insulin aspart oglds for insulin - nave group [table 16 ]. our study reports improved glycaemic control following 24 weeks of treatment with any of the insulin analogues (biphasic insulin aspart ; basal + insulin aspart ; insulin detemir ; insulin aspart) with or without ogld. overall, body weight increased in insulin nave population and decreased in insulin users. though the findings are limited by number of patients, still the trend indicates that insulin analogues can be considered effective and possess a safe profile for treating type 2 diabetes in in agadir, morocco. | background : a1chieve, a multicentric (28 countries), 24-week, non - interventional study evaluated the safety and effectiveness of insulin detemir, biphasic insulin aspart and insulin aspart in people with t2 dm (n = 66,726) in routine clinical care across four continents.materials and methods : data was collected at baseline, at 12 weeks and at 24 weeks. this short communication presents the results for patients enrolled from agadir, morocco.results:a total of 201 patients were enrolled in the study. four different insulin analogue regimens were used in the study. study patients had started on or were switched to biphasic insulin aspart (n = 98), insulin detemir (n = 54), insulin aspart (n = 8), basal insulin plus insulin aspart (n = 8) and other insulin combinations (n = 33). at baseline glycaemic control was poor for both insulin nave (mean hba1c : 10.7%) and insulin user (mean hba1c : 9.1%) groups. after 24 weeks of treatment, both groups showed improvement in hba1c (insulin nave : 2.7%, insulin users : 1.3%). no major hypoglycaemia was observed at 24 weeks. sadrs were reported in 1.5% of insulin users.conclusion:starting or switching to insulin analogues was associated with improvement in glycaemic control with a low rate of hypoglycaemia. |
over the past 30 years, the management of rectal cancer has undergone many significant changes. until the 1980s, surgery was the mainstay of therapy for patients with rectal cancer confined to the bowel and regional lymph nodes. however, local recurrence occurred in approximately 25% to 50% of patients with t3 or lymph node - positive rectal cancer. these local failures, as well as distant metastases, were a serious problem in locally advanced rectal cancer (larc). to reduce these high failure rates, multiple trials evaluated different strategies of adjuvant radiation and 5-fluorouracil- (5-fu-) based chemotherapy [1, 3, 4 ]. trial results demonstrated postoperative adjuvant chemoradiotherapy improved local control and survival compared with surgery alone, leading to the routine integration of adjuvant combined modality therapy into standard practice. at the same time, total mesorectal excision (tme) was introduced and further decreased local failure rates to less than 10%. subsequently, the landmark trial conducted by the german group established superior local control, reduced treatment - related toxicity, and an improved sphincter preservation rate with neoadjuvant chemoradiotherapy compared with adjuvant 5-fu - based chemoradiation. today, although not proven to provide survival advantages (except in the pivotal swedish trial), preoperative chemoradiotherapy with concurrent infusional 5-fu and more recently the oral fluoropyrimidine, capecitabine, followed by tme has become the standard of care for patients with t3 or lymph - node - positive rectal cancer, especially in tumors of the mid- and lower rectum [7, 8 ]. the use of targeted agents in patients with advanced colorectal cancer has led to further improvements in disease - free (dfs) and overall survival (os), and further investigation in various settings is underway [912 ]. these targeted agents are now being studied in the treatment of rectal cancer and are discussed below. targeted therapies block the growth of cancer cells by interfering with specific targeted molecules needed for carcinogenesis and tumor growth. targeted cancer therapies may also be more effective by being potentially less harmful to normal cells. two main categories of targeted therapy exist : small molecules (-nib) and monoclonal antibodies (-mab), both of which can be further subdivided as either signal transduction pathway inhibitors (imatinib mesylate, trastuzumab, cetuximab) or angiogenesis inhibitors (bevacizumab, sunitinib). increasing knowledge of tumor growth and dissemination pathways has turned more attention to the use of targeted agents coupled with chemotherapy in the treatment of metastatic colorectal cancer (mcrc). for these patients, phase iii trials have shown improved disease - free and overall survival rates using epidermal growth factor receptor (egfr) and vascular endothelial growth factor (vegf) inhibitors when combined with conventional chemotherapy [912 ]. in this paper, we have reviewed vegf and egfr receptor inhibitors selectively and how their use may or may not be beneficial in the setting of rectal cancer as a radiosensitizer or in the adjuvant setting of rectal cancer. the majority of novel trials discussed are in phase ii development and are presented here due to their potential benefit in rectal cancer. bevacizumab is a humanized monoclonal antibody that targets the vascular endothelial growth factor (vegf), particularly vegf - a, a ligand with a key role in angiogenesis. angiogenesis is required for tumor growth and malignant progression, and vegf is a crucial regulator of this process. indeed, high vegf expression has been linked to a statistically higher risk of local recurrence and metastasis. thus, the inhibition of vegf is a logical target for the treatment of patients with crc. in addition, anti - vegf antibodies enhance the capacity of radiotherapy to reduce tumor vascular density and interstitial fluid pressure (ifp) in xenografts. these findings taken together support what is known as the vascular normalization hypothesis. according to this hypothesis, an excess of proangiogenic factors within tumors leads to functionally and structurally abnormal vasculature that promotes increased ifp, a known barrier to drug delivery to tumors, and impaired delivery of oxygen and macromolecules, a known barrier to the effective radiation therapy [2022 ]. normalizing this abnormal vasculature, transient antiangiogenic therapy reduces ifp and thereby increases the concentration of oxygen and penetration of cytotoxics, improving the overall effectiveness of combined modality therapy. in the same study, a variety of plasma and circulating cell biomarkers were measured. both vegf and placenta - derived growth factor (pigf) these molecules may prove to be potential biomarkers for anti - vegf therapy, as increases of pretreatment soluble vegf receptor-1 (s - vegfr1) and pigf levels have also been associated with poor pathologic tumor downstaging after preoperative chemoradiation [29, 30 ]. the epidermal growth factor receptor (egfr) is a cell surface 170,000 daltons tyrosine kinase transmembrane receptor of the erbb family, whose members play a critical role in oncogenesis. in particular, egfr has been shown to participate in the progression of crc, as it is essential for tumor growth and division. some crcs have been shown to overexpress egfr, and overexpression of egfr is associated with poorer prognosis [33, 34 ], and with resistance to radiation therapy. egfr has, therefore, become an attractive target for therapy, and two different classes of biologic agents have been evaluated : the egfr monoclonal antibodies (cetuximab and panitumumab) and the small - molecule tyrosine kinase inhibitors (gefitinib and erlotinib). the efficacy of both cetuximab and panitumumab has been clearly demonstrated to depend upon the kras mutation status. multiple analyses have demonstrated that responses to either cetuximab or panitumumab occur exclusively within the 6070% of patients without activating mutations in codon 12 and 13 of kras [23, 24, 28, 35 ]. the activating v600e braf mutation is present in an additional 10% of patients, and it may be predictive of a lack of response to anti - egfr therapy in addition to a clear poor prognostic factor. the anti - egfr monoclonal antibodies and their predictive biomarkers have taken crc treatment another step closer to personalized therapy. however, the recent results of the large uk coin study and a belgian study have not confirmed a benefit in terms of pfs or os from the addition of cetuximab to oxaliplatin - based chemotherapy in wild - type kras patients versus 5-fu and oxaliplatin alone [37, 38 ]. therefore, more thoughtfully designed studies about potential negative predictive factors such as kras, braf, nras, pik3ca mutations, and egfr gene copy numbers would be beneficial. bevacizumab, cetuximab, and panitumumab have been proven to be effective in different combined chemotherapy treatment settings for metastatic colorectal cancer and are briefly described here (tables 1 and 2) [912 ]. bevacizumab, a monoclonal antibody against the vascular endothelial growth factor ligand (vegf - a), has demonstrated efficacy with significant improvement in progression - free survival (pfs) and os in patients with metastatic colorectal cancer [9, 16, 39 ]. results from a pivotal phase iii trial of bevacizumab (5 mg / kg) showed a significantly greater os, pfs, and response rate (rr) when used in combination with irinotecan, bolus 5-fu, and leucovorin (ifl), in 813 patients with previously untreated colorectal cancer. the median os was 20.3 months in the patients treated with bevacizumab, compared to 15.6 months with the placebo - containing regimen (p <.001), and response rates were 44.8% and 34.8%, respectively (p =.004). also of note was the phase iii bevacizumab plus irinotecan in colorectal cancer (bicc)-c trial, consisting of two - arms : folfiri(infusional fl) plus bevacizumab, versus mifl (bolus fl) plus bevacizumab. the median os with the addition of bevacizumab was longer with folfiri than with mifl (28.0 versus 19.2 months ; p =.037) [40, 41 ]. these findings led to the u.s./european union approval of bevacizumab as a first - line - therapy component for mcrc with any 5-fu- based therapy. the most commonly used bevacizumab - based first - line treatment in the usa continues to be folfox plus bevacizumab. it was presumed that the benefit of adding bevacizumab to folfox would be similar to that demonstrated with the ifl regimen, and that the addition of bevacizumab to fu - based combination chemotherapy would result in a significant and clinically meaningful improvement in survival among patients with mcrc. except in cases of major contraindications for bevacizumab, such as severe vascular disease or prior arterial thrombotic events, bevacizumab can be integrated with first - line chemotherapy in patients with metastatic crc. in the event that a bevacizumab - nave patient fails first - line chemotherapy, in fact, the bevacizumab regimens : investigation of treatment effects and safety (brite) observational study confirmed that a notable os benefit was demonstrated in those patients who continued bevacizumab therapy (5 mg / kg every 2 weeks) in combination with chemotherapy, after disease progression following a bevacizumab - based regimen (median os, 31.8 months). the aries study validated the findings of brite and reported a median os of 24.7 months. furthermore, a recent randomized phase iii european trial (tml) suggests continuation of bevacizumab improves os with final results to be reported at a later date. cetuximab plus irinotecan is the standard treatment in irinotecan - refractory patients based on pivotal data from the bowel oncology with cetuximab antibody (bond) trial, which established this as an effective regimen regardless of prior treatment history. this trial confirmed the activity of cetuximab with response rates of 22.9% and 10.8% for combination therapy and monotherapy, respectively. in addition, there was a significantly longer time to tumor progression in favor of the combination arm (4.1 versus 1.5 months). in a subsequent retrospective analysis of patients with kras wild - type tumors, there was an os benefit for patients in favor of cetuximab (9.5 versus 4.8 months). given cetuximab 's demonstrated efficacy in the chemotherapy treatment - resistant setting, several studies were undertaken to evaluate its efficacy in the treatment - naive setting. the randomized, phase iii crystal trial evaluated 1198 patients treated with folfiri chemotherapy either with or without cetuximab, and noted improvement in pfs for the cetuximab - treated group. however, it was also noted that no os benefit was associated with the cetuximab - treated arm, potentially due to the fact that there was a difference in subsequent poststudy anti - egfr therapy (23% in the folfiri arm received subsequent cetuximab therapy, compared to 5.2% in the folfiri + cetuximab arm). retrospective tissue analysis on the crystal study revealed that the benefit from cetuximab was restricted to those patients with kras wild - type tumors and that it improved the pfs to 9.9 months compared to 8.4 months in the control arm. subsequent analysis of the 666 patients with kras wild - type tumors who were enrolled has shown an os improvement from 20 months in the control arm to 23.5 months in the cetuximab arm. the randomized phase ii opus trial demonstrated similar findings when cetuximab was used with oxaliplatin - based chemotherapy ; cetuximab, when combined with folfox-4 chemotherapy, led to an improvement in pfs, when compared to those treated with folfox-4 chemotherapy alone. similarly, the benefit derived from this therapy was limited to those patients with kras wild - type tumors (7.7 versus 7.2 months). however, these results have not been replicated in the phase iii mrc - coin study, where cetuximab was added to folfox or capeox (capecitabine / oxaliplatin) in the first - line setting ; those treated with capecitabine - based therapy fared worse. based on currently available data, it appears to be advantageous when cetuximab is added to irinotecan - based regimens while the advantage of combination with oxaliplatin remains less certain [26, 52 ]. cetuximab can lead cancer cells to g1 or g2/m cell cycle arrest and upregulate several genes involved in proliferation (pik31, cgref1, and plagl1) with a reduction in ki-67. but if only a small proportion of cells arrest in g0/g1 or g2/m, slowing down the cell cycle time may actually increase the amount of time available for dna repair prior to mitosis, and thus could increase resistance to chemoradiation. two phase iii trials have determined the benefit of panitumumab in combination with chemotherapy relative to chemotherapy alone. the final reported results were selected for kras wild - type status as a predictive marker for anti - egfr therapy in both studies. the panitumumab randomized trial in combination with chemotherapy for metastatic colorectal cancer to determine efficacy (prime) was a phase iii randomized trial of folfox with or without panitumumab in previously untreated patients. the pfs time was longer in the panitumumab arm (9.6 versus 8.0 months, p =.02). the role of panitumumab was also investigated in combination with folfiri for second - line treatment. the primary endpoint of a pfs difference (5.9 versus 3.9 months ; p =.004) was fulfilled with the addition of panitumumab but the os endpoint was not met. in the adjuvant treatment of colon cancer, two phase iii trials did not demonstrate any benefit from the addition of bevacizumab to standard oxaliplatin - based chemotherapy in stage ii / iii colon cancer [55, 56 ]. from the results of the nsabp c-08 trial, in which bevacizumab in combination with folfox6 did not improve dfs in the adjuvant setting in patients with stage ii / iii colon cancer, it seems that bevacizumab 's efficacy may be maximal in a setting of more advanced disease. the avant (avastin adjuvant) study compared folfox4 versus folfox4 with bevacizumab versus xelox with bevacizumab in 3,451 patients with stage ii or iii colon cancer. the study did not meet its primary objectives, and survival in the bevacizumab arms was inferior to the chemotherapy - alone arm. two adjuvant trials have evaluated the potential benefit of cetuximab in combination with folfox in a kras wild - type population : no147 and petacc8. there was no improvement in dfs or os with the addition of cetuximab to folfox. the no147 trial compared 12 cycles of the modified folfox6 to the same regimen with cetuximab [58, 59 ]. the 3-year dfs was even better with folfox alone : 75.8% versus 72.3% for folfox plus cetuximab. there was also a trend for 3-year os inferiority : 87.8% with folfox alone versus 83.9% for folfox plus cetuximab. these observations raise questions about the manner of interaction between targeted therapies and chemotherapies and the mechanisms of inducing chemoresistance in some patients. the petacc8 trial aims to increase the dfs in wild - type kras tumor patients. the trial has completed enrollment with final results to be presented at a later date. thus far, we have provided a general overview of the currently fda approved role for targeted agents in metastatic colorectal cancer as well as the investigational status of targeted agents in locally advanced colon cancer. overall, of the targeted agents discussed, many of them have been analyzed to a much smaller extent in rectal cancer versus that of colon cancer in phase recent data from 3 phase iii trials including nsabp r-04, star, and accord 12 [6062 ] have failed to provide a definitive role for oxaliplatin as a radiation sensitizer. targeted agents in combination with fluorouracil - based treatment continue to remain an area of investigation. randomised phase iii trials of neoadjuvant preoperative chemoradiation (crt) in resectable rectal cancer show that the addition of 5-fu to preoperative radiation increases the pathologic complete remission (pcr) rate over radiotherapy alone and improves loco - regional control but has not extended dfs or os [6, 6365 ]. several small phase i / ii studies have since been created in the hopes of improving the pcr rate further by using combined chemotherapy with cytotoxic chemotherapy or targeted agents. it should be noted that pcr remains a common endpoint for many of current and prior trials in development. to date, none of the targeted agents have proceeded to phase iii development as a radiation sensitizer with only one proceeding to the adjuvant setting of rectal cancer. given the data supporting the efficacy of bevacizumab therapy in metastatic colorectal cancer, it was postulated that combining bevacizumab with chemoradiation may increase antitumour efficacy by maximizing inhibition of the vegf pathway. in an early trial of bevacizumab plus infusional 5-fu and radiation in stage ii / iii rectal cancer, bevacizumab administered as a single agent 14 days prior to chemoradiation first reported on a phase i trial of bevacizumab in combination with preoperative 5-fu and radiation. a pcr rate of 16% was reported, and an additional 72% of patients had only microscopic foci remaining after treatment in their phase i / ii study of patients with t3/t4 tumors. reported results of a single institution phase ii trial of neoadjuvant bevacizumab, capecitabine, and radiation in 25 patients with locally advanced rectal cancer. the pcr rate was 32%, with an additional 24% of patients having near - complete responses (less than 10% viable tumor cells). toxicity was generally mild, although wound dehiscence was seen in 12% of patients (table 3). it should be noted that bevacizumab was to be evaluated in the adjuvant setting following definitive chemoradiation therapy in locally advanced rectal carcinoma patients (ecog 5204). however, this study closed prematurely due to slow patient accrual but would have closed eventually given the results of nsabp c-08. data from a variety of experimental models and human tumors suggest that egfr signaling promotes resistance to radiotherapy by activating cell survival signals through akt [73, 74 ]. moreover, retrospective analyses demonstrated shorter dfs and smaller pcr rates in patients with rectal cancers expressing egfr who were being treated with neoadjuvant radiotherapy [75, 76 ]. a landmark randomised phase iii study in patients with locally advanced head and neck cancer showed that cetuximab in combination with radical radiotherapy significantly improved overall survival compared to radiation alone. many mechanisms for this advantage have been proposed, including inhibition of repopulation during the latter phase of radiotherapy [79, 80 ]. treated 20 patients with ultrasound - staged (u)t3 to 4, clinical t4, or locally recurrent rectal cancer with weekly cetuximab and 5-fu during 5.5 weeks of pelvic radiotherapy, followed by an additional 4 weeks of cetuximab. cetuximab in conjunction with 5-fu and radiotherapy was feasible without synergistic or unexpected toxicities ; the pcr rate was 12%. according to the meta - analysis of 13 reports, the addition of cetuximab to fluoropyrimidine - based crt schedules suggest an overall pooled pcr of 9.1%, compared with an overall pcr rate of 13.5% seen with fluoropyrimidine - based chemoradiation schedules in a recent review (table 4). the pcr rates have been disappointingly low, perhaps because anti - egfr therapy is most effective in inhibiting the accelerated repopulation observed after the higher doses of radiotherapy used in patients with head and neck cancer. alternatively, cetuximab - induced inhibition of mitogenic signaling through the extracellular signal - regulated kinase (erk) pathway may mitigate the radiosensitizing effect of s phase - specific chemotherapy. cetuximab can lead to g1 or g2/m cell cycle arrest, and if only a small proportion of cells within the tumour are affected, this decrease in proliferation could impact the chance of achieving a pcr. this process might also affect oxaliplatin, which is mainly active in s phase but would be less likely to affect irinotecan. thus, preclinical data suggests that the sequencing of chemotherapy may have some significance but given the prolonged half - life of cetuximab, it may not necessarily apply to the clinical setting. in light of this hypothesis, a phase i / ii study (xerxes) created to compare a schedule of capecitabine - based chemoradiation with a cetuximab sandwich approach amended their protocol. panitumumab was administered before the start of crt, and every 2 weeks in combination with 5fu - oxaliplatin with concurrent rt(starpan / star-02 study). higher pcr rate in comparison with the results of previous neoadjuvant rectal cancer trials with antiepidermal growth factor receptor monoclonal antibodies supports further studies necessary to understand the possibility of optimal regimens and sequences with crt. though it would appear to be intuitive that a tyrosine kinase inhibitor of the egfr receptor would be of potential benefit, early data for their role in mcrc has not been defined and was determined to be more toxic [67, 86 ]. however, the small molecule tyrosine kinase inhibitors have radiosensitization properties. a phase i trial of a combination of erlotinib and bevacizumab with preoperative chemoradiotherapy of capecitabine demonstrated an impressive pcr(44%). similar preliminary results were also reported of a phase i / ii trial of bevacizumab and erlotinib in combination with 5-fu and pelvic radiotherapy for patients with clinical t3/4 rectal cancer. no dose - limiting toxicities were reported and erlotinib at a dose of 100 mg was chosen as the mtd. at the time of last followup, the reported pcr was 47% and there were no local recurrences reported in patients who completed study therapy. thus, based on these phase i / ii trials, bevacizumab and erlotinib in combination with 5-fu or capecitabine and radiotherapy appear to be well tolerated and a potentially active preoperative regimen for patients with larc. while modern rectal cancer trials with trimodality therapy have reported locoregional recurrence rates of less than 10%, especially with tme, systemic treatment can be delayed for up to 3 - 4 months following the original diagnosis [89, 90 ]. the problem, therefore, remains the persistent high rate of distant metastasis (3035%) in this disease [91, 92 ]. one such reason, the biological response of tumors to radiation, may provide explanation in that tumors that are not completely eradicated undergo accelerated repopulation.thus, integrating more effective systemic therapy into combined modality programs is the challenge and induction chemotherapy has the advantage of earlier administration of systemic therapy and may improve distant control. theoretically, tumour shrinkage with chemotherapy potentially allows improved tumour vascularity and improved oxygenation and higher intratumoural levels of cytotoxic drugs. therefore, newer generation chemotherapeutics as well as targeted agents (cetuximab, bevacizumab) have been incorporated into phase i - ii studies [95, 96 ]. interestingly, early results from the phase iii accord 12/0405-prodige 2 and star trial did not confirm a significant improvement of the primary endpoint, pcr rate, with the addition of oxaliplatin to preoperative 5-fu - based crt [91, 92 ]. however, induction - capecitabine - based ct prior to crt reported eventual pcr rates as high as 24% [97, 98 ]. in a randomized comparison of induction versus adjuvant capecitabine plus oxaliplatin, no difference in clinical outcomes was observed between the two treatment regimens, although the induction regimen resulted in a more favorable safety profile. to date, the 3-year dfs and os remain unchanged versus the control arm. in the avacross study, treatment consisted of bevacizumab and xelox, followed by concomitant radiotherapy plus bevacizumab and capecitabine. pcr was achieved in 36% of the patients but the rate of postsurgical complications and cardiac toxicity was not negligible. most were associated with wound - healing complications with 24% of patients requiring further surgery ; note that patients with a previous history of stable angina, arrhythmia, and coronary syndrome should be excluded. emerging evidence from several phase i - ii trials indicate that induction chemotherapy for rectal cancer patients is feasible and does not compromise crt or surgery, but only an adequately powered phase iii trial will answer the question definitively. an interesting multinational randomised phase ii study, expert - c trial (nct00383695), compares neoadjuvant therapy comprising oxaliplatin, capecitabine, and crt with or without cetuximab in 164 patients. it was designed on the basis of earlier single - arm phase ii study (expert), in which patients received 12 weeks of neoadjuvant capecitabine and oxaliplatin followed by chemoradiotherapy with capecitabine, tme, and 12 weeks of postoperative adjuvant capecitabine. radiological response rate after neoadjuvant chemotherapy and chemoradiotherapy was 89% (93/105) with a reported pcr of 20%. early results of the expert - c trial indicate that improved 3-yr os in patients receiving the investigational arm with crt was 96% compared with 81% among those who received neoadjuvant chemotherapy and chemoradiation alone. in the kras wild - type group, there was no statistically significant difference in pfs (81 versus 80%) and pcr (7 versus 11%). though a phase ii trial, the results of expert - c have potentially renewed interest of investigators for oxaliplatin and cetuximab in the treatment of rectal cancer ; results will need to be validated in a phase iii trial. several novel agents are being studied in phase iii trials for the treatment of metastatic colorectal cancer : aflibercept, ramucirumab, regorafenib, perifosine, and brivanib. of these, aflibercept and ramucirumab are specific vegf - directed therapies, whereas the remaining three are associated with approaches to intracellular signal blockade. aflibercept is a fully human, recombinant fusion protein that functions as a soluble decoy receptor for vegf, with affinity for vegf - a, vegf - b, and placental growth factor. the velour trial is a randomized, placebo - controlled study investigating the combination of folfiri plus aflibercept versus folfiri in the second - line treatment of mcrc. ramucirumab is another human monoclonal antibody directed against vegfr-2, which is considered to be the primary vegfr mediating the process of tumor angiogenesis [105, 106 ]. it is currently in phase iii development in 2nd - line metastatic colorectal cancer in combination with folfiri. regorafenib is a diphenylurea oral multikinase inhibitor that targets a variety of kinases implicated in angiogenic and tumor growth - promoting pathways and has been investigated as a single - agent activity in the phase iii correct trial in refractory mcrc. the final results of the correct trial noted improved os versus best supportive care alone (6.5 versus 5.0 mo, p <.0052). perifosine is an oral alkylphospholipid that inhibits several key signal transduction pathways, including akt, jnk, and nf-b. inhibition of nf-b signaling by perifosine has been reported to restore 5-fu chemosensitivity [110, 111 ]. the xeloda plus perifosine evaluation in colorectal cancer treatment (x - pect) trial with a target enrollment of 430 patients perifosine has been evaluated preclinically in prostate and glioma as a radiation sensitizer [113, 114 ]. brivanib is an oral receptor tyrosine kinase inhibitor that specifically inhibits the vegf and fibroblast growth factor (fgf) signaling pathways [115, 116 ]. recent phase iii data failed to show a benefit in os when brivanib was added to cetuximab in kras - wt metastatic crc patients. brivanib continues to be investigated in combination in a single institutional trial with irinotecan in an enriched patient population with a focus on plasma fibroblast growth factor (pfgf). currently, perifosine appears to be the only targeted agent in phase iii development with some potential as a radiation sensitizer based on preclinical studies. hence, though greater than 50 novel agents are being investigated in phase ii trials, only a small percentage of these agents will proceed to phase iii clinical trial development with a minority eventually investigated for their radiosensitization properties. therefore, it is important to identify predictive biomarkers for chemotherapy and radiation sensitivity, a task that should involve close cooperation and discussion between basic scientists, clinical, and translational investigators. early - phase ii and pivotal - phase iii studies in crc utilizing bevacizumab have identified hypertension (11%32%), bleeding (mainly epistaxis ; 30%53%), proteinuria (10%38%), arterial thromboembolism (ate) (1%10%), gastrointestinal perforation (0.3%2%), and wound healing (1.3%3.7%) as bevacizumab - associated adverse events. hypertension is the most commonly reported associated toxicity of patients treated with bevacizumab [9, 42 ]. the mechanism of bevacizumab - related htn is unclear and has been attributed to possible alterations in the nitric oxide signaling pathway and the endothelial dysfunction of the renin - angiotensin system [48, 119 ]. there are no clear guidelines for the management of hypertension in patients receiving bevacizumab, but patients who develop more than grade 2 htn during bevacizumab therapy should be managed using standard antihypertensive therapy. incidence of grade 3/4 proteinuria in recent phase iii trials with bevacizumab in metastatic crc have been reported in less than 2% of patients [9, 42 ]. the mechanism of proteinuria is not fully understood but may be related to the effects of vegf on the renal glomerular capillaries. it is advisable that patients with proteinuria of more than 2 + on a dipstick should have a 24-hour urine check for quantification of protein. if patients develop proteinuria of more than 2 g/24 hours, bevacizumab administration should be interrupted until proteinuria improves. patients should be evaluated for ace inhibitors or angiotensin - receptor blockers as initial treatment in the case of development of proteinuria with hypertension. bleeding from mucocutaneous membranes is common with bevacizumab and occurs in 20% to 40% of patients, the most significant type being gastrointestinal bleeding (6%) and the most common being self - limited epistaxis (46%). note that the brite study did not exclude patients on antiplatelet therapy or full - dose anticoagulation, so it is relatively safe in terms of risk of serious bleeding complications. thrombosis is a more potentially serious adverse event. in the pivotal phase iii trial in patients with metastatic crc evaluating the ifl regimen with and without bevacizumab, the incidence of all venous and ate was 19.4% versus 16.2% in the nonbevacizumab arm. it has been speculated that the blockade of the vegf receptors leads to apoptosis of endothelial cells in the vasculature, thus exposing the subendothelial collagen and initiating a coagulation cascade resulting in an increased risk for thrombus formation. thus, the food and drug administration (fda) described that the rate of ate is increased in patients with prior events of ate or patients aged more than 65 years during bevacizumab therapy. other less common but serious reported toxicities may include gastrointestinal perforation (2%) and wound - healing complications. patients who underwent surgery within 14 days of receiving bevacizumab are at a higher risk of developing wound healing complications. based on available data, bevacizumab should not be initiated within 30 days after surgery. elective surgeries should be planned 6 to 8 weeks after last dose of bevacizumab, though chemotherapy alone can be continued until 2 to 3 weeks before surgery. furthermore, it has been suggested that extended use of bevacizumab can increase the long - term risk of wound healing complications for up to 6 months after its cessation [55, 123 ]. the incidence of gi perforations may be slightly higher in patients with an intact primary tumor, prior adjuvant radiation therapy in rectal cancer, long - term nonsteroidal anti - inflammatory drug (nsaid) therapy (1 month of use), peptic ulcer disease, diverticulosis, and previous gastrointestinal surgery [9, 100, 124 ]. an extensive meta - analysis reported an incidence of gi perforation under bevacizumab treatment of < 1%, resulting in a mortality of 22%. reversible posterior leukoencephalopathy syndrome (rpls) has been reported in clinical studies (with an incidence of < 0.1%) and in postmarketing experience. rpls is a neurological disorder, which can present with headache, seizure, lethargy, confusion, blindness, and other visual and neurologic disturbances. the most frequently observed toxicity with the use of cetuximab is the development of a rash ; all patients experience some degree of xerodermatitis. the majority will develop a nonscarring, noninfectious, acneiform rash most prominent on the malar aspect of the face, chest, and back ; it is exacerbated by sunlight. but the severity of the rash has been correlated with improved response to cetuximab therapy. in view of this finding, a randomized, phase i - ii study (the everest trial) was developed, with the experimental arm consisting of dose escalation of cetuximab to rash development in those treated patients who failed to develop a grade 2 rash following initial exposure to cetuximab. while a higher radiographic response rate in the experimental arm of this trial (the cetuximab dose escalation to rash) was observed, this did not lead to an improvement in pfs or os advantage. cetuximab has also been frequently associated with renal magnesium wasting leading to hypomagnesemia, with this occasionally becoming symptomatic [129, 130 ]. when symptomatic, repletion with enteral or parenteral magnesium is indicated ; it is not clear that magnesium replacement is beneficial in an asymptomatic patient. paronychial inflammation and fissuring of the distal fingertips are a class effect of these agents. instances of elevated cases of allergic hypersensitivity reactions have been reported regardless of the premedications provided. the development of ige antibodies was noted and varied depending on the geographic location with greater instances in the in the southern usa. in contrast to cetuximab, which is a chimeric monoclonal antibody that may produce severe hypersensitivity reactions in some patients, panitumumab is a fully human monoclonal antibody and is less likely to result in allergic hypersensitivity reaction. pharmacoeconomic studies looking at health care systems have demonstrated that the cost per life - year gained with biologics is relatively high, compared with other interventions [133136 ]. for example, multidrug colorectal cancer treatment regimens containing bevacizumab or cetuximab cost up to $ 30,790 for eight - weeks of treatment, compared with $ 63 for an eight week regimen of 5-fl, the standard treatment until the mid-1990s [137, 138 ]. the cost of care task force of the american society of clinical oncology recently released a guidance statement emphasizing the importance of physician - patient discussion regarding the cost of care and the creation of decision - making tools to allow patients to make informed and educated decisions about their treatment. advances in rectal cancer therapy remain stagnant despite new existing cytotoxic and targeted agents in the treatment of metastatic colorectal cancer. traditionally, agents that have been incorporated into rectal cancer trials are those that have demonstrated promise in advanced and locally advanced colorectal cancer. however, to date, the role of cytotoxic agents as radiation sensitizers (i.e., oxaliplatin) have not demonstrated improved efficacy versus the standard of care, fluorouracil. similarly, targeted agents have a definitive role in the treatment of metastatic colorectal cancer. yet, its role in the treatment of rectal cancer as a radiation sensitizer has yet to be determined. in short, therapeutic developments in rectal cancer may lag behind because rectal carcinoma is not a commonly pursued area of pharmaceutical development. furthermore, whether pcr is the optimal endpoint remains controversial versus the more traditional outcome of dfs or os. retrospective analyses have suggested that pcr is a potential surrogate for dfs or os [71, 72 ]. targeted agents continue to be evaluated in the phase i / ii setting with promising potential but have yet to be proven to be superior to the standard of care, fluorouracil - based radiotherapy. currently, the use of targeted agents is best suited in the setting of a clinical trial. future developments in rectal cancer should focus on the identification of molecular factors that have prognostic and predictive significance to improve treatment outcomes. research efforts focusing on additional biomarkers will refine our ability to use these agents specifically in patient populations that derive a meaningful benefit. with concerns about health care costs, it is also critical to create a pharmacoeconomic framework guiding the clinical use of these agents. the introduction of new therapeutic agents and the discovery and validation of prognostic and predictive markers along with new screening tools will enable oncologists to tailor patient - specific chemotherapy by maximizing drug efficacy and minimizing adverse and possibly severe side effects. | targeted biologic agents have an established role in treating metastatic colorectal cancer (crc), and the integration of targeted therapies into the treatment of crc has resulted in significant improvements in outcomes. rapidly growing insight into the molecular biology of crc, as well as recent developments in gene sequencing and molecular diagnostics, has led to high expectations for the identification of molecular markers to be used in personalized treatment regimens. the mechanisms of action and toxicities of targeted therapies differ from those of traditional cytotoxic chemotherapy. targeted therapy has raised new insight about the possibility of tailoring treatment to an individual 's disease, the assessment of drug effectiveness and toxicity, and the economics of cancer care. this paper covers the last decade of clinical trials that have explored the toxicity and efficacy of targeted agents in locally advanced and metastatic crc and how their role may benefit patients with rectal cancer. future efforts should include prospective studies of these agents in biomarker - defined subpopulations, as well as studies of novel agents that target angiogenesis, tumor - stromal interaction, and the cell signaling pathways implicated in rectal cancer. |
our patient presented with accelerated silicosis starting as gradually progressive shortness of breath for about 1 year, progressing to chronic type 1 respiratory failure. this was followed by development of full blown mctd, with combined features of rheumatoid arthritis, scleroderma and systemic lupus erythematosus with severe arthritis, myositis and joint contractures. this was a case report of a 32-year - old stone crusher presented with gradually progressive breathlessness, initially on exertion and later, also at rest for 1 year. at the same time, he also noticed raynaud 's phenomenon involving the fingers and toes. over the next 6 months, he developed a gradual binding down of the skin over the dorsa of hands, forearms, face and leg. the patient also had severe joint pains predominantly involving all joints of the hands, including the wrist joint and the knees bilaterally. patient had been working as a stone crusher for the past 6 years. on examination, the patient had diffuse hyperpigmentation, visible respiratory distress with a respiratory rate of 34/min with mild pallor. arterial blood gas analysis revealed type 1 respiratory failure with pao2- 54 mmhg, ph - 7.38, paco2- 38 mmhg. testing for immune markers revealed antinuclear antibody (ana) positive-9 (if assay), anti - double stranded - deoxyribonucleic acid positive, anti - scl70 igg antibody positive 86 u / l, anti - u1 ribonucleoprotein (rnp) antibodies positive in high titers, anti - cyclic citrullinated peptide and rheumatoid factor positive. contrast enhanced computed tomography of the chest was suggestive of diffuse intra and interlobular septal thickening with fibrosis and randomly situated nodules [figure 1 ]. transbronchial lung biopsy was compatible with silicosis and revealed focal interstitial fibrosis interspersed with pigment laden macrophages and refractile material. low dose prednisonolone (20 mg od) was given in view of the arthritis. methotrexate at a dose of 25 mg weekly was also started along with proton pump inhibitors and physiotherapy. his hypoxia has also improved on therapy, but he continues to be on supplemental oxygen. some patients have features of more than one rheumatic disease and thus do not fit into traditional classification. patients with combination of clinical finding similar to those of systemic lupus erythematosus, progressive systemic sclerosis, polymyositis, rheumatoid arthritis and with unusually high titers of circulating ana with specificity for nuclear rnp are considered to have mctd. hypotheses implicating modified self - antigens and/or infectious agents in the pathogenesis of mctd have been advanced, but none have been proven yet. crystalline silica (quartz) particles less than 1 are the most pathogenic in silicosis. this constitutes a permanent stimulus for the fibroblasts, which increase their production of collagen leading to cutaneous sclerosis, vascular occlusion and pulmonary fibrosis, which explains the myriad clinical features of scleroderma and mctd. | silica exposure has been implicated with the development of various connective tissue diseases. we report a case of 32-year - old stone crusher who developed silicosis with mixed connective tissue disorder (mctd) 6 years after exposure to silica. this association of silicosis with mctd has never been reported from the indian subcontinent, although the problem of this pneumoconiosis remains rampant. this rare association urges us to report this case. |
