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silane - based self - assembled monolayers (sams) with long organic chains play an important role in surface chemistry as they establish physically, thermally, and chemically stable films on substrates such as silicon oxide and glass. in addition, varying the functionality of the organic chain offers the potential to tailor surface properties in a controllable fashion. while there are many options to deposit a sam from solution or to chemically modify a preformed sam in solution (see, for example, refs (610)), direct gas - phase modification is much more limited. in organic chemistry, c c bond - forming reactions that occur entirely in the gas phase and without an added catalyst are extremely rare, thus there are few options for direct c c bond gas - phase surface modification. despite the limited reactions in the gas phase, solventless reactions are attractive as they are potentially less disruptive to film integrity than dipping a preformed sam into a reaction solution. immersion can reduce the quality of the resultant films, and the penetration and homogeneity are compromised if a solution can not parts of a (sub)microstructure substrate, for example, in the case of microelectromechanical systems (mems), due to microscopic gas bubbles. on the other hand, vapor - phase deposition has been used to form sams on silica and graphite, and we have recently shown that high - quality vinyl - terminated films can be achieved by this approach. the present study aimed to chemically modify the sam in a second gas - phase process by a c c bond - forming reaction. in this context, hexafluoroacetone azine (hfaa) 1 emerged as an appropriate reagent as it has been reported to generate cyclopropanes 2 with olefins such as but-2-ene without any catalyst or coadditive on heating as illustrated in figure 1. the reaction also gave rise to some nitrogen - containing cycloadducts when performed in the gas phase and does not appear to proceed via a discrete (cf3)2c : carbene, but rather the cyclopropane arises from decomposition and nitrogen (n2) extrusion after the addition of 1 to the olefin. clearly, if 1 is activated and long - lived enough in the vapor phase, then it may react directly in the gas phase with the olefinic residues of the vinyl - terminated silanes of preformed sams the goal of the present study was to demonstrate the potential of vinyl - terminated films for direct chemical modification in the gas phase. such an approach offers cleaner reaction conditions than solution - based techniques and would be preferable in many high - end applications. in addition, the selectivity of the reaction would have a clear advantage over the gas - phase plasma treatment of hydrocarbons (e.g., ref (17)). in the present study, fluorine was employed as an xps marker to track the c c bond - forming reaction, rather than a substituent to modify surface properties per se. commercially available n - type, one - side - polished silicon (100) wafers (wacker chemie ag, munich, germany) were used as substrates. vinyl - terminated trichlorosilanes with alkyl chain lengths of 11 carbon atoms (10-undecenyltrichlorosilane, ch2=ch(ch2)9sicl3) and 15 carbon atoms (14-pentadecenyltrichlorosilane, ch2=ch(ch2)13sicl3), referred to as molecules 3 and 4 (figure 2), were prepared according to the literature. the purity of the newly synthesized compounds was similar to values reported in the literature (see si). the methyl - terminated species (referred to as 5, 6, and 7, see figure 2) were obtained from alfa aesar (lancashire, u.k.) : decyltrichlorosilane (ch3(ch2)9sicl3 - 97%), dodecyltrichlorosilane (ch3(ch2)11sicl3 - 95%), and octadecyltrichlorosilane (ch3(ch2)17sicl3 - 95%). hplc - grade toluene (99.9%) hplc - grade dichloromethane was obtained from vwr international (leicestershire, u.k.). hydrogen peroxide (30%), ammonium hydroxide (35%), sulfuric acid (95%), and hydrochloric acid (36%) were all obtained from fisher scientific (leicestershire, u.k.). all experimental steps were performed in a fume hood under an inert atmosphere of nitrogen or argon using oven - dried glassware. the silicon substrates were cleaned before the deposition of the self - assembling monolayers as described in ref (14). once precleaned, the substrates were immediately coated with an organic (silane) film. at the outset, we prepared good - quality methyl- and vinyl - terminated sams on silicon oxide substrates. the methyl - terminated sams served as nonreactive reference substrates, devoid of olefinic groups. freshly prepared, sam - coated silicon substrates were placed in a schott duran bottle (100 ml capacity) equipped with a ptfe sealed cap. the bottle was purged three times with nitrogen before a certain amount of reactive intermediate precursor material hfaa (boiling point : 6768 c) was added to the bottle in a separate, smaller, open - topped vessel (see figure 3). schematic of the setup used for the chemical gas - phase modification of the sams. there was no direct contact between the liquid hfaa 1 reservoir and the organically precoated substrates during the reaction. the vessel was held at fixed temperatures (80, 120, and 160 c) and for defined periods of time (10, 20, and 30 min and 1, 2, 5, and 48 h). after each reaction, the samples were sonicated (15 min) sequentially in toluene, dichloromethane, and then deionized water. the chemically modified silicon substrates were analyzed using contact angle measurements to determine their hydrophilicity / hydrophobicity, ellipsometry to determine the thickness of the (modified) coating, x - ray photoelectron spectroscopy (xps) to determine the chemical composition of the (modified) coating, and atomic force microscopy (afm) to investigate the homogeneity and roughness of the (modified) coating. water contact angles (di water) were measured with a g10 goniometer microscope (krss gmbh, hamburg, germany) under ambient conditions at room temperature. all reported values are the average of three measurements taken from different places on the surface. the thickness of the sams was measured with an m-2000di spectroscopic ellipsometer (j. a. woollam co., inc., thickness values were extracted from fits to the data taken at 45, 50, 55, 60, 65, and 70 over wavelengths from 200 to 1000 nm. the sample surface was modeled as a si substrate with an oxide layer and a cauchy layer. the thickness of the silicon oxide after the oxidative cleaning treatment was 16 1 (average of three samples). the thickness of the monolayer films was calculated with an index of refraction of 1.45. film thickness values are averages of at least three measurements. the observed variability of the thickness of organic films prepared under identical conditions was 2, i.e., the variation of chemically identical films which were prepared at different times (not the same batch). in some cases, we prepared a batch of samples (10 to 15) with the c11 vinyl - terminated film. we determined the film thickness for several samples (typically three to five) rather than for each sample individually. values between 14.5 and 15.5 are reported as 15 (mean value) for all samples from the same batch in the following. the reproducibility (standard deviation) of the thickness for a single film was 0.2 (measured on at least three different spots on the same surface). x - ray photoelectron spectroscopy spectra were obtained using a vg sigma probe (vg scientific ltd., u.k.) and al k radiation (1486.6 ev). during the analysis, the pressure in the test chamber was kept at around 1 10 torr. the xps spectra were corrected for charging by referencing the aliphatic c 1s peak of hydrocarbon to 285.0 ev. elemental compositions of the various surfaces were determined from the area under individual elemental peaks using sensitivity factors provided with the software as well as taking the transmission function of the analyzer into account. casaxps (casa software ltd., u.k.) was used for the analysis. quantitative data is reported assuming a model where all constituents are homogeneously and isotropically distributed in the analyzed volume. the atomic force microscope used was a picospm ii (molecular imaging, usa) with an interchangeable nose scanner. images were recorded under ambient conditions at a scanning speed of 0.8 lines / s and with a nominal contact force on the order of 10 nn. hexafluoroacetone azine (1.0 g, 3.1 mmol, 1 equiv) and 1-decene (0.9 g, 6.2 mmol, 2 equiv) were placed in an autoclave and held at 160 c for 48 h. the reaction mixture was purified by silica gel chromatography (hexane) to give the criss - cross adduct shown in figure 4 as a colorless oil (278 mg, 15%). h nmr (300 mhz, cdcl3) : h 3.623.48 (2h, m, ch(ch2)7ch3), 2.75 (2h, dd, j 13.8, 6.1 hz, (cf3)2ch2 10a), 2.45 (2h, dd, j 13.8, 9.7 hz, (cf3)2cch2 10b), 1.891.75 (2h, m, ch2(ch2)6ch3 8a), 1.371.14 (26h, m, ch2(ch2)6ch3 8b), 0.88 (6h, t, j 6.5 hz, ch3). c nmr (75 mhz, cdcl3) : c 123.7 (q, j 2.8 hz, cf3 12a), 123.5 (q, j 2.9 hz, cf3 12b), 69.0 (m, (cf3)2c), 56.6 (ch : c9), 41.1 ((cf3)2cch2 : c10), 33.0 (ch2(ch2)6ch3 : c8), 32.0, 29.7, 29.6, 29.3, 25.9, 22.8 (ch2, c2c7), 14.2 (ch3). f nmr (282 mhz, cdcl3) : f 67.6 (6f, q, j 18.0, 8.6 hz, cf3 a), 73.6 (6f, q, j 18.0, 8.6 hz, cf3 b). hrmsm / z (ci) : calculated for c26h41f12n2 [m + h ], 609.3073 ; found, 609.3064. the xps survey spectra of all samples with methyl- and vinyl - terminated sams (not shown) showed the presence of si (2s 150.4 ev, 2p 99.2 ev), c (1s 285.0 ev), and o (1s 533.0 ev) as expected. the spectra distinguish between the bulk si (si 2s 150.4 ev, si 2p 99.2 ev) and the silicon oxide (si 2s 154.3 ev, si 2p 102.8 ev). contact angle measurements and the film thickness determination with ellipsometry confirmed densely packed monolayers. the results are summarized in tables 1 and 2. in the first step, the best preparation conditions for the reaction between hfaa and the vinyl - terminated films were determined by testing a range of different temperatures (80, 120, and 160 c) at several defined periods (10, 20, and 30 min and 1, 2, 5, and 48 h). in addition, different amounts of hfaa (50, 100, 150, 200, 300, and 400 l) were employed for modification. the water contact angle values as well as the film thicknesses, before and after the reaction of the vinyl - terminated sams, are listed in table 1. if the evaluation indicated a significant decrease (> 5) in film thickness after the reaction, then we interpreted this as an indication of degradation. those thickness values are reported as < 10 and were not investigated further. for the vinyl - terminated surfaces derived from 3 and 4, organic fluorine on the surface became obvious in the xps xps survey scans of vinyl - terminated sams prepared from 3 after the reaction (20 min) with 1, performed at 80, 120, and 160 c. samples that reacted for times greater than 1 h showed the strongest f signal by xps. xps scans of vinyl - terminated sams prepared from 3 after the reaction with 1, at 160 c for various time periods. the results reported in table 1 and the xps spectra in figure 6 indicate that degradation of the vinyl - terminated sams occurs for reaction periods of more than 30 min. therefore, different amounts of hfaa were tested in combination with a reaction period of 20 min and a temperature of 160 c. xps results obtained from reactions of sams derived from 3 and treated for 20 min at 160 c, varying the levels of 1. although a reservoir volume of 400 l of hfaa resulted in a higher f signal than a volume of 200 l, table 1 indicates that 400 l also caused a slight decrease in both the film thickness and contact angle value. since a volume of 300 l appeared to give very similar results to that of 200 l, without a further increase in the contact angle value, we decided to use a volume of 200 l as the optimum for the preparation of nondegraded modified films. on the basis of the results above a temperature of 160 c, a reaction period of 20 min and a volume of 200 l of hfaa gave the optimum conditions for the modification of the vinyl - terminated sams. the f 1s single - region scan of the films treated with hfaa under these conditions showed one symmetric peak (not shown). in the c 1s single -region scan of hfaa - treated vinyl - terminated films, afm images of the vinyl - terminated films before and after modification with carbene showed very smooth surfaces with rms values for roughness of 75120 pm averaged over an area of 5 m 5 m, in agreement with monolayer coverage. to explore the degree of selective reactivity of hfaa with the c = c double bond versus direct ch2 insertion, several experiments were also performed with methyl - terminated sams of 5 and 7 as an unreactive control. a batch of c10-methyl - terminated films (5) was prepared, and film thickness values were determined in selected cases. the water contact angle values as well as the film thicknesses, before and after exposure of the methyl - terminated sams to hfaa, are listed in table 2. methyl - terminated samples exposed to hfaa under the optimum preparation conditions were further investigated with xps. after their exposure to hfaa, no changes were observed, consistent with their lack of reactivity (see figure 9). xps survey scans of methyl - terminated sams prepared from 5, 6, and 7 after the reaction with hfaa. the spectra showed the presence of si, c, and o. no f signal was observed (reaction conditions : 20 min, 160 c, 200 l hfaa). a control vapor - phase reaction between hfaa and 1-decene was performed under similar reaction conditions to those described above with the vinyl - terminated sams. in the reaction mixture, the methyl - terminated surfaces prepared from 57 remained unmodified for reaction periods of less than 5 h. this is obvious by the absence of any signal in the xps spectra corresponding to the presence of fluorine on the surface. this lack of reaction was also confirmed by film thickness measurements with ellipsometry and water contact angle measurements both before and after the reaction of the precoated substrates. the contact angle values as well as the film thicknesses remained approximately constant before and after exposure to hfaa (table 2). for the vinyl - terminated sams, the optimum reaction conditions produced surfaces with slightly higher contact angles and similar film thickness values to those of the unmodified vinyl - terminated sams (table 1). the reactivity of hfaa and the intermediates formed during reaction and the mechanism of the reaction with a double bond have been a source of discussion for some time. figure 10 illustrates a putative mechanism for the formation of bis(trifluoromethyl)methylene - terminated sams in the vapor phase. putative minimal mechanism for the formation of bis(trifluoromethyl)methylene - terminated sams in the vapor phase. the mechanism is obtained from previous studies on the addition of 1 to organic olefins in reactions carried out in sealed tubes. in those reactions, cyclopropanes were formed, as were dimeric adducts where two molecules of the olefin substrate had reacted with 1 to generate nitrogen - containing heterocyclic products. we did not find any xps evidence for nitrogen - containing products in the surface reactions between vinyl - terminated sams of 3 and 4 and azine 1 for reaction periods of less than 30 min. only after 1 h of reaction with vinyl - terminated films was a small amount of nitrogen detected by xps (see si). the presence of nitrogen might suggest the formation of the criss - cross adduct, but this was apparent only when the sam had undergone degradation, and presumably the steric constraints of the surface became less of a constraint. the control vapor - phase reaction between hfaa and 1-decene showed the criss - cross adduct and some unidentified compounds in the reaction mixture. since reaction on the surface has unique conformational constraints in bringing two olefin groups together, the preferred mode of breakdown of the initial adduct appears to result in products where nitrogen (n2) is extruded and the c3 fragment adds to the double bond (see figure 10). the fact that the xps f 1s single - region scan of the films treated with hfaa showed one symmetric peak suggests a single fluorine environment on the surface, which is assigned to the cf3 group. the carbon region has four different species (figure 8) and is consistent with a bis(trifluoromethyl)methylene cyclopropyl motif as illustrated in figure 11. the carbon peak, showing the largest shift toward higher binding energies from the region of 282.5288.5 ev, was assigned to carbon c1 (288.1 ev). schematic of the proposed reaction product of the vinyl - terminated sams with hfaa, a bis(trifluoromethyl)methylene cyclopropyl motif. the xps c 1s scans indicate four different carbon species. according to the proposed structure, this corresponds to the c(cf3)2 species. a second carbon peak, with a binding energy of 286.6 ev, was assigned to the c2 carbon species (two carbons of the cyclopropane ring). the most intense signal, c3, was assigned to the carbons from the alkyl chain (285.0 ev). the xps spectrum in figure 8 indicates a close to 2:1 ratio of the carbon c2 and c1 species. because of the low intensity of the c1 and c2 signals, note, however, that these signals should be viewed in combination with the clearly observable cf3 signal. the correlation of the three carbon signals supports the assignment and the quantification of the ratios. the ratios are consistent with a predominant cyclopropanation product at the chain terminus. other c3 addition products