in a recent search for offspring of consanguineous matings affected by autosomal recessive diseases, we came across four compound heterozygous patients among 38 affected children. this raised the question of whether this was an unexpectedly high proportion or not. in the past, when we reported about a first compound heterozygous cystic fibrosis (cf) patient with consanguineous parents, we showed that the proportion of affected children with two alleles not identical by descent (non - ibd) can be considerable (ten kate. however, alleles non - ibd may still be identical by state (ibs). so the affected compound heterozygous children are just a subset of the affected children who do not have both alleles ibd notwithstanding parental consanguinity. therefore, we wondered what proportion of non - ibd patients with consanguineous parents represent compound heterozygotes, and what proportion is non - ibd but still ibs. secondly, we wanted to know whether it is possible to calculate the overall pathogenetic allele frequency for an autosomal recessive disorder on the basis of knowledge of the proportion of compound heterozygotes among affected children of consanguineous parents. this might be a useful application as the current global prevalence of consanguineous marriage is estimated at 10.4%, (bittles and black 2009), with much higher percentages in many non - western countries. we start our exploration with the well - known formula to calculate the probability of the presence of a given autosomal recessive disease x in the children of a consanguineous couple (li, 1955). 1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ p(x) = fq + \left ({ 1 - f } \right){q^2 } $ $ \end{document } in this formula, f is the inbreeding coefficient and q is the total frequency of all pathogenic alleles causing disorder x. the fraction fq represents the proportion of affected children whose alleles are ibd, and the fraction (1f)q corresponds with the proportion of affected children whose alleles are not ibd. the fraction (1f)q is composed of two parts one part comprising the compound heterozygotes (ch), and the other part combining all homozygotes non - ibd (hn). the relative frequencies of the two sets within the fraction (1f)q are (in reversed order) 2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ r\left ({ \hbox{hn } } \right) = \sum\limits_{i = 1}^n { \mathop a\nolimits_i^2 }, { \hbox{and } } $ $ \end{document}3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ r\left ({ \hbox{ch } } \right) = 1 - \sum\limits_{i = 1}^n { \mathop a\nolimits_i^2 } $ $ \end{document } in eqs. 2 and 3, ai represents the relative frequency of the i allele. so its square, ai, is the relative frequency of homozygotes of the i allele non - ibd. from eqs. 1 and 3, it follows that the proportion of compound heterozygotes, p(ch), among affected children of consanguineous parents is 4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ p\left ({ \hbox{ch } } \right) = \frac{{\left ({ 1 - \sum\limits_{i = 1}^n { a_i^2 } } \right) \times \left ({ 1 - f } \right){q^2}}}{{fq + \left ({ 1 - f } \right){q^2 } } } $ $ \end{document } we can now calculate the expected proportion of compound heterozygotes, p(ch), if we know f, q, and the relative frequencies of the pathogenic alleles. conversely, knowing p(ch) by observation, as mentioned in the introduction, we can estimate r(ch), r(hn), and p(hn), if we know f and q, as follows : 5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ r\left ({ \hbox{ch } } \right) = \left ({ 1 - \sum\limits_{i = 1}^n { \mathop a\nolimits_i^2 } } \right) = \frac{{p\left ({ \text{ch } } \right) \times \left [{ fq + \left ({ 1 - f } \right){q^2 } } \right]}}{{\left ({ 1 - f } \right){q^2 } } } = \frac{{p\left ({ \text{ch } } \right) \times \left [{ f + \left ({ 1 - f } \right)q } \right]}}{{\left ({ 1 - f } \right)q } }, $ $ \end{document}6\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ r\left ({ \hbox{hn } } \right) = 1 - r\left ({ \hbox{ch } } \right),\,{\hbox{and } } $ $ \end{document}7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ p\left ({ \hbox{hn } } \right) = \frac{{r\left ({ \text{hn } } \right) \times \left ({ 1 - f } \right){q^2}}}{{fq + \left ({ 1 - f } \right){q^2 } } } = \frac{{r\left ({ \text{hn } } \right) \times \left ({ 1 - f } \right)q}}{{f + \left ({ 1 - f } \right)q } } $ $ \end{document } we can also calculate q from (4) or (5) if we know p(ch), f and r(ch) or the relative frequencies of the pathogenic alleles. 8\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ q = \frac{{p\left ({ \text{ch } } \right) \times \left ({ f + q - fq } \right)}}{{\left ({ 1 - f } \right) \times r\left ({ \hbox{ch } } \right)}}{,}\,{\hbox{from}}\;{\hbox{which}}\;q\;{\hbox{can}}\;{\hbox{be}}\;{\hbox{solved}}{. } $ $ \end{document } table 1 shows the dependency of the proportion of compound heterozygotes among affected offspring of consanguineous parents, p(ch), upon the parameters f, q, and r(ch) (see eqs. the examples given illustrate that p(ch) is positively correlated with r(ch) and q, and negatively with f, as expected. table 1expected proportions of compound heterozygotes among affected children of consanguineous parent, p(ch), given some values of f, q, and r(ch), the relative frequency of these compound heterozygotes among non - ibd affected childrenfqr(ch)p(ch)1/80.010.10.0070.50.0330.050.10.0260.50.1301/160.010.10.0130.50.0650.050.10.0430.50.2141/640.010.10.0390.50.1930.050.10.0760.50.380 expected proportions of compound heterozygotes among affected children of consanguineous parent, p(ch), given some values of f, q, and r(ch), the relative frequency of these compound heterozygotes among non - ibd affected children table 2 shows the results of calculations of the frequencies of homozygotes ibd and non - ibd among affected children of first cousins, and the total frequency of pathogenic alleles in the population in case of 10% compound heterozygotes and with different numbers and relative frequencies of pathogenic alleles. as the proportion of compound heterozygotes is fixed at 10% in this table, the row sum of the proportions of homozygotes ibd and non - ibd (third and fourth columns) add up to 90%. the table shows that knowledge of the proportion of compound heterozygotes, the inbreeding coefficient, and the number and relative frequencies of pathogenic alleles (first and second columns) allows one to calculate the total frequency of pathogenic alleles of a gene in the population (fifth column). not unexpectedly, the higher the frequency of the major allele, the higher is the frequency of homozygotes non - ibd and the higher the total frequency of pathogenic alleles in the population for a given frequency of compound heterozygotes among affected offspring of consanguineous matings. the same trend can be observed for children of second cousins (data not shown) and other levels of inbreeding. table 2frequencies of homozygotes ibd and non - ibd among children with an autosomal recessive disease whose parents are first cousins when 10% of these children are compound heterozygotes as well as total frequency of pathogenic alleles in the population for different numbers and relative frequencies of allelesinputoutputallelesfrequencies among affected childrentotal frequency of pathogenic alleles in the populationnumberrelative frequencyhomozygotes ibdhomozygotes non - ibd50.9 ; 0.07 ; 0.02 ; 0.007 ; 0.0030.4580.4420.0790.7 ; 0.2 ; 0,05 ; 0.03 ; 0.020.7860.1140.0180.5 ; 0.3 ; 0.1 ; 0.07 ; 0.030.8450.0550.0120.4 ; 0.3 ; 0.2 ; 0,08 ; 0.020.8580.0420.0110.2 ; 0.2 ; 0.2 ; 0.2 ; 0.20.8750.0250.01030.9 ; 0.07 ; 0.030.4570.4430.0790.7 ; 0.2 ; 0.10.7830.1170.0180.33333 ; 0.33333 ; 0.333330,8500.0500.01220.9 ; 0.10.4440.4560.0830.7 ; 0.30.7620.1380.0210.5 ; 0.50.8000.1000.017 frequencies of homozygotes ibd and non - ibd among children with an autosomal recessive disease whose parents are first cousins when 10% of these children are compound heterozygotes as well as total frequency of pathogenic alleles in the population for different numbers and relative frequencies of alleles since our observation of a compound heterozygous cf patient with consanguineous parents back in 1990, many more observations of compound heterozygotes in consanguineous families have been reported (summarized in petukhova. such patients present a problem to researchers using autozygosity mapping for identification of recessive disease genes. it may comfort parents, who thought or were told that their consanguinity was causally related to the disorder in their children, to learn now that their consanguinity can not be blamed for it. the same applies to some extent for parents who can be told that there is a considerable chance that the homozygosity in their affected child is not caused by alleles ibd. moreover, the mere existence of compound heterozygotes and the presence of non - ibd homozygotes among affected offspring of consanguineous parents may be of help in disputes with opponents of consanguineous matings, who are abundantly present in countries where consanguineous matings are not customary. as we have shown here, we can also learn more from the frequency of compound heterozygotes, as this frequency is related to the inbreeding coefficient, the number and relative frequencies of alleles, and their total frequency. while preparing the manuscript of this communication, we came across the paper of petukhova. these authors developed a formula to calculate the frequency of compound heterozygotes in the presence of inbreeding as we did, but unfortunately assumed equal frequencies of disease - causing mutations. as we have shown here, this is a serious omission and, moreover, far from realistic. a second difference with their paper is that we did not only calculate the frequency of compound heterozygotes, but turned the problem upside down by looking for inferences following from observed frequencies of compound heterozygotes. if f is known in a certain (sub)population, then the most straightforward way to estimate q would be via the prevalence of the disease in that (sub)population. in practice, however, f and the prevalence of the disease in a population are seldom known with any certainty. most of the times, they are unknown or the estimates are debatable because of large variances or possible biases. arriving at accurate and dependable estimates of both parameters takes a lot of effort and resources. for this reason, any method to estimate q from other sources, such as the one we describe, is an improvement. while estimating f in a population requires knowledge of the prevalence of consanguineous matings and the distribution of different degrees of consanguinity among them, estimating f from a small number of consanguineous families known to a laboratory in general is less of a challenge. once the total frequency of pathogenic alleles is known, the frequency of an autosomal recessive disease in a population, p(d), can be inferred from the total frequency of disease - causing alleles, especially when the frequency of consanguineous matings, c, is known as well, using the equation 9\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ p(d) = \left ({ 1 - c } \right){q^2 } + c\left [{ fq + \left ({ 1 - f } \right){q^2 } } \right ] $ $ \end{document } others have taken a different approach to calculate the frequency of a disease in the population by looking at the proportion of consanguineous parents among affected children and inferring from there, taking into account the frequency of consanguineous matings, the total pathogenic allele frequency and the total frequency of recessives in the general population (romeo. this method might result in a biased estimate if the presence of consanguinity of the parents alerts the physician to think of the possibility of a recessive disorder and diagnose accordingly, while this might not have been the case in the absence of consanguinity. starting from the proportion of compound heterozygotes gives an unbiased estimate and therefore at least represents an additional tool to determine disease frequency in the general population. firstly, inferences can only be made about the population to which the cases belong. if a population is non - homogeneous as to the frequency of consanguineous matings, population stratification has to be taken into account. secondly, for any recessive disorder, the number of compound heterozygotes among affected children of consanguineous parents will be limited. this means that estimates of the proportion of compound heterozygotes will tend to have rather wide confidence intervals, which will persist in derived figures. nevertheless, a provisional estimate of the frequency of pathogenic alleles using our method can be useful before embarking on larger studies, or as a check when other data are already available. | this short communication deals with the questions of how to calculate the expected proportion of compound heterozygous patients among affected offspring of consanguineous parents, and how, from an observed proportion of compound heterozygotes, to calculate both the proportion of homozygotes not identical by descent and the frequency of pathogenic alleles in the population. this estimate of allele frequency may be useful when dealing with populations with a considerable number of consanguineous matings. |
kaya or decoction is an ayurvedic dosage form, prescribed based on the stage of the disease according to the principles of ayurveda. this dosage form is traditionally prepared fresh and consumed on the same day but for the sake of convenience ; the process of preparation has been modified so that it can be stored with longer shelf life, easy availability and produced in large quantities. there is a need to understand the implications of this modification in terms of chemical changes. this work attempted to check the phytochemical profile of both freshly prepared decoction and commercially available decoction with reference to some analytical parameters like ph, total soluble solids, phenols, alkaloids, potassium and to assess the changes in the thin layer chromatography profiling of the decoction. the results showed that phenols and potassium are found to be two fold higher in freshly prepared decoction, compared to commercially available decoction diluted to dosage in practice (1:4 ratio). however, the total alkaloid content was found to be approximately ten fold higher in commercially available decoction. it was observed that the thin layer chromatography profile of decoctions was extracted into petroleum ether and chloroform was similar and consistent with different batches though the bands in commercially available decoction were slightly more intense compared to freshly prepared decoction. the total soluble solids in commercially available decoction were four times higher than freshly prepared decoction. the study reveals that there are differences in the phytochemical profiles of the freshly prepared decoction and commercially available decoction of the same formulation. however, the significance of these differences can be determined only by further clinical studies. on the other hand, the study lends support to the practice of diluting the commercially available decoction to make it equivalent to freshly prepared decoction. |
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a geographic region is indeed a complicated symbol of physical, biological and cultural processes. by analyze its elements and characteristics one can define the possible diseases that may occur by identifying a small place such as a house or a backyard, or through identifying the immigration route of birds and the viruses that are dispersed by them. increasing the world population and changing the world economy increase the risk of emergence of diseases, or the immunity against them. geographical variations have been studied under the intermediate fields of geographical pathology, medical ecology, medical topography, geographical epidemiology, geomedicine, etc. geographical perspective and method in studying health and illnesses ecological perspective of diseases or health maintained its scientific importance, even up to the mid-19 century that the microbic theory was developed. 18 and 19 centuries physicians (fennec-1792 - 1795, fosesh-1853, hiresh (1883 - 1886) used the term medical geography for the 1 time and had great efforts for describing and organizing data about human diseases, cultures and environments, continued the old method of hippocrates, indeed. characteristic skin lesions (keratosis, hyperpigmentation and hypopigmentation) are the hallmark of high exposure to arsenic, and nowadays, pose a public health problem in the world. these lesions may be used as an indicator of high exposure and are quite distinctive in contrast to other clinical manifestations of arsenic intoxication including weakness, conjunctiva congestion, edema, portal hypertension, and hepatomegaly. these skin lesions generally develop 5 - 10 years after exposure commences, although shorter latencies are possible. emerging evidence shows that the ingestion of inorganic arsenic may also lead to nonmalignant respiratory effects. limited epidemiological studies in chile (not mentioned in other countries, i.e., japan and taiwan) have previously suggested an association between arsenic and nonmalignant respiratory effects, and more recently another study from west bengal, india has suggested that long - term arsenic ingestion can cause pulmonary effects. they used samples of water, and soil then interpolate this data and compute the correlation of arsenic concentration and number of diseases. in this paper, we present respiratory system findings from a landmark epidemiological study that was conducted in iran, focusing on the relation between arsenic spatial dispersion and dyspnea diseases which were assessed in the survey. in this study, the first attempt was to gather resources and scientific and applied research in this field. we used information such as statistics of the dyspnea diseases in isfahan prepared over 3 years by isfahan province health center, map of the distribution of arsenic in isfahan province generated from combination of the mineral exploration organization maps and remote sensing technique. this map were prepared for the years that the statistics produced by dyspnea diseases then by using the statistics for ten common dyspnea diseases in isfahan, and entering this information by geographic information system (gis) in isfahan information maps the spatial distribution of common dyspnea diseases in the province was drawn and the created maps were adapted and compared with the map of the distribution of arsenic in isfahan province. remote sensing, using satellite image to extract data, provide an instrument to monitor the environment in general and particularly detect pollutions. in this research, we used remote sensing data and techniques to detect arsenic distribution in soil and also in ground waters. base on kirchhoff law in physic each material has a unique reflectance in the electromagnetic spectrum ranging from gamma to radio. this unique feature is called spectral signature and using this we can detect and separate different material from each other. arsenic is an element in the environment that can be found naturally in rocks and soil, water, air, and in plants and animals. although sometimes found in its pure form as a steel grey metal, arsenic is usually part of chemical compounds. these compounds are divided into two groups : inorganic compounds (combined with oxygen, iron, chlorine, and sulfur)organic compounds (combined with carbon and other atoms). inorganic compounds (combined with oxygen, iron, chlorine, and sulfur) organic compounds (combined with carbon and other atoms). inorganic arsenic compounds are found in industry, in building products (in some pressure - treated woods), and in arsenic - contaminated water. this is the form of arsenic that tends to be more toxic and has been linked to cancer. organic arsenic compounds are much less toxic than the inorganic arsenic compounds and are not thought to be linked to cancer. base on thornton (1996) the main compounds of arsenic are arsenates 60%, sulfides 20% and sulfosalts and the remaining 20% includes arsenic, oxides, and silicates. difference between spectral signatures of arsenic compounds there are different type of satellite images including hyperspectral, multispectral and radar each one has its pros and cons. hyperspectral sensors collect data with varied features such 20 - 100 m special resolution and in 50 - 200 spectrums, compare this with human eye which collect data in just three spectrum to understandability of those sensors to detect and identify different materials. multispectral sensors collect data in 3 - 10 spectrum and radar sensors use high wavelength, which can penetrate in surface. in this research, we used target detection algorithms to find arsenic distribution in isfahan. in the most cases, there is a mixture of materials in the area and the reflectance of each pixel is related to the proportion of material in that pixel. by knowing the spectral signature of a specific substance, target detection algorithms can detect proportion of that substance in each pixel. we implement the following target detection methods on esfahan images : constrained energy minimization, spectral angle mapper, adaptive coherence estimator, match filter, spectral similarity mapper and normalized euclidean distance then combined the result of those algorithms to achieve maximum accuracy. for this purpose, each method trained independently by using the map of arsenic mines then each pixel considered as contaminated if 4 of 6 algorithms show highly amount of arsenic and so on. figure 2 shows the result of target detection algorithms and density of arsenic in soil. density of arsenic in soil (g / kg) in the next step, we have to compute the amount of arsenic in underground waters. it is possible to create this map by using a dense sampling network and interpolating data. due to lockage of field data in this research we create the arsenic concentration in groundwater by using a model and calibrate it by available data. the researchers show that the flow length index and the presence of organic - rich sediments and alluvial deposits are the variables most positively correlated with the arsenic content, while the terrain altitude is highly negatively correlated. based on these researches we used linear regression to calibrate a model between flow length index [figure 3 ], terrain altitude and amount of arsenic in soil, computed by target detection method, as independent variables and ground water contamination as dependent variable. flow length index computed from digital elevation model using arc geographic information system ground water contamination with arsenic computed by model chronic arsenic (as) poisoning has become a worldwide public health issue. most human as exposure occurs from the consumption of drinking water and respiration containing high amounts of inorganic as (ias). chronic effects of ias exposure via drinking water and respiration include pulmonary lesions and dyspnea disease, neurological effects, hypertension, peripheral vascular disease, cardiovascular disease, diabetes mellitus, and malignancies including skin cancer. arsenic is an element in the environment that can be found naturally in rocks and soil, water, air, and in plants and animals. although sometimes found in its pure form as a steel grey metal, arsenic is usually part of chemical compounds. these compounds are divided into two groups : inorganic compounds (combined with oxygen, iron, chlorine, and sulfur)organic compounds (combined with carbon and other atoms). inorganic compounds (combined with oxygen, iron, chlorine, and sulfur) organic compounds (combined with carbon and other atoms). inorganic arsenic compounds are found in industry, in building products (in some pressure - treated woods), and in arsenic - contaminated water. this is the form of arsenic that tends to be more toxic and has been linked to cancer. organic arsenic compounds are much less toxic than the inorganic arsenic compounds and are not thought to be linked to cancer. base on thornton (1996) the main compounds of arsenic are arsenates 60%, sulfides 20% and sulfosalts and the remaining 20% includes arsenic, oxides, and silicates. there are different type of satellite images including hyperspectral, multispectral and radar each one has its pros and cons. hyperspectral sensors collect data with varied features such 20 - 100 m special resolution and in 50 - 200 spectrums, compare this with human eye which collect data in just three spectrum to understandability of those sensors to detect and identify different materials. multispectral sensors collect data in 3 - 10 spectrum and radar sensors use high wavelength, which can penetrate in surface. in this research, we used target detection algorithms to find arsenic distribution in isfahan. in the most cases, there is a mixture of materials in the area and the reflectance of each pixel is related to the proportion of material in that pixel. by knowing the spectral signature of a specific substance we implement the following target detection methods on esfahan images : constrained energy minimization, spectral angle mapper, adaptive coherence estimator, match filter, spectral similarity mapper and normalized euclidean distance then combined the result of those algorithms to achieve maximum accuracy. for this purpose, each method trained independently by using the map of arsenic mines then each pixel considered as contaminated if 4 of 6 algorithms show highly amount of arsenic and so on. figure 2 shows the result of target detection algorithms and density of arsenic in soil. density of arsenic in soil (g / kg) in the next step, we have to compute the amount of arsenic in underground waters. it is possible to create this map by using a dense sampling network and interpolating data. due to lockage of field data in this research we create the arsenic concentration in groundwater by using a model and calibrate it by available data. the researchers show that the flow length index and the presence of organic - rich sediments and alluvial deposits are the variables most positively correlated with the arsenic content, while the terrain altitude is highly negatively correlated. based on these researches we used linear regression to calibrate a model between flow length index [figure 3 ], terrain altitude and amount of arsenic in soil, computed by target detection method, as independent variables and ground water contamination as dependent variable. flow length index computed from digital elevation model using arc geographic information system ground water contamination with arsenic computed by model chronic arsenic (as) poisoning has become a worldwide public health issue. most human as exposure occurs from the consumption of drinking water and respiration containing high amounts of inorganic as (ias). chronic effects of ias exposure via drinking water and respiration include pulmonary lesions and dyspnea disease, neurological effects, hypertension, peripheral vascular disease, cardiovascular disease, diabetes mellitus, and malignancies including skin cancer. the population includes 16,238 medical records to patients suffering from dyspnea diseases in the isfahan province. the period studied, according to the number of samples is sufficiently reliable, and over 3 years (from 2009 to early 2011) was considered. this figure showed that the cities of isfahan, ardestan, kashan, naeein, and natanz have been allocated the highest dyspnea diseases. as shown in figure 5, the rate of dyspnea diseases, consistent with the distribution of arsenic in the province, and we can say those two parameters are related with square function and dyspnea diseases is reduced by leading away from areas containing arsenic. spatial distribution of dyspnea diseases in case study per 10,000 health criteria values for inorganic arsenic are summarized in table 1. the oral index dose (i d oral) in table 1 is based on the uk drinking - water standard for arsenic. this dose is estimated to be associated with an excess lifetime respiratory disease risk of around 40 - 400 in 100,000. an i d oral derived on the basis of minimal risk in accordance with the principles described in the toxicological framework report would lie in the range of 0.0006 - 0.003 g / kg bodyweight (bw) day-1. however, the uk drinking water standard for arsenic of 10 g / l is equivalent to a higher intake of approximately 0.3 g / kg bw day-1 [table 2 ]. recommended health criteria values for inorganic arsenic cities of isfahan province with the most consumed chemical fertilizers during 2009 - 2010 as it can be realized from the above table, most of the fertilizers are in the regions that the rate of arsenic dispersion is higher when compared to other areas. this could explain the reason for such frequency, to some extent [table 3 ]. spatial distribution dyspnea diseases commonly found in isfahan province and the most common diseases in isfahan province showed that the distribution dyspnea diseases have been consistent with the scatter of the arsenic in these points and these findings could represent a connection between the arsenic and the frequency of dyspnea diseases [figure 6 ]. these are caused by industrial pollutants, fertilizers and other agricultural items into the environment by humans. the distribution of arsenic in the areas where the population and human activities are higher, will be more evident and the main reason can be contaminated by industrial pollution from vehicles, used in producing gasoline, lead - acid batteries, electronic components, cable sheathing, ammunition, glass, ceramics, lead pipes, paints, alloys, connections and joints in roofs as a sealant for protection against rain, cited, inadvertently enter the environment. barrett1 in 1980 depicting the spatial distribution and etiology of the disease began surveying the course of the disease explained. he said for the 1 time that representing a geographic area is a place, and a symbol of the complex physical, biological and cultural process. if someone finds out, it is able to analyze the elements and patterns, it is possible that the disease can be determined, and spatial distribution of the disease can also be traced. this fact is true everywhere, identification of a small place like home to the intercontinental migration of birds and the viruses that are released by them. the world population growing up and the world economy changing, increase disease risk or protection since the most basic principles of combat and noncommunicable diseases, including dyspnea diseases, is the change in lifestyle, it seems for achieving this important result, providing cheap and reliable data, using remote sensing and gis, for decision maker is necessary. in order to create an environment that promotes appropriate behavior and healthy lifestyles and in the community with proper intervention, many factors can influence the risk of reduced or destroyed. even the minor changes can bring big benefits to be had, (butterfly effect). prevention through community intervention against risk factors for dyspnea diseases is possible by identification of harmful elements in the environment and by their distribution or the distribution of environmental contaminants or at least use them as well as other environmental variables. reduction of arsenic in products industries, improvement of work environments and health education among the people is necessary. similarly, for situations such as isfahan collaboration between geographical and medical sciences, health workers, legislators and the community is needed. government and professionals must ensure to be punctual and have the scientific and practical problems in dealing with the difficulties. to solve the global problem, the soil resources should be evaluated and tested periodically. in the case of toxic poisoning of the soil to such elements, preventive measures should be considered such as using the filters, lime and other things to prevent the entry of sewage into rivers and freshwater resources and the soil, up to the desired stage that its value can be compared and accepted to the standards defined by the world health organization and prevent further spread of disease from arsenic poisoning. cultivation of plants that absorb toxic elements that are suspended in air or soil, effectively, for example, borage or ox tongue plant, one element of soil containing arsenic, is refined and in these areas should not be used in food. the use of air filters can be very effective in cleaning the air in the house. correlation between arsenic concentration in soil (x - axis) and proportion of patients to population (y - axis) the findings of this study underscore the importance of preventing the exposure to arsenic, and to control arsenic products industries, to improve work environmental health and to increase the health professionals and public knowledge in this regard. in this research, we used combination of remote sensing and field date to extract arsenic concentration map and then used this map and water samples of wells to model arsenic concentration in underground waters. in esfahan province there are several structural project including two industrial town and a huge water canals and as we shown in our study there is a relation between the number of repository disease and arsenic contamination so at first we recommended authorities to consider arsenic concentration map in their decision making to prevent health problem and then restrict or increase population in polluted area. | background : as geographic science discusses the analysis of environment, human beings and their mutual relations, thus the field of medical geography consists of being inspired from the relations between these two factors, analyzing environmental factors, their identification them and the state of their effects on human health, as well as determining the location of these factors. some hazards that threat human health are the results of environmental factors and the relevant pollutions. some important categories of diseases including (shortness of breath or, dyspnea) have considerable differences in various places, as observed in their spatial prevalence and distribution maps.methods:the record of patients with dyspnea diseases were prepared for this descriptive research, for the period of 2009 - 2011, from the provincial health center, with the questionnaires were excluded patients with a family history of disease and the spatial diagram for disease prevalence was drawn according to the prepared data. the arsenic geographical distribution diagram in isfahan province was also prepared and then the relation between an element of arsenic in the province and the dyspnea diseases were analyzed.results:the analyses showed that the highest rate of arsenic is entered the soil via fertilizers to come eventually into the food cycle of humans. by analyzing the amount of used fertilizers in isfahan province and the dispersion diagram of arsenic in isfahan province, it was found that the highest frequency of arsenic is in places having agricultural base. the spatial dispersion of dyspnea diseases also showed that the spreading of dyspnea diseases is greater in places with higher scale of arsenic.conclusions:this study is a logical justification between the two diagrams to confirm the hypothesis regarding the effect of arsenic on dyspnea. |
onychomycosis is any nail plate infection caused by dermatophytes, yeasts and non - dermatophyte molds (ndms). the prevalence of onychomycosis is low in children when compared to adults due to reduced exposure to infected environments, less trauma due to smaller and thinner nail surface and faster linear nail growth. nevertheless, onychomycosis should be considered in differential diagnosis of nail plate disorders in children and even newborns. fusarium oxysporum is a ndm that has been described as a pathogen causing onychomycosis only in adults [46 ]. predisposing factors include family history of onychomycosis, habit of walking barefoot, hyperhidrosis, close contact with soil, frequent emersion of hands in water, hot humid climate, systemic immunossupression and diabetes. onychomycosis by fusarium oxysporum was found with high prevalence in immunocompetent patients in a study performed in brazil. we describe a case of onychomycosis by fusarium oxysporum diagnosed in a 60-days - year - old child whose placenta, umbilical cord and amniotic membranes were also infected. as far as the authors are concerned, this is the first case of fusarium oxysporum onychomycosis in a new born, acquired in utero. a 60-days - old caucasian girl weighting 4850 g was admitted at a pediatric infectology division (day 60) with fever without source and nail dystrophy with leukonychia of all nails (that her mother described being present since birth). the childs mother was an 18-year - old vertically hiv infected asymptomatic primigravida with immunosuppression (tcd4 lymphocytes at delivery of 101 cell/l). the mother was being treated with high active antiretroviral therapy (haart), which included zidovudine, lamivudine, tenofovir and lopinavir / ritonavir, but she stopped treatment 30 days before delivery. the baby was born (day 0) by cesarean section with intact membranes at 37 weeks of gestation, weighed 2655 g with apgar scores of 8 at 1 min and 9 at 5 min, and screening for other congenital infections were negative (syphilis, hepatitis b and c, toxoplasmosis). she received azt prophylaxis at birth and the first viral load (day+30) was undetectable. because all tests resulted negative and the fever persisted, she was clinically diagnosed with onychomycosis by candida and probable candidemia, when oral fluconazole (6 mg / kg / d) was started. a clipping of the fingernail showed hyphae on microscopic analysis, but culture was not performed by the time she was evaluated. two days after admission the baby was afebrile and then discharged being addressed to pediatric dermatology division. after 30 days of fluconazol (day+94) all nails have improved, but all the fingernails and the 1st and 2nd toenails bilaterally still presented yellow - white discoloration and thickening with involvement of the distal surfaces (fig. a second nail clipping was performed for microscopic analysis and again fungal elements were seen (day+94). as congenital fungal onychomycosis was under consideration microscopic evaluation of placenta, umbilical cord and amniotic membranes was made and revealed multiple fungal elements in all structures (fig. judging from nested polymerase chain reaction using specie - specific oligonucleotides and sequencing of its amplicons generated from total dna of nail clippings, placenta and umbilical cord, we confirmed fusarium oxysporum from samples collected on day-0. a tree was built using the its sequences which were deposited in genbank (fo01 km001721, fo02 km001722, fo03 km001723) which were compared with reference strains of fusarium genus (fig. total improvement of nails occurred after 60 days of fluconazole and 30 days of ciclopirox nail laquer 8% (day+124). the prevalence ranging between 1.45% and 45% and this great variation may be secondary to increasing numbers of immunosuppressed individuals and geographic differences in mold distribution. ndms are fungi that live in soil and plant debris and they are considered to be plant pathogens. they are not keratolytic and live on unkeratinized intercellular cement so they need previous keratin destruction by dermatophytes, trauma or any nail disease, but can also invade healthy nails. the clinical manifestations vary with the host immunity, as well as with the form of contamination. tosti and cols found that onychomycosis due to ndm resulted in proximal subungual nail alterations associated with painful periungual inflammation of the proximal nailfold. involvement of the nail is less severe in immunocompetent hosts and may lead to long evolution without complications, but disseminated infection has been described in immunosuppressed individuals. described here the nail plate invasion by the fungus was confirmed by microscopic analysis of the nail clipping but molecular study was needed for the identification of fusarium oxysporum. the incidence of human infection by this mold has been increasing in recent years, both in imunossupressed and immunocompetent patients. all ndms are not keratinolytic so they are usually regarded as a secondary invader of the nail plate. a predisposing factor such as immunosuppression or trauma may allocate the nail plate as a favorite location for invasion by the fungi. ahmadi refers that in some adult patients the only factor that may have increased the risk of infection by this type of fungus was working with water for a long period of time. moisture from the intrauterine environment and amniotic fluid contaminated by this agent may have been the determinant factors for nail infection by fusarium oxysporum in our patient. to the best of our knowledge this is the first case of onychomycosis by fusarium oxysporum in a newborn. in a previous study, infection by fusarium represented 0.09% of the onychomycosis in a 15 years period in tunsia. in another one, fusarium oxysporum was identified in 20% (34 cases), and the youngest patient was 20-years - old. ndms are difficult to eradicate by using combining systemic treatments, and fusarium onychomycosis was eradicated in only 40% of adult patients in a study performed in italy. the fact that the child was not infected with hiv may have contributed to good treatment response. in addition, the smaller and thinner nail surface and faster linear nail growth seen in children might have helped to eradicate the fungus. we wonder if the resolution would occur despite treatment, as it is known that azoles do not have activity against fusarium species or if the ciclopirox could have accelerated the spontaneous clinical resolution. we believe the same factors that determine the infection being restricted to the nail in congenital candidiasis can be extrapolated to congenital ndm infection. because we were able to detect fusarium oxysporum by molecular data, both in the placenta and the nail, and because signs of onychomycosis were present since birth, we assume the fusarium oxysporum, just like in the case of candida infection, was present in the mothers vagina and ascended to uterine cavity, invading amniotic membranes, contaminating amniotic fluid and finally invading placenta and umbilical cord, even the mother being asymptomatic. the mother was not treated for the fungal infection because of lack of symptoms during pregnancy and even at the time of the detection of the fungus in the placenta. in the present case, candida infection was first considered because onychomycosis by this agent was more probable in the clinical setting of the baby. additionally, candidiasis is frequent in hiv infected patients and her mother was hiv positive and immunosuppression was present at delivery. congenital candidiasis usually presents as a skin rash but can also affect skin appendages. in one case this infection was confined to nail plates and contamination was attributed to chorioamnionitis and transient amniotic fluid contamination. a 6-week - old female with nail candidiasis was described and onychomadesis induced by the stress of a breech presentation during preterm labor and candidiasis limited to the bands of onychomadesis was attributed to weakened nail plate substrate that increased susceptibility to candida infection. there were onycholysis, yellowish discoloration and roughness of the nail plates, hyperkeratosis and paronychia. involvement of several fingernails was present in all patients and several toenails in 2 cases. nail changes secondary to congenital candida infection were present at birth but also appeared between 2 and 6 weeks of life and there were no signs of immunosuppression in any of these cases. the authors stated that the direct contact of the fetus with the infected material may be the cause of congenital cutaneous candidiasis and the limited nail infection was attributed to flexion of fingers and fist that can result in entrapment of infected amniotic fluid around the nail bed. in conclusion, we describe the first case to our knowledge of congenital onychomycosis in a child caused by fusarium oxysporum. the infection being acquired in utero was proven by molecular methods with the identification of the fungus both in the nail and placenta, most probably as an ascending contamination / infection in a hiv - positive, immunosuppressed mother. we highly recommend considering ndm infection, including fusarium oxysporum, as the differential diagnosis of suspected congenital fungal infection in hiv exposed or immunosuppressed newborns. special attention should be given to the evaluation of the nails of hiv exposed newborns, for the purpose of early detection of potential life - threatening fungal infections. | fusarium oxysporum has been described as a pathogen causing onychomycosis, its incidence has been increasing in immunocompetent and disseminated infection can occur in immunosuppressed individuals. we describe the first case of congenital onychomycosis in a child caused by fusarium oxysporum. the infection being acquired in utero was proven by molecular methods with the identification of the fungus both in the nail and placenta, most probably as an ascending contamination / infection in a hiv - positive, immunosuppressed mother. |
according to the american cancer society, more than 178,000 women are affected by breast cancer every year ; international statistics reports estimated 1,152,161 new cases annually. this form of the disease is the leading killer of women between 40 and 55 years old and is the second leading cause of death overall in women. due to this, screening techniques allowing early detection and diagnosis have been studied in order to increase the chances of survival using less aggressive treatment [2, 3 ]. among the screening techniques currently available, mammography is the most often used, considered as the gold standard to breast tumor detection. however, this procedure is less effective when investigating dense breasts due to relatively high false negative rates. moreover, the number of unnecessary biopsies is very large and can lead to changes in the parenchyma making it difficult to read subsequent mammographic images. in recent years, ultrasonography has proven a valuable technique used as an adjunct to conventional mammography for the detection and classification of breast lesions. this procedure has been used to obtain additional diagnostic information, in order to reduce the number of unnecessary biopsies and assist with more accuracy the diagnosis of simple cysts (around 96100% of efficacy when both of these techniques are used together). an additional advantage of ultrasound is that it does not use ionizing radiation and therefore is useful especially for younger patients who tend to have dense breasts. the detection of abnormalities in medical images is a procedure prone to errors, even for qualified radiologists, due to the subjectivity in defining boundaries, overlap between benign and malignant characteristics, and the presence of artifacts that may confuse the diagnosis. in order to increase diagnostic accuracy and minimize such errors, computational tools have been developed to provide a second opinion for the specialist and assist in early detection of breast cancer. in this context, this work aims to develop a tool to aid the diagnosis based on the automatic detection of lesions in ultrasound images and the consequent classification of such finding as clinically suspicious (malignant) or not (benign) considering an analysis of their morphological characteristics. the database used in this research consisted of two distinct sets of breast ultrasound images. the first corresponds to images from breast phantoms and the second from conventional clinical examinations. the phantom images were acquired from tests performed by the group of innovation in medical instrumentation and ultrasound (giimus) from university of so paulo, brazil. the phantoms used in this research were bb-1 model (breast biopsy phantom, ats laboratories) and models previously developed by vieira.. all the phantoms were made of an acoustically tissue mimicking material and have a shape similar to the breast of an adult woman. all these phantoms were submitted to ultrasound beams from a ge logic - book xp portable device, operating in the frequency range of 110 mhz. a total of 144 phantom images in b - mode were acquired as those illustrated in figure 2. two medical centers of imaging diagnosis at so carlos, sp, brazil, provided clinical ultrasound images. four devices were used in the process of obtaining these images, siemens g50, medison x8, toshiba nemio 30, and general electric logiq p5, considering a broadband linear transducer of 7.510 mhz frequency range. figure 3 shows some examples of b - mode ultrasound images acquired with such different equipment. for each image, an experienced radiologist performed the analysis in order to detect lesions with suspicious appearance and then selected the regions of interest (rois). therefore, such a procedure resulted in 173 rois corresponding to actual clinical ultrasound images and 144 from phantoms images. in order to remove noise and to smooth the image components, rois have been preprocessed by a wiener filter, followed by the contrast enhancement (image equalization) and median filter. based on the variability in segmentation techniques, the efficacy was evaluated taking into account some techniques applied in order to highlight the lesion : active contour, region growing, fuzzy c - means, k - means, and som neural network [18, 19 ]. a postprocessing technique was then applied aiming to improve the segmentation quality since many pixels were verified to be disconnected from the actual lesion after the use of some of the techniques mentioned above. as a consequence, this effect has produced a more spiculated and noisier appearance than the nodule actually had. therefore, artifacts disconnected from the object of interest were eliminated and the internal valleys have joined the region. feature extraction in digital images is a critical step for identifying objects. in most cases, the use of more than one measure is required in order to decide to which class the pattern belongs. the most common is to extract from each sample several measures and then represent them through a vector, which will serve as an input to the classifier. in general, thus, based on the shape of the lesion as previously reported, 24 morphological features were extracted from each one. however, due to the large number of features considered, gaussian distribution curves were used regarding this features set optimization, so that it could accurately describe the identified object. in this procedure, each descriptor is normalized and gaussian curves are generated based on the distribution of values presented for each class (or just two for this work 's purposes). the most important is the level of curves intersection : the smaller its occurrence, the higher the probability of such feature representing such a category. furthermore, the distribution range of the values in the abscissa axis must be checked in order to determine the optimum distance between the classes. multilayer perceptron (mlp) neural network is a tool frequently used in differentiating between benign and malignant lesions. its topology consists of sensory units which include the input layer, one or more hidden layers (also known as intermediary), and an output layer. a supervised learning algorithm analyzes, through comparative actions between inputs and the desired output, the training data and produces an inferred function, which can be used for mapping new examples. an optimal scenario will allow for the algorithm to correctly determine the class labels for unknown instances. after performing comparisons in the learning method (the backpropagation), the synaptic weights are adjusted continuously reaching for convergence. in this step, the discrepancy between the responses produced by the network and the desired signal is evaluated. the network adjusts the values of the synaptic weights and this process is finished only when the error assumes an acceptable value. this procedure was performed through random partitioning of the dataset into two subsets : training and test. the training was accomplished only in phantom images due to the low number of malignant cases in actual clinical exams. thus, 144 rois from phantoms images were used for classification, 72 corresponding to benign and 72 to malignant images. from these 144 images, the first computational test was performed on a set of 80 rois from phantom images and 50 from actual clinic ones. after the roi selection, the following preprocessing techniques were applied : wiener filter, equalization, and median filter. figures 5 and 6 show some examples of this preprocessing effect on phantom and clinical images. in the second step, some segmentation techniques (as those previously mentioned in section 3) were applied in order to precisely delineate the lesions with smooth and regular edges. however, it was necessary to apply opening and closing morphological operators after segmentation to smooth the segmented lesion edge. manual segmentation provided by an experienced radiologist as well as some examples of the effects of segmentation techniques application and postprocessing on phantom images can be seen in figure 7. in order to confirm the validation of these procedures also to actual clinical images, some corresponding results are shown in figure 8. due to the visual subjectivity in evaluation to find the most accurate detector, measures were determined to quantify the distance between the edge automatically defined and that manually delineated by an experienced radiologist. hence, ten measures were evaluated, according to the descriptions in [2426 ]. the calculated values for the phantoms images dataset are shown in table 1 while table 2 reports those values but they were calculated for the actual breast images dataset. based on these data, the advantage of segmentation by active contours and by the som neural network can be noted in comparison to the others. in addition, when comparing both methods with the delimitation by the radiologist, they reported greater accuracy and low error rates. thus, both detectors were tested individually to gather the best classification results with phantoms as well as actual clinical images. the first step was the extraction of 24 morphological features only for the 144 phantoms images (72 corresponding to benign signals and 72 to malignant ones). then, the most relevant features were selected by means of gaussian distribution curves. among the 24 curves produced, those not evidencing appropriate visual results, that is, those with fully or partially overlapping areas, were discarded. just 8 gaussian distributions provided good partition for both the active contour segmentation and som, as shown in table 3. each of the 8 selected features by the gaussian distribution curves was individually introduced to mlp, but the results achieved were not significant. the amount of neurons in the single hidden layer varied from 1 to 9, and the learning rate was constantly adjusted between 0.1 and 0.9. during this step, the topology that achieved the best result for each detector is described in table 4. the accuracy rate in classification when the lesion was segmented by som was 94.2% and it was 95.6% when the lesion was segmented by active contour. figure 9 illustrates the respective gaussian distribution curves used for determining each descriptor selected by their overlapping analysis. after completing the classification procedure, we obtained the values of true positive (tp), false positive (fp), false negative (fn), and true negative (tn), as shown in table 5. in figure 10, roc curves regarding the classification of phantom images with mlp are shown as for segmentation by som network (a) as well as by active contour (b). the values calculated for the areas under the curve (auc) are displayed in their respective graphics. the performance of this classifier was compared to others previously described by correlate literature with similar purposes. table 6 shows the data previously presented by each one of those studies in comparison to our results mainly in terms of accuracy, sensitivity, and specificity rates. both classification proposals, with the contour segmented by som (case 1) and active contour (case 2), have yielded high sensitivity and specificity in breast lesions classification, similar to the works considered in table 6. the same accuracy was achieved for both cases and it was higher than all the results presented by such works. in our study, the sensitivity was higher in case 1, while the specificity was higher in case 2. additionally, it is important to stress that in our study the training and validation procedures were performed with phantom images, due to the small set of clinical images corresponding to the class malignant. these phantom images had structures of interest with a relatively regular shape and in some cases they were easily segmented. this aided in achieving high accuracy in both cases (for benign and for malignant simulated structures). in order to evaluate whether the classifier is able to generalize and reach similar results when applied to actual clinical images, tests with the second dataset were performed, taking into account the fact that it was trained with phantom images which have slightly different intrinsic characteristics. with this purpose, an experienced radiologist determined 173 rois which were automatically segmented by both techniques (i.e., active contour and som). then, measures of solidity, area ratio, and form factor were extracted from each roi. finally, these data were used by the neural network classifier with the topological configuration providing the best result for each detector (shown in table 4). the high fp rates are largely related to morphological differences between phantom images and actual ones, mainly the simulated structures in the phantoms under test with more rounded shapes. as a consequence, even with these morphological differences between the types of images, the classifier achieved good data generalization, reaching 100% of sensitivity and 78% of specificity when using segmentation by som. in the classification after segmentation by active contour, the sensitivity rate decreased significantly (only 63%) though specificity was almost the same : 79%. this difference is evident when the area under the curve is calculated ; corresponding results are shown in figure 12. the overlap of benign and malignant characteristics in interpreting ultrasound images turns the process subjective and tends to complicate the diagnosis of breast lesions. for this reason, cad schemes have emerged to improve the analysis of the radiologist by means of computerized characterization. some flaws however often arise in many cad schemes when evaluating images from breast ultrasound acquisitions mainly due to speckle noise influence on the lesions boundaries definition. testing five detectors techniques and measuring how close they were relative to the ground truth, only the som network and the active contour yielded significant accuracy rates. a differential of active contour technique is not requiring the preprocessing and the use of morphological operators to smooth edges. nevertheless, this final smoothing caused some changes in lesion limits, influencing the classification step. moreover, its algorithmic complexity requires many numerical operations and iterations until convergence of data has been reached. consequently, this leads to a high computational cost, making the processing relatively slow : about 30 seconds for each roi. the segmentation by som network on the other hand produced smoother contours and faster outcome results allowing a better understanding of the morphological differences between benign and malignant lesions. based on such a result, the classification by mlp was performed for both detectors. after extensive tests and topological changes, the classification taking breast phantom images with detection by active contour was more than 95% accurate, a rate higher than that for detection by som network (94.24%). however, the index of the classification with detection by active contour decreased significantly when applied to actual clinical images, which has registered an accuracy of 77.5%. therefore, the detection using the neural som network allowed better accuracy and data generalization when new images were introduced to the classifier. another important feature to be stressed is the contribution that breast phantoms ultrasound images have made to this investigation, since they constituted a manageable dataset with known materials and morphological characteristics. furthermore, as large variations related to the simulated lesion morphology can be provided, the dataset becomes useful for the network training before the tests with actual us images, especially with the new guidelines and recommendations for clinical use of ultrasound elastography. the system described here has an intuitive and easy interface, providing fast and accurate responses to the specialist. the tool is fully automated but allows, if necessary, user intervention to improve the segmentation process, due to manual change in the maximum and minimum parameters in the training. it is considered that the system has produced good results, acting as an important tool for the aid in diagnosis in breast ultrasound. | this research presents a methodology for the automatic detection and characterization of breast sonographic findings. we performed the tests in ultrasound images obtained from breast phantoms made of tissue mimicking material. when the results were considerable, we applied the same techniques to clinical examinations. the process was started employing preprocessing (wiener filter, equalization, and median filter) to minimize noise. then, five segmentation techniques were investigated to determine the most concise representation of the lesion contour, enabling us to consider the neural network som as the most relevant. after the delimitation of the object, the most expressive features were defined to the morphological description of the finding, generating the input data to the neural multilayer perceptron (mlp) classifier. the accuracy achieved during training with simulated images was 94.2%, producing an auc of 0.92. to evaluating the data generalization, the classification was performed with a group of unknown images to the system, both to simulators and to clinical trials, resulting in an accuracy of 90% and 81%, respectively. the proposed classifier proved to be an important tool for the diagnosis in breast ultrasound. |
with age, men experience decreases in important health indicators like muscle amount, physical activity, and sexual drive. furthermore, lower urinary tract symptoms (luts) are highly prevalent in males, and it has been clearly shown that the prevalence of luts increases with age. the incidence of erectile dysfunction (ed) also increases with age, as does the incidence of cardiovascular disease risk factors, such as hypertension, obesity, and diabetes. additionally, ed has a significant negative impact on quality of life (qol). satisfaction with sexual life and the existence of luts severely affect men 's qol and plague middle - aged men 's health. numerous studies have assessed qol in men with luts, benign prostatic hyperplasia (bph), and ed, but an estimation of middle - aged men 's health in comparison to other comprehensive clinical parameters has not been reported previously in korea. questionnaires are one of the most commonly used techniques for collecting health - related information in clinical studies because of their ease and simplicity. the international prostate symptom score (ipss) questionnaire is used worldwide to measure luts. to evaluate various voiding symptoms, perineal or suprapubic pain, and sexual disturbance, the national institutes of health chronic prostatitis symptom index (nih - cpsi) is used to assess chronic prostatitis (cp)/chronic pelvic pain syndrome. the androgen deficiency in aging males (adam), premature ejaculation diagnostic tool (pedt), and internal index of erectile function-5 (iief-5) questionnaires are used worldwide to evaluate men 's sex lives. by using these five questionnaires to evaluate detailed clinical problems, we proposed to research several factors affecting middle - aged men 's general health. we evaluated the relationship of various clinical parameters that are related to men 's general health and the symptom scores of five widely used questionnaires for detecting men 's health problems. we determined the predictive abilities of two obesity indexes and other clinical parameters for screening for luts and sexual dysfunction in middle - aged men. we believe that this study may not only provide a clearer figure of the suffering caused in middle - aged men 's qol, but also widen the public 's understanding of middle - aged men 's general health. between march and september 2013, a total of 1910 healthy korean men aged 40 to 59 years who participated in a health examination were included. all men underwent detailed clinical evaluations by use of the ipss, nih - cpsi, adam, iief-5, and pedt questionnaires. blood and urine samples were obtained for serum prostate - specific antigen (psa) and testosterone measurement between 0700 and 0900. the estimated glomerular filtration rate (egfr) was calculated and metabolic syndrome (mets) was evaluated. the egfr was calculated by using the abbreviated four - variable modification of diet in renal disease equation : gfr=186.3(serum creatinine)-1.154(age)-0.203(0.742 if female)(1.210 if black) mets was defined by using the criteria established by the national cholesterol education program - adult treatment panel iii published in 2005. mets was diagnosed when at least three of the following criteria were present : (1) waist circumference greater than 90 cm or body mass index (bmi) higher than 25 kg / m, (2) systolic blood pressure 130 mmhg or greater or diastolic blood pressure 85 mmhg or greater or use of antihypertensive medication, (3) fasting blood sugar greater than 110 mg / dl or self - reported diabetes medication use, (4) triglyceride greater than 150 mg / dl, and (5) high - density lipoprotein cholesterol less than 45 mg / dl or use of lipid medication. thereafter, all of the participants underwent a digital rectal examination (dre) to screen for prostate enlargement or nodules suggesting prostate cancer. next, transrectal ultrasound (trus) was performed and ultrasonographic inspection of the prostate was performed in each subject by using a 3.5-mhz transducer (voluson 730, general electronic, milwaukee, wi, usa). prostate volume was measured by using the prostate ellipsoid formula, multiplying the largest anteroposterior (height, h), transverse (width, w), and cephalocaudal (length, l) prostate diameters by 0.524 (hxwxlxii/6) by using trus. bmi was calculated as the weight (kg) divided by the square of the height (m), and waist - to - hip ratio (whr) was determined as the waist circumference divided by the hip circumference. waist circumference was measured by placing a tape measure snugly at the horizontal level between the subcostal margin and pelvic brim. exclusion criteria included presence of pyuria (white blood cells>5 on urinalysis), the presence of neurogenic bladder dysfunction, confirmed prostate cancer, a history of recurrent urinary tract infections or bladder stones, or any other history of lower urinary tract disorder influencing urination. in addition, individuals who used medications affecting prostate growth, such as anti - androgens and 5--reductase inhibitors, or who had abnormal findings on the dre were excluded from the study. t - tests were conducted to compare the differences in men 's general health scores according to other parameters, such as psa, testosterone level, egfr, and total prostate volume (tpv). chi - square or fisher exact test were conducted to compare the differences in mens ' general health scores according to mets, obesity, and central obesity. univariate and multivariate logistic regression analyses were performed for each group to determine the significance of age, psa, testosterone, egfr, and tpv as predictors of men 's health problems. the study protocol was approved by the institutional review board in national police hospital (seoul, korea ; irb registration number-11100176 - 201412-hr-008). between march and september 2013, a total of 1910 healthy korean men aged 40 to 59 years who participated in a health examination were included. all men underwent detailed clinical evaluations by use of the ipss, nih - cpsi, adam, iief-5, and pedt questionnaires. blood and urine samples were obtained for serum prostate - specific antigen (psa) and testosterone measurement between 0700 and 0900. the estimated glomerular filtration rate (egfr) was calculated and metabolic syndrome (mets) was evaluated. the egfr was calculated by using the abbreviated four - variable modification of diet in renal disease equation : gfr=186.3(serum creatinine)-1.154(age)-0.203(0.742 if female)(1.210 if black) mets was defined by using the criteria established by the national cholesterol education program - adult treatment panel iii published in 2005. mets was diagnosed when at least three of the following criteria were present : (1) waist circumference greater than 90 cm or body mass index (bmi) higher than 25 kg / m, (2) systolic blood pressure 130 mmhg or greater or diastolic blood pressure 85 mmhg or greater or use of antihypertensive medication, (3) fasting blood sugar greater than 110 mg / dl or self - reported diabetes medication use, (4) triglyceride greater than 150 mg / dl, and (5) high - density lipoprotein cholesterol less than 45 mg / dl or use of lipid medication. thereafter, all of the participants underwent a digital rectal examination (dre) to screen for prostate enlargement or nodules suggesting prostate cancer. next, transrectal ultrasound (trus) was performed and ultrasonographic inspection of the prostate was performed in each subject by using a 3.5-mhz transducer (voluson 730, general electronic, milwaukee, wi, usa). prostate volume was measured by using the prostate ellipsoid formula, multiplying the largest anteroposterior (height, h), transverse (width, w), and cephalocaudal (length, l) prostate diameters by 0.524 (hxwxlxii/6) by using trus. bmi was calculated as the weight (kg) divided by the square of the height (m), and waist - to - hip ratio (whr) was determined as the waist circumference divided by the hip circumference. waist circumference was measured by placing a tape measure snugly at the horizontal level between the subcostal margin and pelvic brim. exclusion criteria included presence of pyuria (white blood cells>5 on urinalysis), the presence of neurogenic bladder dysfunction, confirmed prostate cancer, a history of recurrent urinary tract infections or bladder stones, or any other history of lower urinary tract disorder influencing urination. in addition, individuals who used medications affecting prostate growth, such as anti - androgens and 5--reductase inhibitors, or who had abnormal findings on the dre were excluded from the study. t - tests were conducted to compare the differences in men 's general health scores according to other parameters, such as psa, testosterone level, egfr, and total prostate volume (tpv). chi - square or fisher exact test were conducted to compare the differences in mens ' general health scores according to mets, obesity, and central obesity. univariate and multivariate logistic regression analyses were performed for each group to determine the significance of age, psa, testosterone, egfr, and tpv as predictors of men 's health problems. the study protocol was approved by the institutional review board in national police hospital (seoul, korea ; irb registration number-11100176 - 201412-hr-008). among 1,910 participants, 1,899 patients completed urological and psychiatric evaluations and did not meet the previous exclusion criteria. the median age of the patients was 53 years, and the median values of bmi and whr were 24.8 kg / m and 0.93, respectively. bmi was categorized into two groups : nonobese (< 24.8 kg / m) and obese (24.8 kg / m) because 24.8 kg / m was the median value in the healthy volunteers. we also divided the patients into two groups by their whr, either above or below 0.93, the median value in the healthy volunteers. the data on the patients ' ages, serum psa levels, and testosterone levels and the results of the anthropometric measurements are summarized in table 1. in addition, the median scores on the ipss, nih - cpsi, adam, iief-5, and pedt are summarized in table 1. we examined men 's general health by using several clinical parameters, including obesity - related parameters. to analyze differences in men 's general health according to various clinical parameters, we divided the five questionnaire groups into two subgroups each depending on the degree of symptom severity (table 2). in this study, moderate to severe luts was defined as an ipss score8, and the group with moderate to severe luts had relatively greater tpv than did the group with no or mild luts (p=0.001). other clinical parameters were not significantly different between the two groups divided by severity of luts. moderate to severe nih - cpsi was defined as a score over 14, and inversely, those men showed a lower prevalence of mets (p=0.023) than did men in the group with no or mild cpsi. other clinical parameters, including obesity - related parameters, did not differ significantly between the two groups divided by severity of nih - cpsi. to evaluate the sex life of the patients, we divided the subjects by iief-5 score. moderate to severe ed was defined as a score less than 12. in this study, the group with moderate to severe ed showed a relatively higher prevalence of mets (p=0.012) and central obesity (measured by whr0.93) (p<0.001). there were no statistically significant differences in other clinical parameters between the two groups divided according to the severity of their iief-5 scores. for the two remaining questionnaires, adam and pedt, there were no significant differences between subgroups. we evaluated the predictive value of several factors for moderate to severe luts by looking at ipss scores over 7. multiple logistic regression analyses revealed that age and total tpv were independent predictors of moderate to severe luts in the multivariate analysis (p=0.001, odds ratio [or ], 1.034 ; 95% confidence interval [ci ], 1.014 - 1.056 ; p=0.010, or, 1.019 ; 95% ci, 1.004 - 1.033 ; respectively) (table 3) we evaluated the predictive value of several factors for moderate to severe cp - like symptoms by nih - cpsi scores over 14. as determined by logistic regression analysis, mets was the only significant negative predictive factor for moderate to severe cpsi scores (p=0.022 ; or, 0.747 ; 95% ci, 0.581 - 0.959) (table 4). we evaluated the predictive value of several factors for moderate to severe aging male 's symptoms by adam scores over 36. multiple logistic regression analyses revealed that age was the only significant independent predictor for moderate to severe adam score (p=0.018 ; or, 1.024 ; 95% ci, 1.004 - 1.045). the degree of ed was considered moderate to severe when the score on the iief-5 questionnaire was 11 or lower. the logistic regression analysis showed that age and mets were independent predictive factors for moderate to severe ed in the multivariate analysis (p<0.001, or, 1.121 ; 95% ci, 1.087 - 1.156 ; p=0.036, or, 1.323 ; 95% ci, 1.018 - 1.720 ; respectively). whr had a statistically significant value for predicting moderate to severe ed in the univariate analysis (p=0.016 ; or, 47.683 ; 95% ci, 2.080 - 1,093.027) (table 6). thus, higher age, existence of mets, and central obesity measured by whr had predictive ability for ed. among 1,910 participants, 1,899 patients completed urological and psychiatric evaluations and did not meet the previous exclusion criteria. the median age of the patients was 53 years, and the median values of bmi and whr were 24.8 kg / m and 0.93, respectively. bmi was categorized into two groups : nonobese (< 24.8 kg / m) and obese (24.8 kg / m) because 24.8 kg / m was the median value in the healthy volunteers. we also divided the patients into two groups by their whr, either above or below 0.93, the median value in the healthy volunteers. the data on the patients ' ages, serum psa levels, and testosterone levels and the results of the anthropometric measurements are summarized in table 1. in addition, the median scores on the ipss, nih - cpsi, adam, iief-5, and pedt are summarized in table 1. we examined men 's general health by using several clinical parameters, including obesity - related parameters. to analyze differences in men 's general health according to various clinical parameters, we divided the five questionnaire groups into two subgroups each depending on the degree of symptom severity (table 2). in this study, moderate to severe luts was defined as an ipss score8, and the group with moderate to severe luts had relatively greater tpv than did the group with no or mild luts (p=0.001). other clinical parameters were not significantly different between the two groups divided by severity of luts. moderate to severe nih - cpsi was defined as a score over 14, and inversely, those men showed a lower prevalence of mets (p=0.023) than did men in the group with no or mild cpsi. other clinical parameters, including obesity - related parameters, did not differ significantly between the two groups divided by severity of nih - cpsi. to evaluate the sex life of the patients, we divided the subjects by iief-5 score. moderate to severe ed was defined as a score less than 12. in this study, the group with moderate to severe ed showed a relatively higher prevalence of mets (p=0.012) and central obesity (measured by whr0.93) (p<0.001). there were no statistically significant differences in other clinical parameters between the two groups divided according to the severity of their iief-5 scores. for the two remaining questionnaires, adam and pedt, there were no significant differences between subgroups. we evaluated the predictive value of several factors for moderate to severe luts by looking at ipss scores over 7. multiple logistic regression analyses revealed that age and total tpv were independent predictors of moderate to severe luts in the multivariate analysis (p=0.001, odds ratio [or ], 1.034 ; 95% confidence interval [ci ], 1.014 - 1.056 ; p=0.010, or, 1.019 ; 95% ci, 1.004 - 1.033 ; respectively) (table 3) we evaluated the predictive value of several factors for moderate to severe cp - like symptoms by nih - cpsi scores over 14. as determined by logistic regression analysis, mets was the only significant negative predictive factor for moderate to severe cpsi scores (p=0.022 ; or, 0.747 ; 95% ci, 0.581 - 0.959) (table 4). we evaluated the predictive value of several factors for moderate to severe aging male 's symptoms by adam scores over 36. multiple logistic regression analyses revealed that age was the only significant independent predictor for moderate to severe adam score (p=0.018 ; or, 1.024 ; 95% ci, 1.004 - 1.045). the degree of ed was considered moderate to severe when the score on the iief-5 questionnaire was 11 or lower. the logistic regression analysis showed that age and mets were independent predictive factors for moderate to severe ed in the multivariate analysis (p<0.001, or, 1.121 ; 95% ci, 1.087 - 1.156 ; p=0.036, or, 1.323 ; 95% ci, 1.018 - 1.720 ; respectively). whr had a statistically significant value for predicting moderate to severe ed in the univariate analysis (p=0.016 ; or, 47.683 ; 95% ci, 2.080 - 1,093.027) (table 6). thus, higher age, existence of mets, and central obesity measured by whr had predictive ability for ed. because qol has improved in recent years, more attention is being paid to diseases that influence qol rather than to only life - threatening diseases. luts, ed, and premature ejaculation are such diseases that affect qol in elderly men. to further our understanding about the seriousness of middle - aged men 's health, we assessed the severity of men 's urinary tract symptoms, sexual function, and premature ejaculation by using validated questionnaires. over the past decades, many different groups have investigated the influence of obesity on the development of bph and luts with conflicting results. most of these groups concluded that overall obesity, abdominal obesity, and whr can increase the risk of bph and luts, at least to some degree. in our study, to minimize the effect of comorbidity by senility, the age of patients was confined to 40 to 59 years, representing middle - aged men. a total of 1899 healthy korean men aged 40 to 59 years were included, and the age range was selected because it encompassed the aged most closely associated with prostatitis and ed. first, we showed that clinical parameters including obesity - related parameters did not differ significantly between the groups divided by ipss, nih - cpsi, iief-5, adam, and pedt scores, except for tpv and mets. our results indicated that the group with moderate to severe luts had a relatively greater tpv. second, the group with moderate to severe nih - cpsi scores had a lower prevalence of mets. third, according to iief-5, the group with moderate to severe ed showed a relatively higher prevalence of mets and central obesity as measured by whr. fifth, mets was the only significant negative predictive factor for moderate to severe cpsi scores. we could not find a definite reason for the negative relationship between mets and cp - like symptoms in our study. however, we suspect that effects of dietary and healthy behaviors could be important issues in this relationship. suggested that cp / chronic pelvic pain syndrome (cpps) is a multifactorial disease. cigarette smoking, a high - calorie diet with low fruit and vegetable consumption, constipation, slow digestion, a sexual relationship with more than one partner, and coitus interruptus were more likely in patients with cp / cpps than in controls. in addition, zhang. reported that higher leisure - time physical activity was associated with a lower risk of cp / cpps. patients diagnosed with mets often give a lot of attention to their eating habits and lifestyle. further studies are needed to better characterize the etiopathogenesis in individuals to define the optimal approach to diagnosis and treatment. sixth, we revealed that age was the only significant independent predictor for moderate to severe adam scores. finally, we revealed that aging, the existence of mets, and central obesity measured by whr had predictive ability for ed. interestingly, we found no evidence that higher bmi in middle age is associated with an increased risk of luts, ed, or other related men 's health concerns. overall, there are clear advantages of using whr ; it is convenient and simple to measure and an easy metric in diagnosing not only the sequelae normally associated with mets, but also voiding dysfunction. whr may therefore be both useful and important as a measure of success for possible future treatment options for patients with ed. active reduction of waist circumference via treatment of mets in addition to treatment of pelvic inflammation and ischemia may lead to durable and material improvement in ed. other studies have also shown that weight loss significantly improves insulin sensitivity, erectile function, and testosterone levels in men. to our knowledge, this is first study simultaneously assessing several clinical parameters including obesity - related parameters and the five questionnaires for assessing middle - aged men 's health. we studied a large number of subjects in a population - based study, and there are few asian studies in which more than 1,500 subjects were analyzed. this is the first large - scale study of korean middle - aged men using the five validated questionnaires. the number of patients in this study was large enough to achieve a representation of middle - aged men. although several studies have shown an association of luts and obesity, there are few studies regarding the effects of the five validated questionnaires representing men 's urinary symptoms and sexual function according to several clinical parameters. our data showed median values and several functional status indexes of middle - aged korean men. although the five questionnaires that we used were validated, studies based on questionnaires have limitations in that they do not include basic disease development and limited information can be obtained, particularly regarding diagnostics. because we only used the five questionnaires to assess luts and sexual function, we lacked the detailed medical and social history of the patients. another limitation of this study was that the data were cross - sectional and did not provide information regarding the temporal cause - effect relationship between the clinical parameters and middle - aged men 's general health. the baseline demographics of the men enrolled in this study were not fully inspected or randomized ; thus, the baseline comorbidity of each patient may have affected the patients ' responses to the questionnaires. because this study consisted of data from a single institution, there may be a potential selection bias as well. in the future, local population - based studies and randomized controlled trials it is meaningful that this study showed a relationship of several clinical parameters with middle - aged men 's general health in a population - based study. our data showed that tpv is a significant predictor of luts, and our results indicated that age and tpv were associated with the severity of luts symptoms. it is suggested that patients with mets are not prone to cp - like symptoms. also, we showed that aging, the existence of mets, and central obesity measured by whr had predictive ability for ed. whr is a convenient and simple measurement, so it may therefore be useful and important as a measure of success for future treatment. | purposethere is no reported evidence for an anthropometric index that might link obesity to men 's sexual health. we evaluated the ability of an anthropometric index and the symptom scores of five widely used questionnaires to detect men 's health problems. we determined the predictive abilities of two obesity indexes and other clinical parameters for screening for lower urinary tract symptoms and sexual dysfunction in middle - aged men.materials and methodsa total of 1,910 middle - aged men were included in the study. participants underwent a detailed clinical evaluation that included recording the symptom scores of five widely used questionnaires. the participants ' body mass index and waist - to - hip ratio were determined. serum prostate - specific antigen, urinalysis, testosterone, estimated glomerular filtration rate, evaluation of metabolic syndrome, and transrectal ultrasonography were assessed.resultsby use of logistic regression analysis, age and total prostate volume were independent predictors of lower urinary tract symptoms. metabolic syndrome was the only significant negative predictive factor for chronic prostatitis symptoms. age and metabolic syndrome were independent predictive factors for erectile dysfunction. waist - to - hip ratio had a statistically significant value for predicting erectile dysfunction.conclusionsour data showed that total prostate volume is a significant predictor of lower urinary tract symptoms, and central obesity has predictive ability for erectile dysfunction. metabolic syndrome was the only significant negative predictive factor for chronic prostatitis - like symptoms. the management of correctable factors such as waist - to - hip ratio and metabolic syndrome may be considered preventive modalities against the development of men 's health problems. |