may also be relevant as illustrated in the box in figure 10 ; however, they fit much less well into the integrated carbon ratios from xps as their carbon c2 to c1 ratio is 1:1. the corresponding ratios obtained from the c11-vinyl 3 and the c15-vinyl 4 sams based on the c 1s and f 1s single - region scans figure 8 also demonstrates that the experimentally observed ratio of the aliphatic c 1s to the cf3 signal is as high as 20:1, while the theoretical ratio is 7:1 if all vinyl groups of the sam derived from 3 react. on the basis of the intensity of the f 1s signal and the sum of intensities of the c 1s signals and taking into account the attenuation of the latter, the reaction conversions of hfaa with c11-vinyl 3 and c15-vinyl 4 can be estimated to be 27 and 23%, respectively. for temperatures above 120 c, surface degradation competed with chemical modification over the time period of 30 min to 48 h, as was evident from a reduced carbon signal in xps (see figure 6), a reduced film thickness, and the decrease in water contact angle values (see table 1). a comparison of the vinyl- and the methyl - terminated films (tables 1 and 2) suggests that entire surfactant molecules are most likely removed, i.e., cleavage at the si o interface occurs because otherwise the methyl - terminated films should also show signs of degradation under these conditions. the f signals in the survey scans of the vinyl - terminated films indicate that some f - containing species are present, which might be expected to lead to an increase in the contact angle (figure 6). however the vinyl - terminated films are less well packed than their methyl - terminated counterparts, which makes it easier for hydroxyl or potentially fluoride species to access the surfactant substrate interface and to attack the si o bond. a possible route for degradation is then via cleavage of the si o bond. c addition reaction is competing with the removal of entire molecules, which is reflected by the increasing f signal but decreasing c signal in the xps survey spectra for periods of between 30 min and 5 h (figure 6). from a certain point on, however, all remaining surfactants are modified and further removal of surfactants will then lead to a decrease in the f signal (figure 6, spectrum for 48 h). this is also supported by the appearance of a nitrogen signal in the xps spectra for periods of between 1 and 5 h (see si), indicating the establishment of the criss - cross product followed by a decrease in the n signal if modified molecules are removed by further degradation. it has been suggested that similar films decompose via c c bond cleavage ; however, this is inconsistent with our observations. elucidating the mechanism of degradation we observed indications of degradation in methyl - terminated films only after a reaction period of 5 h (see table 2). at this time this observation is consistent with that reported in ref (23) that hfaa is stable at elevated temperatures. however, after 5 h at 160 c hfaa may start to decompose, and the various species formed from its decomposition may initiate sam degradation. after this time, heating of the methyl - terminated films to 160 c led to a degradation in control experiments. for a reaction period of 20 min there was no degradation if less than 300 l of hfaa was used, while the presence of fluorine atoms at the surface was clearly evident from xps. at a constant reaction time of 20 min and a temperature of 160 c the amount of hfaa 1 precursor in the reservoir evaporated completely from the liquid reservoir. there is a clear correlation between the amount of hfaa 1 precursor in the vapor phase and the level of conversion. at this relatively short reaction time the resulting films were smooth when analyzed by afm (the rms roughness varied from 75 to 120 pm over a scan area of 5 m 5 m) and showed no significant change in the film thickness and a slight increase in the contact angle value, supporting the integrity of high - quality films. the modified films are likely to be slightly more disordered than the vinyl - terminated layers due to the bulky bis(trifluoromethyl)cyclopropane group. with a reaction yield of around 25%, this indicates that the processes that occur are complex and depend on several parameters such as exposure dose and temperature. it appears that the chemical modification is determined by an activation energy and the kinetics of the competing reactions that are involved. in this article, we describe a gas - phase surface modification reaction that is performed by heating in standard glassware under standard conditions, and there is no need for a mixed monolayer to ensure that the functional groups are particularly exposed. the reaction of hfaa with terminal olefins generates a bis(trifluoromethyl)cyclopropyl product, which constitutes a rare c c bond - forming reaction in the gas phase.. a future challenge will be to extend the range of precursors that can generate suitable reactive species for such modification. it is also a challenge to offset the chemically induced thermal degradation of the surface layer for longer reaction periods. | vinyl - terminated self - assembled monolayers (sams) on silicon oxide substrates were chemically modified by the addition of a bis(trifluoromethyl)methylene group in a rare gas - phase c c bond - forming reaction to directly generate films carrying terminal cf3 groups. the vinyl - terminated films were treated with hexafluoroacetone azine (hfaa) for modification. the films were characterized with ellipsometry, contact angle measurements, atomic force microscopy (afm), and x - ray photoelectron spectroscopy (xps). in this study, we find that for optimized conditions clean reactions occur on a surface between sams with terminal olefins and hfaa, and the product is consistent with bis(trifluoromethyl)cyclopropanation formation after nitrogen extrusion. |
a 60-year - old woman, 135 cm in height and 39 kg in weight, was scheduled for right thyroid lobectomy due to thyroid cancer. she had severe kyphoscoliosis, and surgery was planned under a sternotomy because the thyroid was located below the sternum. she tolerated light ordinary life activity, but she felt short of breath during exercise and sleeping. at those times, she had been taking inhaled oxygen intermittently. on preoperative examination, she did not complain of respiratory difficulty, and both lungs had clear sounds. however, her neck was very short, and movement limitations were noted (fig. chest computed tomography showed severe kyphoscoliosis with destructive vertebral body fusion in the cervical and upper thoracic spine. the trachea was deviated at an angle of 90 degrees with stenosis just below the deviated area, with the smallest diameter being 6.5 13 mm (fig. 2). a pulmonary function test revealed the presence of severely restricted pulmonary disease. the measurements demonstrated fev1 of 0.55 l (35% of predicted), fvc of 0.7 l (35% of predicted) and fev1/ fvc 78%. the arterial blood gas analysis in room air was pao2 62.6 mmhg, paco2 44.8 mmhg and ph 7.406. other preoperative laboratory tests including echocardiography and thyroid function test were unremarkable. when the patient entered the operating room, the routine monitoring systems, including electrocardiogram, pulse oximetry, and non - invasive blood pressure were started. proper bag - and - mask ventilation was assured and rocuronium 30 mg was administered to facilitate endotracheal intubation. under direct laryngoscopy, no laryngeal structures could be identified due to the limited neck movement. subsequently, we used the glidescope videolaryngoscope (saturn biomedical systems inc., burnaby, british columbia, canada) and were able to obtain a proper laryngeal view however, the tube could not be advanced more than 3 cm beyond the vocal cords due to resistance despite the good laryngeal view. we attempted intubation again with 5.5 and 5.0 mm tubes, but the same problem was encountered. other attempts with various methods, such as full rotation of the tube tip, applying a lubricant, and using fiberoptic bronchoscopy, also failed due to same problem. intubation was tried with a 7.0 mm tube, but the location of the cuff between the vocal cords resulted in gas leakage around the tube despite ballooning. ultimately, intubation was accomplished with an 8.0 mm tube without cuff ballooning and saline - soaked gauzes were packed around the tracheal inlet to minimize gas leakage and to fasten the tube in the trachea. then, positive pressure ventilation could be performed successfully, and dexamethasone 5 mg was administered to minimize airway edema. anesthesia was maintained with sevoflurane in 50% oxygen / air, and a ventilator was set to deliver peak airway pressure of 22 cmh2o at a respiratory rate of 12 to 14 breaths / min. the tidal volume was maintained around 300 ml, and oxygen saturation by pulse oximetry remained above 95% throughout the anesthesia. the thyroid gland was very difficult because it was unusually located on the back of the trachea and adhered to neighboring soft tissues. after the surgery was complete, muscle relaxation was reversed with sugammadex 200 mg, and extubation was uneventful. the operation time was 3 h 30 min, and the anesthesia time was 5 h 5 min. after the operation, the patient was transferred to the intensive care unit and was kept for 5 days because of newly developed atelectasis and pleural effusion in the right lung. chest physiotherapy such as coughing, deep - breathing exercise, and ambulation was also performed. her pulmonary condition gradually improved, and she was discharged on postoperative day 20 without sequelae. the abnormal thoracic cage geometry limits the vital capacity and chest wall compliance, generally resulting in restrictive respiratory disease. this induces alveolar ventilation / perfusion mismatch, worsening of gas exchange, and desaturation. pulmonary hypertension is the result of increased pulmonary vascular resistance caused by compressed lung vasculature and the response to arterial hypoxia. the patient complained of dyspnea intermittently, the arterial blood gas analysis showed mild hypoxemia, and pulmonary function tests revealed the presence of severely restrictive pulmonary disease. however, the patient had been taking medications for dyspnea, and the disease was well - controlled without pulmonary hypertension. rather, an important issue in this patient was the anatomic deformity of the airway, which was anticipated to make endotracheal intubation difficult. first, her neck was very short, and her neck movement was limited due to kyphoscoliosis with destructive vertebral body fusion this problem was overcome by using a relatively new airway device : the glidescope videolaryngoscope. compared with direct laryngoscopy, the glidescope videolaryngoscope has been shown to provide a superior laryngoscopic view for endotracheal intubation. we were able to obtain a proper laryngeal view using this device and could insert a tracheal tube between the vocal cords easily. her trachea was severely deviated to an angle of 90 degrees and stenosed below the deviated area. the tracheal deviation could be caused by various factors including a thyroid goiter, a tumor in the neck and thorax, trauma, congenital cardiac disease, lung collapse and, like this patient, spinal deformities such as kyphoscoliosis. whatever the cause may be, in most previous reports, authors tried to advance a tracheal tube below the deviated area and the results were successful. but davies had reported a case of intubation failure in a patient with combination of marked tracheal deviation and a tracheal diverticulum. in that case, the airway was secured using a laryngeal mask airway during the operation. in the present case, we also tried insert a tracheal tube through the deviated area with various methods. but the tube could not be advanced more than 3 cm beyond the vocal cords despite the proper laryngeal view. the use of supraglottic airway devices was not considered in this patient because they could have been relatively unsafe during the sternotomy and dissection of the thyroid gland which was unusually located on the back of the trachea. in addition, if the supraglottic airway device needed to be adjusted, it might have been difficult because the patient 's neck was very short. therefore, we ultimately decided to place the tube tip above the resistive tracheal area and to additionally pack saline - soaked gauzes around the tracheal inlet to minimize gas leakage and to fasten the tube in the trachea. then, mechanical ventilation was performed successfully, and the operation proceeded uneventfully. in conclusion, severe kyphoscoliosis could be associated with difficult airway management due to a distorted airway anatomy. if a tracheal tube could not advance easily in a patient with this disease, one should not force the passage of a tube against resistance. depending on the surgical procedure, using supraglottic airway devices or positioning a tracheal tube above the area of resistance may be useful alternatives. | we report on a case of difficult endotracheal intubation in a patient with marked tracheal deviation at an angle of 90 degrees combined with stenosis due to kyphoscoliosis with vertebral body fusion. after induction of general anesthesia, a proper laryngeal view was easily obtained using a videolaryngoscope. but a tracheal tube could not be advanced more than 3 cm beyond the vocal cords due to resistance, despite various attempts, including the use of small size tubes, full rotation of the tube tip, and fiberoptic bronchoscopy. ultimately, the airway was successfully secured by placing a tube tip above the area of resistance and by additionally packing saline - soaked gauzes around the tracheal inlet to minimize gas leakage and to fasten the tube in the trachea. |
mos1 alleles were selected by visual screening in wormbase (www.wormbase.org) for appropriately located transposon insertions and provided by the nemagenetag consortium. mos1 alleles were identified in wormbase (www.wormbase.org) and requested from the nemagenetag consortium (http://elegans.imbb.forth.gr/nemagenetag/). the presence of the mos1 insertion was verified with gene - specific primers, annealing inside and outside of the mos1 element. the mos1 element was crossed into the unc-119(ed3) background to make an injection strain and verified by pcr analysis. targeting constructs were designed to contain (1) a right homology region (adjacent to the mos1 element, (2) a left homology region (distant from the mos1 element) and (3) a c. briggsae - unc-119(+) rescue region. right homology regions were comprised of approximately 2.0 kb of homologous dna immediately adjacent to the mos1 insertion site. left homology regions were comprised of 2 to 3 kb of homologous dna, which specifies the endpoint of the targeted deletion. these regions were selected to avoid repetitive dna sequences, in particular short inverted repeats, which are likely to anneal and reduce the frequency of correct deletions. a c. briggsae unc-119(+) rescue fragment was chosen because it is smaller than the c. elegans unc-119 gene. an injection mix was made that contained the targeting plasmid (50 ng / ul), mos1 transposase helper plasmid pjl43.114 (50 ng / ul), mcherry co - injection markers pgh810 (prab-3::mcherry, 10 ng / ul), pcfj90 (pmyo-2::mcherry, 2.5 ng / ul), and pcfj10410 (pmyo-3::mcherry, 5 ng / ul). the injection strain was maintained at 15c on hb101 bacteria. young adult animals were mounted on an agarose pad under halocarbon oil and injected following standard techniques. injected worms were left to recover at 15c for a couple of hours and then transferred three at a time to hb101 or op50 seeded ngm plates and placed at 25c. in our hands, approximately 70 % of all injected worms resulted in transgenic progeny. we found that growing worms on hb101 bacteria at 15c significantly improved the health of unc-119 animals. after approximately 7 days each plate was screened for deletion mutants. screening was greatly facilitated by allowing the plate to starve out completely because unc-119 animals can not form dauers and are therefore selected against. strains with an unc-119(+) insertion were identified on a fluorescence dissection microscope as l1 animals that move like wild - type animals but have none of the fluorescent co - injection markers. a single rescued, non - fluorescent animal was picked to a new plate and allowed to propagate for one generation. in the case of obvious phenotypes (for example, dpy-13) a single mutant animal was picked from the progeny ; in cases where the phenotype was wild - type ten animals were picked to individual plates and tested for homozygosity. it takes approximately two weeks from injecting the strain to recovering a homozygous deletion animal. we calculated the distance of every protein coding gene in the ws205 referential release of wormbase (http://ws205.wormbase.org15) to all current mos1 insertion alleles using a state machine algorithm written in perl. the closest mos1 element was defined as the distance from the insertion site to the atg start codon. the number of mos1 elements in the vicinity of each gene was determined by extracting a sequence segment upstream and downstream of the atg and summing the number of elements contained within that span. the number of intervening genes between a given gene and its nearest mos1 element was determined by extracting the segment ranging from the atg to the insertion site and tallying the number of genes present, including