dengue epidemiology in rio de janeiro in 2008 - dengue transmission in rio de janeiro is strongly associated with summer (december - april), when higher temperatures and intense rainfall are frequent (honrio., the city of rio de janeiro suffered one of its most severe dengue outbreaks when denv-2 and denv-3 co - circulated. between january - april 2008, more than 155,000 cases and 110 deaths were reported and an incidence of 2,453.7/100,000 inhabitants was observed (teixeira. the epidemic reached its peak in march and april, with 86,036 and 90,945 human cases reported, respectively, and had ended by june (malho. aegypti survival in the laboratory - to estimate the effect of dengue infection on ae. aegypti longevity and how that effect may vary with mosquito size, we reanalysed data reported by sylvestre. (2013), who gave a full description of experimental conditions and methods. briefly, we orally challenged females with denv-2 at a viral titre of 2 x 10 tcid50. aegypti populations : paea (a population maintained as laboratory colony at oswaldo cruz institute, rio de janeiro, for almost 13 years) (sylvestre. 2013) and a field population from rio de janeiro. 2013) reported that 93.3% of females challenged in this way had detectable dengue in the head, suggesting disseminated infection. after being orally infected, females were maintained individually in plastic vials (6.5 cm height, 2.5 cm diameter) with cotton soaked in a 10% sucrose solution ad libitum and an oviposition substrate at the bottom for egg laying. during the longevity study one blood meal on an anesthetised mouse [oswaldo cruz foundation ethical committee for animal use (ceua l-0007/09) ] was offered to mosquitoes once a week. after death, wing length (axillary incision to wing tip, excluding fringe) (alto. aegypti field collections - mosquito collections started in march 2008 during the dengue outbreak and continued until june 2009. aegypti were collected in 23 districts that encompassed three municipalities in metropolitan region of rio de janeiro (supplementary data). district selection was done together with the health municipal secretary, which directed collectors to neighbourhoods in which dengue transmission had recently become intense. at each field - site, we randomly choose 10 - 20 houses per day for mosquito collection using backpack aspirators or bg - sentinel traps (clark. mosquito collections began after explaining the objectives of this research project to householder and receiving their oral consent to use an aspirator or a mosquito trap to collect mosquitoes. aspirating was performed for 15 - 20 min per house, depending on the premise area, and was also done in the peridomestic environment. bg - sentinel traps were installed inside houses and remained in the same house for a 24-h trapping period. mosquito preparation - all mosquitoes collected in the field (1,586 males and 1,213 females) were brought to the laboratory for identification using taxonomic keys (consoli & loureno - de - oliveira 1994). aegypti females had their wing length measured from axillary incision to wing tip excluding the fringe (alto. 2008b). each adult adult females from all 23 sites were individually macerated in 1 ml of leibovitz l-15 medium (sigma) plus antibiotics (penicillin - streptomycin, 10,000 units - invitrogen) and centrifuged (6,000 rpm at 4c for 30 min). supernatant was transferred to an eppendorf tube containing 100 ml of streptomycin / fungizone and penicillin, kept in an ice bath for 1 h and centrifuged (3,000 rpm at 4c for 15 min). supernatant was transferred to an eppendorf tube containing 0.3 ml of foetal calf serum (fcs) (invitrogen) and frozen (-70c) (de castro. virus isolation - virus isolation was performed by inoculation into monolayers of c6/36 ae. albopictus cells (igarashi 1978) in leibovitz l-15 medium (sigma) supplemented with 2% fcs (invitrogen) and 0.2 mm of nonessential amino acids (invitrogen). cells were incubated at 28c for five - seven days and observed for cytopathic effects (de castro. isolates were identified by indirect fluorescent antibody test (ifat) using dengue serotype - specific monoclonal antibodies (gubler. 1984) and infected supernatant was clarified by centrifugation and virus stocks stored in 1-ml aliquots at -70c. rna extraction and reverse transcriptase polymerase chain reaction (rt - pcr) - viral rna was extracted using qiaamp viral rna mini kit (qiagen) following the manufacturer s instructions. rt - pcr for detecting denv was performed as described previously (lanciotti. 1992). statistical analysis - laboratory - for the laboratory longevity study, time to death was analysed by a proportional hazards model with classification effects of population (field, lab) and dengue infection (yes, no) and continuous effects of wing length (mm) and wing length(mm) (proc phreg) (sas 2011). we initially tested a model with all interactions of the class variables and wing length and wing length. all interactions were far from significant (p > 0.15) and this full model yielded a poorer information theoretic index [akaike s information criterion with a correction for small sample sizes (aicc) ] (burnham. similarly, effects of population on longevity were also far from significant (p > 0.15) and a model with population yielded a poorer aicc ; hence our final analysis was a reduced model including only dengue infection, wing length and wing length. this same model also was the endpoint of using the stepwise option in proc phreg (sas 2011). aegypti were collected from march to early june 2008, corresponding to epidemic period reported by the ministry of health (malho. aegypti field collected during this epidemic period, testing effects of date of collection (expressed as days post 1 march 2008) and (date of collection) and the random effect of site for 545 individuals from all 23 sites. we used a mixed model anova (sas proc mixed) (sas 2011). aegypti females and we analysed only these sites for frequency of dengue infection because we could not be sure dengue was active at a site unless we detected it. we analysed the relationship between frequency of dengue infection and wing length (mm) and (wing length) across these 13 sites. all models tested included date of collection (days post 1 march 2008) (date of collection) and the random effect of site for 492 individuals from these 13 positive sites. we used a mixed effects generalised linear model (proc glimmix) (sas 2011), employing an information theoretic approach to model comparison (anderson 2008) using aicc. a major advantage of this approach is quantification of weight of evidence (wi) for different models (anderson 2008, burnham. we tested three models that included wing length as both linear and quadratic terms, wing length as a linear term only and no wing length term. we used a binary distribution of the dependent variable dengue (yes, no), with a logit link function (sas 2011). we used multimodel estimation (anderson 2008) to estimate weighted average consensus predicted probabilities of infection and their weighted standard errors (se), as a function of wing length and date of collection based on all the models evaluated and weighting model contributions by wi (anderson 2008). for all 23 sites, including those with no dengue - positive females, we determined mean female size (wing length) and proportion of females infected. we then tested for a relationship across sites of proportion dengue - infected and mean female size per site using pearson s correlation (proc corr) (sas 2011). laboratory - sizes of the 134 females used in the laboratory study ranged from 2.10 - 3.20 mm wing length, with a mean se of 2.89 0.01 mm. infection with dengue significantly shortened the life of females [= 7.71, degrees of freedom (df) = 1, p = 0.0055 ], with a hazard ratio of control / infected of 0.560, indicating that controls had nearly half the hazard of death as infected individuals. both wing length (= 4.06, df = 1, p = 0.0439) and (wing length) (= 4.81, df = 1, p = 0.0283) were significantly related to the hazard of death. the linear term (se) was negative (-20.81 10.30), indicating that hazard of death initially declines with adult size. the quadratic term was positive (4.09 1.90), indicating that hazard of death increases as larger sizes are reached. thus this analysis describes a hazard function of size that is significantly concave upward. predicted survival curves for infected and uninfected females of wing lengths = 2.10, 2.70 and 3.30 mm (fig. 1) indicated that longevity peaked at 2.70 mm and was actually least at 3.30 mm. fig. 1 : predicted survivorship curves for adult female aedes aegypti in the laboratory that are not infected with dengue (a) or infected with dengue (b). statistical analysis reported in the results section. plotted wing lengths were chosen to span the range of sizes of females included in the experiment. field - sizes of 545 field collected females ranged from 1.60 - 3.95 mm wing length, with a mean se of 2.69 0.01 mm. wing length varied significantly among sites (z = 2.24, p = 0.0124), but was unrelated to date of collection, as a model omitting date of collection, and only including site yielded the best aicc. frequency of dengue infection was best predicted by a model with wing length as a linear effect (table i) and this linear effect was positive (slope se = 1.24 0.56, t 476 = 2.20, p = 0.0285). wi indicated that the model with linear + quadratic effects of wing length also had some support, given this data set, based on aicc [and, indeed, any of the other information theoretic indices reported by sas institute (2011) ]. for the quadratic model, the linear effect of wing length was again positive (linear coefficient se = 9.33 7.33, t 475 = 1.27, p = 0.2034), whereas the quadratic effect was negative (quadratic coefficient se = -1.46 1.32, t 475 = 1.11, p = 0.2688). the model omitting wing length entirely was substantially less likely as a model generating these data (table i). predicted frequencies of dengue infection for these two most likely models, holding the random site effect constant at the value for the valqueire collection (supplementary data, fig. 2a, b, table ii) show that in both cases predicted frequency of dengue infection increases from wing lengths of 2.0 mm to about 3.5 mm. at wing lengths from 3.5 - 4.0 mm the quadratic model predicts frequency of dengue infection declines, though never to the low levels observed for the smallest females (fig. we expected frequency of dengue infection to be related to day of collection and the relationship proved to be quadratic, with frequency first increasing to a peak in the middle of the epidemic, then declining (fig. table imixed effect generalised linear models for the relationship of wing length to frequency of dengue infectionmodel effectsaiccaiccexp[-0.5(aicc)]wi wing, day, day, site301.87010.5608wing, wing, day, day, site302.470.600.74080.4155day, day, site308.206.330.04220.0237sum--1.7830-all considered models included linear and quadratic effects of date of collection and the random effect of site. weight of evidence (wi) expresses exp[-0.5(aicc) ] as a proportion of the sum of that column and indicates the evidence for the model (i.e., the probability that the model is the correct one). aicc : akaike s information criterion with a correction for small sample sizes. all considered models included linear and quadratic effects of date of collection and the random effect of site. weight of evidence (wi) expresses exp[-0.5(aicc) ] as a proportion of the sum of that column and indicates the evidence for the model (i.e., the probability that the model is the correct one). aicc : akaike s information criterion with a correction for small sample sizes. 2 : predicted frequencies of dengue infection among field collected female aedes aegypti from rio de janeiro, brazil. all models included a random effect for collection site (n = 13 dengue positive sites). the predicted relationships are shown holding site effect constant at the value for collections from valqueire (supplementary data, table ii). statistical assessment of support for linear (a) and quadratic (b) models of frequency vs. wing length (mm) is given in table i. uncertainty of predicted frequencies given in table ii. table iiestimates of standard error (se) of prediction for models of the relationship of frequency of dengue infection to wing lengthwing (mm)modelmodel (wi)predicted frequency (dengue)conditional se predictedmodel selection uncertaintyaverage predicted frequency (dengue)unconditional se predicted2.0wing, wing 0.41550.0780.0620.0010.1120.335wing0.56080.1450.0860.011 - 0.02370.2750.1160.0553.0wing, wing 0.41550.3990.1470.0740.2990.547wing0.56080.3680.1410.059 - 0.02370.2750.1160.0224.0wing, wing 0.41550.2200.3520.0040.3480.590wing0.56080.6670.2080.260 - 0.02370.2750.1160.014all models include date and date. for prediction, the random effect of site was held constant at the value for valqueire (supplementary data), which had an intermediate frequency of dengue - positive females (0.1719) and date was held constant at 21 april (middle of the epidemic). unconditional se includes that uncertainty plus model selection uncertainty weighted by evidence (wi). for prediction, the random effect of site was held constant at the value for valqueire (supplementary data), which had an intermediate frequency of dengue - positive females (0.1719) and date was held constant at 21 april (middle of the epidemic). unconditional se includes that uncertainty plus model selection uncertainty weighted by evidence (wi). for details multimodel consensus prediction of frequency of dengue infection, based on the weighted averages of the three models (table ii) indicated that frequency of dengue infection likely increased monotonically across the range of wing lengths from 2.0 - 4.0 mm. multimodel predictions, particularly at the largest wing lengths, had high uncertainty (table ii) due to both inherent error of prediction and model selection error (anderson 2008). site - wide frequency of dengue infection was not significantly correlated with mean wing length for a site (fig. 3), but highly variable and unlikely to be linear. fig. 3 : relationship of frequency of female aedes aegypti infected with dengue [standard error (se), binomial formula ] and mean female body size (se) for 23 sites in rio de janeiro, brazil, and vicinity during the dengue epidemic of 2008. our data support the competition - longevity hypothesis and suggest that the competition - susceptibility hypothesis does not account for patterns of dengue infection of these vectors in nature. however, a central point of this paper is that the frequency of dengue infection in the field may have a complex relationship to vector body size. thus, it is important to determine that empirical relationship and to understand the ecological processes that cause heterogeneity in frequency of infection of vectors. though our data refute the competition - susceptibility hypothesis, we do not reject the physiological mechanism underlying that hypothesis (i.e., physiological effects of competition and body size on probability of dengue infection given an infectious blood meal). that mechanism has been demonstrated in laboratory studies of infection of aedes with dengue (alto. our data indicate, however, that the demographic effects of competition (longevity of adults) are more important in nature than these physiological effects of competition (susceptibility to infection). in the epidemic in rio aegypti indicates that longevity is not linearly related to size, but rather shows a quadratic relationship, with maximal predicted longevity at intermediate sizes (wing lengths of 2.7 mm) and lower longevity at smaller or larger sizes. typically mosquito ecologists assume, based on some data (e.g., hawley 1985, reiskind & lounibos 2009), that adult aedes longevity is positively related to body size. some field studies have failed to find a relationship (e.g., scott. 2000, maciel - de - freitas. 2007) and others find evidence for non - monotonic relationships (e.g., hawley 1985). our laboratory study of longevity had a relatively limited range of female wing lengths represented (2.1 - 3.2 mm) compared to our field collected females (1.6 - 4.0 mm) ; nevertheless this range was sufficient to show the nonlinear relationship of size to longevity. in the field, frequency of dengue infection increases with female size and the linear model was the best description of our field data. even if we accepted the quadratic model of frequency of dengue, it yields a peak predicted frequency of dengue for females between 3.0 - 3.5 mm wing length (relatively large females) (fig. wing lengths 3.5 mm represented less than 1% of all females collected for this study, which indicates that for the vast majority of females in the field in rio de janeiro during the epidemic, frequency of dengue infection increases with size for both the linear and quadratic models (fig. if the decline in frequency of dengue among this largest 1% of females is real, its cause remains unknown. that decline may result from low crowding of larvae during development, yielding very large, less physiologically susceptible adults, as observed by alto. (2008a, b). alternatively, low crowding may yield very large, but short - lived adults, as observed in our laboratory analysis. reduced longevity of small ae. aegypti adults from crowded larval conditions was evident at adult body sizes at or below 2.6 mm mean wing length (reiskind & lounibos 2009), which suggests that the very small females < 2.5 mm wing length, which constituted 25% of our field sample, may be expected to have significantly reduced longevity relative to larger females. in contrast, based on logistic regressions, estimated physiological susceptibility to dengue infection and disseminated infection in the laboratory decline rapidly with increasing size for female ae. aegypti across the range of wing lengths from 2.0 - 3.8 mm (alto. this wing length range encompasses about 99% of the field collected females in our study. as we observe increased frequency of dengue infection across virtually all of that size range, we conclude that the field patterns we observe are not consistent with the competition - susceptibility hypothesis and that the demographic mechanisms postulated in the competition - longevity hypothesis are most important in producing patterns of dengue infection with body size in the field. aegypti clearly vary considerably in size in nature and this variation is related to probability of infection at the individual level. we found little evidence that inter - site variation in size, which was significant, was related to local frequency of dengue infection at the site level. thus, individual variation does not obviously relate to larger - scale site - level patterns of risk of dengue. whether spatial variation in dengue risk is associated with female body size and the ecological conditions that produce that size variation at smaller spatial scales (e.g., the urban block or the house level) remains to be tested it is also well established that susceptibility to dengue infection has a genetic component (black. aegypti varies due to environmental factors beyond larval density and competition [e.g., temperature (muturi & alto 2011) ]. thus, for genetically competent populations, we expect larval density and other environmental factors to foster heterogeneity in susceptibility to dengue infection among adult females. whether genotypic variation in dengue susceptibility interacts with environmental effects remains to be investigated. beyond the present study, our results indicate that there are important patterns of frequency of dengue infection among ae. larger females appear to be the most dangerous dengue vectors during the outbreak in rio de janeiro. this suggests that larval conditions that yield large, robust adult females are those that are most likely to produce the most dangerous individual vectors of dengue. uncrowded aquatic habitats for larvae, with ample food, thus seem likely to be sources of vectors that pose the greatest threat. this indicates an important future direction for research in vector - borne disease : quantifying factors creating heterogeneity of disease vectors. not all individual vectors are alike in their likelihood of infection and disease transmission and approaches to limit vector - borne disease need to consider the ecological conditions, particularly during larval development for vectors with complex life cycles that produce the most dangerous individuals. a similar concept is already enshrined in general ecology of host - pathogen interactions (lloyd - smith. 2012) where some individual hosts are considered superspreaders because of their disproportionately high tendency to spread the infection to other hosts. similar effects are likely for insect vectors of human and animal disease, even though those effects may be more difficult to detect among the large numbers of individuals in vector populations. even more important than understanding the lack of homogeneity of vectors (or hosts) for contributions to disease transmission is the identification of measurable traits, such as body size, that can be used to identify those individuals that are the most likely to transmit pathogens. this can enable us to identify the ecological conditions that are likely to produce those most dangerous vectors and thus may aid in limiting the transmission of vector - borne disease. | two hypotheses for how conditions for larval mosquitoes affect vectorial capacity make opposite predictions about the relationship of adult size and frequency of infection with vector - borne pathogens. competition among larvae produces small adult females. the competition - susceptibility hypothesis postulates that small females are more susceptible to infection and predicts frequency of infection should decrease with size. the competition - longevity hypothesis postulates that small females have lower longevity and lower probability of becoming competent to transmit the pathogen and thus predicts frequency of infection should increase with size. we tested these hypotheses for aedes aegypti in rio de janeiro, brazil, during a dengue outbreak. in the laboratory, longevity increases with size, then decreases at the largest sizes. for field - collected females, generalised linear mixed model comparisons showed that a model with a linear increase of frequency of dengue with size produced the best akaike s information criterion with a correction for small sample sizes (aicc). consensus prediction of three competing models indicated that frequency of infection increases monotonically with female size, consistent with the competition - longevity hypothesis. site frequency of infection was not significantly related to site mean size of females. thus, our data indicate that uncrowded, low competition conditions for larvae produce the females that are most likely to be important vectors of dengue. more generally, ecological conditions, particularly crowding and intraspecific competition among larvae, are likely to affect vector - borne pathogen transmission in nature, in this case via effects on longevity of resulting adults. heterogeneity among individual vectors in likelihood of infection is a generally important outcome of ecological conditions impacting vectors as larvae. |
canine intestinal parasites have recently become a more important pathogen for humans, as some parasites, e.g., giardia (g.) intestinalis, toxocara (t.) canis and others, have become potential public health hazards due to zoonotic transmission. in particular, private household dogs that have relatively close contact with humans represent a serious potential source of direct parasitic transmission. although human parasitic diseases are well controlled in japan, many people are still contract zoonotic parasites. epidemiological data from dogs can undoubtedly contribute to preventing direct zoonotic transmission from dogs to humans via the control of infectious animals. in japan, recent reports have indicated that the prevalence of intestinal parasites in private household dogs kept in the same area has declined in comparison with previous years [1,12 - 14 ]. however, the epidemiological background is extremely important for the interpretation of results relating to canine intestinal parasites, since prevalence differs significantly according to the profiles of surveyed dogs [3 - 7 ]. unfortunately, previous long - term surveillance studies in japan [1,12 - 14 ] have lacked data analysis based on multifactorial considerations in regard to epidemiological backgrounds. previous our reports [3 - 7 ] suggest that the considering of high detective factor may be helpful for the comparison of data among differentiated studies. the aim of this investigation was to determine the prevalence of g. intestinalis and other intestinal parasites (isospora spp., t. canis, ancylostoma (a.) caninum, trichuris (t.) vulpis and strongyloides (s.) stercoralis) in private household dogs of hachinohe region in aomori prefecture, japan in 1997, 2002 and 2007. data were analyzed based on consideration of epidemiological backgrounds such as canine ages and origin. fresh fecal specimens were collected from 420, 350 and 335 private household dogs presented to individual veterinary clinics regardless of illness history and living in the hachinohe area of tohoku region in aomori prefecture of japan, in 1997, 2002 and 2007, respectively. hachinohe is situated in the northern part of japan, with approximately 300,000 people living in this area. parasitic agents in stools were detected using the formalin - ethyl acetate sedimentation technique as previously described. obtained data was analyzed by considering the high detective factor complex for each parasitic agent, and statistical significance of results was calculated using fisher 's exact probability test. values of p 15.0%) were recorded every year. and t. canis also had no changes in the high detective factor complex group, with the prevalence > 10.0% every year. until the present study was performed, the authors believed that the recent regional prevalence of intestinal parasites in private household dogs had decreased compared to 10 years ago, since the knowledge of owners regarding canine intestinal parasites and parasitic zoonoses has been increased provably. several papers in japan have actually shown that the prevalence of intestinal parasites in private household dogs had decreased compared to past years [1,12 - 14 ]. unexpectedly, the prevalence of all intestinal parasites studied had not changed over the past 10 years in the present study, when data was analyzed based on the concept of epidemiological high detective factor complex. the present results indicate that profiles in surveyed dogs are important for the interpretation of data from epidemiological studies on canine intestinal parasites. although the prevalence of isospora spp. protozoa in the present study outwardly showed a significant decline associated with the studied year in terms of overall prevalence, the real prevalence under the consideration of epidemiological factors showed no changes. differentiation of surveyed canine populations may produce different results, as many factors related to dogs are mixed, and comparison under identical conditions is impossible. data on canine intestinal parasites obtained without considering canine profiles seems to supply only limited information, and sometimes induces meaningless but apparently significant differences among the results. when zoonoses are discussed, epidemiological backgrounds, including information of dog - human contact are more important in public health. even if the prevalence of zoonotic parasites in dogs is high, the problem will be minimal if contact between infected dogs and humans is infrequent. in contrast, a serious problem is obviously present for humans if there is frequent close contact, even if prevalence is low. certain indicators are needed to compare epidemiological data on canine intestinal parasites from differential populations. the concept of high detective factor complex, which is supported by the results of previous reports [3 - 7 ]. a surprising finding was that the protozoan g. intestinalis in the high detective factor complex group (under 1 year old, derived from pet shops / breeding kennels and kept indoors) recorded a high prevalence continuously over the past 10 years, with a peak of 26.2% in 2007. although there has been no clear evidence of giardia transmission from dogs to humans, there have been reports of suspected human cases of giardiasis from a canine source. moreover, household dogs have the possibility of higher prevalence of g. intestinalis infection than seen in the present results. this is because recent previous studies have demonstrated that enzyme - linked immune absorbent assay and polymerase chain reaction techniques are more sensitive to detecting giardia infection than the convenient microscopic examination method such as used in the present study. however more sensitive methods were unused in the present study, because the certain technique was needed to evaluate the prevalence among the differentiated years. emphasis should be placed on the fact that g. intestinalis infected dogs are often in very close contact with humans, creating a scenario for direct transmission. t. canis is most famous nematode among the canine intestinal parasites, and the potential for zoonotic transmission is commonly recognized around the world. since numerous anthelmintic drugs for t. canis are sold in pet shops and home centers in japan, owners are easily able to obtain and treat their animals. however, the prevalence in the group with a high detective factor complex (under 1 year old, bred by private owners and kept outdoors) has been steady (> 10%) over the past 10 years. the risk of transmission through the contamination of t. canis eggs in the field has clearly not been reduced in the area of the present study. the importance of regular fecal examination in household dogs needs to be recognized more among owners and clinical veterinarians. although s. stercoralis is also a zoonotic nematode in close contact with humans according to the epidemiological profile, prevalence has been low over the past 10 years. s. stercoralis was detected to a limited extent in dogs derived from pet shops / breeding kennels under 1 year old. this suggests that s. stercoralis transmission may occur continuously among young dogs in pet shops / breeding kennels. infections, in that the infected dogs were almost all young dogs from pet shops / breeding kennels. a. caninum and t. vulpis are mainly detected in older outdoor dogs, and the prevalence has not changed over the past 10 years. contamination of fields via a. caninum and t. vulpis eggs is suspected to have contributed to the lack of change in the region of the present study. in conclusion, the present results indicate that the prevalence of g. intestinalis and other intestinal parasites in private household dogs has not always decreased, and the potential for parasitic zoonotic transmission from dogs to humans seems to remain at a higher level than we initially presumed. we recommend careful surveillance of intestinal parasites and aggressive use of anthelminthic in private household dogs under considering the epidemiological factors. | an epidemiological study on canine intestinal parasites was undertaken to evaluate changes in the prevalence among private household dogs from the hachinohe region of aomori prefecture, japan, in 1997, 2002 and 2007, using the formalin - ethyl acetate sedimentation technique. the risk of zoonotic transmission from household dogs to humans was also discussed. all intestinal parasites detected in the present study (giardia intestinalis, isospora spp., toxocara canis, ancylostoma caninum, trichuris vulpis and strongyloides stercoralis) showed no changes in prevalence over the past 10 years based on analysis considering canine epidemiological profiles. in particular, prevalence of giardia intestinalis in dogs under 1 year old, derived from pet shops / breeding kennels and kept indoors was unchanged, remaining at a high level of > 15.0% at each time point. toxocara canis also showed no changes in the group of dogs under 1 year old, bred by private owners and kept outdoors, and the prevalence was > 10.0% every year. the present results indicate that the prevalence of giardia intestinalis and other intestinal parasites in private household dogs has not always decreased, and the potential for direct parasitic zoonotic transmission from dogs to humans may be relatively high level, than from the environment (indoors and outdoors). we recommend careful surveillance of intestinal parasites and aggressive use of anthelminthic in private household dogs under considering the epidemiological factors. |
there are more than 150,000 bariatric surgeries performed in the united states each year.1) about two - thirds of patients presenting for bariatric surgery have a history of some psychiatric disorder,2) and around 4.0% of bariatric candidates suffer from bipolar disorder.2,3) over one - third of patients who undergo bariatric surgery take some psychiatric medication.2,4) lithium continues to be used as a first - line treatment for bipolar disorder and, given its narrow therapeutic window, changes in lithium pharmacokinetics following bariatric surgery is of particular interest. we report a case of lithium toxicity after sleeve gastrectomy that illustrates the potential need for closer monitoring of lithium levels following bariatric surgery. the established association between morbid obesity and psychiatric disease2,3,5) makes understanding how bariatric surgery affects the pharmacokinetics of psychotropic drugs of utmost importance. an 18-year - old female with a history of bipolar disorder, anxiety disorder, and bulimia nervosa, initially presented to the emergency department complaining of fatigue, diarrhea, and tremors. the patient was taking 900 mg of lithium daily (300 mg morning, 600 mg evening). she was also taking citalopram 30 mg once daily and ziprasidone 160 mg once daily. the patient had lost 70 pounds since starting her bariatric surgery consultation program 6 months prior to presentation. since her surgery, she had intermittent symptoms of vomiting and nausea, which contributed to poor fluid intake. the patient was admitted for dehydration and acute kidney injury, and aggressive intravenous hydration was initiated. lithium level was found to be 2.7 mmol / l, and lithium toxicity was diagnosed. mmol / l, potassium 3.1 mmol / l, chloride 96 mmol / l, bicarbonate 20 mmol / l, blood urea nitrogen (bun) 53 mg / dl, creatinine (cr) 2.19 mg / dl and hemoglobin (hb) of 11.7 g / dl. the patient s condition deteriorated dramatically on the second day of admission and she became very lethargic and obtunded. despite having received 5 l intravenous fluid, the lithium level remained elevated at 2.6 mmol / l (bun 44 mg / dl and cr 1.50 mg / dl). serum sodium, potassium, chloride and bicarbonate all normalized, while the hb level declined to 10.3 g / dl. on examination, two courses of hemodialysis were subsequently performed, and by the third day of her admission her serum lithium was 1.4 she continued to exhibit agitation, restlessness, and confusion with inability to follow 1-step commands. chest and abdominal computed tomography (ct) scan showed no significant pathology ; head ct showed no evidence of bleeding or stroke. hospital course was further complicated by thyroid storm, nephrogenic diabetes insipidus, and pneumonia. the patient was discharged 19 days after admission at which time no neurologic sequelae were noted. although not well studied, concerns over alterations in the pharmacokinetics of lithium following bariatric surgery have been documented in the literature. notably, there have been two cases of lithium toxicity following roux - en - y gastric bypass.6,7) the mechanism behind our patient s lithium toxicity is likely multifactorial. the patient had acute kidney injury, as evidenced by her increased creatinine, as well as likely secondary decreased fluid intake and dehydration. one study found the incidence of acute kidney injury in individuals who underwent bariatric surgery to be 5.8%.8) given that lithium is primarily renally excreted, it is clear that an alteration in hydration status affecting kidney function would lead to decreased lithium clearance. dehydration occurs frequently during the six months following bariatric surgery, owing to the mechanically restricted stomach limiting fluid intake. additionally, weight loss itself can contribute to changes in the pharmacokinetics of lithium. in one study, the clearance of lithium in obese individuals was found to be significantly greater than that of a control group (33.97.0 ml / min versus 23.06.2 ml / min ; p=0.005).9) the 31.75 kg weight loss our patient experienced in just a few months may have impaired her lithium clearance. furthermore, the possibility of increased lithium absorption after bariatric surgery has been raised previously in the literature. in one in vitro model, investigators studied the dissolution of psychotropic drugs in media environments that mimic the gastrointestinal tract of a post - roux - en - y patient and compared to that of a preoperative patient. while most of the drugs studied were found to have decreased or unaffected dissolution, the dissolution of lithium increased significantly (> 200%) in the post bariatric surgery model.10) stomach ph increases significantly following both roux - en - y and vertical sleeve gastrectomy,11) which may facilitate the deprotonation of carbonate salt and result in an increased dissolution of lithium ions. given its narrow therapeutic window, careful monitoring and perhaps dose reductions may be necessary for patients during the pre- and postoperative period. although some institutions have protocols in place to monitor lithium levels before and after bariatric surgery,12) there are currently no widely accepted guidelines. a significant gap still exists in the literature concerning the pharmacokinetics of psychotropic medications following bariatric surgery. further research on the pharmacokinetic changes of high - risk medications like lithium following bariatric surgery seems needed. | we are presenting the first documented case of lithium toxicity after vertical sleeve gastrectomy surgery in an 18 year - old female with psychiatric history of bipolar disorder who was treated with lithium. this case illustrates the need for closer monitoring of lithium levels following bariatric surgery. both psychiatrists and surgeons should be aware of the potential risk of lithium toxicity following bariatric surgery, as well as the need to judiciously monitor lithium level and possibly adjust the dose of some medications. |
neuroendocrine tumors are derived from neuroendocrine cells and the most common site is the bronchopulmonary system. theoretically, primary neuroendocrine tumors do not occur in the breast due to the lack of endocrine cells. although there are many published studies regarding this rare pathological entity, there are a few reports that also include radiology findings. however, most of the published studies of patients with radiology findings are of the small cell type of tumor, and the characteristic imaging features are not described well (1 - 7). we report the imaging findings in a case of primary neuroendocrine tumor (large cell neuroendocrine carcinoma with atypical carcinoid features) found in the breast. a 42-year - old female was presented with a lump in her right breast, which she had been aware of for several weeks. on physical examination, an approximately 5-cm, firm, movable lump was palpated in the upper outer subareolar region of her right breast. mammography revealed a high - density mass with an ill defined margin in the 11 o'clock direction of the right breast subareolar area. targeted sonography of the right breast revealed an irregularly shaped, lobulated marginated, heterogeneous echotextured mass with posterior enhancement. heterogeneous echotexture was noted, especially in the center of the mass, and increased vascular flow was noted in the mass on color doppler scanning. there was also lymphadenopathy in the right axilla with cortical thickening and loss of fatty hilum (fig. the mass was categorized as breast imaging reporting and data system category 5, highly suggestive of malignancy. fine needle aspiration cytology revealed highly cellular smears with monotonous hyperchromatic atypical small cells, suggesting high - grade invasive ductal carcinoma. the mass appeared to be of heterogeneously low signal intensity on t1 weighted images and a few parts of the mass showed intermediate to high signal intensity on t2 weighted images. the t2 non - high signal area showed rapid enhancement within the first two minutes of gadolinium injection, and washout was noted (fig. the patient underwent a modified radical mastectomy and axillary dissection of the right breast. on the cut surface, the tumor appeared as a fairly well - defined but partially infiltrative, gray - white, solid mass with morphology similar to that of commonly seen, invasive ductal carcinoma. histopathologically, several, large, mucoid, necrotic areas with foamy macrophages were surrounded by dark, solid, and trabecular pattern tumor cells. the tumor was composed of a mostly solid growth of large to intermediate, polygonal or occasionally spindle cells, which showed organoid, nesting, trabecular, rosette - like, and palisading patterns. immunohistochemically, the tumor cells were diffusely positive for synaptophysin and were focally positive for chromograin a, although they were negative for cd 56., we considered this tumor to be a large cell neuroendocrine carcinoma with atypical carcinoid features (histologic grade 3, t2n1m0, estrogen receptor [er ] [+ ], progesterone receptor [pr ] [+ ], human epidermal growth factor receptor 2 [- ]). positron emission tomography - computed tomography after surgery showed an absence of abnormal uptake, suggesting that the breast was the only primary site. neuroendocrine tumors are derived from the neuroendocrine cells and the most common site is the bronchopulmonary system, where the endocrine cells are constantly present. in the breast, hyperplastic or benign neoplastic neuroendocrine lesions have never been reported, and this lead to the hypothesis that neuroendocrine differentiated breast tumors probably do not arise from the pre - existing endocrine cells, but are rather the result of the differentiation process within breast carcinoma (8). focal neuroendocrine differentiation can be found in diverse histological types of breast carcinoma. in one case series, most neuroendocrine cells were found with invasive ductal carcinoma or with mixed carcinoma showing mucinous differentiation or signet - ring cells (9). however, the term, neuroendocrine tumor, is defined by the diffuse expression of neuroendocrine markers, such as chromogranin - a, neuron - specific enolase, synaptophysin, and cd56, in more than 50% of the cell population. neuroendocrine breast tumors are very rare, comprising of approximately 2 - 5% of all breast cancers, and most of these patients are in the sixth or seventh decade of life (10). different classifications, according to the morphological features of histopathology, have been proposed for neuroendocrine tumors, arising within the breast. perhaps the most pragmatic is the who classification, which defines three principle tumors, i.e., solid neuroendocrine carcinoma, small cell / oat cell carcinoma, and large cell neuroendocrine carcinoma (10). it should be noted, however, that not all breast tumors that exhibit neuroendocrine features completely fit into the who classification categories. there are no specific differences in the clinical features of neuroendocrine tumors from those of the other breast malignancy, and endocrine hormone - related syndromes are extremely rare. of interest is the increase in neuroendocrine markers, such as chromogranin a, in the blood (10). as metastatic neuroendocrine tumors of the breast are more common than that of primary neuroendocrine tumors of the breast, it is, therefore, important to differentiate primary breast neuroendocrine tumor from metastatic disease to the breast because of the differences in treatment focus. primary neuroendocrine tumor of the breast can be diagnosed if the presence of a non - mammary primary site can be clinically ruled out or if an in situ component is histologically detected, or both. the treatment of primary neuroendocrine tumors is highly variable, although surgery should always be considered as the first line of treatment. recent reports, which only deal with small cell neuroendocrine carcinoma, indicate that the size, stage of disease at the time of diagnosis, expression of the er and pr, and the ki-67 index are important determinants of the prognosis (11 - 13). there is no relevant report about the prognosis of primary large cell neuroendocrine carcinoma seen in our case. however, the prognosis of our case would be poor, considering tumor size, axillary metastasis, and histologic grade. previously published case reports with radiology findings (1 - 7) have also described the imaging features of neuroendocrine tumors. (1) described that the common radiological features of breast neuroendocrine tumor include, a high - density mass with predominantly spiculated or lobulated margins on mammography and mostly irregular or microlobulated, homogeneously hypoechoic masses with normal sound transmission on sonography., the number of cases with radiology findings has been too small to allow generalization of the imaging features, and there is no case report with radiology and pathology findings similar to ours. in this report we present a case of primary neuroendocrine tumor (large cell neuroendocrine carcinoma with atypical carcinoid features) of the breast, together with its mammographic, sonographic, and mri features. however, the radiologic findings are hard to differentiate from those of much more commonly seen invasive ductal carcinoma. reports of new cases will be necessary in order to determine the radiologic presentation of primary neuroendocrine tumor of the breast. | focal neuroendocrine differentiation can be found in diverse histological types of breast tumors. however, the term, neuroendocrine breast tumor, indicates the diffuse expression of neuroendocrine markers in more than 50% of the tumor cell population. the imaging features of neuroendocrine breast tumor have not been accurately described due to extreme rarity of this tumor type. we present a case of a pathologically confirmed, primary neuroendocrine breast tumor in a 42-year - old woman, with imaging findings difficult to be differentiated from that of invasive ductal carcinoma. |
trochlear nerve palsy may be clinically characterized by vertical diplopia, incomitant hypertropia that increases upon head tilt toward the paralyzed site (positive bielschowsky 's test), excyclotropia, and head tilt. the differentiation between acutely acquired palsy vs. the decompensation of a congenital palsy is important for an accurate diagnosis. the presence of acute unilateral trochlear nerve palsy with claude bernard - horner syndrome represents a rare clinical condition. in a review of 215 cases of trochlear nerve palsy examined over a period of 23 years, the current report describes a patient with trochlear nerve palsy associated with claude bernard - horner syndrome and contralateral hemiparesis, with onset following a hypertensive crisis. a 35-year - old caucasian woman with a history of chronic headache and systemic arterial hypertension was admitted with sudden acute intense bitemporal headache, diplopia, dysarthria, and left hemiparesis. the patient 's blood pressure was lowered with antihypertensive drugs, including sodium nitroprusside, and she regained consciousness the next day. a neurological examination showed right trochlear nerve palsy, slight ipsilateral claude bernard - horner syndrome (fig. 1), and left hemiparesis. at rest, the patient presented cyclovertical diplopia when her head was tilted down and to the right. magnetic resonance imaging (mri) of the brainstem and cerebellum, acquired on the sixth day, showed multiple small areas localized mainly to the pons and mesencephalon that were distinguished by t1 and t2 hyperintense signals and no contrast enhancement (fig. an mri in the fourth week showed remission of the previous lesions ; the patient was asymptomatic at that time and returned to her daily activities. the most frequent causes of trochlear nerve palsy are traumas, surgical injuries, tumors, demyelinating and inflammatory diseases such as meningitis and cysticercosis. brainstem stroke represents a rare cause of trochlear nerve palsy, in which infarcts may be extensive or restricted to the microvascular territory [4, 5 ]. the patient presented in the current report developed sudden - onset clinical manifestations associated with arterial hypertension. these clinical features were suggestive of a vascular etiology, which was supported by the neuroimaging findings (fig. 2). most likely, small bleedings at the brainstem and cerebellum occurred as consequence of increased blood pressure. this clinical picture has previously been described in patients with trochlear nerve palsy [5, 6, 7 ]. the right - side claude bernard - horner syndrome and left - side hemiparesis observed in this patient may be related to the right - side pons lesion that was observed (fig. in addition, the right - side trochlear palsy was possibly a result of the left dorsal mesencephalic lesion (fig. after exiting the trochlear nucleus, which is ventrolateral to the cerebral aqueduct, the nerve fascicles course posteroinferiorly around the cerebral aqueduct and decussate in the dorsal midbrain [1, 5 ]. therefore, in the current patient, the involvement of the trochlear nerve must have occurred before the decussation, resulting in contralateral palsy. the literature also contains descriptions of contralateral trochlear nerve palsy with ipsilateral horner 's syndrome. guy. describe nuclear or fascicular involvement of the trochlear nerve prior to decussation in the superior medullary velum and impairment of the adjacent sympathetic fibers. in the current report, the multiplicity of lesions observed in the patient likely was responsible for the unusual clinical presentation. in the case presented here, progressive improvement was observed, and the patient was asymptomatic at the end of the fourth week. similar outcomes have been described in patients with trochlear nerve palsy related to minor vascular lesions. however, there is not sufficient evidence to draw definitive prognostic conclusions in this rare situation [3, 4, 6 ]. | the association of unilateral trochlear nerve palsy with claude bernard - horner syndrome represents a rare clinical condition. we present the case of a patient with this unusual presentation. the investigation performed implicated cerebrovascular disease as the underlying cause of the condition in this patient. |