genes that partially reside within the interval. the analysis was repeated against all genes lacking a deletion allele from either the c. elegans gene knockout consortium in the u.s. and canada (ok alleles) and the national bioresource project in japan (tm alleles). genomic dna from worms was isolated with the gentra puregene tissue kit (qiagen) following the manufacturer s supplementary protocol : purification of archive - quality dna from nematodes or nematode suspensions using the gentra puregene tissue kit. dna labeling, sample hybridization, image acquisition, and determination of fluorescence were all performed as previously described12. we used a 3x high - density (hd) chip divided into three 720 k whole genome sections for all experiments. all microarrays were manufactured by roche - nimblegen with oligonucleotides synthesized at random positions on the arrays. for all experiments, normalization of intensity ratios were performed with a loess regression as previously described12. three strains, eg5810 (25 kb deletion), eg5620 (15 kb) and eg5621 (15 kb), were tested against wild - type dna. all samples also showed two identical untargeted deletions : an approximately 8 kb deletion of pgp-6 and pgp-7 on chromosome x and an approximately 4 kb deletion of the telomeric region ctelx3.1 at the left end of chromosome v. we verified by cgh that these deletions were present in the parent strain (eg5003) and the deletions therefore do not represent second site mutations caused by the mosdel technique. targeting vectors typically consist of three distinct fragments : a right homology region, a positive selection marker (cb - unc-119(+)) and a left homology region. in some cases the positive selection marker was flanked by the fluorescent marker punc-122::gfp, which is dimly expressed in the coelomocytes. all targeting vectors were made using the multisite gateway three - fragment vector construction kit (invitrogen, catalog no. 12537 - 023). to verify deletions by pcr we designed oligos that would amplify short genomic dna fragments inside and outside the targeted regions. these reactions were reproducible and robust ; pcr amplification was successful on crude genomic lysates from 510 worms or from high quality dna samples prepared with a gentra puregene kit (qiagen). dyn-1 heterozygous verification primers : complete dyn-1 deletions were verified at the 5 end by pcr amplification with the three primers : ogh154, ogh133 and ocf125. oligos ogh154 + ogh133 give a 2.7 kb pcr product when the wild - type copy of dyn-1 is present. oligos ogh154 + ocf125 give a 3.4 kb pcr product when the dyn-1 locus is deleted. the deletion was verified at the 3 end by pcr with oligos ogh155, ogh156 and ocf400. oligos ogh155 + ogh156 produce a 2.5 kb pcr product when the wild - type dyn-1 locus and the ttti14024 mos1 element are present. oligos ogh155 + ocf400 produce a 3.0 kb pcr product when the dyn-1 gene is deleted. all molecular biology analysis and design was done with the program ape (a plasmid editor) that is freely available at http://www.biology.utah.edu / jorgensen / wayned / ape/. all oligos and plasmids are listed in supplementary table 3. mos1 alleles were selected by visual screening in wormbase (www.wormbase.org) for appropriately located transposon insertions and provided by the nemagenetag consortium. mos1 alleles were identified in wormbase (www.wormbase.org) and requested from the nemagenetag consortium (http://elegans.imbb.forth.gr/nemagenetag/). the presence of the mos1 insertion was verified with gene - specific primers, annealing inside and outside of the mos1 element. the mos1 element was crossed into the unc-119(ed3) background to make an injection strain and verified by pcr analysis. targeting constructs were designed to contain (1) a right homology region (adjacent to the mos1 element, (2) a left homology region (distant from the mos1 element) and (3) a c. briggsae - unc-119(+) rescue region. right homology regions were comprised of approximately 2.0 kb of homologous dna immediately adjacent to the mos1 insertion site. left homology regions were comprised of 2 to 3 kb of homologous dna, which specifies the endpoint of the targeted deletion. these regions were selected to avoid repetitive dna sequences, in particular short inverted repeats, which are likely to anneal and reduce the frequency of correct deletions. a c. briggsae unc-119(+) rescue fragment was chosen because it is smaller than the c. elegans unc-119 gene. an injection mix was made that contained the targeting plasmid (50 ng / ul), mos1 transposase helper plasmid pjl43.114 (50 ng / ul), mcherry co - injection markers pgh810 (prab-3::mcherry, 10 ng / ul), pcfj90 (pmyo-2::mcherry, 2.5 ng / ul), and pcfj10410 (pmyo-3::mcherry, 5 ng / ul). the injection strain was maintained at 15c on hb101 bacteria. young adult animals were mounted on an agarose pad under halocarbon oil and injected following standard techniques. injected worms were left to recover at 15c for a couple of hours and then transferred three at a time to hb101 or op50 seeded ngm plates and placed at 25c. in our hands, approximately 70 % of all injected worms resulted in transgenic progeny. we found that growing worms on hb101 bacteria at 15c significantly improved the health of unc-119 animals. after approximately 7 days screening was greatly facilitated by allowing the plate to starve out completely because unc-119 animals can not form dauers and are therefore selected against. strains with an unc-119(+) insertion were identified on a fluorescence dissection microscope as l1 animals that move like wild - type animals but have none of the fluorescent co - injection markers. a single rescued, non - fluorescent animal was picked to a new plate and allowed to propagate for one generation. in the case of obvious phenotypes (for example, dpy-13) a single mutant animal was picked from the progeny ; in cases where the phenotype was wild - type ten animals were picked to individual plates and tested for homozygosity. it takes approximately two weeks from injecting the strain to recovering a homozygous deletion animal. we calculated the distance of every protein coding gene in the ws205 referential release of wormbase (http://ws205.wormbase.org15) to all current mos1 insertion alleles using a state machine algorithm written in perl. the closest mos1 element was defined as the distance from the insertion site to the atg start codon. the number of mos1 elements in the vicinity of each gene was determined by extracting a sequence segment upstream and downstream of the atg and summing the number of elements contained within that span. the number of intervening genes between a given gene and its nearest mos1 element was determined by extracting the segment ranging from the atg to the insertion site and tallying the number of genes present, including genes that partially reside within the interval. the analysis was repeated against all genes lacking a deletion allele from either the c. elegans gene knockout consortium in the u.s. and canada (ok alleles) and the national bioresource project in japan (tm alleles). genomic dna from worms was isolated with the gentra puregene tissue kit (qiagen) following the manufacturer s supplementary protocol : purification of archive - quality dna from nematodes or nematode suspensions using the gentra puregene tissue kit. dna labeling, sample hybridization, image acquisition, and determination of fluorescence were all performed as previously described12. we used a 3x high - density (hd) chip divided into three 720 k whole genome sections for all experiments. all microarrays were manufactured by roche - nimblegen with oligonucleotides synthesized at random positions on the arrays. for all experiments, normalization of intensity ratios were performed with a loess regression as previously described12. three strains, eg5810 (25 kb deletion), eg5620 (15 kb) and eg5621 (15 kb), were tested against wild - type dna. all samples also showed two identical untargeted deletions : an approximately 8 kb deletion of pgp-6 and pgp-7 on chromosome x and an approximately 4 kb deletion of the telomeric region ctelx3.1 at the left end of chromosome v. we verified by cgh that these deletions were present in the parent strain (eg5003) and the deletions therefore do not represent second site mutations caused by the mosdel technique. targeting vectors typically consist of three distinct fragments : a right homology region, a positive selection marker (cb - unc-119(+)) and a left homology region. in some cases the positive selection marker was flanked by the fluorescent marker punc-122::gfp, which is dimly expressed in the coelomocytes. all targeting vectors were made using the multisite gateway three - fragment vector construction kit (invitrogen, catalog no. 12537 - 023). to verify deletions by pcr we designed oligos that would amplify short genomic dna fragments inside and outside the targeted regions. these reactions were reproducible and robust ; pcr amplification was successful on crude genomic lysates from 510 worms or from high quality dna samples prepared with a gentra puregene kit (qiagen). dyn-1 heterozygous verification primers : complete dyn-1 deletions were verified at the 5 end by pcr amplification with the three primers : ogh154, ogh133 and ocf125. oligos ogh154 + ogh133 give a 2.7 kb pcr product when the wild - type copy of dyn-1 is present. oligos ogh154 + ocf125 give a 3.4 kb pcr product when the dyn-1 locus is deleted. the deletion was verified at the 3 end by pcr with oligos ogh155, ogh156 and ocf400. oligos ogh155 + ogh156 produce a 2.5 kb pcr product when the wild - type dyn-1 locus and the ttti14024 mos1 element are present. oligos ogh155 + ocf400 produce a 3.0 kb pcr product when the dyn-1 gene is deleted. all molecular biology analysis and design was done with the program ape (a plasmid editor) that is freely available at http://www.biology.utah.edu / jorgensen / wayned / ape/. all oligos and plasmids are listed in supplementary table 3. (note : large dataset, too large to include in joined pdf file.) | we have developed a method, mosdel, to generate targeted knock - outs of genes in c. elegans. we make use of the mos1 transposase to excise a mos1 transposon adjacent to the region to be deleted. the double - strand break is repaired using injected dna as a template. repair can delete up to 25 kb of dna and simultaneously insert a positive selection marker. |
labetalol, a combined alfa and beta - adrenergic receptor antagonist, is an antihypertensive drug frequently used to treat systemic hypertension in acute care settings, including in obstetrics. we present an unusual case characterized by a rise in blood pressure (bp), rather than hypotension, and temporary neural dysfunction following inadvertent intrathecal administration of labetalol during obstetric anesthesia. a 25-year - old woman, parity status p1 + 0, weighing 50 kg, presented in early labor at term with a history of loss of fetal movement and cesarean section in the earlier pregnancy. on admission, she was fully oriented with a pulse rate of 80/min and bp of 130/70 mmhg, and the fetal heart sounds were equivocal. she was put up for emergency lower segment cesarean section under spinal anesthesia and premedicated with ranitidine 50 mg by intramuscular (i m) route and metoclopramide 10 mg i m in the labor room before being brought into the operation theater (ot). in the ot, her vitals were taken which showed pulse 76/min, bp 140/72 mmhg, and oxygen saturation 98% in room air. intravenous (iv) cannulation was done with an 18 g cannula and ringer lactate infusion started. spinal anesthesia was administered, in the sitting posture, at the l2-l3 interspace with a 25 g quincke 's spinal needle by the anesthesia junior resident. the intended drug was hyperbaric bupivacaine 0.5% w / v, and the volume administered was 2.6 ml. immediately upon assuming the supine position, the patient started complaining of acute pain in both lower limbs along with intense burning sensation. the situation was rescued by converting to general anesthesia by propofol 100 mg iv, intubating with the help of succinylcholine 50 mg iv and maintaining with nitrous oxide 60% and oxygen 40% inhalation. muscle relaxation was continued with atracurium 25 mg iv in two divided doses (20 mg + 5 mg). the cesarean section was completed without problems and a 2.4 kg live baby was delivered. relaxation was reversed by neostigmine 2.5 mg iv with glycopyrrolate 400 mcg iv, and nitrous oxide was withdrawn after recovery of spontaneous respiration. the patient was extubated uneventfully and did not complain of any further leg pain or burning sensation after recovery from anesthesia. vital signs remained normal postoperatively, and the patient was shifted to the postnatal ward. the case was not reported to the hospital administration as there is no system of medication error reporting in the institution. the mix - up had occurred in picking up the vial from the anesthetic injection tray on which multiple ampoules and vials were kept. an intern had picked up the ampoule, broken the top and held it while the resident drew up the injection in the syringe without reading the label. the ampoules [figure 1 ] were similar in size and amber colored although the labeling was distinct. the ampoule of labetalol from which the injection was drawn shown side by side with an ampoule of spinal bupivacaine from which it should have been drawn. they are similar in appearance though labeling is distinct a medication error is any failure in the medicine selection, prescription, dispensing, handling, and use process that may harm the patient. like adverse drug reactions (adrs), medication errors are an inevitable consequence of pharmacotherapy, although, unlike adrs, they are often preventable through individual and institutional measures. though recognized as an important cause of patient mortality and morbidity globally, particularly in operative, emergency, critical care, and other such high pill burden settings, medication errors remain a neglected phenomenon in india, particularly in public hospital context. it may be related to professional practice, products, procedures, environment or systems ; may involve prescribing and ordering ; dispensing and distribution ; preparation and administration ; labeling, packaging, and nomenclature ; communications and education ; or use and monitoring of treatment. commonly implicated are medication factors (e.g., similar sounding names, low therapeutic index, polypharmacy), patient factors (e.g., impaired cognition, vision problems and other physical disabilities, poor renal or hepatic function) or health system - related factors (e.g., use of inappropriate abbreviations and decimal points on prescriptions, failure to read drug labels, overburdening, and fatigue of healthcare providers). in our case, the reasons were keeping similar looking ampoules together, failure on the part of the intern to read the label and failure on the part of the resident to cross - check that the correct medication was being injected. labetalol is a dual alfa - and beta - adrenergic antagonist that is often used to treat systemic hypertension in acute care settings. iv overdose of labetalol has been reported to cause hypotension and bradycardia as would be expected from the receptor profile of its activity. therefore, the acute rise of bp, tachycardia, and the intense pain and burning are difficult to explain. theoretically, the unopposed beta - adrenergic blockade can lead to rise in bp by peripheral vasoconstriction mediated through alfa1-adrenergic receptors on vascular smooth muscle. however, this is not expected of labetalol since it simultaneously blocks alfa - receptors. nevertheless, the network of neurotransmission in the central neuraxis is complex and it is possible that a large dose of labetalol may have unexpected receptor activity (for instance blockade of central alfa2-autoreceptors will increase noradrenaline release from adrenergic synapses) that transiently caused these events. finally, it is to be noted that a system of medication error reporting does not exist in this hospital, or for that matter, most hospitals in india, and this may lead to such events recurring in future. introducing such a system can be worthwhile and would be an important step toward assessing and reducing the medication error burden. unfortunately, we anticipate that any attempt at the introduction of such a system would be met by resistance out of fear of being identified and subsequently penalized for negligence. two approaches to human error have been described : (1) the person approach and (2) the systems approach and they are also applicable to the issue of medication errors. the systems approach accepts that humans are fallible, and therefore, errors may occur regardless of the competence of individuals working within the system. rather than focusing on the individual, it focuses on the conditions under which individuals work and how those conditions can predispose to errors. it is now widely acknowledged that although professionals must take responsibility for their actions, blaming doctors, pharmacists, or nurses for errors does not encourage a culture of reporting or learning. serious errors are often caused by wider defects in the system that lie dormant (latent conditions) until they combine with human error to precipitate a serious incident. in order to reduce risk, a system needs to understand its defects so that it can minimize them by building appropriate defenses and safeguards. defects can only be identified if there is a commitment to an open culture of reporting throughout the system. | labetalol, a combined alfa and beta - adrenergic receptor antagonist, is used as an antihypertensive drug. we report a case of an acute rise in blood pressure and lower limb pain due to the inadvertent intrathecal administration of labetalol, mistaking it for bupivacaine, during obstetric anesthesia. the situation was rescued by converting to general anesthesia. the cesarean delivery was uneventful, and mother as well as newborn child showed no ill - effect. this particular medication error was attributable to a failure on the part of the doctors administering the injection to read and cross - check medication labels and the practice of keeping multiple injections together. in the absence of an organized medication error reporting system and action on that basis, such events may recur in future. |