leiomyomas, also known as fibroids, are the most frequent benign uterine tumors to develop during a woman s reproductive years ; occurrence tends to regress after menopause.1 malign transformation of uterine leiomyoma (ul) seems to be a very rare event,2 but they are symptomatic in 20%30% of cases and are the main reason for approximately one - third of all hysterectomies in developed countries.3,4 symptoms related to ul are menstrual disorders, mainly menorrhagia, pelvic pain, and infertility, which can adversely affect pregnancy outcomes.5,6 ul disproportionately affects black women.4,7,8 the etiology of ul is unknown, but it can be influenced by genetic liability,9 hormonal environment,10,11 and local tissue mediators.10,12 definitive prevalence of ul in the general female population is also unknown, but differs by ethnicity, age, and method used for diagnosis.13 in clinical practice, transvaginal ultrasonography (tvs) is the most widespread method for evaluating ul, with an estimated accuracy of 87%92%.1416 the objective of this study was to determine the prevalence of ul, diagnosed by tvs, at a private health care setting located in the central eastern part of portugal, and to explore the demographic and clinical factors related to diagnosis and symptomatology. the study retrospectively analyzed the medical files of all female patients who consecutively attended a gynecological consultation at a private health care setting located in covilh, portugal, from december 231, 2010. excluded from the study were virgins, patients who had previously undergone a hysterectomy, or those pregnant at the time of the consultation. all included patients were caucasian, except three, who were african. the same gynecologist (jaf), who has more than 20 years of gynecological experience and competence in the practice of gynecologic ultrasound, attended all patients. policies of the private clinic include recording of demographic and clinical data from every patient in a standard digital database. in all cases, the sonographic equipment used was a nemio 17 (ssa-550a) with a pvm-651vt 6 mhz endovaginal probe (toshiba corporation, tokyo, japan). for the anonymous use of data from patient files, written consent was obtained from the clinical director of the private health care clinic. lsb and dgt analyzed the files of 624 patients who met the inclusion criteria, and from each file they retrieved age, weight, height, age at menarche, number of pregnancies and deliveries, marital status, menstrual cycles characteristics, and contraceptive method at time of consultation. the presence, number, and largest dimension of the uls were retrieved from tvs records. for analysis, ultrasound records were analyzed for the presence or absence of ul, their number, location and largest dimension. the data were organized and analyzed in statistical package for the social sciences software ([spss ] v 20.0 for windows ; ibm corporation, armonk, ny, usa), by smn. using tvs, ul was diagnosed in 161 (25.8%) of those 624 cases included in the study. from those 161 patients with an ultrasound - based diagnosis of ul, 99 (61.5%) had previous knowledge of the disease. in three patients, the mean age was 39.6 years ; 66.2% of the women were married ; 68.2% reported having had at least one pregnancy and 64.2% at least one delivery ; 31.6% were asymptomatic. ul was diagnosed by ultrasound in 11.0% of all women aged 20 to 39 years old, in 45.4% of all women aged 40 to 59 years, and in 19.5% of all women aged 60 years or more. metrorrhagia and menorrhagia were the most usual presenting symptoms, and were recorded in 11.3% of all cases and in 22.3% of all women with an ultrasound diagnosis of ul. uls larger than 3.0 cm were identified through ultrasound imaging in 24.2% of the cases. in order to uncover significant demographic and clinical differences between women with and without ultrasound diagnosis of ul, significant factors associated with an ultrasound - based diagnosis of ul were the following : 40 to 59 years of age ; married ; excessive body weight (obese) ; prior pregnancy or delivery ; non - use of the combined pill for contraception ; and symptoms of menorrhagia. however, after adjustment by multivariate logistic regression analysis, age 40 to 59 years remained the single demographic and clinical factor found to be statistically associated with ultrasonographic diagnosis of ul. when menorrhagia was the presenting symptom, an ultrasound imaging diagnosis of ul was made in 73.3% of cases (table 3). plotting a univariate analysis, metrorrhagia and menorrhagia as presenting symptoms were significantly related to the tvs diagnosis of multiple uls, but not with the largest ul dimension (tables 4 and 5). although lacking statistical significance, patients diagnosed with smaller uls seemed to be more symptomatic (table 5). ul prevalence has been evaluated by different methods, such as population surveys,17 pathologic study of surgical specimens of hysterectomy,18 postmortem of women who died without gynecological diseases,19 and pelvic ultrasonography,13,20,21 with, as expected, different results. kratochwil introduced ultrasound imaging of feminine pelvic organs in 1972. in 1980, muram defined criteria for ul identification with tvs as a relatively spherical mass that is echogenically different from the surrounding myometrium. since then, tvs has become the most noninvasive, lowest - cost, and widespread method for evaluation of uterine pathology, with highly accurate performance.1416 the main disadvantage of ultrasound is that it is an operator - and device - dependent technique.24 in order to improve ul diagnosis accuracy, saline contrast sonohysterography and three - dimensional ultrasound have been proposed, although with conflicting results.24,25 nowadays, two - dimensional pelvic ultrasonography remains the best method for routine detection of uls.24,25 ultrasonography has been shown to be an insufficient method of myoma mapping, and magnetic resonance imaging should preferred for surgical therapy planning.25 in the present study, we found a 25.8% overall prevalence of ul, with the highest prevalence (45.4%) in women aged 3950 years, which is in accordance with previous ultrasound - based studies.13,20,26 multiple uls were present in 50.1% of cases, which is lower than that reported in surgical specimens by cramer and patel (84%).18 in contrast to cramer and patel s findings, only 49.1% patients had a ul larger than 20 mm. a previous diagnosis of ul had been made in 99 (61.5%) patients, who were in watchful waiting management, which has been considered as a safe management option for asymptomatic ul.27,28 except in three cases submitted to resectoscopy for treatment of symptomatic submucous myomas, the main component of the leiomyomas was intramural. a purely intramural myoma represents a temporary status, and its growth is dependent on the existing hormonal conditions of the woman.29 most of the knowledge about demographic and clinical risk factors for ul is based on results of large populations survey studies. risk factors such as age, ethnicity, education, hormonal factors, diet, physical exercise, oral contraceptives use, childbirth, smoking and tissue injury has been reported, with conflicting results. in accordance with other ultrasound - based studies,13,20,26 after multivariate statistical analysis, we do nt found any demographic or clinical risk factor for ul, with exception of age. the most common symptom of ul is abnormal uterine bleeding, especially menorrhagia.5 the mechanism of leiomyoma - associated menorrhagia is unknown, but submucous localization, vascular defects, and impaired endometrial hemostasis have been suggested as possible explanations.30,31 even small uls without distortion of the endometrial cavity can alter myometrial vascularization and contractibility. a meta - analysis conducted by benecke reported that intramural uls may have a negative impact on fertility. in our series, menorrhagia was significantly associated with multiplicity of uls, but not with the volume of the largest one, suggesting the importance of factors other than those associated with distortion of the myometrium and endometrial cavity. extracellular matrix that embed ul cells influence both normal myometrial and ul cells, promoting proliferative activity and decreasing apoptosis.33,34 previous ultrasound - based studies2035 have reported that the presence, number, and volume of leiomyomas are unrelated to abnormalities in the menstrual cycle. other studies,36,37 but not all,38 suggest that menstrual symptoms are related to tissue layer location in the uterine wall and with the axial position of leiomyomas. in our study, multiplicity of uls was significantly related to abnormal uterine bleeding (menorrhagia and metrorrhagia) as a presenting symptom. currently, most women with menstrual disorders and multiple uls are scheduled for medical therapy followed by hysterectomy or myomectomy.39 hysterectomy seems a good option when childbearing wishes have been fulfilled;39 but it is not devoid of risks, morbidity and mortality as for any surgical procedure. myomectomy is indicated for those women who wish to retain their fertility.39 the major disadvantage of surgical myomectomy is leiomyoma recurrence. in next 5 years, more than one - third of that women submitted to myomectomy will need additional surgical intervention for treatment of newly developed uls.40 minimally invasive procedures, such as laparoscopic myolysis, uterine artery embolization, uterine fibroid embolization, and magnetic resonance - guided focused ultrasound, as standard practice for treatment of symptomatic multiple uls are currently limited.41 it seems obvious that new treatments better than surgical approaches are needed for the management of symptomatic ul. in recent years the development of uls has been shown to be dependent of sex steroids, especially progesterone,42,43 and many leiomyoma - related growth factors have been identified, including epidermal growth factor, platelet - derived growth factor, transforming growth factor beta, insulin - like growth factor, activin, and myostatin. we speculate that, in patients with multiple uls and menstrual disorders, each ul can produce different kinds of growth factors or other biological mediators that may have additive negative effects on the myometrial and endometrial environment. discovery of selective progesterone receptor modulators (sprms) has provided a new paradigm for treatment of symptomatic ul. antiproliferative, growth factors down - regulation, and proapoptotic actions of sprms had been reported in vitro studies over cultured leiomyoma cells.43 randomized, placebo - controlled clinical trials have demonstrated that the sprm ulipristal can suppress menstrual bleeding, restore normal levels of hemoglobin, reduce ul volume, and improve leiomyoma - related symptoms.44,45 the main objective of symptomatic ul treatment is to regress symptomatology, because expectant management is suitable for most asymptomatic leiomyomas.27,28 it is expected that new therapies for the treatment of ul - associated symptomatology will be developed, based on the growing knowledge of functional aspects and regulatory mechanisms of leiomyoma biology and uterine homeostasis. to our knowledge, this is the first population study conducted in portugal to assess the prevalence and demographic and clinical factors associated with the diagnosis of ul by ultrasound. in our portuguese population, the prevalence of uterine leiomyoma increases with age, and was similar to that reported in literature for white women. menorrhagia and metrorrhagia were the more distressing symptoms, especially in patients with multiple uls diagnosed by tvs. in patients with abnormal uterine bleeding, special attention should be taken to search for multiple leiomyomas during pelvic ultrasound examination. further studies are needed to assess the biology and symptomatic impact of multiple versus single uls, in order to find more efficient therapeutic approaches. | purposeto determine the prevalence of uterine leiomyomas, diagnosed by ultrasound, in a private health care setting located in the central eastern region of portugal, and to explore the demographic and clinical factors related to diagnosis and symptomatology.patients and methodsthe files of 624 patients attending a private clinic in covilh, portugal, from january 2 to december 31, 2010 were retrieved for evaluation. pelvic ultrasound record, age, weight, height, age at menarche, number of pregnancies and deliveries, marital status, menstrual cycles characteristic, and contraceptive method at consultation were included in the analysis.resultsuterine leiomyoma (ul) was diagnosed by ultrasonography in 161 (25.8%) patients. a single ul was diagnosed in 80 (49.7%) patients. in 79 (49.1%) patients, the largest leiomyoma had a dimension < 20 mm. prevalence of ul was age dependent : at 11.0% for women 2039 years old ; 45.4% for those aged 4059 years ; and 19.5% for women 60 years or older. metrorrhagia was the most distressing presenting symptom. when menorrhagia was the presenting symptom, the probability of having an ultrasound diagnosis of ul was 73.3%. metrorrhagia or menorrhagia, as presenting symptom, was significantly related to the ultrasound diagnosis of multiple uls.conclusionul was especially prevalent in women aged between 40 and 59 years. patients with multiple uls had significantly more abnormal uterine bleeding. in patients with menorrhagia or metrorrhagia, special attention should be taken in searching for the presence of multiple uls during ultrasound. |
acute urticaria is caused by various factors including foods, chemical agents, infections, and insect bites. when a clinician is trying to determine the cause of a patient 's urticaria, it is important to ask about the patient 's food intake, possible chemical agent exposure, and symptoms that may indicate an infection. here, we report the cases of five patients with acute urticaria. although four of the patients did not describe experiencing a bee sting at their presentation, the subsequent examination detected anti - bee - specific ige antibodies. the ages of the five japanese patients ranged from 33 to 86 years (median : 61). the onset of urticaria was 614 days (median : 10) after a bee sting. all five patients presented at our hospital in the 3-month period from july to september. only one patient had a local skin reaction at the sting site, 2 days before the onset of urticaria. the type of bee involved was paper wasp in three cases and yellow jacket in two cases. venom - specific iges for paper wasp, yellow jacket and honeybee were examined in four cases. the three patients stung by a paper wasp were positive for paper wasp and yellow jacket but not for honeybee. the urticaria was well controlled by a type 2 antihistamine antagonist in three patients ; the other two patients required oral prednisone. although most anaphylactic reactions caused by insect stings occur shortly after the sting, they occasionally have a delayed onset. in the study by the american academy of allergy and immunology committee, there were only three patients in whom the reaction started more than 48 h after the sting in the recorded 2,219 patients with allergic reaction by insect sting. so, it may be difficult to suspect bee sting as cause of allergic reaction if it shows a delayed onset. four of our five patients took no notice of a relationship between their acute urticaria and their bee sting history. importantly, an examination detected ige antibodies for bees in all four examined cases with a cross - reactivity between paper wasp and yellow jacket in three of them. a history of a bee sting should thus be part of the medical interview sheet for patients with acute urticaria, especially in the summer to early autumn. if a patient has incurred a bee sting within 2 weeks prior to his or her presentation, an examination of ige for bees may help prevent a severe bee - related anaphylactic reaction in the future. moreover, individuals who are stung by a bee should be advised that their antibodies for bees should be examined if a general or local eruption with pruritus occurs within approximately 2 weeks after the sting. a question is raised regarding the mechanism of late onset of acute urticaria in our cases. reisman and livingston reported 10 patients with allergic reactions 12 weeks after bee stings. four of them had serum sickness - type reactions with joint swelling, arthralgia and urticaria, three had only generalized urticaria, two had severe symptoms of anaphylaxis with urticaria and the remaining one had a large local swelling. all patients had venom - specific ige and four had serum venom - specific igg which relates to serum sickness reaction. however, sickness - type reactions were observed only in one of four patients with venom - specific igg. reisman and livingston suggested that the late reaction ranges from typical anaphylaxis to serum sickness, probably related to simulation of ige antibodies and subsequent reaction with persisting venom antigen. although the mechanism of late onset of acute urticaria without rapid reaction is unclear, our cases might show delayed urticaria caused by a reaction of the bee antigen - specific ige antibody to the antigens remaining in the body, after the first sensitization. it suggests that bee antigens can remain for 12 weeks in the body after a bee sting, because the bee antigen - specific ige antibody can not react without bee antigens. | here we report the cases of five patients with a late onset of acute urticaria after a bee sting. the ages of the five japanese patients ranged from 33 to 86 years (median : 61). all patients had no history of an allergic reaction to bee stings. the onset of urticaria was 614 days (median : 10) after a bee sting. although four of the patients did not describe experiencing a bee sting at their presentation, the subsequent examination detected anti - bee - specific ige antibodies. so, we think a history of a bee sting should thus be part of the medical interview sheet for patients with acute urticaria, and an examination of ige for bees may help prevent a severe bee - related anaphylactic reaction in the future. |
yeast strains and constructs the following yeast strains were used : by4741 (mata his31 leu2 met15 ura3) and ndy257 (by4741 rtn1::kanmx4 rtn2::kanmx4 yop1::kanmx) (6). strains expressing gfp fusions to the chromosomal alleles of yop1 and rtn1 were obtained from invitrogen. the plasmid encoding sec63-gfp (pjk59) has been previously described (12). to make the plasmid encoding rtn1-gfp (pcv19), the rtn1 gene, including 400 bp upstream of the start site, was pcr - amplified from yeast chromosomal dna and inserted into the same sites. ha - rtn3c was cloned by pcr amplifying rtn3c (ncbi accession number : bc036717) from mouse cdna with primers containing an n - terminal ha tag and inserted into pcdna3.1d (invitrogen). for rtn4a - gfp, human rtn4a was pcr - amplified from rtn4a - myc (described in a previous study (6)) and ligated into the pacgfp - n1 backbone (clontech) using the xhoi and kpni restriction sites at the 5 and 3 ends, respectively. for gfp - rtn3c, rtn3c was pcr - amplified from ha - rtn3c and ligated into the pacgfp - c1 backbone (clontech) using the xhoi and ecori restriction sites. to clone gfp - rtn4hd, the region encoding amino acids 9611192 was pcr - amplified from human rtn4a - myc and inserted into pacgfp - c1 using the xhoi / ecori restriction sites. gfp - dp1 was subcloned by pcr - amplifying mouse dp1 from ha - dp1 (described in a previous study (6)) and inserting into pac - gfp c1 using saci / bamhi restriction sites. for gfp - climp63, climp63 was pcr - amplified from mouse cdna and cloned into pacgfp - c1 using the xhoi / ecori sites. climp63lum - gfp was cloned by pcr amplifying the region encoding amino acids 1115 (as described in (13)) from gfp - climp63 and inserted into pacgfp - n1 using xhoi / ecori restriction sites. lbr - gfp was pcr - amplified from plasmid containing human lbr (14) and cloned into pacgfp - n1 using the xhoi / bamhi restriction sites. for gfp - sec61, human sec61 was pcr - amplified from the pcdna3.1/gfp - sec61 construct described previously (6), and inserted into pacgfp - c1 using the bglii / ecori restriction sites. rfp - sec61 was subcloned from gfp - sec61 using the same restriction sites as above and inserted into an mrfp1 vector (pegfp - c1 vector backbone where pegfp has been replaced with mrfp1). microscopy of yeast yeast strains were grown in synthetic complete medium (0.67% yeast nitrogen base and 2% glucose) and imaged live at room temperature using an olympus bx61 microscope, uplanapo 100/1.35 lens, qimaging retiga ex camera, and iplabs version 3.6.4 software. screen for mutations in yeast rtn1 that affect localization error - prone pcr on rtn1 was performed using the genemorphii random mutagenesis kit (stratagene). the product of this reaction and pjk59 cut with xbai and xhoi were used to transform wild - type yeast. cells were grown at 37 c with 5% co2 in dulbecco 's modified eagle 's medium containing 10% fetal bovine serum and subcultured every 23 days. after 5 h of transfection, cells were split onto acid - washed no. 1 coverslips and allowed to spread for an additional 2436 h before being processed for indirect immunofluorescence. for immunofluorescence, transfected cells were fixed in pbs containing 4% paraformaldehyde (electron microscopy sciences) for 15 min, washed twice, and permeabilized in 0.1% cells were washed twice again and then probed with primary antibodies for 45 min in pbs containing 1% calf serum, at the following concentrations : rat anti - ha antibody (roche applied science) at 1:200 dilution ; mouse anti-tubulin (sigma) at 1:500 dilution ; and rabbit anti - calreticulin antibody (abcam) at 1:500 dilution. cells were washed three times in pbs, and then incubated with various fluorophore - conjugated secondary antibodies for an additional 45 min (alexafluor 488 or 555 anti - mouse at 1:250 dilution, alexafluor 647 anti - rabbit 1:500 dilution, and alexafluor 488 anti - rat 1:200 dilution (all from invitrogen)). cells were then washed and mounted onto slides using fluoromount - g mounting medium (southern biotech). all imaging for indirect immunofluorescence was captured using a yokogawa spinning disk confocal on a nikon te2000u inverted microscope with a 100 plan apo numerical aperture 1.4 objective lens, and acquired with a hamamatsu orca er cooled charge - coupled device camera using metamorph 7.0 software. for image presentation, brightness and contrast were adjusted across the entire image using adobe photoshop 7.0, and images were converted from 12 to 8 bits. cos-7 cells expressing gfp - rtn4hd were sorted in a moflo cell sorter (cytomation). the resulting cell pellet was fixed for 1 h in a mixture of 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 m sodium cacodylate buffer (ph 7.4), washed in 0.1 m cacodylate buffer, and postfixed with a mixture of 1% oso4 and 1.5% kfecn6 for 30 min. the pellet was then washed in water and stained in 1% aqueous uranyl acetate for 30 min followed by dehydration in grades of alcohol (50%, 70%, and 95%, 2 100%). next, the pellet was infiltrated in a 1:1 mixture of propylene oxide and taab peon (maria canada inc.) for 2 h, placed in pure taab epon in a silicon - embedding mold, and polymerized at 65 c for 48 h. ultrathin sections (6080 nm) were cut on a reichert ultracut - s microtome, placed onto copper grids, and stained with 0.2% lead citrate. specimens were examined on a tecnai g spirit biotwin transmission electron microscope, and images were acquired with a 2k amt charge - coupled device camera. transfected cos-7 cells were imaged in phenol red - free hyq dme (hyclone) supplemented with 25 mm hepes, ph 7.4, and 1% fetal bovine serum. frap experiments were conducted on a zeiss lsm 510 nlo laser scanning inverted microscope using a plan - neofluor 100/1.3 oil objective with argon laser line 488 nm (optical slices < 1.2 mm for cos-7 and 4.2 m for yeast). mammalian cell experiments were done at 37 c using an objective heater (bioptechs) and an enclosed stage incubator (zeiss). lsm 510 software version 3.2 was used for image acquisition and analysis. for all mammalian experiments, cos-7 cells were treated with 0.5 m nocodazole, and all data were collected during the first 530 min of nocodazole addition. for photobleaching all constructs, except for lbr - gfp, the tubular er was magnified using the 3 zoom function so that individual tubules could be seen clearly. for lbr - gfp images taken for 5-s prebleaching, whereupon a region of interest of 65 65 pixels was photobleached at 100% laser power. after the photobleaching, images were taken at 1-s intervals for 75300 s. yeast cells were treated similarly except that the region of interest was 17 17 pixels, and images were taken every 24 s at room temperature., the fluorescence intensity of three regions of interest was measured : the photobleached region (pr), a region outside of the cell to check for overall background fluorescence (br), and a region within the cell that was not photobleached to check for overall photobleaching and fluorescence variation (cr), for the entire course of the experiment. microsoft excel was used to normalize the relative fluorescence intensity, i, for each individual frap experiment using equation 1. 1)\documentclass[10pt]{article } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{pmc } \usepackage[euler]{upgreek } \pagestyle{empty } \oddsidemargin -1.0 in \begin{document } \begin{equation}\;i=[({\mathrm{pr}}_{{\mathrm{t}}}-{\mathrm{br}}_{{\mathrm{t}}})/({\mathrm{pr}}_{{\mathrm{t}}0}-{\mathrm{br}}_{{\mathrm{t}}0})^{{^\ast}}100]{\times}[(100-{\mathrm{cr}}_{{\mathrm{t}}}/{\mathrm{cr}}_{{\mathrm{t}}0})/100 + 1]\;\end{equation}\end{document } for data presentation, the mean averages of the normalized data for each set of frap experiments were plotted using graphpad prism 5.0, and fluorescence recovery curves were shown for the first 80140 s of each experiment. estimated half - times of recovery and mobile fraction values were calculated using the standard michaelis - menten equation. sucrose gradient centrifugation for yeast sucrose gradient analysis, crude membranes were isolated from yeast strains expressing gfp - fused proteins at endogenous levels as follows : 200 ml of culture were grown to od 1, pelleted and then resuspended in tkmg lysis buffer (50 mm tris, ph 7.0, 150 mm kcl, 2 mm mgcl2, 10% glycerol, 1 mm edta, 1 mm pmsf, 1 mm 4-(2-aminoethyl)benzenesulfonylfluoride hydrochloride), flash frozen in liquid nitrogen, and ground using a mortar and pestle. cell debris was separated from the lysate by low speed centrifugation for 5 min at 2,000 g. membranes were then pelleted by ultracentrifugation for 15 min at 100,000 g and solubilized in 200 l of tkmg buffer containing 1% digitonin. solubilized lysate was centrifuged for 10 min at 12,000 g to separate out any remaining cell debris. 100-l of lysate were run on 530% w / v sucrose gradients for 4 h at 166,000 g at 25 c on a beckman tls55 rotor. twenty gradient fractions were collected from top to bottom and analyzed by sds - page and immunoblotting with anti - gfp antibody (roche applied science). 50 mg of apoferritin, catalase, and aldolase was used as molecular weight standards. xenopus washed membrane fractions were prepared in mwb (50 mm hepes, ph 7.5, 2.5 mm mgcl2, 250 mm sucrose, and 150 mm potassium acetate) as previously described (6), incubated for 60 min at 25 c in mwb containing 200 mm kcl and 0.5 mm gtp, and then solubilized for 30 min at 25 c with either 2% nonidet p-40 or 1.25% digitonin. samples were pelleted for 15 min at 12,000 rpm, and the soluble fraction was loaded onto a 1030% w / v sucrose gradient made with mwb containing 200 mm kcl, 0.1 mm gtp, and either 0.1% nonidet p-40 or 0.1% digitonin, respectively. sixteen gradient fractions were collected and analyzed by sds - page and immunoblotted with antibody against xenopus rtn4 (described in a previous study (6)). for mammalian sucrose gradient analysis, cos-7 cells transiently transfected with ha - dp1 or gfp - sec61 were harvested by scraping and then lysed and solubilized in hkme buffer (25 mm hepes, ph 7.8, 150 mm potassium acetate, 2.5 mm magnesium acetate, 1 mm edta, and 2 mm pmsf) containing 1% digitonin for 1 h. the lysate was clarified by centrifugation at 10,000 g for 10 min, and 100 l of clarified lysate was sedimented on 530% w / v sucrose gradients under the same conditions as yeast. fractions were analyzed by sds - page and immunoblotting with anti - ha antibody or anti - sec61 antibody (described in a previous study (15)). chemical cross - linking experiments yeast crude membrane fractions were resuspended in buffer containing 50 mm hepes, ph 7.0, 150 mm kcl, and 1 mm pmsf. ethylene glycobis(succinimidylsuccinate) (egs, pierce), was dissolved in anhydrous dmso and diluted to the desired concentration. 1 l of egs was added into every 20 l of protein - containing sample for 30 min at room temperature. the reactions were quenched for 15 min with 2 l of 1 m tris, ph 7.5. samples were analyzed on a 420% sds - page and immunoblotted using standard procedures with mouse anti - his or rat anti - ha antibody conjugated to peroxidase (sigma). for mammalian cross - linking experiments, transfected cos-7 cells were grown in a 10-cm plate to 80% confluency and then lysed using a standard hypotonic lysis protocol. briefly, cells were harvested in pbs, washed, incubated in hypotonic buffer (10 mm hepes, ph 7.8, 10 mm potassium acetate, 1.5 mm magnesium acetate, 2 mm pmsf) for 10 min, and then passed through a 25-gauge syringe ten times. nuclei and any remaining intact cells were separated from the lysate by centrifugation for 5 min at 3,000 g, and the supernatant was then centrifuged for 10 min at 100,000 g to pellet the membrane fraction. the membrane pellet was washed in hkm buffer (25 mm hepes ph 7.8, 150 mm potassium acetate, 2.5 mm magnesium acetate, and 2 mm pmsf), repelleted at 100,000 g, and resuspended to a final volume of 60 l in hkm buffer. 10-l membrane aliquots were used for each cross - linking reaction using the same conditions as above. samples were analyzed on a 420% sds - page and immunoblotted using standard procedures with anti - ha antibody. figure 1.rtn1p and yop1p have slow diffusional mobility in the er of yeast cells. a, typical frap of sec63-gfp or rtn1-gfp in s. cerevisiae cells expressed at endogenous levels. b, fluorescence intensities normalized to prebleach values of frap analyses on yeast sec63-gfp, rtn1-gfp, and yop1-gfp were plotted over time. c, fluorescence intensities normalized to prebleach values plotted over time of frap analyses on yeast rtn1p in atp - depleted (green) or non - depleted (orange) cells, compared with that of sec63p - gfp (atp depleted in blue ; non - depleted in red). rtn1p and yop1p have slow diffusional mobility in the er of yeast cells. a, typical frap of sec63-gfp or rtn1-gfp in s. cerevisiae cells expressed at endogenous levels. b, fluorescence intensities normalized to prebleach values of frap analyses on yeast sec63-gfp, rtn1-gfp, and yop1-gfp were plotted over time. c, fluorescence intensities normalized to prebleach values plotted over time of frap analyses on yeast rtn1p in atp - depleted (green) or non - depleted (orange) cells, compared with that of sec63p - gfp (atp depleted in blue ; non - depleted in red). atp depletion experiments for yeast experiments, atp was depleted by the addition of 10 mm 2-deoxy - d - glucose and 10 mm sodium azide (both from sigma) for 25 min, and frap experiments were performed using the same parameters as described above. similarly, for mammalian cell experiments, cos-7 cells were depleted of atp as follows : transfected cells were washed twice in opti - mem serum - free media (invitrogen) and then incubated with 50 mm 2-deoxy - d - glucose and 0.02% sodium azide in glucose - free imaging buffer (50 mm hepes, ph 7.4, 150 mm potassium acetate, 2.5 mm magnesium acetate, and 1% fetal bovine serum). frap experiments were conducted in the same medium and completed within 530 min of treatment using the same parameters as above. yeast rtn1p and yop1p are immobile proteins in the er membrane the reticulons and dp1/yop1p may promote tubule formation in the er by assembling into structures that surround the membrane and deform it (16). we predicted that such structures might be immobile in the lipid bilayer. to test whether the reticulons and yop1p differ in their diffusional mobility from normal er membrane proteins, we used fluorescence recovery after photobleaching (frap). this technique determines the diffusional kinetics of integral membrane proteins in living cells (17, 18). as a control, we analyzed sec63-gfp, a 75-kda resident er protein with three transmembrane segments. a small portion of the cortical er was photobleached, and fluorescence recovery was measured over time (fig. rtn1p and yop1p both displayed relatively slow and restricted diffusional properties compared with sec63p (fig. these frap data show that rtn1p and yop1p diffuse significantly more slowly in the membrane than normal er proteins. rtn1p and yop1p form oligomers in the membrane rtn1p and yop1p may diffuse slowly within membranes because they are tethered to the cytoskeleton or because they oligomerize (6, 1921). we ruled out that low mobility is caused by association with actin filaments, the major cytoskeletal element responsible for yeast er dynamics (12, 22), because no significant difference in mobility was seen in cells treated with latrunculin (data not shown). therefore, we wondered if oligomerization of rtn1p and yop1p caused their low mobility in the er membrane, because it had been previously shown that the reticulons and dp1/yop1p can interact with themselves and with each other (6, 1921). a, membranes isolated from wild - type or rtn1rtn2yop1 (ndy257) yeast cells expressing wild - type or mutant forms of rtn1-gfp at endogenous levels were solubilized in 1% digitonin, fractionated by 530% w / v sucrose gradient centrifugation, and analyzed by sds - page and immunoblotting with anti - gfp antibody. note that the mutant forms of rtn1p have peak migrations at a smaller molecular weight than the wild - type protein. b, membranes isolated from wild - type or rtn1rtn2yop1 yeast cells expressing wild - type yop1-gfp at endogenous levels were solubilized in 1% digitonin and were analyzed as in a. molecular weight standards in the sucrose gradient are as indicated. c, isolated membranes of yeast cells expressing rtn1-his were treated with increasing concentrations of egs and analyzed on sds - page, and oligomers were visualized by immunoblotting with anti - his antibody. d, membranes of yeast cells expressing yop1-ha were analyzed as in c and immunoblotted with anti - ha antibody. asterisks, number of monomeric copies of the specified protein as seen by sds - page. a, membranes isolated from wild - type or rtn1rtn2yop1 (ndy257) yeast cells expressing wild - type or mutant forms of rtn1-gfp at endogenous levels were solubilized in 1% digitonin, fractionated by 530% w / v sucrose gradient centrifugation, and analyzed by sds - page and immunoblotting with anti - gfp antibody. note that the mutant forms of rtn1p have peak migrations at a smaller molecular weight than the wild - type protein. b, membranes isolated from wild - type or rtn1rtn2yop1 yeast cells expressing wild - type yop1-gfp at endogenous levels were solubilized in 1% digitonin and were analyzed as in a. molecular weight standards in the sucrose gradient are as indicated. c, isolated membranes of yeast cells expressing rtn1-his were treated with increasing concentrations of egs and analyzed on sds - page, and oligomers were visualized by immunoblotting with anti - his antibody. d, membranes of yeast cells expressing yop1-ha were analyzed as in c and immunoblotted with anti - ha antibody. asterisks, number of monomeric copies of the specified protein as seen by sds - page. preliminary evidence for the idea that oligomerization of rtn1p causes its slow diffusional mobility was obtained with cells depleted of atp. yeast cells expressing rtn1-gfp were depleted of atp with 10 mm 2-d - deoxyglucose and 10 mm sodium azide and subjected to frap analysis. rtn1-gfp became essentially immobile, whereas the control membrane protein sec63-gfp showed no significant change in diffusional mobility (fig. the simplest interpretation of these results is that rtn1p forms oligomers whose size increases in the absence of atp, perhaps because atp is required for the disassembly of the oligomers. to provide more direct evidence for oligomerization, we used sucrose gradient centrifugation. membranes purified from yeast cells expressing rtn1-gfp at endogenous levels were solubilized in 1% digitonin. the detergent extracts were subjected to sucrose gradient centrifugation, and fractions were analyzed on sds - page and immunoblotting with gfp antibodies (fig. 2a, top panel). a second band consistent in size with an sds - resistant dimer was also observed, but its intensity varied in different experiments, perhaps because of slight differences in expression levels. the protein peaked at a molecular mass of 300350 kda when compared with molecular weight marker proteins, indicating that these complexes may contain 4 or 5 monomers. when rtn1-gfp was solubilized with the harsher detergent nonidet p-40, it ran at a lower molecular weight (supplemental fig. the majority of yop1-gfp migrated in the density gradients at a size of 300400 kda (fig. 2b), indicating that this protein is present in a complex of a similar size to those containing rtn1p. a prominent sds - resistant dimer form was also seen. both rtn1-gfp and yop1-gfp sedimented at a similar position when expressed in yeast cells lacking endogenous rtn1p, rtn2p, and yop1p (fig. the relative amounts of rtn1-gfp and yop1-gfp in the higher molecular weight fractions in the rtn1rtn2yop1 cells were more prominent (fig. 2a, top two panels, and fig. 2b) than in wild - type cells, suggesting that these proteins may form larger complexes when they homo - oligomerize. next, we used chemical cross - linking to determine the oligomeric state of rtn1p and yop1p in intact membranes. membranes were isolated from yeast cells expressing rtn1-his or yop1-ha, treated with egs, a bifunctional cross - linker, and then analyzed by sds - page and immunoblotting using his or ha antibodies. with increasing egs concentrations, a ladder of cross - linked bands became apparent whose molecular sizes are consistent with homo - oligomers of rtn1-his or yop1-ha (fig. 2, c and d). up to four cross - linked monomers could be easily distinguished with sds - page, consistent with the size estimate of the major protein population in our sucrose gradient centrifugation experiments. in addition, a higher molecular weight smear can also be seen, suggesting that larger complexes can also form. together, these data suggest that rtn1p and yop1p can form homo - oligomeric complexes containing at least four and probably more monomers mutations in rtn1p that affect its localization in vivo have oligomerization defects we wished to determine whether the exclusive localization of rtn1p to the tubular er correlates with its ability to form oligomers. we reasoned that mutants in rtn1p that have lost their specific localization would have defects in oligomerization. thus, we first screened for mutations in yeast rtn1-gfp that cause it to localize not only to the peripheral tubular er but also to the nuclear envelope. the rtn1 portion of the rtn1-gfp fusion was mutated by error - prone pcr and introduced into a low copy plasmid by homologous recombination. approximately 2000 transformants were visually screened, and two independent mutants, mutant 1 and mutant 2, were isolated that showed nuclear envelope staining in addition to cortical er localization (fig. there were five mutations in mutant 1, three of which are localized to the intervening region. no single mutation was the cause of this relocalization, suggesting a synergistic relationship amongst several mutations. for mutant 2, however, site - directed mutagenesis showed that the lysine to isoleucine mutation in the first membrane - embedded region, k48i, is responsible for the mislocalization. next, we determined whether these mutant proteins had a decreased ability to form immobile oligomers. when analyzed using frap, mutant 1 and 2 were significantly more mobile than wild - type rtn1-gfp (fig. interestingly, the mobility of the rtn1p mutants was not affected by atp depletion (data not shown), in contrast to that of the wild - type protein (see fig. the mutants also appeared to have defects in oligomerization in sucrose gradient centrifugation experiments, because they migrated at lower molecular weight compared with the wild - type protein (fig. because these proteins are associated with a detergent micelle, it is unclear whether they were completely monomeric. these findings suggest that oligomerization is indeed responsible for the exclusive localization of rtn1p in the tubular er and for its immobility in the membrane. we noticed that the rtn1p mutants were not entirely non - functional : when expressed on a cen plasmid in a rtn1rtn2yop1 triple knock - out mutant (which has only sheet - like er), they were able to regenerate the tubular er similar wild - type rtn1p (fig. indeed, cross - linking with a bifunctional reagent still produced a ladder of rtn1p bands (data not shown). by combining the mutations in mutants 1 and 2, we were able to generate a mutant (mutant 91) that no longer restored the tubular er morphology when expressed in rtn1rtn2yop1 cells (fig. 3e, far right panels). however, while mutant 91 diffused rapidly in the membrane (fig. 3d) and migrated at a lower molecular weight in sucrose gradient centrifugation (fig. 2a, bottom panel), it still seemed to oligomerize, as seen in cross - linking experiments (not shown). thus, oligomerization may not be the only feature required for the ability of rtn1p to form tubules. mammalian rtn4a and dp1 are also immobile in the membrane we next determined whether the mammalian homologs of yeast rtn1p and yop1p also oligomerize and diffuse slowly in the er membrane. we performed analogous frap experiments on a number of mammalian gfp - fused er proteins expressed in cos-7 cells (indicated in fig. we previously had found that mammalian myc - tagged rtn4a and hemagglutinin (ha)-tagged dp1 localize exclusively to the tubular er (6). gfp - dp1 also formed perinuclear aggregates in cells highly expressing this protein (data not shown). at moderate expression levels, rtn4a - gfp and gfp - dp1 localized to the tubular er and were absent from the ne and peripheral sheets in cos-7 cells (fig. 5, a and b). in contrast, rfp - fused sec61 (rfp - sec61), a normal resident er membrane protein, was seen in all er domains. to analyze the diffusional mobility of mammalian rtn4a - gfp and gfp - dp1 in the tubular er by frap, we first selected a small portion of the er in the periphery of a cell, where a single layer of tubular network can be visualized. in our initial frap experiments, we found that the er tubules were highly dynamic, often growing into the photobleached region within seconds (data not shown). the fluorescence recovery seen was thus not that of protein diffusion in the membrane, but of er tubule movement itself. to remedy this and effectively freeze the microtubule - dependent er tubule movement (23), transfected cells were first treated with 0.5 m nocodazole, a concentration that inhibited microtubule dynamics and partially depolymerized the cytoskeleton. all subsequent frap experiments were conducted in the presence of nocodazole and performed within 530 min upon addition of the drug. figure 3.a yeast rtn1p mutant with defects in localization and oligomerization also can not shape tubules in vivo. a, mutations in rtn1 mutant 1, mutant 2, and mutant 91 that affect the ability of the protein to localize exclusively to the tubular er. the residues altered in mutant 1 are shown in blue, and those in mutant 2 are shown in green. d, fluorescence intensities normalized to prebleach values plotted over time from frap analyses of rtn1-gfp mutant 1 (b), mutant 2 (c), or mutant 91 (d) expressed in wild - type s. cerevisiae cells. e, the indicated gfp fusions were constitutively expressed under their endogenous promoters in rtn1rtn2yop1 cells. the er was visualized by focusing on either the center or periphery of the cell. note that wild - type, mutant 1, and mutant 2 rtn1-gfp restore tubular er structure, whereas mutant 91 and the control protein sec63-gfp do not. a yeast rtn1p mutant with defects in localization and oligomerization also can not shape tubules in vivo. a, mutations in rtn1 mutant 1, mutant 2, and mutant 91 that affect the ability of the protein to localize exclusively to the tubular er. the residues altered in mutant 1 are shown in blue, and those in mutant 2 are shown in green. b d, fluorescence intensities normalized to prebleach values plotted over time from frap analyses of rtn1-gfp mutant 1 (b), mutant 2 (c), or mutant 91 (d) expressed in wild - type s. cerevisiae cells. e, the indicated gfp fusions were constitutively expressed under their endogenous promoters in rtn1rtn2yop1 cells. the er was visualized by focusing on either the center or periphery of the cell. note that wild - type, mutant 1, and mutant 2 rtn1-gfp restore tubular er structure, whereas mutant 91 and the control protein sec63-gfp do not. figure 4.mammalian gfp - fused membrane proteins and their known lateral mobilities used for frap analysis. mammalian gfp - fused membrane proteins and their known lateral mobilities used for frap analysis. upon photobleaching, both rtn4a - gfp and gfp - dp1 diffused relatively slowly into the bleached area, much more slowly than the control membrane protein, gfp - sec61 (fig. 5c). quantitating the fluorescence intensities normalized to prebleach values showed that the half - times of recovery were 3- to 4-fold slower than that of the control membrane protein gfp - sec61 (fig. 5, d and e). moreover, although gfp - sec61 showed almost maximal recovery to prebleach levels, gfp - dp1 and rtn4a - gfp only recovered to a maximum of 50 and 60%, respectively. the diffusional kinetics of rtn4a - gfp and gfp - dp1 were similar to that of lamin b receptor (lbr), a protein that has restricted mobility due to its association with the nuclear lamina (fig. 5, d and e) (24). the mobilities of both rtn4a and dp1 were also similar to that of another relatively immobile er protein, climp-63 (fig. gfp - climp63 diffuses slowly because it oligomerizes extensively with itself via its large coiled - coil luminal domain (13). a mutant climp-63 lacking this luminal domain, gfp - climp63lum, diffuses rapidly (13) with similar kinetics to that of gfp - sec61 (fig. these results indicate that both rtn4a - gfp and gfp - dp1 have slow and