hplc system (waters 2489, milford, usa) consisting of quaternary gradient pump (tm 600), rheodyne manual injector with 20 l loop, column oven and uv detector was employed for analysis. working standards of atenolol and lercanidipine were provided by emcure pharmaceuticals, pune, india. a tablet containing 50 mg ate and 10 mg ler (lotensyl at ; hplc grade acetonitrile and orthophosphoric acid were obtained from merck india limited, mumbai. analytical grade hydrochloric acid, sodium hydroxide pellets and hydrogen peroxide solution 30% (v / v) were obtained from ranbaxy fine chemical (new delhi, india) and 0.45 m membrane filter was obtained from pall life sciences (mumbai, india). high purity deionised water was obtained from millipore, milli - q (milford, usa) purification system. phenomenex gemini c18 (2504.6 mm, 5 m) column used as a stationary phase. the isocratic mobile phase consisting of acetonitrile and buffer (20 mm potassium dihydrogen phosphate, ph 3.5 adjusted with orthophosphoric acid) in the ratio of (55:45, v / v) was used throughout the analysis. standard stock solution containing ate (500 g / ml) and ler (100 g / ml) was prepared by transferring 50 mg ate and 10 mg ler working standard into a 100 ml volumetric flask. a 40 ml portion of diluent (acetonitrile - water 50:50, v / v) was added, sonicated and cooled to room temperature. standard solution containing ate (100 g / ml) and ler (20 g / ml) was prepared by pipetting 10 ml stock solution into a 50 ml volumetric flask and diluting to volume with diluent. the tablets were crushed with a mortar and pestle for 10 min. a portion of powder equivalent to the weight of one tablet was accurately weighed and transferred to a 100 ml volumetric flask. approximately 50 ml diluent was added and the mixture was sonicated for 15 min with intermittent shaking. the contents were restored to room temperature and diluted to volume with diluent to furnish stock test solution. the stock solution was filtered through 0.45 m membrane filters and 10 ml of the filtered solution was transferred to a 50 ml volumetric flask and diluted to volume with diluents to give test solution containing 100 g / ml ate and 20 g / ml ler. tablet powder equivalent to the weight of one tablet was transferred to 250 ml round bottomed flask and treated under acidic, alkaline, oxidizing, thermal and photolytic stress conditions. when degradation was complete, the solution were left to equilibrate to room temperature and diluted with diluents to furnish solutions of concentration equivalent to 100 g / ml ate and 20 g / ml ler. was heated under reflux with 1 m hydrochloric acid for 30 min at 80 on oil bath and the drug was treated with 0.1n naoh at room temperature for 2 h in alkaline degradation. the drug was treated with 2% (v / v) h2o2 at room temperature for 2 h in oxidative degradation. thermal degradation was performed by exposing the solid drug to dry heat in a convection oven at 70 for 72 h and photolytic degradation was performed by exposing the drug to sunlight for 72 h. in the system suitability standard solution of 100 g/ ml ate and 20 g / ml ler (n=5) was prepared and injected. then system suitability parameters like retention time, theoretical plates, peak asymmetry and resolution were calculated from the chromatogram. the specificity of the method was evaluated by assessing interference from excipients in the pharmaceutical dosage form prepared as a placebo solution. the specificity of the method for the drug was also established by checking for interference with drug quantification from degradation products formed during the forced degradation study. test solutions for assessment of the linearity of the method where prepared at seven concentrations from 40 to 160% of the analyte concentration in the assay (i.e. 40, 60, 80, 100, 120, 140 and 160 g / ml for ate and 8, 12, 16, 20, 24, 28 and 32 g / ml for ler). the precision of the method, as intra - day repeatability was evaluated by performing six independent assays of the test sample preparation and calculating the rsd %. the intermediate (interday) precision of the method was checked by performing same procedure on different days by another person under the same experimental conditions. accuracy was studied by adding three different amounts (corresponding to 50, 100 and 150% of the test preparation concentrations) of ate and ler to the placebo preparation and comparing the actual and measured concentrations. for each level, three solutions were prepared and each was injected in duplicate. the robustness was studied by evaluating the effect of small but deliberate variations in the chromatographic conditions. the conditions studied were flow rate (altered by 0.1/min), mobile phase composition (by using 57:43 and 53:47 v / v acetonitrile : buffer ph 3.5), buffer ph (altered by 0.2) and use of hplc columns from different batches. sample solution stability was evaluated by shorting the solution at ambient temperature and at 2 - 5 and analysis after 6, 12, 24, 36 and 48 h. the responses from the aged solutions were compared with those from freshly prepared standard solution. phenomenex gemini c18 (2504.6 mm, 5 m) column used as a stationary phase. the isocratic mobile phase consisting of acetonitrile and buffer (20 mm potassium dihydrogen phosphate, ph 3.5 adjusted with orthophosphoric acid) in the ratio of (55:45, v / v) standard stock solution containing ate (500 g / ml) and ler (100 g / ml) was prepared by transferring 50 mg ate and 10 mg ler working standard into a 100 ml volumetric flask. a 40 ml portion of diluent (acetonitrile - water 50:50, v / v) was added, sonicated and cooled to room temperature. standard solution containing ate (100 g / ml) and ler (20 g / ml) was prepared by pipetting 10 ml stock solution into a 50 ml volumetric flask and diluting to volume with diluent. the tablets were crushed with a mortar and pestle for 10 min. a portion of powder equivalent to the weight of one tablet was accurately weighed and transferred to a 100 ml volumetric flask. approximately 50 ml diluent was added and the mixture was sonicated for 15 min with intermittent shaking. the contents were restored to room temperature and diluted to volume with diluent to furnish stock test solution. the stock solution was filtered through 0.45 m membrane filters and 10 ml of the filtered solution was transferred to a 50 ml volumetric flask and diluted to volume with diluents to give test solution containing 100 g / ml ate and 20 g / ml ler. tablet powder equivalent to the weight of one tablet was transferred to 250 ml round bottomed flask and treated under acidic, alkaline, oxidizing, thermal and photolytic stress conditions. when degradation was complete, the solution were left to equilibrate to room temperature and diluted with diluents to furnish solutions of concentration equivalent to 100 g / ml ate and 20 g / ml ler. was heated under reflux with 1 m hydrochloric acid for 30 min at 80 on oil bath and the drug was treated with 0.1n naoh at room temperature for 2 h in alkaline degradation. then resulting solution the drug was treated with 2% (v / v) h2o2 at room temperature for 2 h in oxidative degradation. thermal degradation was performed by exposing the solid drug to dry heat in a convection oven at 70 for 72 h and photolytic degradation was performed by exposing the drug to sunlight for 72 h. in the system suitability standard solution of 100 g/ ml ate and 20 g / ml ler (n=5) was prepared and injected. then system suitability parameters like retention time, theoretical plates, peak asymmetry and resolution were calculated from the chromatogram. the specificity of the method was evaluated by assessing interference from excipients in the pharmaceutical dosage form prepared as a placebo solution. the specificity of the method for the drug was also established