restricted diffusional mobility within the membrane, consistent with the frap data obtained in yeast. the reticulon homology domain is sufficient for localization to tubules and for diffusional immobility all reticulons possess a reticulon homology domain (rhd), which contains the two membrane - embedded hairpins, but the length and sequence of the n terminus differ greatly among different mammalian isoforms (25). to test whether the conserved rhd is sufficient to localize the reticulons to er tubules, we deleted the n - terminal region from rtn4a and expressed the remaining segment (rtn4hd) as a gfp fusion in cos-7 cells. like the full - length rtn4a protein, gfp - rtn4hd localized to tubules, but was absent from the peripheral er sheets and the nuclear envelope (fig. an exclusively tubular er localization was also seen with gfp - fused rtn3c, another reticulon isoform expressed from a different gene, which also consists of only the rhd (fig. these results indicate that the conserved rhd mediates the exclusive tubular localization of the reticulons in mammalian cells. next, we tested whether the rhd is also sufficient for the slow lateral mobility of the reticulons. frap experiments were done on the two reticulon isoforms, gfp - rtn4hd and gfp - rtn3c. for these experiments, we selected cells expressing these proteins only at moderate levels, because these proteins were found enriched in aberrant ribbon- or globule - like membrane structures in highly expressing cells. these structures did not colocalize with endogenous er or golgi markers and were reminiscent of crystalloid membranes in transmission electron microscopy (supplemental fig. frap analyses showed that both gfp - rtn4hd and gfp - rtn3c diffuse slowly compared with gfp - sec61 and have similar diffusional kinetics to lbr - gfp (fig. these results support a model in which the highly conserved rhd mediates both their tubular localization and their slow diffusional properties. this is consistent with our results on yeast rtn1p, which also contains little more than the rhd. reticulons and dp1 oligomerize in higher eukaryotes as in yeast, we obtained initial evidence for oligomerization of the reticulons and dp1 by performing frap analysis on cells depleted of atp. cos-7 cells expressing the gfp - fused proteins were treated with 50 mm 2-d - deoxyglucose and 0.02% sodium azide, and frap experiments were conducted in the first 30 min of treatment. atp depletion did not have gross effects on tubular er morphology (data not shown), but it decreased the mobility of rtn3c, rtn4hd, and dp1 (fig. 7a and supplemental fig., the mobility of the control membrane protein, gfp - sec61, remained unchanged (fig. these results suggest that the reticulons and dp1 form oligomers, the size of which is regulated by atp. direct evidence for oligomerization was obtained by sucrose gradient centrifugation and chemical cross - linking. the membranes were solubilized in either digitonin or nonidet p-40, and the detergent extracts were subjected to sucrose gradient centrifugation (fig. we also found that digitonin - solubilized ha - dp1 expressed in cos-7 cells migrated in sucrose gradient centrifugation at a molecular weight consistent in size with larger multimeric complexes (supplemental fig. digitonin efficiently solubilized membrane proteins, as gfp - sec61 monomers sedimented at lower molecular weight fractions (supplemental fig. figure 5.mammalian rtn4a - gfp and gfp - dp1 have slow, restricted lateral mobilities in er tubules. a and b, rtn4a - gfp and gfp - dp1 (both in green) expressed in cos-7 cells localize only to the tubular er, whereas a normal resident er membrane protein, rfp - sec61 (red) localizes to all er subdomains, including the nuclear envelope and peripheral sheets. c, fluorescence recovery of gfp - dp1 compared with the control membrane protein gfp - sec61. images were taken before and then after the photobleach for the times indicated. the boxed region shows the area that was photobleached. d, quantitation of frap analyses of rtn4a - gfp (blue ; n = 7 cells) compared with gfp - sec61 (green ; n = 6) and lbr - gfp (red ; n = 3), where the average fluorescence intensity normalized to prebleach values was plotted over time. e, quantitation of frap analyses of gfp - dp1 (blue ; n = 9) compared with gfp - sec61 (green) and lbr - gfp (red). f, quantitation of frap analyses of gfp - climp63, an er membrane protein known to be immobile through homo - oligomerization (blue ; n = 6), compared with gfp - sec61 (green) and lbr - gfp (red). g, quantitation of frap analyses of climp63lum - gfp, a mutant form of climp63 lacking its luminal homo - oligomerization domain (blue ; n = 5), compared with gfp - sec61 (green) and lbr - gfp (red). mammalian rtn4a - gfp and gfp - dp1 have slow, restricted lateral mobilities in er tubules. a and b, rtn4a - gfp and gfp - dp1 (both in green) expressed in cos-7 cells localize only to the tubular er, whereas a normal resident er membrane protein, rfp - sec61 (red) localizes to all er subdomains, including the nuclear envelope and peripheral sheets. c, fluorescence recovery of gfp - dp1 compared with the control membrane protein gfp - sec61. images were taken before and then after the photobleach for the times indicated. the boxed region shows the area that was photobleached. d, quantitation of frap analyses of rtn4a - gfp (blue ; n = 7 cells) compared with gfp - sec61 (green ; n = 6) and lbr - gfp (red ; n = 3), where the average fluorescence intensity normalized to prebleach values was plotted over time. e, quantitation of frap analyses of gfp - dp1 (blue ; n = 9) compared with gfp - sec61 (green) and lbr - gfp (red). f, quantitation of frap analyses of gfp - climp63, an er membrane protein known to be immobile through homo - oligomerization (blue ; n = 6), compared with gfp - sec61 (green) and lbr - gfp (red). g, quantitation of frap analyses of climp63lum - gfp, a mutant form of climp63 lacking its luminal homo - oligomerization domain (blue ; n = 5), compared with gfp - sec61 (green) and lbr - gfp (red). membranes of cos-7 cells transiently transfected with ha - rtn3c were isolated, treated with increasing concentrations of the bifunctional cross - linker egs, and analyzed by sds - page and immunoblotting. as in yeast, a multimeric ladder was observed, with the step size being consistent with homo - oligomers (fig. these data indicate that the rhd of the reticulons is sufficient for their ability to oligomerize. homo - oligomer formation was also observed when ha - dp1 was cross - linked (fig. these results indicate that the reticulons and dp1/yop1p form oligomers in yeast and mammalian cells, suggesting that they do so in all eukaryotic cells. mammalian reticulons or dp1 can stabilize er tubules in the absence of microtubules we hypothesized that, if reticulon proteins form oligomeric structures that directly shape and stabilize the tubular er, then overexpressing these proteins might stabilize the tubular shape of membranes in vivo under conditions where the tubular er is normally disrupted. prolonged depolymerization of microtubules is known to cause peripheral er tubules to retract to the cell center, resulting in unstructured, cisternal er (26). when cos-7 cells expressing the control membrane protein gfp - sec61 were treated with 1 mm nocodazole for 30 min to completely depolymerize all dynamic microtubules, the tubular er converted into mostly sheet - like structures (fig. however, when we subjected cos-7 cells overexpressing gfp - rtn3c to the same treatment, large parts of the tubular er remained intact, with many tubules connected by three - way junctions present (fig. some of the protein also accumulated bright punctae that colocalized with other endogenous er markers (data not shown). similar results were seen in cells overexpressing rtn4a, rtn4hd, or dp1 (data not shown). thus, it appears that increased levels of the reticulons or dp1 can stabilize the tubular er in the absence of the cytoskeletal network in mammalian cells, consistent with their proposed role in structurally stabilizing er tubules. a and b, exclusive tubular er localization of gfp - rtn4hd and gfp - rtn3c (both in green), two reticulon isoforms containing only the conserved reticulon homology domain, compared with the general membrane protein rfp - sec61 (red) when expressed in cos-7 cells. c, typical frap experiments performed in cos-7 cells comparing the fluorescence recovery of gfp - rtn3c to gfp - sec61 illustrates that reticulons containing only the rhd is relatively immobile. d, quantitation of frap analyses of gfp - rtn3c (blue ; n = 6) compared with gfp - sec61 (green) and lbr - gfp (red), where fluorescence intensities normalized to prebleach values were plotted over time. e, quantitation of frap analyses of gfp - rtn4hd (blue ; n = 14), compared with gfp - sec61 (green) and lbr - gfp (red). a and b, exclusive tubular er localization of gfp - rtn4hd and gfp - rtn3c (both in green), two reticulon isoforms containing only the conserved reticulon homology domain, compared with the general membrane protein rfp - sec61 (red) when expressed in cos-7 cells. c, typical frap experiments performed in cos-7 cells comparing the fluorescence recovery of gfp - rtn3c to gfp - sec61 illustrates that reticulons containing only the rhd is relatively immobile. d, quantitation of frap analyses of gfp - rtn3c (blue ; n = 6) compared with gfp - sec61 (green) and lbr - gfp (red), where fluorescence intensities normalized to prebleach values were plotted over time. e, quantitation of frap analyses of gfp - rtn4hd (blue ; n = 14), compared with gfp - sec61 (green) and lbr - gfp (red). here we demonstrate that the reticulons and dp1/yop1p diffuse only slowly in the membrane in both yeast and mammalian cells. rather, immobility appears to be caused by their oligomerization that is evident in our sucrose gradient centrifugation and cross - linking experiments. the conserved rhd containing the two hydrophobic segments is sufficient for reticulon oligomerization ; this domain alone causes slow lateral mobility in the membrane and forms homo - oligomers in cross - linking experiments. this conclusion is supported by the isolation of mutants of yeast rtn1p that have amino acid changes in the rhd ; they oligomerize less extensively according to sucrose gradient sedimentation experiments, and they diffuse rapidly in the membrane. the same mutants also no longer localize exclusively to the tubular er, suggesting that oligomerization of the reticulons and dp1/yop1p is required for their proper localization. we presume that oligomerization is also required for the ability of the reticulons and dp1/yop1p to shape the tubular er. in support of this notion, we found that the overexpression of the mammalian reticulons and dp1 stabilized er tubules that would normally collapse in the absence of microtubules. however, oligomerization may not be sufficient, because rtn1p mutant 91, which is defective in forming er tubules, still shows oligomers in cross - linking experiments (data not shown). figure 7.the reticulons and dp1 form oligomers in higher eukaryotes. a, quantitated frap analyses of mammalian gfp - rtn3c under atp - depleted (red, n = 7) or non - depleted (blue, n = 14) conditions. b, mammalian sec61-gfp under atp - depleted (red, n = 9) or non - depleted (blue, n = 6) conditions. frap data from atp - depleted cells were compared with those from non - treated cells from fig. c, xenopus membranes solubilized in either 1.25% digitonin or 2% nonidet p-40 were fractionated by 1030% w / v sucrose gradient centrifugation, analyzed by sds - page, and immunoblotted with antibody against xenopus rtn4. d, isolated membranes of cos-7 cells expressing ha - dp1 or ha - rtn3c were treated with increasing concentrations of egs, analyzed on sds - page, and immunoblotted with anti - ha antibody. a, quantitated frap analyses of mammalian gfp - rtn3c under atp - depleted (red, n = 7) or non - depleted (blue, n = 14) conditions. b, mammalian sec61-gfp under atp - depleted (red, n = 9) or non - depleted (blue, n = 6) conditions. frap data from atp - depleted cells were compared with those from non - treated cells from fig. c, xenopus membranes solubilized in either 1.25% digitonin or 2% nonidet p-40 were fractionated by 1030% w / v sucrose gradient centrifugation, analyzed by sds - page, and immunoblotted with antibody against xenopus rtn4. d, isolated membranes of cos-7 cells expressing ha - dp1 or ha - rtn3c were treated with increasing concentrations of egs, analyzed on sds - page, and immunoblotted with anti - ha antibody. asterisks, number of monomers cross - linked. how oligomerization causes the reticulons and dp1/yop1p to localize to and induce the tubular er is not yet clear, but other proteins that shape membranes into tubules also oligomerize. examples include the caveolins that coat caveolae, flask - like invaginations at the plasma membrane, the ehd and pch proteins that are involved in endocytosis, and the dynamins and dynamin - like proteins, gtpases that participate in vesicle budding and in shaping mitochondria (2731). these proteins all oligomerize, and many are known to form spirals around membranes. based on these precedents, one may speculate that the reticulons and dp1/yop1p would use their propensity to oligomerize in a similar manner. however, the majority of the oligomers detected by sucrose density gradients and cross - linking experiments contained only four or five monomers, which would be insufficient to form a spiral around a membrane tubule. our data do not exclude that larger oligomers are actually present in vivo, because they may dissociate upon solubilization in detergent and may not be detectable by cross - linking experiments. however, smaller oligomers could still shape tubules if they formed arc - like structures around the tubule. arc - shaped oligomers may explain why relatively small oligomers of the reticulons and dp1/yop1p may still diffuse slowly in the membrane : such structures would diffuse in a one - dimensional way along the tubule and would be hindered to pass one another or do so only very slowly. by contrast, globular particles of similar mass would diffuse much faster, because they can move two - dimensionally and would not be obstructed by one another. finally, arc - shaped oligomers may also explain the localization of reticulons and dp1/yop1p ; it may be energetically most favorable for arcs to be in curved structures such as tubules. we propose that the reticulons and dp1/yop1p not only form arc - like scaffolds around membrane tubules, but also use their central domain, including the two hydrophobic segments, to form a wedge - like structure in the membrane. the use of both wedging and scaffolding mechanisms would be analogous to how other proteins generate high curvature in membranes. for example, n - bar domains wedge a hydrophobic segment into the outer leaflet of the lipid bilayer and form banana - shaped scaffolds (32). figure 8.overexpression of a reticulon stabilizes the tubular er in the absence of microtubules in mammalian cells. a, a typical cos-7 cell expressing gfp - sec61 (green), where microtubules have been depolymerized by nocodazole treatment (immunostained with anti-tubulin, red), has collapsed, sheet - like peripheral er. the lower row of panels shows an enlargement of the boxed region from the top panels. b, a typical cos-7 cell expressing gfp - rtn3c (green) after microtubule depolymerization (stained in red) leads to an accumulation of bright foci containing rtn3c but also retains much more tubular er. the lower row of panels shows an enlargement of the boxed region from the top panels. overexpression of a reticulon stabilizes the tubular er in the absence of microtubules in mammalian cells. a, a typical cos-7 cell expressing gfp - sec61 (green), where microtubules have been depolymerized by nocodazole treatment (immunostained with anti-tubulin, red), has collapsed, sheet - like peripheral er. the lower row of panels shows an enlargement of the boxed region from the top panels. b, a typical cos-7 cell expressing gfp - rtn3c (green) after microtubule depolymerization (stained in red) leads to an accumulation of bright foci containing rtn3c but also retains much more tubular er. the lower row of panels shows an enlargement of the boxed region from the top panels. scale bar, 10 m. we discovered that the diffusional mobilities of the reticulons and dp1 decrease upon atp depletion. this finding suggests that the oligomers continuously form and disassemble in vivo, with the disassembly requiring atp. surprisingly, however, atp depletion did not increase the size of the rtn1-gfp oligomers in sucrose density gradient or cross - linking experiments (data not shown). although it is therefore possible that atp depletion affects the diffusion of the reticulons and dp1 in other ways, it is tempting to speculate that the dynamic nature of the oligomers would allow them to continuously form and disassemble at discrete but random sites throughout the er. they could thus tubulate a larger surface of the er than they physically occupy at any given moment. in fact, although the reticulons and dp1/yop1p are some of the most abundant membrane proteins in the er, they are not present in amounts that would allow them to cover the entire surface of all er tubules. the postulated arc shape of the oligomers and their spacing on the tubules would allow other er proteins to freely diffuse in the membrane. it is also possible that an energy - driven disassembly is not the only mechanism by which the size of reticulon and dp1/yop1p oligomers is restricted. we have found that homo - oligomers of these proteins tend to be larger than hetero - oligomers. homo - oligomerization may therefore lead to excessive amounts of these proteins in a given tubule. because high concentrations of the reticulons and dp1/yop1p have been shown to decrease the diameter of the tubule and displace resident luminal proteins (11, 33), different hetero - oligomeric combinations of the reticulons and dp1/yop1p and their various isoforms it has recently been shown that overexpression of rtn3c in mice leads to large sds - resistant oligomers in neurons, and these mice exhibit neuropathies similar to that of alzheimer disease (20). although it is unclear whether these aggregates are present in the tubular er, it is appealing to speculate that this pathology results from excessive and unregulated oligomerization of the reticulons. | we recently identified a class of membrane proteins, the reticulons and dp1/yop1p, which shape the tubular endoplasmic reticulum (er) in yeast and mammalian cells. these proteins are highly enriched in the tubular portions of the er and virtually excluded from other regions. to understand how they promote tubule formation, we characterized their behavior in cellular membranes and addressed how their localization in the er is determined. using fluorescence recovery after photobleaching, we found that yeast rtn1p and yop1p are less mobile in the membrane than normal er proteins. sucrose gradient centrifugation and cross - linking analyses show that they form oligomers. mutants of yeast rtn1p, which no longer localize exclusively to the tubular er or are even totally inactive in inducing er tubules, are more mobile and oligomerize less extensively. the mammalian reticulons and dp1 are also relatively immobile and can form oligomers. the conserved reticulon homology domain that includes the two membrane - embedded segments is sufficient for the localization of the reticulons to the tubular er, as well as for their diffusional immobility and oligomerization. finally, atp depletion in both yeast and mammalian cells further decreases the mobilities of the reticulons and dp1. we propose that oligomerization of the reticulons and dp1/yop1p is important for both their localization to the tubular domains of the er and for their ability to form tubules. |
alagille syndrome (algs) is an autosomal dominant disorder caused by heterozygous mutation in the jag1 (jagged 1) gene on chromosome 20p12. its estimated prevalence is about 1 out of 70,000 births, when ascertained by the presence of neonatal jaundice. it has been traditionally defined by a paucity of intrahepatic bile ducts, in association with five main clinical abnormalities : cholestasis, cardiac disease, skeletal abnormalities, ocular abnormalities, and a characteristic facial phenotype. abnormalities were reported in different systemic organs : in the eye, posterior embryotoxon and retinal pigmentary changes were observed ; in the heart, pulmonic valvular stenosis as well as peripheral arterial stenosis ; in the bones, abnormal vertebrae (butterfly vertebrae) and decrease in interpediculate distance in the lumbar spine ; in the nervous system, absence of deep tendon reflexes and difficulty learning ; in the faces, characteristic traits such as broad forehead, deep - set eyes, prominent nose, pointed mandible and chin, bulbous tip of the nose, and varying degrees of fingers foreshortening. no gene mutations associated with craniosynostosis was found, therefore, jag1gene was suggested to play a possible role in cranial suture formation. furthermore, severe hypodontia was described in a 3-year - old asian boy with algs. twenty - two percent of the patients with algs had spontaneous or post - procedure bleeding in various organs and were subjected to special risk for bleeding. therefore, it can be speculated that abnormalities in the jag1-gene may impair hemostatic function. nine percent had vascular anomalies or events, and vascular accidents accounted for 34% of mortality. vascular anomalies including basilar and middle cerebral artery aneurysms, internal carotid artery anomalies, aortic aneurysms, and co - arctation of the aorta were documented in algs patients. furthermore, latex - hypersensitivity and severe susceptibility to dental decays or decalcifications on the posterior deciduous teeth were described. this report describes a combined surgical - orthodontic procedure for guided - eruption of a deeply impacted macrodontic maxillary central incisor in young algs patient. although an interdisciplinary approach was needed, surgical procedure may be complicated by the severe risk of increased bleeding and vascular events. a 10-year - old male was presented to our dental office complaining of delayed eruption of a maxillary central incisor [figure 1 ]. his medical history indicated a diagnosis of algs with no previous systemic treatment or complication. clinical examination revealed characteristics algs traits : broad forehead, deep - set eyes with eye - socket, pointed mandible and prominent chin ; maxillary hypoplasia, maxillo - mandibular retrusion ; and increased vertical facial dimension were also noted. he presented a class i molar relationship on both side, a correct overjet and a deep overbite (90%). absence of maxillary right central incisor with no adequate space in the maxillary arch was noted. an intra - oral examination revealed the presence of several decalcifications on deciduous teeth with previous conservative treatment on first permanent molars and maxillary first deciduous molar on right side. a 10-year - old male with history of alagille syndrome, presence of macrodontic central incisor vertically retained were shown cephalometric analysis indicated a class i skeletal pattern with retrognathic maxilla and mandible, prominent chin, and slight hyperdivergent tendency. treatment objective was to create space in the maxillary arch and allow the eruption of the impacted central incisor. surgical exposure and orthodontic traction with a fixed appliance was planned. risk of increased bleeding, vascular events, and latex hypersensitivity were the major concern, requiring the use of latex - free materials, controlled hemostatic procedure, and antibiotic prophylaxis as recommended by the cardiologist. bands on deciduous second molars and begg 's brackets on permanent incisors and deciduous canines were placed. after alignment and levelling were completed using 0.014 nickel titanium archwire, a relatively rigid stabilizing wire (0.018 round australian wire) was placed, and a coil spring was used to create adequate space between the central incisor and the right lateral incisor. an access flap was elevated to expose the impacted tooth and a lingual button connected to a ligature wire was bonded to the palatal surface. a closed surgical exposure technique was chosen. an elastic module for applying an apically directed force once crown of the maxillary incisor adequately appeared in the maxillary arch, a begg bracket was bonded to the vestibular surface and alignment and levelling were completed using 0.014 nickel titanium wire, and later replaced by 0.018 round australian wire [figure 3 ]. closed surgical exposure and vertical traction of the central incisor post - treatment photographs 9 months later. macrodontic incisor caused gingival margin and incisal border incongruence with the adjacent teeth the impacted maxillary right central incisor was brought into proper alignment with the adjacent teeth. once alignment was achieved, greater height and crown width of the macrodontic incisor caused gingival margin and incisal border alterations [figure 3 ]. conservative treatment and minimal restorative interventions, accessory periodontal treatment, and dental reshaping procedures may be indicated to re - establish the proper size and dimension of the macrodontic incisor. a 10-year - old male was presented to our dental office complaining of delayed eruption of a maxillary central incisor [figure 1 ]. his medical history indicated a diagnosis of algs with no previous systemic treatment or complication. clinical examination revealed characteristics algs traits : broad forehead, deep - set eyes with eye - socket, pointed mandible and prominent chin ; maxillary hypoplasia, maxillo - mandibular retrusion ; and increased vertical facial dimension were also noted. he presented a class i molar relationship on both side, a correct overjet and a deep overbite (90%). absence of maxillary right central incisor with no adequate space in the maxillary arch was noted. an intra - oral examination revealed the presence of several decalcifications on deciduous teeth with previous conservative treatment on first permanent molars and maxillary first deciduous molar on right side. a 10-year - old male with history of alagille syndrome, presence of macrodontic central incisor vertically retained were shown cephalometric analysis indicated a class i skeletal pattern with retrognathic maxilla and mandible, prominent chin, and slight hyperdivergent tendency. treatment objective was to create space in the maxillary arch and allow the eruption of the impacted central incisor. surgical exposure and orthodontic traction with a fixed appliance was planned. risk of increased bleeding, vascular events, and latex hypersensitivity were the major concern, requiring the use of latex - free materials, controlled hemostatic procedure, and antibiotic prophylaxis as recommended by the cardiologist. bands on deciduous second molars and begg 's brackets on permanent incisors and deciduous canines were placed. after alignment and levelling were completed using 0.014 nickel titanium archwire, a relatively rigid stabilizing wire (0.018 round australian wire) was placed, and a coil spring was used to create adequate space between the central incisor and the right lateral incisor. an access flap was elevated to expose the impacted tooth and a lingual button connected to a ligature wire was bonded to the palatal surface. a closed surgical exposure technique was chosen. an elastic module for applying an apically directed force once crown of the maxillary incisor adequately appeared in the maxillary arch, a begg bracket was bonded to the vestibular surface and alignment and levelling were completed using 0.014 nickel titanium wire, and later replaced by 0.018 round australian wire [figure 3 ]. closed surgical exposure and vertical traction of the central incisor post - treatment photographs 9 months later. macrodontic incisor caused gingival margin and incisal border incongruence with the adjacent teeth the impacted maxillary right central incisor was brought into proper alignment with the adjacent teeth. once alignment was achieved, greater height and crown width of the macrodontic incisor caused gingival margin and incisal border alterations [figure 3 ]. conservative treatment and minimal restorative interventions, accessory periodontal treatment, and dental reshaping procedures may be indicated to re - establish the proper size and dimension of the macrodontic incisor. the major concern in treating this patient was the cardiological and vascular risk following surgical periodontal procedure, such as dental implant insertion, since high impaction position of a retained tooth may often require a traumatic surgery. increased bleeding, vascular events, and latex hypersensitivity were previously reported in algs patients, therefore, the use of latex - free materials and controlled hemostatic procedure were mandatory, even if these patients reported no previous medical history of specific hypersensitivity or hemostatic dysfunction. in agreement with cardiological recommendations, it was decided that antibiotic prophylaxis was not required since surgical procedure was not considered a risk for bacterial endocarditis. in accordance, first, closed - eruption technique may often avoid a second surgical procedure or re - intervention, second, it usually provides the most esthetically pleasing result since it preserves as much as possible gingival margin contour of the central incisor and adequate zone of attached gingival. open surgical exposure has the disadvantage of producing a non - keratinized vestibular gingival margin during vertical traction. using this technique, papillary apical positioned partial - thickness flap during the orthodontic traction may be needed to achieve an esthetic gingival margin contour over the central incisors and provide the teeth with an adequate zone of attached gingival labial to the crown. furthermore, because of the relatively high prevalence of gingival defects in some studies, adjunctive post - orthodontic periodontal surgery might be required in many patients treated with this method. an impacted maxillary central incisor in a child often represents, in addition to a disturbing esthetical problem, the parent 's main concern according which a patient can be referred to the orthodontist to evaluate the possibility of a disturbance eruption. orthodontic treatment has not been usually postponed until the complete eruption of the permanent dentition especially when the problem can be treated in the early mixed dentition stage. however, it is important to properly inform the patient about the possibility of failure before undertaking extensive measures necessary for the management of a severely impacted tooth. factors suggesting clinical success may be considered on the basis of position and orientation of the impacted tooth, amount of root formation, and degree of root dilacerations. and sabri reported that movement of an impacted central incisor could be impossible because of ankylosis and external root resorption. ho and liao reported that unsuccessful predictors for impacted central incisors were older age, highimpacted tooth, and dilacerated incisors. in this case, high impaction in the maxillary bone was the only negative factor increasing the likelihood of unsuccessful treatment result. however, central incisor has been successfully brought into proper position after having provided sufficient space in the maxillary arch, but an unavoidable unesthetic higher gingival margin in respect to the adjacent teeth was present after alignment ; this was due to the different crown size of the macrodontic central incisor. in fact, problems related to macrodontic tooth can include difference in size and shape, space deficiency, interproximal contact points with the adjacent teeth, and incongruence in gingival margin or incisal border. nevertheless, this case can not be considered as a true macrodontia ; when true macrodontia occurs in the incisal region, it can often be confused with conjoined teeth or fusion of teeth, since the union of two or more teeth results in a single large tooth during the odontogenesis. the main difference is that size reduction of macrodontic central incisors by proximal grinding is not possible without jeopardizing tooth vitality. in this case, reshaping procedure or minimal restorative interventions such as indirect composite veneers may be indicated. the esthetic outcome of these procedures can be excellent without unnecessary loss or teeth extraction. knowledge of the use of indirect composite veneers can provide another treatment option for esthetic problems in the management of tooth anomalies, including macrodontia. in conclusion this case report demonstrates that is possible, controlling carefully the procedure, to perform a combined surgical and orthodontic treatment in an algs patient. | this case report describes the surgical - orthodontic guided - eruption of a deeply impacted macrodontic maxillary central incisor in a 10-year - old patient with alagille syndrome (algs). in the first stage, orthodontic treatment with fixed appliance on deciduous teeth allowed to create enough space for the eruption of the maxillary right central incisor. the second stage included closed surgical exposure and vertical traction. after impacted tooth erupted in the proper position, accessory periodontal treatment and dental reshaping procedures may be indicated to camouflage macrodontic incisor with the adjacent teeth. this is the first report that presents a patient with algs undergoing orthodontic and surgical treatment. |
all the patients of breast cancer and lung cancer undergoing chemotherapy in day care centre during the study time period were recruited. patient receiving anticancer agents either as single drug regimen or in combination of two or more drugs, aged > 18 years were included in the study. adverse events caused by administration errors, noncompliance or overdose were excluded from the study. this observational prospective study was designed to assess the safety of chemotherapy drugs used in lung and breast cancer patients. the study was performed in post graduate institute of medical education and research, chandigarh. during the study period, the prescription pattern of oncologists was observed accordingly and patients were interviewed for the occurrence of adrs in the presence of their healthcare team i.e. nurses or doctors. adrs were collected by filling the suspected adr form for each patient who experienced adr. during the patient interview ; patient information, drug information, past medical history, laboratory investigations were accessed. after collecting the information on adr, causality assessment was done with the help of pharmacovigilance expert (pharmacovigilance centre coordinator). this observational prospective study was designed to assess the safety of chemotherapy drugs used in lung and breast cancer patients. the study was performed in post graduate institute of medical education and research, chandigarh. during the study period, the prescription pattern of oncologists was observed accordingly and patients were interviewed for the occurrence of adrs in the presence of their healthcare team i.e. nurses or doctors. adrs were collected by filling the suspected adr form for each patient who experienced adr. during the patient interview ; patient information, drug information, past medical history, laboratory investigations were accessed. after collecting the information on adr, causality assessment was done with the help of pharmacovigilance expert (pharmacovigilance centre coordinator) the study sample comprised of 174 patients out of which 101 breast cancer and 73 lung cancer patients receiving chemotherapy were screened for adverse drug reactions. out of 174 patients 152 (87.36%) patients experienced at least one adr and 22 (12.64%) patients did not develop any adr. a summarized table is given below (table 1) : among the different types of anticancer drug therapy the patients of breast cancer were prescribed different drug regimens of fluorouracil+doxorubicin+cyclophosphamide (41.58%), paclitaxel (22.77%), docetaxel (10.89%), fluorouracil+epirubicin+cyclophosphamide (9.90%), and some other regimens, which involve docetaxel+carboplatin+trastuzumab (3.96%), trastuzumab (2.97%), epirubicin+docetaxel (1.98%), fluorouracil+docetaxel+cyclophosphamide (1.98%), cisplatin+vinorelbine (0.99%), cisplatin+vinblastin (0.99%), gemcitabine+cisplatin (0.99%), docetaxel+doxorubicin (0.99%) ; fig. 1 illustrates the prescription pattern in breast cancer group of patients : percentage of adr in breast and lung cancer prescription pattern for patients having breast cancer. number of patients treated () and number of patients experienced adr (). out of 101 patients of breast cancer 91 (90.09%) patients experienced at least one adr but 10 (9.90%) did not experience any adr at all. the combined regimen of fluorouracil, doxorubicin and cyclophosphamide was prescribed to a total of 42 (41.58%) patients out of which 36 (85.71%) developed adverse events. the number of patients receiving paclitaxel alone was 23 (22.77%) out of which 21 (91.30%) patients experienced adverse events. docetaxel treatment therapy was received by 11 (10.89%) patients and 10 (90.90%) of them have experienced adr. a combined drug regimen of fluorouracil+epirubicin+cyclophosphamide was also prescribed to 10 (9.90%) patients out of whom 9 (90%) patients experienced adr. some other drug regimens in combination as well as single drug regimens were also prescribed like trastuzumab+docetaxel+carboplatin to 4 patients, epirubicin+docetaxel to 2 patients, fluorouracil+doxorubicin+cyclophosphamide to 2 patients, trastuzumab to 3 patients. less frequently used drug regimens like docetaxel+doxorubicin, cisplatin+gemcitabine, cisplatin+vinblastine, and cisplatin+vinorelbine were prescribed to 1 patient each of the study population, which developed atleast one single adr including alopecia, breathlessness, cough, diarrhoea, nausea, neuropathy, pain and restlessness etc. percentage of patients experienced different adrs are tabulated below (table 2) : out of total 73 patients of lung cancer, pemetrexed+cisplatin, docetaxel+cisplatin, irinotecan+cisplatin were administered to 24.65, 30.14 and 17.81% patients, respectively. on the other hand, some other regimens as docetaxel (6.85%) and paclitaxel with cisplatin (6.85%). while docetaxel and paclitaxel in combination with cisplatin and/or carboplatin were also prescribed to some patients. 2 illustrates the prescription pattern of patients of lung cancer : adr profile of different prescribed drug regimens of breast cancer patients prescription pattern for patients having lung cancer. number of patients treated () and number of patients experienced adr (). out of 73 patients of lung cancer 61 (83.56%) patients experienced adverse events and rest of them did not develop any adr. during treatment, 24.65% patients were prescribed combined regimen of pemetrexed+cisplatin out of which 72.22% patients experienced adverse events like dysgeusia (69.23%), anorexia (38.46%), constipation (38.46%), neuropathy (30.77%) and fever (30.77%). docetaxel+cisplatin was another regimen, which was prescribed to 30.14% lung cancer patients out of whom 86.36% experienced adverse drug events such as diarrhoea (63.16%), alopecia (63.16%), anorexia (42.10%), dysgeusia (31.58%), oral ulceration (31.58%). irinotecan+cisplatin was prescribed to 17.81% patients out of those patients 84.61% patients experienced at least one adr like nausea, constipation (45.45%) anorexia, diarrhoea, dysgeusia (36.36%). docetaxel was prescribed to 6.85% out of which 60% patiets experienced adr and paclitaxel+cispaltin was prescribed to 6.85% patients and all the 100% patients experienced adr. carboplatin in combination with pemetrexed was given to 4.17% patients and 2.78% patients were on combination of paclitaxel with carboplatin. patients treated with these regimens had constipation, fever, dysgeusia, neuropathy, diarrhoea, rashes, oedema, pain, insomnia, oral ulceration, nail discoloration and peeling of skin. table showing the adr profile is given below (table 3) : in the present study we have identified the prescription pattern of oncologist. in which we found that combined drug regimens were commonly used for the treatment of breast and lung cancer. while single drug regimens were also prescribed to some patients. adr profile of different prescribed drug regimens of lung cancer patients after performing the causality assessment it has been found that most of the adrs falls in probable or possible categories of who 's scale of causality assessment suggesting the real existence of adrs in sample studied, which has been shown in the table given below (table 4). some premedications like ranitidine and ondansetrone were administered intravenous bolus prophylactically to prevent the adr. reporting of new adrs is to be encouraged but one should not ignore already reported and well known adrs because there might be a pattern shift of adrs over the time. clinical trials of new drug therapies provide information about the nature and number of serious adverse events (saes). as these trials are conducted in very controlled environment rare and delayed adrs may fail get to get reported or reported less and sometimes may not accurately simulate the experiences in general population. studies have demonstrated that saes that do not occur during clinical trials can occur after a drug is approved for marketing and consumed by the public. in fact, a study of saes identified that after approval more than 20 cancer drugs were found linked with saes. adrs associated with chemotherapeutic drugs decrease the quality of life, and increases the mortality as well as the healthcare budget. it has been found that the adr profile of cancer chemotherapeutics is very less reported and the situation is even worse in india. the reason might be that the data collected by regulatory authorities and pharmaceutical industries is inaccessible to public. the total contribution of india in adverse drug reaction information is only 1% in global data, which shows the under - reporting and/ or under - detection as physicians are not so aware about pharmacovigilance or do not have adequate knowledge about adr monitoring and reporting, due to which the exact incidence of adr is still unknown. as a result of which, indian regulatory decisions are generally based on mimicking the decisions. withdrawal of pioglitazone by drugs controller general of india (dcgi) is a recent example but now the ban has been revoked. in the present study, prescription pattern of oncologists was studied and it was found that they have used single, double and/or triple regimen in the treatment therapy of breast and lung cancer. our study is also in conformation with earlier studies showing commonly occurring adrs in patients undergoing chemotherapy. in our study 152 (87.36%) our finding is in contrast to a study by malik., which shows 42% incidence and a study by goyal. our results show that dysgeusia, diarrhoea, alopecia, anorexia, nausea and vomiting were found to be very common adrs usually associated with every chemotherapeutic regimen. chemotherapy induced nausea and vomiting in cancer patients were found common, which is similar to other study findings previously published. in our study female patients of breast cancer were more prone to adrs 90% against lung cancer male patients 83%. the variation may be due to difference in medications and treatment guidelines followed for treating the cancer in different set up. dermatological reactions like alopecia 43.39%, nail discolouration 32.24%, were more frequently experienced adrs by the patients. other dermatological adrs like hives 1.97%, hyper pigmentation of skin 7.98%, itching 3.95% and rashes 5.26% were also experienced. gastrointestinal adrs like 19.18% of total study population experienced oral ulceration, out of which 23.08% in breast cancer patients, which is in comparison to males 13.11% is more and it is supported by sanches.. in comparison to the previous studies, which showed that adrs were more common in females as compared to males, which is supported by a study from nepal. pheniramine maleate and hydrocortisone were the common drugs used to manage the adrs like restlessness, breathlessness and rash while granisetron hydrochloride and ondansetron were used to manage nausea with or without vomiting experienced during chemotherapy cycles. in the present study in which we found that combined drug regimens were commonly used for the treatment of breast and lung cancer. while single drug regimens were also prescribed to some patients. adrs associated with chemotherapy were seen very often even in single drug regimens. after comparing the different regimens i.e. single and combined, it was observed that there was no significant difference in the adr profiles. since the study time period was only 2 months, the number of patients screened was less due to which we could not apply the extensive statistics therefore, further long duration studies covering large population are needed to validate the data. | adverse drug reactions associated with the use of anticancer drugs are a worldwide problem and can not be ignored. adverse drug reactions can range from nausea, vomiting or any other mild reaction to severe myelosuppression. the study was planned to observe the suspected adverse drug reactions of cancer chemotherapy in patients aged > 18 years having cancer attending postgraduate institute of medical education and research, chandigarh. during the study period, 101 patients of breast cancer and 73 patients of lung cancer were screened for occurrence of adverse drug reactions during their treatment with chemotherapy. about 87.36% patients experienced adverse drug reactions, 90.09% and 83.56% of breast and lung cancer patients experienced at least one adverse drug reaction respectively. in breast cancer patients, 41.58% patients were prescribed fluorouracil+doxorubicin+cyclophosphamide while paclitaxel was prescribed to 22.77% patients. alopecia (54.94%), nail discolouration (43.96%), dysgeusia (38.46%), anorexia (30.77%), nausea (29.67%), and neuropathy (29.67%) were found to be very common in breast cancer patients treated with single / combined regimen. in lung cancer group of patients, cisplatin with docetaxel, cisplatin with pemetrexed and cisplatin with irinotecan were prescribed to 30.14, 24.65 and 17.81% patients, respectively. dysgeusia (40.98%), diarrhoea (39.34%), anorexia (32.77%) and constipation (31.15%) and alopecia (31.15%) were commonly observed adverse drug reactions having lung cancer patients. causality assessments using world health organization causality assessment scale showed that observed adverse drug reactions were of probable (64.67%) and possible (35.33%) categories. alopecia, dysgeusia, anorexia, constipation diarrhoea, nausea, nail discoloration were more prevalent amongst the cancer patients undergoing chemotherapy. |
dopamine - producing cells in the brain start to diminish, and thus the concentration of dopamine in the brain diminishes. the loss of dopamine results in motor dysfunction, changes in behavior, and other burdensome effects to patients and caregivers. very little is known about the cause of pd, and there is no cure or way to prevent progression. current treatment focuses on alleviating motor symptoms, with very few options for treating non - motor symptoms such as psychosis.1 pimavanserin, manufactured by acadia pharmaceuticals, inc., was the first atypical antipsychotic drug approved by the food and drug administration (fda) for the treatment of hallucinations and delusions associated with parkinson s disease psychosis (pdp). pimavanserin was approved after several successful preclinical studies on april 29, 2016, after being granted breakthrough status in 2014.2 as per the fda, a breakthrough drug is one that treats a serious or life - threatening condition and demonstrates substantial improvement over current therapies for that condition. the fda usually expedites development and review of the drug, leading to a faster approval.3 before the approval of pimavanserin, pdp was treated with the same medications indicated to treat schizophrenia and bipolar disorder. clozapine and quetiapine are the second - generation antipsychotics (sgas) usually utilized for pdp, but quetiapine has not been shown in placebo - controlled studies to be efficacious.4 clozapine has been shown to be effective, but it also has additional monitoring requirements that can be burdensome on patients and caregivers. in addition, all sgas have a black box warning from the us fda. this states that all patients with dementia - related psychosis treated with sgas are at an increased risk of death. this is a class - wide distinction for all sgas, and not all drugs in this class have been proven to have this effect. in several studies, it was found that the death rates of those taking sgas versus placebo were 4.5% compared to 2.6%.5 this means that physicians must do a more thorough evaluation of their patients prior to prescribing any antipsychotics, especially in those with a history of cardiac or metabolic disorders. this limitation has been influential in the search for novel drugs that may be safer in this population. in a study to identify mortality risk factors in pd, psychosis at baseline this shows that the disease itself has a mortality risk, and the risk is not solely due to addition of dopaminergic therapy. in addition to psychosis at baseline, higher age, male sex, and decreased cognitive function led to worse prognosis in patients presenting with pd.6 in a 12-year, prospective longitudinal cohort study from norway, researchers found that 59.5% of the patients had developed pdp at some time during the course of the disease. it was also noted that 41.9% of the patients without a history of pdp had an incidence of pdp.7 advancing age is often a risk factor for pd, and the world s elderly population is increasing. it is hypothesized that this will increase to 16.7% in 2050.8 it seems necessary that new drugs to treat this prevalent disease state be approved, given that the likelihood of pd will increase. many sgas are hypothesized to work at least partially by blocking dopamine d2 receptors in the limbic system. used to treat pdp (3%10% of the usual dose used in schizophrenia), d2 receptors are blocked enough to cause some motor symptoms. since the d2 receptors are only partially blocked, but the psychosis did subside, it is hypothesized that blocking the d2 receptors in pdp may not be the reason for the therapeutic effect seen with sgas. since clozapine is effective at low doses, and quetiapine has not been proven to be, other receptors that may cause pdp were examined. this led to the hypothesis that the serotonin (5-ht)2a receptor was implicated in pdp, rather than the d2 receptor. this reasoning led researchers to investigate pimavanserin, a selective serotonin2a receptor inverse agonist, which was subsequently shown to treat pdp without adversely effecting motor functions.4 due to its novel drug status and quick approval, more post - marketing clinical trials are warranted to prove long - term safety and efficacy.2 this drug will allow clinicians to have more therapeutic options when it comes to treating pdp. the etiology of the disease is still not known, but now it is considered to be a mixture of environmental and genetic factors. though many chemicals have been tested, such as pesticides, metals, and neurotoxins, a single causative chemical has not been observed. it is noted that patients who smoke cigarettes may be at a decreased risk of pd. in addition, patients who drink coffee or tea may also be at a decreased risk. it is very difficult to find a reproducible study on the prevalence and incidence of pd. according to a multinational study from the tzu chi medical journal, the burden of diseases such as pd is greatly underestimated. the prevalence of pd in europe is estimated to be between 65.6 and 12,500 per 100,000 patients. it is noted that the prevalence is lower in asian countries, although it was not determined if this was due to genetic or environmental factors or both.9 it is also estimated that over 1 million usa citizens have pd. it is said to affect approximately 1% of people over 65 years old and 2.5% of people over 80 years old. the general age range of diagnosis is between 55 and 65 years old.10 it is considered the second - most prevalent and burdensome age - related neurodegenerative disorder behind alzheimer s disease.11 pd has historically been diagnosed by motor symptoms. james parkinson first described these clinical indicators in 1817, and since then no cure has been found. patients often present with bradykinesia, muscular rigidity, resting tremor, postural instability, or a combination of these symptoms. diagnosis is sometimes very difficult because symptoms will present differently and at varying times in patients. practitioners will diagnose a patient with pd if they present with bradykinesia or asymmetry prominence and also respond to dopaminergic therapy.10 the etiology and pathology of pd are not completely understood, but numerous advances over the past 100 years have shown some insight. it was discovered that a loss of neurons in the substantia nigra could be the cause for the symptoms experienced by patients.12 during the 1950s, additional data about the depletion of dopamine from the basal ganglia helped create a theory of the pathophysiology of the disease.13 it has been shown that the number of neurons in the substantia nigra, both pigmented and non - pigmented, decreases by 66% and 24%, respectively, in patients with pd.12 many hypotheses have been generated from this information, but it is clear that further studies must be done in order to truly understand the pathogenesis of this disease. it is known that the world s percentage of elderly persons is increasing, which may result in diseases that generally affect the elderly population becoming more prevalent with time. analysis of the 2015 global census data leads to estimates that the percentage of adults over 65 will increase from 8.5% to 16.7% by 2050. also, the number of persons over 80 is likely to triple (126.5 million to 446.6 million), and in some countries quadruple by 2050.8 although there is no evidence that any risk factor is more likely to lead to a pd diagnosis than another, it is likely that global incidence will increase with advancing age. pd affects people globally, with differences in prevalence likely due to multiple factors such as genetics, average life expectancy in certain countries, and availability of health care. this shows that pd, and potentially pdp, is a global concern that is likely to increase. in addition to increasing prevalence, patients who currently have the disease are living longer, which means that they are exposed to the disease and therapeutic agents longer.14 health care providers and researchers should be informed about current and future treatments for these chronic and debilitating diseases, and they should be prepared to see increasing number of patients with the disease and long - term effects of therapy. as the number of cases of pd increases, it is also being observed that the treatment of motor symptoms is no longer sufficient. non - motor symptoms, such as depression, dementia, and psychosis, are reported to be more burdensome than motor symptoms in patients with pd. these symptoms often result in a more disabling disease with worse outcomes.15 these symptoms put further strain on patients and their caregivers, and they are the primary reason that pd patients are admitted to nursing homes.16 pdp, like parkinson s disease, is not fully understood. questions remain as to whether it is a symptom of the disease, a response to dopaminergic therapy given to alleviate motor symptoms, or a completely independent pathology. the pdp in drug - treated patients presents in a similar fashion to schizophrenia (hallucinations and delusions), but the manifestation of the hallucinations is often starkly different. most commonly, pdp patients present with visual hallucinations, with a lifetime prevalence of visual hallucinations being 50% or higher.17 possibly, one - third of individuals with pd experience visual hallucinations ; however, up to 75% of patients will develop such phenomena over a 20-year period.18 these hallucinations have been reported to often involve children or animals, or they can be simpler visual disturbances, such as a quick flashing light.19 it has been observed that these visual hallucinations often occur during the evening or when lighting is dim.18 in schizophrenia, auditory hallucinations are most common.17 these auditory hallucinations, which occur in 60%80% of schizophrenic patients, are commonly voices (either internal or external).20 these auditory hallucinations are perceived by the patient as clearly as actual perceptions do.21 delusions also present differently in both disease states. they are not as common in pdp, and they may present as delusions of abandonment or infidelity.19 schizophrenic delusions can present in many ways ; they can be grandiose, persecutory, nihilistic, somatic, or any combination of these. they could also present as bizarre, where the hallucination is completely implausible or referential.21 schizophrenia has historically been considered a disease in which there is an excess of dopaminergic signaling, and it has been treated with drugs that reduce this signaling.17 since most of the therapies used in pd increase dopaminergic transmission in the brain, these therapies have been implicated in causing psychosis in pd patients. contrary to these studies, some patients on dopaminergic therapy never experience psychosis.15 it is now hypothesized that medications are not the only factor that determines whether a pd patient experiences hallucinations. in numerous studies, there has been no correlation between dose of levodopa and whether or not patients experienced psychosis. also, high - dose levodopa given intravenously has not been proven to induce visual hallucinations. it is currently not possible to predict which patients will experience pdp, but it is likely that most patients will experience visual hallucinations at least once over the course of their disease.18 it is essential that clinicians have therapeutic options for the treatment of psychosis with high efficacy and minimal intolerability. since a decrease in dopamine is implicated as the cause of the disease, current therapies for pd center around increasing dopaminergic signaling in the brain. there are several mechanisms by which this can be done, such as supplementation with a drug that produces dopamine in the body (levodopa), inhibiting the enzymes that break down dopamine (monoamine oxidase b inhibitors [mao - b inhibitors ]), and directly stimulating the receptors in the brain (dopamine agonists [das ]). it is often used in combination with carbidopa (or benserazide in canada and europe), which helps increase efficacy and reduce side effects (which are primarily gastrointestinal). mao - b inhibitors such as selegiline and rasagiline have side effects similar to levodopa, which include nausea, headache, and difficulty falling asleep. they also have a specific drug interaction with certain antidepressants that may cause dangerous increases in blood pressure. das such as pramipexole and ropinirole are used in younger patients to conserve the most effective treatment for later in the progression of the disease. das can be used to postpone levodopa therapy, which prolongs the time until patients will experience levodopa - associated motor complications. das have other undesirable side effects, including sedation, swelling of lower extremities, and decreased impulse control. both levodopa and das cause an increase in dopaminergic transmission, and there is the potential for them to cause psychosis, hallucinations, and delusions.22 another class of drugs helps mainly by increasing the effectiveness of dopaminergic drugs. catechol - o - methyl transferase inhibitors, such as tolcapone and entacapone, help enhance levodopa effects and are used to treat wearing off periods. tolcapone has been shown to increase liver enzymes, so it is not commonly used in practice. anticholinergic drugs such as benztropine and trihexyphenidyl are often beneficial in younger patients whose primary symptom is tremor. amantadine, originally an antiviral drug, has also shown efficacy in treating mild dyskinesias. though these drugs work primarily through other mechanisms, not dopamine or its receptors, they all have potential side effects of confusion and hallucination.22 it is known that all of these medications can lead to psychosis in pd patients, with das being the most commonly implicated. it appears that medications may be a risk factor for developing pdp, but it is not an absolute risk. there are some patients on therapy who will never experience psychosis, and some patients will develop psychosis before pd medications are initiated. other observations correlated with psychosis include changes in visual processing, increased sleep disturbances, and abnormalities in other neurotransmitters such as serotonin and acetylcholine. these can occur in the elderly population and may also be causes for perceived psychosis.15 prior to april 2016, there was no fda - approved treatment for pdp. sgas (atypical antipsychotics) have often been used off - label by clinicians to reduce the symptoms of these patients. of these current agents, there are few medications that have been proven to be both effective and safe in this population through clinical trials. the most commonly used agent, clozapine, has been shown to be effective for pdp, requiring doses ~10% of the normal dose used in schizophrenia to reduce psychosis symptoms without causing a relapse in motor symptoms.23 new research has shown that the indication - specific target plasma concentration for clozapine is between 15 and 141 ng / ml.24 sgas, such as clozapine, are thought to treat hallucinations by blocking dopamine d2 receptors in the brain. at 10% of the dose typically administered in schizophrenia, clozapine shows selectivity for serotonin 5-ht2a and histamine h1 receptors. since antagonizing h1 receptors results in drowsiness, but not clinical efficacy, it has been hypothesized that blocking 5-ht2a is the actual mechanism by which pdp is ameliorated using clozapine.23 this raises questions regarding whether the psychosis is actually due to excess dopamine - mediated signaling in the brain, or if there is some other mechanism causing these symptoms in pdp. it has the potential to cause rare, but life - threatening, agranulocytosis.25 it is likely that clozapine is underutilized due to its many rare side effects and required blood monitoring. this monitoring for agranulocytosis (an absolute neutrophil count below 500/mm) was initiated after a study in 1975. in this study, 17 of 2,660 (0.7%) patients were diagnosed with agranulocytosis, and 8 of these patients died from infection after diagnosis.26 it has been shown that agranulocytosis may occur at any dose ; therefore, patients taking even the smaller doses used in pdp must be closely monitored. during the first 6 months of therapy, patients are required to have blood drawn weekly to check for neutrophil count, and without these results, pharmacies in the usa can not dispense the medication. as can be expected, this creates a huge burden for both the patient and the caregiver. for this reason, although it is the only antipsychotic with proven efficacy in multiple clinical trials, many doctors will not prescribe it for their patients or continue it when a patient is admitted to a nursing home. quetiapine has a mechanism similar to clozapine ; therefore, a number of clinicians have moved to using this antipsychotic instead. although its safety profile is preferential to clozapine, it has not had many clinical trials confirming its efficacy.23 clinicians typically use doses that are far below the normal dosing for schizophrenia, and quetiapine does have safety concerns of its own, most specifically metabolic syndromes.27 typical antipsychotics (first - generation antipsychotics) should not be used since trials showing their efficacy are lacking, while multiple studies have shown side effects, including an increase in motor symptoms, risk of stroke, cognitive decline, and death.27 typically, no other atypical antipsychotics are used besides clozapine and quetiapine. risperidone and olanzapine often result in worsening motor symptoms, in addition to multiple metabolic and cerebrovascular risks.25 aripiprazole, an atypical antipsychotic that acts on serotonergic and dopaminergic receptors, has been shown to be effective in psychosis. it may also result in decreased movement and function, so is unlikely to be beneficial in pd patients.19 the recent approval of pimavanserin could be a vitally important development needed by clinicians and patients, but more data are required. several searches were conducted to find the most up - to - date information on pimavanserin. we have also included some of the data used by acadia pharmaceuticals in support of its new drug application. the centers for disease control and prevention (cdc), the us fda, and the world health organization (who) also provided excellent starting ground in order to learn more about the prevalence of disease and current therapy difficulties. the approval of pimavanserin by the fda was supported by 21 completed studies and 4 ongoing studies. of the 1,237 subjects estimated to have taken pimavanserin (as of january 2016), there were four placebo - controlled studies confirming safety and efficacy that led to the approval of pimavanserin. all of these were multicenter, randomized, double - blind, placebo - controlled studies. in the concept phase 2 study (study 006), 60 subjects were followed for 4 weeks, and they were allowed a flexible titration of 17, 34, or 51 mg. phase 2b/3 studies (studies 012 and 016) increased the study time to 6 weeks and attempted to see if lower doses (8.5 mg) were effective in addition to previous strengths used. the pivotal phase 3 trial (study 020) was also 6 weeks but focused on the eventually approved dose of 34 mg versus placebo.28 in the monograph by hunter, the authors review studies 006 and 020.1 two phase 2b/3 trials (studies 012 and 014) were not published as full manuscripts. it is from the data of these studies that the phase 3 trial was developed and designed. these patients were randomly assigned in a 1:1:1 manner to receive either pimavanserin 8.5 mg, pimavanserin 34 mg, or placebo every day for 6 weeks. these patients were spread over 72 sites in the usa, india, and europe. to be included in the study, the patients had to be at least 40 years old, be diagnosed with pd for a minimum of 1 year, and have had hallucinations or delusions within the 4 weeks before screening. patients were not enrolled if they had previous psychotic disorders prior to or at diagnosis of pd.28 this study had a high placebo response, which researchers think may have confounded results. because no results could be determined from this study, they reevaluated their methods that led to the first improvement in the phase 3 trial design. the primary endpoint was an improvement in scale for the assessment of positive symptoms hallucinations and delusions (saps - h+d) score from baseline. secondary endpoints were changes in clinical global impression improvement (cgi - i) and clinical global impression severity (cgi - s) scores. it was noted during this trial that pimavanserin 34 mg patients had higher improvement in saps - h+d scores in the usa, but not in the other countries. interestingly, it was also observed that placebo patients had higher improvement in saps - h+d scores in india and europe. it was hypothesized that the high rate of response in the placebo group may have shadowed statistically significant clinical effects in the pimavanserin group, but this conclusion can not be proven with the current data. secondary endpoints were numerically higher in the pimavanserin 34 mg patients, but these results were not statistically significant. although the outcomes of the study were not encouraging, it did lead to a better design for the phase 3 trial. this was accessed using the unified parkinson s disease rating scale (updrs) ii + iii composite score.28 study 014 was structurally similar to study 012. researchers concluded that they had similar study design problems as the previous trial, so it was stopped early and only enrolled 123 of the anticipated 280 patients. using the data collected from the shortened trial, the primary endpoint was not found to be statistically significant. similar to the previous trial, this trial showed no negative influence on the motor symptoms of pd caused by pimavanserin. the 17-mg pimavanserin patients improved in cgi - i scores more than placebo patients (0.66 ; 95% ci 1.21 to 0.11 ; p=0.020).28 although these trials did not prove efficacy, they did lead to the improvement of the phase 3 trial. first of all, the data did show some positive trends, so the researchers were able to determine which doses may be successful. also, they did show that pimavanserin did not have a negative impact on motor symptoms in pd diagnosed patients. the phase 3 trial added a 2-week placebo lead - in focused on psychosocial therapy. this was included in order to help improve the validity of the study by removing patients who were not candidates for drug therapy based on their positive response to non - pharmacological therapy alone. the scale used to assess the primary endpoint was improved to make it more specific to the pdp population. instead of using the saps - h+d scale, which is commonly used for schizophrenia, the saps - pd is a rating scale of 9 items, decreased from the 20-item form used in schizophrenia (saps - h+d). it assesses if the patient has positive psychotic symptoms, specifically hallucinations and delusions specific to pdp. the new scale removed items most commonly associated with schizophrenia, not pdp.28 the hallucination items removed were voices commenting and olfactory hallucinations. the delusion items removed were delusions of guilt or sin, grandiose delusions, religious delusions, somatic delusions, delusions of being controlled, delusions of mind reading, thought broadcasting, thought insertion, and thought withdrawal.29 the removal of these items allowed the new scale to appropriately access only the most commonly associated symptoms of pdp. the phase 3 trial did significantly show an improvement in this score while using the 34 mg dose (equivalent to 40 mg pimavanserin tartrate) versus placebo. a decrease in score by at least 2.33 points on the saps - pd scale was deemed clinically significant. in this trial, patients taking pimavanserin had a mean reduction in score over placebo by 3.06 points (ci 4.91, 1.20). this reduction was shown in 65.3% of pimavanserin - treated patients versus 42.2% of patients receiving placebo. also, complete remission was seen in 13.7% of the pimavanserin group, compared to 1.1% in the placebo group.30 this study also confirmed that pimavanserin is not associated with negative motor function capabilities in pd patients. in addition to short - term efficacy and safety studies, studies have also been done to show the long - term safety of pimavanserin. long - term studies include over 300 patients taking the drug for at least 6 months, over 270 patients taking the drug for at least a year, and over 150 patients taking the drug for at least 2 years. the most common adverse drug events found over placebo were peripheral edema (7% versus 2%) and confusional state (6% versus 3%).30 another long - term safety study showed the major adverse drug events to be fall, urinary tract infections, and hallucinations. risk factors for these adverse drug events include increased age and time participated in the study. in previous studies, different adverse drug events were reported, so further clarification of true adverse drug effects versus worsening of pdp is needed. since levodopa is considered the best drug available to treat pd, most patients either currently take it or will take it as their disease progresses. since there is no drug interaction, clinicians can feel safe prescribing both medications concomitantly if necessary. pimavanserin is also not known to be either a cytochrome p450 (cyp 450) inhibitor or inducer. cyp 450 is a family of enzymes that aid in the metabolism of drugs through the liver. pharmacokinetic data show that dose adjustments should be made if pimavanserin will be used with drugs that are considered cyp 450 inhibitors or inducers. when given concomitantly with a cyp 450 inhibitor, it is recommended to reduce the dose by 50%. when given with a cyp 450 inducer, clinicians are encouraged to watch for decreased efficacy and consider a dose increase if necessary.30 qt prolongation (the elongation of the space between the q and t sections on an electrocardiogram) was seen in trials, but the largest observed mean for the 34 mg dose was 9.6 milliseconds.30 a qt interval of 500 ms is usually associated with fatal arrhythmias, such as torsades de pointes, but there are little data on how much the interval can prolong from normal to induce these arrhythmias.31 generally, in order to prevent complications, clinicians are encouraged to avoid multiple drugs that can cause qt prolongation together. the american journal of health - system pharmacists recommends not to use pimavanserin in patients who are currently on drugs that increase the qt interval, have preexisting qt prolongation, or have a history of cardiac arrhythmia.32 the only other statistically significant event was orthostatic hypotension. the placebo group had a higher percentage of patients experiencing this side effect compared to the pimavanserin group (5.2% versus 1.0%). there was no significant change in sedation - related events, metabolic syndrome, or blood dyscrasias. urinary tract infection (13%), fall (11%), peripheral edema (7%), hallucination (7%), and confused state (6%) were reported side effects in the group that took at least one dose of pimavanserin. nausea was reported equally between both groups (6%), and headache was reported more in the placebo group (5% versus 2%) than in the pimavanserin group.30 after review of the literature, the approval of pimavanserin for the treatment of hallucinations and delusions associated with pdp seems appropriate. according to the package insert, prescribers are encouraged to use two 17 mg tablets (34 mg total) per day, which was the dose most effectively used in the clinical trials.33 special dosing requirements are necessary when there are strong cyp p450 interactions, but not with mild - to - moderate renal impairment. no studies were done in patients with severe renal or hepatic impairment, so currently this drug is not recommended in these populations. like all other antipsychotic drugs, pimavanserin has a black box warning on its package labeling in the usa, which is mandated by the fda for other drugs in this class for increasing mortality in elderly patients with dementia - related psychosis. since this is a class - wide warning, it is likely to be precautionary only. the labeling also precautions practitioners against using this drug in combination with others that potentially cause qt prolongation. pimavanserin showed several side effects in its clinical trials, but it showed no negative effect on motor symptoms in patients with pdp. while the new drug application for pimavanserin was approved on april 29, 2016, there are still multiple post - market trials taking place.34 post - marketing commitments still required are a randomized withdrawal trial comparing pimavanserin to placebo, at least one randomized placebo - controlled trial with predominantly frail and elderly subjects that exposes at least 500 patients to pimavanserin 34 mg daily for a minimum of 8 weeks, an in vivo drug drug interaction study measuring the effect of strong cyp p450 inducers and recommending a maximum dose with this combination, and a microscopic reevaluation of lung tissue samples to detect collagen from data collected in the 2-year rat study.35 assuming positive results from each of these additional studies, pimavanserin may replace current antipsychotic therapy (clozapine and quetiapine) for the treatment of patients diagnosed with pdp. | parkinson s disease (pd) has a prevalence of nearly 1 million people in the usa, with increasing incidence in the elderly population. generally, the age of presentation is between 55 and 65 years, with the likelihood of diagnosis increasing as patients reach the age of 80 years or above. some of the common treatments for pd increase dopamine levels in the brain. dopaminergic therapy helps to improve motor and non - motor symptoms, but it is not without risks. dopaminergic therapy can cause confusion, delirium, and psychotic - like behavior. it is recommended that these agents are used cautiously in patients with a history of psychosis due to the risk of exacerbation. it is unclear whether parkinson s disease psychosis (pdp) is due to the disease itself, the treatment, or a combination of both, but it is clear that a safe, effective treatment is necessary. second - generation (atypical) antipsychotics are the current choice of therapy for pdp. all of these agents have a black box warning from the us food and drug administration (fda) for elevated risk of mortality in elderly patients with dementia - related psychosis. pimavanserin (nuplazid) received its novel drug approval by the fda on april 29, 2016, to treat hallucinations and delusions associated with psychosis experienced by some people with pd. we review in this article the new research that led to this approval as well as its potential place in therapy. |
in appreciating this corrigendum it deserves to be remembered how paedophilia and cognate terms entered clinical parlance. this warrants a slightly broader look at how notions of age - specificity and age - exclusivity in erotic attraction figure in the medical history of sexuality. paedophilia has been consistently signalled out as a discrete entity under the shifting headings of psychosexual perversion (1890s, as referenced below), sexual deviation or pathologic behaviour (195267 : dsm - i and ii ; 1965 : icd-8 : code 302.2), psychosexual disorder (1979 : icd-9-cm), paraphilia (1980 : dsm - iii through iv - tr), disorder of sexual preference (1992 : icd-10 : code f65.4), finally like other sexual deviations, paedophilia entered the dsm and the who s international classification of diseases (icd) without attribution, reference, definition or diagnostic criteria. apart from paedophilia few words pertinent to erotic age - preference ever trickled down into the oxford english dictionary (oed) and only paedophilia / pederosis ever entered the dsm / icd indexes. where heterosexuality was named and conceptualised in the earliest context of the naming of and apology for homosexuality, the clumsy, gender - neutral neologisms adultophilia and teleiophilic preference (ordinary attraction to adults) appeared long after an initial rush on pathological and gender - specific terms, and strictly on forensic occasions. note that with teleiophilia the reader is already in the twenty - first century. historically, definitions of morbid age of attraction, age of consent and mean or modal age of puberty show an approximate and a priori alignment. definitions of all of these are at a fundamental level conventional, and their alignment around 1896 required an intertwining of as much moral and legal as psychiatric sensibility. proposed definitions of paedophilia have continued to waver between ill - defined physiological terms such as puberty, related somatoscopic (tanner) stages, and ages - of - man categories such as child and adolescent. revealingly comparable to age - of - consent legislation worldwide, they have also varied and shifted in stipulated minimal age and minimal age difference requirements for diagnosis. the dsm-5 concept of normophilia, or non - paraphilia, balances a naturalistic with a legal reference to age where it is defined in terms of the sexual maturity and the legal ability to consent of the normophile s preferred partner. moreover, nosological suggestions have consistently wrestled with the relation of mental disorder to the contingency of offences. it was child endangerment that had been the most immediate connotation of the us legal rubric of sexual psychopath, which was current from the late 1930s to the early 1950s, and certainly of its 1990s successor, the sexually violent predator. it has been a peculiarity of these rubrics that they have an opportune, not a necessary, footing in psychiatric diagnoses, and that the former particularly appealed to residual (not otherwise specified) categories in the dsm - iii - r and subsequent revisions. paraphilia has concentrated on paedophilia and psycho - diagnostic notions developed largely around child sex offenders, notably, since 1984, that of cognitive distortion. diagnostic criteria guided research only from the dsm - iii onward. to date, these have importantly conformed to a criterion template applied to all these observations will prove of increasing significance to historians of the sexological present, given the rising importance of age to global definitions and representations of sexual justice since the weaning from until the 1973, 7th printing of the dsm - ii and the 1992 icd-10, anglophone psychiatric taxonomies still listed homosexuality and paedophilia under the common heading of sexual deviation. historians of homosexuality note that between these dates, the paedophile came to replace the homosexual in the anglo - american experience as a paragon of sexual danger to youth. in post - war psychology, a growing body of research evidence was building up a dividing wall between the non - dangerous homosexual and the pederast who did threaten the young and whom it was still thought necessary to include in the dangerousness framework. during the 1980s, the century - old trope of nineteenth century french dictionaries defined pdrastie [pederasty ] only vaguely as a criminal passion either between men or between men and jeunes garons [young boys ]. yet, even in the european nineteenth century, homosexuality importantly answered to a popular presumption, indeed a forensic pattern, of age difference. this may have reflected the likelihood of crimes being reported. in any case, associations with child abuse plagued the earliest apologetic outlines of homosexuality. as discussed below, these associations inform the coinage and etymology of some of the earliest terms for homosexuality. where most early terms denoting age preferences were subsequently coined to classify homosexuals, a definitive reversal in the order of mobilising parameters is achieved only in the dsm - iii, which illustratively advises the specification of pedophilia where the diagnosed person is found to be sexually attracted to males, females or both. moreover, the strong philological and philhellenic orientation to ancient paiderastia of even early twentieth century advocates of same - sex love necessarily married reflections on gender and age orientation. greek love had perhaps been more central to the modern assimilationist politics and acceptance of homosexuality than sympathetic historians may have cared to stress. well into the 1950s, many apologists for homoeroticism wrestled extensively with the spectres of greek love and pedagogical eros, in efforts to distance themselves from the nascent sciences of crime and mental health. writings celebrating the beauty of ageless boys and youth often deployed philosophical and anthropological arguments against medico - physiological and legal sensibilities around age and age difference. in the 1900s, anthropologically oriented sexologists (bloch and ellis) and also early ethnologists of homosexuality including ferdinand karsch - haack, either explicitly rejected or ignored emergent nosological frameworks for paedophilia / knabenliebe. the forensic identification of paedophilia only very gradually came to spearhead the new and dual scientific parlance of these key notions were to assume an obvious architectural relation to today s ubiquitous, triple psychiatricisation, which was, importantly, already converging in the mid-1890s, of the paedophile, the child sexual abuse victim and the erotic age preference assumed more sustained relevance in the nineteenth century context of naming, categorising, theorising and defending gender deviance. paedophiles still figured importantly in terms of recognizably different categories among adult male homosexuals. robertson suggests that, in the us, by 1950, the media had begun to split homosexual offenders away from pedophiles and to present them as a problem in their own right, a new moral menace to our youth, as the title of an article in coronet magazine trumpeted. in the netherlands too, the word paedophilia was rarely used before 1945, but shows up regularly in the medical records after 1950 ; homosexuality and paedophilia only began to be separated from that time onwards. the first book - length forensic studies on paedophilia, in the netherlands, canada, germany and denmark, were published only from 1960 onward, at the time of the decriminalisation of homosexuality across europe. a lecture by michel foucault given on 19 march 1975, gestured toward a genealogy of paedophilia, arguably the first of such gestures. since then, substantial contributions to paedophilia s medico - forensic prehistory have been few, fragmentary and recent. like homosexual, the label invites a historicisation of the labeller at least as much as of the labelled. the broad outline offered below focuses primarily on the few, but diverse, references to age - specificity in nineteenth century west - european sexological nosology. historians of homosexuality have observed an intricate cross - fading of the ramifications of pagan custom, sin and crime with those of mental disorder and social identity. notwithstanding, medical as well as lyrical conceptions of homosexuality remained critically embroiled with those of ancient greek paiderastia well into the twentieth century. knabenliebe der griechen und entarteten rmer [boy love of the greeks and degenerate romans ] as among the sexuellen verirrungen des alterthums [sexual aberrations of antiquity ]. discussion of this ancient custom was to remain a central point of reference in the sexual psychopathology here anticipated. the closest to an ancient medical pronouncement on boy - love had arguably been plutarch s ethico - moral distinction, in his moralia, between approved and untoward. christian disparagement of featured a different, but still only moral, neologism. child / boy seducer or corrupter) appeared in numerous early christian, as well as anonymous greek ascetic texts. stereotyped lists of sins apologetically or polemically levelled against the surrounding greco - roman world. part of a stock arsenal of accusations to be used in debates with pagans. it occurs in various enumerations of sins, and it is a matter of dispute whether the word can be taken to denote all homosexuality, all seduction of youth or both. the constitution of the apostles (late fourth century ad) expanded the sixth commandment on adultery with : do not abuse boys (oude paidophthorseis) : for this vice is against nature and had its beginning in sodom []. among these earliest sources to specify sex offenders against the synod of elvira (305 - 306 ce) says of stupratores puerorum that they shall not be admitted to communion, not even on their deathbeds. the term knabenschndung (knabenschnde, knabenschnderei, knabenschnderey) [violation of boys ] cross - faded from the sixteenth to early - nineteenth century from an ecclesiastical to a medico - legal relevance, often subsumed with bestiality under the legal header of sodomie (sodomiterey). the term long retained its scriptural ring : the luther bibel and the zrcher bibel (both sixteenth - century) translate the arsenokoitai of 1 corinthians 6:9 and 1 timotheus 1:10 as knabenschnder. although the gender - neutral term kinderschnder child violator is of early - eighteenth - century origin and encountered in early - nineteenth - century legal medical textbooks, krafft - ebing is notably one of the first, in 1896, to use it in an indisputably psychiatric context. where the early christian neologisms were intended to reframe greek boy - love, the earliest mid - nineteenth - century psychiatric terms proposed by heinrich kaan (puerorum amor [boy love ] or paederastia) and claude - franois micha (amour grec [greek love ], including male philopdie) were undistorted echoes of and direct references to ancient greece, retaining a philological connotation of pederastic age preference. philopdie and pdophilie were among the very few modern sexological terms with cognate terms actually attested in ancient greek. lexicographically, philopdie appears to have been the first of modernity s philias to figure alongside the earlier monomanies rotiques [erotic monomanias ]. at that time the phil-/-philus prefix / suffix was scientifically familiar in entomology (1838 : sitophilus), medicine (1828 : haemophilie) and phrenology (1815 : philoprogenitiveness). denoting a psychiatric condition, philologically, lifelong [friendship ] between men was the ideal corollary of an initial paiderastic tutorship. the superordinate term paraphilie, incidentally, would not be coined until 1903, and here only as a counterpoint to medicalising alternatives. from an etymological point of view, the late - nineteenth through to twentieth century juggling of terms (philia, sex, eros, perversion, disorder, orientation) amounted to considerable wortsalat. to mid - nineteenth - century forensic authorities, the matching of ancient terms and medico - legal cases had already proved problematic. the ancient distinction between edifying love and problematic behaviour, specifically between pdophilie and pderastie, had been revived deliberately in the late 1830s by the pioneering researchers into ancient greek sexual mores julius rosenbaum, moritz hermann eduard meier and heinrich hli. however, like johann ludwig casper, all three notably used the terms mnnerschndung (fr. : andrrastie) and mnnerliebe alongside the juxtaposed terms of knabenschndung and knabenliebe / paedophilie. right up to krafft - ebing s forensic appropriation of the latter term in 1896, as evidenced in work by albert von schrenck - notzing, this contrast between pdophilie (pedophily in the 1895 english translation) and pderastie for many was still of purely philological significance and remained without definite nosological intentions. krafft - ebing s paedophilia erotica, in this light, was a careful, but still awkward, reworking of known, indeed ancient, terms. following french nosological terms including monomanie rotique and folie rotique [erotic madness ], krafft - ebing coined the phrases fetischismus eroticus in 1891 (adapting from alfred binet s 1887 expression ftichisme dans lamour, itself a diversion from fetishism s previous anthropological uses) and zoophilia erotica (next to bestialitt and zooerastie) in 1894. it is in this terminological journey that krafft - ebing coined pdophilia erotica in 1896 and, for purely taxonomical purposes, gerontophilie in 1901. in texts on attentats aux moeurs [sexual offences ] of the second half of the nineteenth century by casper and subsequently by authorities such as devergie, tardieu, pnard, brouardel, bernard, toulmouche and thoinot virtually all attention was given to the physical and venereological (ie. while the fourth, 1887, edition of paul moreau s des aberrations du sens gnsique [aberrations of the sexual instinct ] is full of pre - adolescent nymphomaniacs and incestuous fathers and brothers, one finds moral denunciations of odieux attentats [odious crimes ] but not yet a specification of offender types. of the mdchenschneider [girl - cutter ], mdchenstecher [girl - stabber ], and knabengeissler [boy - flogger ] cases subsequently discussed by krafft - ebing, some had long appealed to typological sensibilities ; but they were to be subsumed under the rubric of sadism not paedophilia. nineteenth- and early - twentieth - century evidence of slang applied to frequenters of child prostitutes, and to connoisseurship of young male beauty or girlhood innocence may be too scant to empirically consolidate a link with the kind of identity positions associated with paedophile activism, which may be traced back to the theme becoming a matter of discussion and schism in the late-1950s west - european homophile movement. considered symptomatologically, reports of paedophilia remain exceedingly rare and casuistic until the 1890s. in 1840, american phrenologist w. byrd powell mentioned a man, george kennedy, hanged for rape of a ten - year - old girl who, according to a memorandum, while standing on the drop, with the rope about his neck, [] informed the spectators that such [sexual ] commerce with female children had been the governing passion of his life. clinical research and organic theories of paedophilia seem to have been consolidated in, but also largely limited to, an 1864 article by powell, discussing craniological similarities of at least five men (three executed, several incarcerated) said to have had an intemperate desire for commerce with female children. this empirical basis confirmed powell s earlier impression that as inveterate masturbators, and in contrast to nymphomaniacs, such men turned out to have underdeveloped organs of animal sensibility and overdeveloped amatory organs. however, various independent nosological identifications of paedophilia can be seen at the close of the nineteenth century. this entails the passage from the incidental symptom of indecent behaviour to a sui generis mental affliction, that is, to an inborn, primary orientation that is not strictly either an occasional symptom of senility, dementia, imbecility, epilepsy or an occasional corollary of alcoholism, impotence or libertine brutality. an 1881 conference paper by freiburg psychiatry resident ludwig kirn, published in 1883, already offered most of this differential diagnosis, interestingly without elaborating its eponymous suggestion that heterosexual unzucht mit kindern unter vierzehn jahren [indecency with a child under fourteen years ], as widernatrliche unzucht and sodomie [sodomy ], could be an expression of perverse sex drive. in 1890, italian sexologist guglielmo cantarano included tendenza verso persone impuberi [propensity for prepubescents ] in a classification of sexual aberrations, as an example of perversion due to a failing sense of pleasurable reciprocity. in 1891, french criminal anthropologist mile laurent, in a short chapter on les amoureux des enfants [child - lovers ], discussed those, unlike elderly offenders and libertines, who are so to say born with the passion and, furthermore, those whose amour slides into exclusive sexual obsession. in that same year, in the first major monograph on homosexuality, albert moll would discuss neigung zu unreifen mdchen und knaben [inclination to immature girls and boys ], liebe zu kindern [love for children ] and liebe zu jnglinge [love of youths ], beside neigung zu alten mnnern, as distinct perversions and complications of that condition. in 1893 sexual perversion, a class of cases where the criminal has no desire for female adults, but for female children only. by 1894, spanish author jos de letamendi identified pederastia (amor nios [love of children ]) as an erotic aberration (parafrodismo as opposed to lamour des hommes pour les impubres [men s love for prepubescents ] outside both normal uranism and normal heterosexuality. eine krankhafte disposition, eine psychosexuale perversion [a morbid disposition, a psychosexual perversion ] in an 1896 aetiological paper on unzucht, excluding those the term entered his textbook on psychiatry first in its sixth, 1897 edition, his psychopathia sexualis in the tenth german edition of 1898, the english language in that edition s 1899 translation, the french language (as pdophilie rotique) in 1900 and the italian language (pedofilia erotica) about 1902. influential medico - legal textbooks, notably dating before the first edition of psychopathia sexualis, provided case studies krafft - ebing went on to consider indicative of paedophilia. another forensic case study, singled out by krafft - ebing for this purpose, dealt with an exclusive age preference for boys aged between six and twelve, with accompanying horror feminae and horror puellarum. its authors stressed that their case evidenced an element of sensual and sentimental adoration, not just carnal interest. dgnr atteint de perversion du sense gnital [degenerate affected by perversion of the sexual instinct ] and referred to an asile dalins [insane asylum ]. with his new diagnosis, krafft - ebing described the case a year later as eine spezielle anomalie innerhalb des rahmens der homosexualitt [a special anomaly within the frame of homosexuality ]. early - twentieth - century comments on paedophilia were few, and hardly deferential to krafft - ebing. most psychiatrists including kraepelin and bleuler dedicated only a few lines to child molestation in their textbooks ; both, like freud, mentioned not sexual perversion but the older differential diagnoses of epilepsy, dementia senilis (altersbldsinn) and mental retardation. havelock ellis s brief use of paidophilia in 1905/6, denoting a prevalent inflection of erotic symbolism, was credited neither to krafft - ebing nor to saint - paul (discussed below) ; ellis would explicitly doubt the existence of a paedophilic perversion. auguste forel coined a competing term pderosis in 1905, again without reference to krafft - ebing, to mean a argentine physician, jos ingegnieros, also leaves krafft - ebing uncited where discussing a bizarre case of contemplative, morbidly paedophilic fugue. krafft - ebing briefly alluded to an associationist aetiology of the eigenthmliche art von fetischismus des alters or altersfetischismus [peculiar kind of age fetishism ] which he had named. he coined the word gerontophilie when returning to this hypothesis in 1901, but did not discuss actual cases. in 1905, both laurent and ellis described paedophilia in poorly elaborated terms of fetishism (laurent : ftichisme des juvnilits [juvenilities - fetishism ]), neither with reference to krafft - ebing. more intricate theoretical approaches to age - specific attraction, as a purported dimension of either psychical hermaphroditism or psychosexual infantilism, were being offered by albert moll (see below), later by early students of psychoanalysis (hirschfeld, otto juliusberger, max marcuse) and finally in monographic studies by wilhelm stekel and arthur kronfeld. in psychoanalysis, civilisational, economy of familial libidinal investments. yet, of the five complexes involving incestuous tendencies toward children proposed in subsequent psychoanalytic texts apropos the oedipus and electra complex (so named in 1910 and 1913, respectively), not one gained a psychoanalytic foothold. the reported case load of paedophilia erotica at that time, it needs to be stressed, was small, combining those of krafft - ebing (about ten by 1899), schrenck - notzing (two), ellis (one), forel (one) and, avant la lettre, laurent (two). as it appears from a late edition of his der hypnotismus [hypnotism ], forel claimed acquaintance with many cases but reported treating only one case by hypnosis (outcome uncured). typological attention to age is evident in the work of two early apologists for homosexuality, neither of whom was medically trained. in an 1869 pamphlet karl - maria kertbeny made a distinction between boy - loving homosexuality native to southern countries and man - loving homosexuality native to northern countries. at the same time, epithets including pderast / knabenliebhaber [pederast / boy lover ] and allegations of knabenverfhrung [seduction of boys ] were countered in a plea for social acceptance of adult male homosexuality. comparably, in his first of twelve famed pamphlets karl heinrich ulrichs coined the term urnische liebe specifically to establish a contradistinction to the popular connotation of knabenliebe. ulrichs initial terminology had modern homosexuality emerge right out of pausanias speech in plato s symposium that provided the (dissenting) distinction between an inferior love belonging to common aphrodite (borne from zeus and dione) oriented toward women as much as boys [paides] on the one hand, and on the other, a love informed by an older, heavenly aphrodite (borne from uranus) of younger men but not boys before the stage when their intelligence begins to develop, which is near the time when they begin to grow a beard. ulrichs later urning typology, in his memnon essays, blended a gender habitus and an age orientation schema : the weibling loved drauci [strong young men ], the zwischen - urning men aged between eighteen and twenty - three and the mannling pueri [youths ]. this early theoretical imbrication of gender and age preference, on the basis of a shared physical habitus between women and male youths from the perspective of the male urning, was followed by theorists of sexual inversion of the 1890s. however, the terms themselves fell into disuse despite being used by john addington symonds and havelock ellis in the early- and mid-1890s, in the posthumous reprint of memnon in 1898, and in allusion by magnus hirschfeld in the 1899, maiden issue of his jahrbuch fr sexuelle zwischenstufen [yearbook for sexual intermediates ]. in his subsequent, 1869 pamphlets incubus and argonauticus, ulrichs would further reflect on the relative incidence and pathological nature of geschlechtsneigung [geschlechtsliebe ; geschlechtslust ] zu unreifen knaben [sexual inclination / love / lust directed at immature boys ] in urnings and dionings (heterosexuals), in connection with a widely publicised forensic case (fall zastrow) of alleged sadistic rape of a five - year - old boy in that year. the accused apparently possessed and endorsed ulrichs memnon, and has been identified as the second person in this period, after ulrichs, to publicly announce an attraction to men. the case led to a sensational criminal trial and was timed so as to impact on important discussions later that year of possible reform of the prussian anti - sodomy law. significantly, in his discussions ulrichs makes boy - love and man - love mutually exclusive (wer knaben liebt, liebt nicht mnner ; und umgekehrt [who loves boys, does not love men and vice versa ]) and qualifies the former consistently as ulrichs 1868/9 verdict was perhaps the earliest instance of an age - centric, rather than gender - centric, nosological conception of knabenliebe at least an early instance where the metaphor of disease was applied strictly on the basis of age. an absolute schism between pathological boy - love and non - pathological, inborn man - love can thus be traced back to the earliest apologetic representations of homosexuality, and arose in the context of a criminal case, in an attempt to pre - empt popular conflations of homosexuality with violent child abuse. northern homosexualist would notably have to argue the same case for at least a century to come. for ellis / symonds and later moll, following ulrichs, age - preference came to speak to the nascent theorem of sexual inversion, albeit briefly. male youths shared the feminine features of women, such that only attraction to robustly adult males would indicate a complete inversion, and thus full pathology, of gendered sensibilities in males. this symptomological significance of age is not found in krafft - ebing s early stufen - theory. on the basis of many case studies, moll alluded to the possibilities that age - attraction is a graded symptom of either developmental stagnation (hemmung) or even a standstill in the differentiation of sexual drive, or an admixture (mischung) of otherwise properly gendered, inborn forms of sexual receptivity (komplexe von reaktionsfhigkeiten). both erotic age- and gender - preference would thus be the outcome of sexual differentiation and had the tendency to appear as such transitional states would be the rule, not the exception. in 1899, moll pointed out that in even in germany full sexual inversion which would mean men s attraction to men above the age of thirty was rarer than attraction to those below twenty, and that until recent times a full degree of inversion may have been unknown as it appeared to be in the americas and the orient. here, homosexualitt mit neigung zu ganz oder halb unreifen knaben [homosexuality with an inclination to completely or semi - immature boys ], would have been widely acknowledged at the time. a comparable taxonomical suggestion in mid-1890s sexology maintained that unlike innate, effeminate and passivist (masochistic) uranists, acquired and active pederasts, the only true pederasts, are attracted by immature youths (gytons) of feminine aspect. of a half dozen loose schemas of age - specificity in erotic attraction found in the 18961914 period, all were occasioned by similar, mostly theoretical, attempts to differentiate between homosexuals, and mostly by authors seeking to qualify sexual inversion s pathological status. most prominently, they occur in writings by georges saint - paul in 1896, by ludwig frey (pseud ?) in 1898, in a 1904 theoretical typology by dutch physician von rmer published under editorial care of hirschfeld, in a 1906 essay and 1914 book by hirschfeld and finally in a book on urban vice and gay subculture by catalan pedagogue max bembo, published around 1912. only hirschfeld s male schema, which borrowed all terms without attribution from krafft - ebing and saint - paul, proved of some utility to twentieth - century sexologists, although it never found broad cultural resonance. of further note, the earliest identifications of gerontophilia, which krafft - ebing only encountered among saint - paul had used the same term, paidophilie, in the same year as krafft - ebing, but used it in reference of a homosexual love for young phbes, not prepubescent enfants, that is, as phbophilie, a word also coined here for contrastive purposes and later to be reintroduced by hirschfeld without attribution. saint - paul classified oscar wilde, in the year after his trials, as un inverti paidophile [a paedophilic invert ]. paedophilia in or seduction by inverts was less, or at least not more, prevalent than among heterosexuals. already in 1891, complete analogy. both hirschfeld, in 1914, and moll, in 1921, ventured estimations of the respective incidence of age - preference categories. these estimations honoured a by now firmly established division between the first - order category of homosexuality and second - order questions of perversion or age - preference. yet, the title of one of krafft - ebing s last articles flagellatio puerorum als ausdruck des larvirten sadismus eines paedophilen contrrsexualen illustrates the concomitant problem of describing complex cases in terms of co - morbidity, especially in relation to the increasingly arguable aberration of sexual inversion. was the defendant at root an invert, a paedophile, a sadist or a flagellantist ? the question clearly mattered to a world in which many, including krafft - ebing, had begun to de - pathologise sexual intermediates. one empirical contribution to hirschfeld s jahrbuch fr sexuelle zwischenstufen claimed that 100 out of a nonclinical sample of 550 cases of kontrrsexualismus proved to be anomalies in sexual preference, about fifteen cases by pdophilie, the latter in turn considered there has been an elaborate early modern legal discourse on the nature of child sex offenders, but at least in england this did not include the notion of a sexual perversion. even by the late-1850s, tardieu signalled out pederasty where habitually committed on boys aged six to twelve years but could at that time only provisionally refer to kaan and casper in considering pederasty the corollary of a possible perversion morale. in two 1894 book chapters marking the beginning of american forensic interest in the area of under - age sex crimes, neither of the authors specified perversion in relation to offences against children, although both alluded, as tardieu, to the emergent european availability of such a specification. forensic psychology of child sex offenders is of twentieth - century, particularly psychoanalytic, origin, and at first relies minimally on the notion of paedophilia. one could ask why a specifically forensic definition of a paedophilic predilection was not forthcoming until the 1890s, despite decades of alienist and forensic study of child sex offenders. harry oosterhuis characterises krafft - ebing s medical forensic coinage of paedophilia as coterminous with a shift which was only gradual from a psychiatric perspective in which deviant sexuality was explained as a derived, episodic and more or less singular symptom of a more fundamental mental disorder, to a consideration of perversion as an integral part of a more general, autonomous and continuous sexual instinct. early forensic observations on victim age were largely concerned with hypotheses at the environmental and demographic level. tardieu correlated offender and victim age, observing a general inverse relation, an empirical law not contradicted in research by alexandre lacassagne, paul bernard and paul brouardel. for brouardel, it fitted the suggestion that impotence was a central predisposing factor in sex offences. lombroso connected an apparent nineteenth century rise in offences against children, as opposed to adults, not to an idiopathic perversion but to insatiability with regard to pleasure in the cases of individuals of high culture, together with the abundance of opportunity. alienist attention to moral offences against children into the twentieth century focused mainly on possible grounds for defendants reduced capacity. defendants, for their part, were likely to cite circumstantial and incidental, not preferential, factors. exculpatory statements were scrutinised as possible clues to the state of their mental faculties. a case of predominantly homosexual child abuse reported in 1843, for instance, had alienists puzzling over whether the defendant s apologia a primary school teacher referring to lexemple de socrate et dalcibiade [the example of socrates and alcibiades ] and contesting medical consensus about onanism entailed la corruption du cur ou la perversion de lintelligence [a corruption of the heart or a perversion of the intellect ]. legal students including ulrichs and krafft - ebing spelled out that any distinct morbid sexual orientation in recidivist cases of notzucht [rape ], unzucht [indecency ], verfhrung [seduction ], blutschande / inzucht [incest ] or schamverletzung whether involving men, women or minors, would not in itself indicate mental alienation. krafft - ebing s coinage of paedophilia erotica was only to supplement a long - known list of etiological factors (senility, alcoholism, epilepsy, degeneracy and mental retardation) that in themselves might, or might not (libertinism), imply diminished responsibility in sex offenders. in all of his own cases, except a seeming platonic one, krafft - ebing found signs of degeneration. still, without a third factor (neurasthenia, dementia paralytica) present, acting upon a paedophilic orientation would not suggest diminished capacity. corruption and abuse had long informed the linked epidemiologies of onanie [onanism ], knabenschnde and verfhrung. they assumed more precise aetiological significance to psychiatrists from the mid - nineteenth century onward, in informed assumptions about acquired pederasty (by casper, from 1852) and in binet s 1887 associationalist aetiology of fetishism. the broadening of this significance by freud in 1895/6 to hysteria, obsessional neurosis and paranoia (seduction s promotion to the status of caput nili der neuropathologie, in freud s terms) was famously dismissed by krafft - ebing as a scientific fairy - tale. in other words, at the time of the naming of a paedophilic orientation, there was only a limited and disputed basis for the psychiatric relevance of sexual victimhood. paedophilia was clearly relevant both to narrowly - defined and expanded sexual aetiologies of mental illness, but certainly not critical. the label did occur in the very same year as the publication of freud s three seduction theory papers, a coinage suspended between edward tylor s exogamy and edward westermarck s incest avoidance theories and the germination of freud s oedipus complex. freud s rethinking of the issue has been considered the central event in the discovery of psychoanalysis, both in freud s own account and in that of his biographers. in the early 1980s, historical unearthing of the abandoned seduction hypothesis figured prominently in an epochal reassertion of that inference. freud notably signalled out three groups of seducers : nursemaids, governesses, domestic servants, and teachers ; older children and siblings ; and adult strangers. not a single accusation of a parent or the term paedophilia does not appear in the minutes of the vienna psychoanalytic society (190618). freud never used it in print or correspondence, and would only briefly reflect on the child as erotic object of choice in 1905. equally, in perhaps the earliest aetiology - centred quantitative study of child sex offenders, published in that year, the existence of any such mental disposition as paedophilia (eines kinderschndungstriebes) was considered most questionable and no mention was made of either krafft - ebing or his term. wulffen s voluminous textbook der sexualverbrecher [the sex offender ] of 1910 still had little to add to krafft - ebing s provisional, descriptive account. in 1912 hermann rohleder would claim to be the first to suggest that incest could be read as a corollary of paedophilia or parthenophilia erotica, matched with the suggestion that it should be punished only where involving depravity against children. the first to offer substantial discussions of paedophilia and gerontophilia, incidentally, had fallings - out with freud moll and stekel. as hebephilia, a new empirical gaze on erotic age preference is seen in the mid- to late-1950s, and soon became tied to phallometry or penile plethysmography (the measurement of erectile circumference and/or volume in response to usually visual stimuli). diagnosis of sexual deviation, in particular the differential diagnosis between homosexuality and heterosexuality. it is here, in a series of czech articles by freund, that both sex - preference (including homosexuality) and age - preference (including pedophilia, ephebophilia / hebephilia, and androphilia) are first, and simultaneously, diagnosed in a psychophysiological test. age of attraction soon became the mobilising research parameter for phallometrists. the mid-1960s goal was to turn a supposed diagnosis arising from legal precedent (recidivism) and/or self - admitted preference into a pretenders and more broadly with a largely clinical distinction hitherto mostly associated with homosexuality, between situational and preferential offenders. in the us, distinctions such as this would inform civil commitment procedures, especially from 1990, with the first sexually violent predator law in california. it is indeed only in the 1990s mostly the 2000s that academic neologisms like ganyphilia, hebephilia, adolescentofilie (in czech) and juventofilia (in spanish) were seen aiming to designate discrete paraphilias. chronophilia, john money s idiosyncratic term coined in 1986 to replace his slightly earlier classificatory nudges at fixation on age disparity and age - discrepancy paraphilia, has remained an entirely speculative and normative gesture. chronophilia family (coining infantophilia / nepiophilia), and repeatedly complained about the absence of terms such as twentiophilia, thirtiophilia and so on, stating that such terms would enrich nosology and inform diagnosis of people s lovemap. for a clinician highly sensitive to the medicalisation and politicisation of sex, this seems careless use of terminology : money reported no cases and no evidence supporting the existence of a chronophilic subgroup of paraphilias, nor data that would establish the conceptual validity of chronophilia (in evidence when the paraphile s sexuoerotic age is discordant with his / her actual chronological age and is concordant with the age of the partner), nor, finally, did he empirically consolidate the status of mental disorder for any so - called paraphilia (to date, paraphilias lack biomarkers). the 2013 passage from paraphilias - as - mental - disorders to paraphilic disorders did little to solve these basic, nineteenth - century problems. if anything, the lived metaphors of disease, trauma and therapy seem to have become more fundamental to western responses to sex crime than ever. greatly exceeding the boundaries of a strictly medical history, psychiatric and psychoanalytic notions (sexualtrauma [sexual trauma ] ; verdrngung [repression ]) have been greeted with a massive socio - political and legal appropriation. both in the uk and the us, this development seems traceable with some precision to media coverage of sexual minorities and sex crimes in 1977/8. paedophilia, within the resulting culture of truth - unearthing, memory - recovering and conspiracy - revealing, figures quite diffusely as a mental, institutional and cultural morbidity. legal, psychiatric and culture - critical dimensions of the term are today rarely cleanly distinguished, surprisingly even in many legal, clinical and historical communications. | a scientific nomenclature of erotic age preferences informed the mid- through late nineteenth century joint appearance of homosexuality and sexual abuse of minors on the medico - legal scene. yet, even in the twenty - first century, legal, psychiatric and culture - critical dimensions of related terms are rarely cleanly distinguished. review of primary sources shows the ongoing western suspension of notions of sick desire, alongside and beyond the medicalisation of homosexuality, between metaphor, legal interdiction and postulated psychopathology. virtually all early attention to erotic age preference occurred in the context of emergent attention to erotic gender preference. age of attraction and age difference centrally animate modern homosexuality s pre - modern past ; its earliest psychiatric nomenclature and typologies (184469) ; its early aetiologies stipulating degrees of sexual differentiation (1890s) ; its concomitant sub - classification (18961914) ; its earliest psychophysiological tests (1950s) ; and, finally, its post - psychiatric, social scientific typologies (1980s). several identifications of paedophilia were seen throughout the 1890s but as a trope it gained cultural momentum only during, and as a seemingly intriguing corollary of, the progressive depsychiatricisation of homosexuality across the anglo - european world (late 1950s through 1980s). early twentieth century sources varied in having it denote (1) a distinct perversion, thus possible complication of sexual inversion (2) a discrete corollary of psychosexual differentiation akin to gender preference (3) a distinct subtype of fetishism, thus a likely imprint of early seduction (4) a more intricate expression of erotic symbolism or psychosexual complex or (5) a taste answering to culture, a lack of it, or a libertine disregard for it. |
outofhospital cardiac arrest (ohca) is a major public health problem affecting 300 000 people in the united states each year.1 past studies demonstrate that there are sex differences in survival from cardiac arrest. studies have consistently found more favorable arrest characteristics in men, including younger age and a higher frequency of initial shockable rhythm, witnessed arrest, and bystander cardiopulmonary resuscitation (cpr).2, 3, 4, 5 however, there is conflicting evidence regarding patient outcomes (survival and neurological outcome). whereas some studies have reported improved survival in women,2, 3, 6 particularly those of childbearing age,7 others show no difference or even lower survival in women.4, 5, 8, 9 the implications for differences in care also remain undefined. the majority of past studies included patients treated before regional systems of care and advanced postresuscitation management, including widespread adoption of early percutaneous coronary intervention (pci) and targeted temperature management (ttm). a recent metaanalysis of these studies reported increased survival in women, but outcomes may be very different with current management approaches.10 the 2014 academic emergency medicine consensus conference cardiovascular resuscitation working group identified ohca epidemiology as a research priority, noting that exploring sexspecifics in ohca can allow targeted improvements in resuscitation and outcomes.11 the goal of this study was to evaluate sex differences in ohca characteristics, interventions, and outcomes, including survival to hospital discharge and survival with good neurological outcome, in a large regional cardiac system of care with established pci and ttm protocols. this is a retrospective study of registry data from the los angeles (la) county emergency medical services (ems) system. the study was reviewed and approved with exemption of informed consent by the institutional review board. la county is a large metropolis, comprising 88 cities spanning over 4000 square miles with a population of 10.2 million. la county ems operates a regional cardiac system of care for patients with stsegment elevation myocardial infarction (stemi) and/or ohca that has been previously described.12, 13 ems providers transport patients resuscitated from ohca to 1 of 35 currently designated cardiac arrest receiving centers. these centers are capable of providing immediate coronary angiography and primary pci 24 hours per day, 7 days per week, and have cardiovascular surgeons available. in addition, all centers are required to have a robust quality improvement program and internal policies for pci, fibrinolysis, and ttm. cardiac arrest receiving centers submit data on all adult patients with return of spontaneous circulation (rosc) postohca to a single registry maintained by the la county ems agency. data abstraction from prehospital and hospital records is completed by registered nurses (rns) in the departments of emergency medicine, cardiology, and/or quality improvement. completeness and accuracy of the entered data is reviewed by the ems agency with verification performed during site visits. study variables included age, sex, race / ethnicity, initial cardiac rhythm, arrest location, witness, bystander cpr, electrocardiogram (ecg) findings (stemi or no stemi), length of stay, induction of hypothermia, and whether the patient received coronary angiography and pci. stemi was indicated in the database if stemi was identified on the ecg, either in the field by software interpretation or in the emergency department by physician interpretation. the primary outcome of the study was survival to hospital discharge with good neurological outcome in women compared to men, as defined by a cerebral performance category (cpc) score at hospital discharge of 1 or 2. a cpc of 1 corresponds to a return to normal or mildly impaired cerebral function and independence with activities of daily living (adl). a cpc of 2 corresponds to moderate cerebral disability but sufficient function to remain independent with the adl. the cpc scores documented by physician, nurse, or occupational therapy assessment at the time of discharge were abstracted from the medical record and recorded in the database. patient characteristics, interventions, and outcomes were evaluated by sex for the entire cohort as well as the subgroup of witnessed cardiac arrest with initial shockable rhythm. the adjusted odds ratio (or) for survival with good neurological outcome all data were entered into microsoft excel (microsoft corporation, redmond wa) and transferred to sas software (version 9.4 ; sas institute inc., we report the neurologically intact survival rates as proportions with risk differences and exact binomial confidence intervals. adjusted ors and their p values variables in regression, which included patient characteristics (age, sex), arrest characteristics (initial rhythm, witness, and bystander cpr), and interventions (coronary angiography, pci, and ttm), were selected based on previous knowledge of their contribution to cardiac arrest outcomes. in order to specifically evaluate the confounding effects of hospital treatment on the association of sex with outcome, we constructed 2 primary models : the first limited to baseline patient and arrest characteristics without hospital interventions and the second inclusive of all variables above entered simultaneously into the model. in addition, we accounted for clustering by hospital with generalized estimating equations (gees) using the proc genmod option in sas 9.4. given that there is evidence that women are less likely to undergo treatments, such as coronary angiography and ttm,8, 14, 15, 16 as part of a sensitivity analysis, the sexeffect estimate was evaluated in an additional model, one in which the interaction terms between sex and ttm and between sex and coronary angiography were included. there were 5174 ohcas in the registry during the study period, including 3080 males and 2094 females. women were older, median 71 (interquartile range [iqr ], 5982) versus 66 years (iqr, 5578). despite a similar frequency of witnessed arrest, women were less likely to present with a shockable rhythm (22% vs 35% ; risk difference [rd ], 13% ; 95% ci, 1115) compared to men. characteristics, treatment and outcome by sex (n=5174) cpc indicates cerebral performance category ; cpr, cardiopulmonary resuscitation ; iqr, interquartile range ; pci, percutaneous coronary intervention ; stemi, stsegment elevation myocardial infarction ; ttm, targeted temperature management. regarding interventions, women were less likely to have stemi on the ecg (23% vs 32% ; rd, 13% ; 95% ci, 711), receive emergent coronary angiography (11% vs 25% ; rd, 14% ; 95% ci, 1216), pci (5% vs 14% ; rd, 9% ; 95% ci, 711), or ttm (33% vs 40% ; rd, 7% ; 95% ci, 410). in the unadjusted comparison, women had decreased survival to hospital discharge (33% vs 40% ; rd, 7% ; 95% ci, 410) and a lower proportion survived with good neurological outcome (16% vs 24% ; rd, 8% ; 95% ci, 610). among patients with a witnessed cardiac arrest with an initial shockable rhythm shown in table 2, of whom there were 974 males and 390 females, sex differences were similar, with a few exceptions. in addition, survival to hospital discharge was the same as compared to men. however, survival with good neurological outcome among women remained lower (38% vs 45% ; rd, 7% ; 95% ci, 113). characteristics, treatment, and outcome by sex for patients with witnessed arrest and initial shockable rhythm (n=1364) cpc indicates cerebral performance category ; cpr, cardiopulmonary resuscitation ; iqr, interquartile range ; pci, percutaneous coronary intervention ; stemi, stsegment elevation myocardial infarction ; ttm, targeted temperature management. tables 3 and 4 show the results of the regression analyses. with adjustment for age and arrest characteristics, there is a borderline statistically significant association between female sex and worse neurological outcome (or, 0.8 ; 95% ci, 0.71.0 ; table 3). however, after adjustment for age, arrest characteristics (initial rhythm, witness, and bystander cpr), and interventions (coronary angiography, pci, and ttm), female sex was not associated with decreased survival with good neurological outcome (or, 0.9 ; 95% ci, 0.81.1 ; table 4). the sensitivity analysis, shown in table 5, including the interaction terms between sex and ttm and between sex and catheterization, demonstrated similar results with a wider confidence interval (or, 1.0 ; 95% ci, 0.71.4). that is, taking into account the potential differing effects of hospital interventions in males and females, when coronary angiography and ttm are performed, there is no association of sex on outcome. in both models odds ratios for survival to hospital discharge with good neurological outcome adjusting for age and arrest characteristics and accounting for clustering by hospital (n=4504) cpr indicates cardiopulmonary resuscitation ; or, odds ratio. odds ratios for survival to hospital discharge with good neurological outcome adjusting for age, arrest characteristics and interventions, and accounting for clustering by hospital (n=4477) cpr indicates cardiopulmonary resuscitation ; or, odds ratio ; pci, percutaneous coronary intervention ; ttm, targeted temperature management. odds ratios for survival to hospital discharge with good neurological outcome adjusting for age, arrest characteristics, and interventions including sextreatment interaction terms and accounting for clustering by hospital (n=4477) cpr indicates cardiopulmonary resuscitation ; or, odds ratio ; pci, percutaneous coronary intervention ; ttm, targeted temperature management. at coronary angiography and ttm in this contemporary cohort of consecutive patients treated in a large, regional care system, we found significant sex differences in ohca. overall, men were more likely to present with favorable arrest characteristics (initial shockable rhythm) and more likely to receive interventions (emergent coronary angiography and ttm). however, there was no association with sex and neurologically intact survival after adjustment for these factors. our data support underlying differences in the presentation and management of cardiac arrest between men and women as the cause for noted outcome disparities. kim evaluated sex 's modification of the effect of therapeutic hypothermia on neurological outcome and reported an overall lower mortality in men. the researchers noted that the favorable arrest characteristics, including increased frequency of initial shockable rhythm, witnessed arrest, and bystander cpr, were a potential reason.4 a large study in japan reported better unadjusted survival in men compared to women and attributed this to a higher proportion of initial shockable rhythm and witnessed arrest.3 the differences in arrest characteristics were also noted in a subsequent analysis of the lucas in cardiac arrest trial. similar to our study, women were older and less frequently presented with an initial shockable rhythm, although there was no difference in the frequency of bystander witness arrests or cpr. with adjustment for these factors, there was no association between sex and survival to hospital discharge or at 6 months.5 our study differs from much of the past literature in the population and setting. the majority of past studies included in the recent metaanalysis by bougouin, which noted improved survival in women, spanned a time frame when current postresuscitation management was not available.10 in addition, much of the more recent studies are from cohorts in asia and scandinavia and inclusive of all ohca resuscitations.3, 4, 14, 17, 18 our us study population, with racial and ethnic diversity, is limited to those patients with rosc, regardless of whether sustained, because that is the entry criteria into the data registry. despite these dissimilarities, the differences in arrest characteristics between men and women are consistent across these various populations. interestingly, earlier studies suggested protective benefits of female sex,2, 6, 7 whereas in later studies that account for the current standards of patient care the results are less clear.9, 14, 17, 18 several population studies have continued to find a survival advantage to female sex within strata of shockable and/or nonshockable rhythm ; however, these studies have not reported frequency of interventions in postresuscitation care.19, 20 changes in resuscitation care may differentially affect men and women. in la county, advanced postresuscitation care capabilities, including pci and ttm, are required at the cardiac arrest receiving centers. in addition to the differences in arrest characteristics, we found differences in frequency of these interventions between men and women. in our cohort, women were less likely to receive interventions, including emergent coronary angiography and ttm, even among those most likely to have good neurological outcome, with witnessed arrest and initial shockable rhythm. yet, the sensitivity analysis accounting for effect modification between sex and interventions, further confirmed that female sex was not associated with differences in neurological outcome. other studies have noted similar sex disparities in interventions.8, 14, 15, 16 given the retrospective nature of this study, the reason for this difference is not known. it is likely attributable, in part, to a higher frequency of stemi on the ecg and initial shockable rhythm for men. aggressive postresuscitation care, including early coronary angiography, is more likely in patients with favorable arrest characteristics. as such it is notable that, among those patients who received early coronary angiography, men and women had a similar proportion of pci. furthermore, although both crude analysis and initial adjustment for patient and arrest characteristics alone showed worse outcomes for women, once accounting for interventions in the regression model, we did not find a difference in survival with good neurological outcome between men and women. these results raise the possibility of an effect of disparities in treatment shifting outcomes from ohca. compared to national surveillance data of ohca, women make up a similar proportion of our cohort (40% vs 39%), but la county data have a much higher proportion of hispanics (22% vs 5%).1 there was also a higher proportion of witnessed arrest (84% vs 47%), bystander cpr (41% vs 33%), and initial shockable rhythm (32% vs 24%) in these patients transported to specialty centers. similarly, in comparison to the study population evaluated by daya, representing 139 different ems agencies across the united states, our population had a similar median age, proportion of women, and bystander cpr rate but a higher proportion of witnessed arrest (84% vs 49%) and initial shockable rhythm (32% vs 22%).21 this study has several additional limitations. this is a retrospective cohort. as such, we can not determine causality, and the frequency of interventions is subject to selection bias. our analysis is not inclusive of all patients with cardiac arrest in la county ; the registry includes patients with ohca who achieve rosc, at least transiently, and are treated at a cardiac arrest receiving center. this likely explains the higher proportion of witnessed arrests and initial shockable rhythm compared with national studies. patients who were not transported (met termination of resuscitation criteria) and those transported to a hospital other than a cardiac arrest receiving center were not included in this analysis. we were unable to include adjustment for downtime, which is known to affect outcomes, because this was missing in the majority of patients and is likely to be inaccurate when reported.22, 23 by limiting analysis to complete subjects, we make the assumption that data are missing completely at random, which, if untrue, can lead to bias or incomplete control of confounders. however, because values were missing less than 10% of the time, we believe that imputation or other means of adjusting for missing values would not make a substantive difference in the results. in addition, given the retrospective methodology, there are other potential confounders that remain unmeasured. specifically, the database does not include information on comorbid conditions, arrest etiology, and other risk factors that may differ between men and women presenting with ohca. these factors could not be accounted for in this analysis and causality can not be determined. | backgroundthe purpose of this study was to evaluate sex differences in outofhospital cardiac arrest (ohca) characteristics, interventions, and outcomes.methods and resultsthis is a retrospective analysis from a regionalized cardiac arrest system. data on patients treated for ohca are reported to a single registry, from which all adult patients were identified from 2011 through 2014. characteristics, treatment, and outcomes were evaluated with stratification by sex. the adjusted odds ratio (or) for survival with good neurological outcome (cerebral performance category 1 or 2) was calculated for women compared to men. there were 5174 outofhospital cardiac arrests (ohcas ; 3080 males and 2094 females). women were older, median 71 (interquartile range [iqr ], 5982) versus 66 years (iqr, 5578). despite similar frequency of witnessed arrest, women were less likely to present with a shockable rhythm (22% vs 35% ; risk difference [rd ], 13% ; 95% ci, 1115), have stsegment elevation myocardial infarction (23% vs 32% ; rd, 13% ; 95% ci, 711), or receive coronary angiography (11% vs 25% ; rd, 14% ; 95% ci, 1216), percutaneous coronary intervention (5% vs 14% ; rd, 9% ; 95% ci, 711), or targeted temperature management (33% vs 40% ; rd, 7% ; 95% ci, 410). women had decreased survival to discharge (33% vs 40% ; rd, 7% ; 95% ci, 410) and a lower proportion of good neurological outcome (16% vs 24% ; rd, 8% ; 95% ci, 610). in multivariable modeling, female sex was not associated with decreased survival with good neurological outcome (or, 0.9 ; 95% ci, 0.81.1).conclusionssexrelated differences in ohca characteristics and treatment are predictors of survival outcome disparities. with adjustment for these factors, sex was not associated with survival or neurological outcome after ohca. |
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