by checking for interference with drug quantification from degradation products formed during the forced degradation study. test solutions for assessment of the linearity of the method where prepared at seven concentrations from 40 to 160% of the analyte concentration in the assay (i.e. 40, 60, 80, 100, 120, 140 and 160 g / ml for ate and 8, 12, 16, 20, 24, 28 and 32 g / ml for ler). the precision of the method, as intra - day repeatability was evaluated by performing six independent assays of the test sample preparation and calculating the rsd %. the intermediate (interday) precision of the method was checked by performing same procedure on different days by another person under the same experimental conditions. accuracy was studied by adding three different amounts (corresponding to 50, 100 and 150% of the test preparation concentrations) of ate and ler to the placebo preparation and comparing the actual and measured concentrations. for each level, three solutions were prepared and each was injected in duplicate. the robustness was studied by evaluating the effect of small but deliberate variations in the chromatographic conditions. the conditions studied were flow rate (altered by 0.1/min), mobile phase composition (by using 57:43 and 53:47 v / v acetonitrile : buffer ph 3.5), buffer ph (altered by 0.2) and use of hplc columns from different batches. sample solution stability was evaluated by shorting the solution at ambient temperature and at 2 - 5 and analysis after 6, 12, 24, 36 and 48 h. the responses from the aged solutions were compared with those from freshly prepared standard solution. in this work hplc method with uv detection for analysis of ate and ler in a tablet formulation was developed and validated. the analytical conditions were selected after testing the different condition effecting hplc analysis, for example diluent and buffer composition, buffer concentration, organic solvent in the mobile phase, buffer to organic solvent ratio and other chromatographic conditions. preliminary trials with mobile phases comprising mixtures of water with methanol or acetonitrile did not give good peak shape. the best peak shape was obtained by use of 0.02 m potassium dihydrogen phosphate buffer, adjusted to ph 3.5 with phosphoric acid and use of mobile phase of composition 55:45 (v / v) acetonitrile-0.02 m potassium dihydrogen phosphate buffer. acetonitrile chosen as a organic constituent of the mobile phase to reduce retention times and buffer was chosen to reduce peak asymmetry and achieve good peak shape. the optimized mobile phase enabled good resolution of ate and ler and of compounds generated during forced degradation. the newly developed analytical method was validated according to the ich guidelines and its updated international convention, usp and aoac international. typical chromatogram of tablet extract, showing peaks of ate and ler system suitability was verified by measurement of peak asymmetry (a3.0) and number of theoretical plates (n>2000) after chromatography of standard solution. the specificity of the method was determined by checking for interference with the analytes from placebo components by measuring peak purity for ate and ler during the forced degradation study. ate showed extensive degradation in oxidative conditions while extensive degradation of ler occurred in acidic conditions as shown in (fig. table 1 indicated the extent of degradation results of ate and ler under various stress conditions. chromatographic separation of ate and ler from their oxidative degradation products chromatographic separation of ate and ler from their acidic degradation products chromatographic separation of ate and ler from their alkaline degradation products forced degradation study data ate and ler showed linearity in the range of 40 - 160 g / ml and 8 - 32 g / ml, respectively. the linear regression equations were y=16456x+11776, correlation coefficient 0.9995 for ate and y=47672x+7888, correlation coefficient 0.9993 for ler, where x is the concentration in g / ml and y is the peak area in absorbance units. the limits of detection and quantification were evaluated by serial dilution of ate and ler stock solution until the signal - to - noise ratios were 3:1 for lod and 10:1 for loq. the lod for ate and ler were 0.02 and 0.05 g / ml, respectively ; the loq were 0.05 and 0.1 g / ml, respectively. the relative standard deviations were 0.47% for ate and 0.27% for ler, which are well within the acceptable limit of 2.0%. the rsds for intermediate precision were found to be 0.21% for ate and 0.30% for ler. mean recovery was 98.54 - 101.54% for ate and 98.81100.86% for ler (table 2). in all deliberately varied conditions, the rsd of peak areas of ate and ler were found to be well within the acceptable limit of 2.0%. the tailing factor and asymmetry for both the results obtained from study of the stability of the test preparation, it was concluded the test solution was stable for up to 48 h at 2 - 5 and at ambient temperature, because difference between measured and original values were < 2.0%. proposed hplc method is specific, accurate and precise for the simultaneous determination of atenolol and lercanidipine hydrochloride from pharmaceutical dosage form. the described method is suitable for routine analysis and quality control of pharmaceutical preparations containing atenolol, lercanidipine, nifedipine, indapamide, hydrochlorothiazide and amlodipine either as such or in combination with atenolol. the method can be used to separate these drugs from their degradation products and excipients found in the tablet dosage form. | a simple, rapid, precise and accurate isocratic reversed phase stability indicating hplc method was developed and validated for the simultaneous determination of atenolol and lercanidipine hydrochloride in commercial tablets. the chromatographic separation was achieved on phenomenex gemini c18 (2504.6 mm, 5 m) column using a mobile phase consisting of acetonitrile and buffer (20 mm potassium dihydrogen phosphate ph 3.5) in the ratio of (55:45, v / v) at a flow rate of 1.0 ml / min and uv detection at 235 nm. the linearity of the proposed method was investigated in the range of 40 - 160 g / ml (r2=0.9995) for atenolol and 8 - 32 g / ml (r2=0.9993) for lercanidipine. degradation products produced as a result of stress studies did not interfere with the detection of atenolol and lercanidipine and the assay can thus be considered stability - indicating. |
central odontogenic fibroma (cof) is a very rare proliferation of mature odontogenic mesenchyme. it is sufficiently rare that locations and sex and age distributions can not be accurately determined. the lesion may evolve from a dental germ (dental papilla or follicle) or from the periodontal membrane, and therefore is invariably related to the coronal or radicular portion of teeth. radiographically the tumor sometimes produces an expansile multilocular radiolucency similar to that of the ameloblastoma. rarity of this tumor excludes it from most differential lists, and when diagnosed, it is an unexpected finding that usually requires expert second opinions for confirmation. most presentations will suggest the more common radiolucent odontogenic cysts and tumors such as an odontogenic keratocyst, an ameloblastoma or an odontogenic myxoma, as well as an ameloblastic fibroma in children and teenagers. in younger individuals, the presentation will also suggest a central giant cell tumor. a 16-year - old female patient presented with a painless swelling on the right side of the lower jaw. extraoral examination revealed facial asymmetry with a hard swelling on the right side of lower jaw, extending from zygomatic arch superiorly to the lower border of mandible inferiorly and from the corner of mouth anteriorly to the angle of mandible posteriorly [figure 1 ]. on intraoral examination, a swelling extending from distal of right mandibular canine back to retromolar area was noticed. occlusion was deranged with displacement of right lower premolars and first molar without relevant mobility [figure 2 ]. there was obliteration of lingual and buccal sulci, and the overlying mucosa was patchy in appearance with red and yellow patches. preoperative front view preoperative intraoral view orthopantomogram (opg) showed a large multilocular radiolucent lesion of the right mandible, extending from right second premolar to the angle region [figure 3 ]. incisional biopsy under local anesthesia was done and a creamish white mass firm in consistency was obtained. the report of this incisional biopsy was inconclusive of a definite pathology and a deeper section was advised. biopsy showed numerous collagen bundles and some fiboblasts with extravasation of blood at few places. the tumor was excised under general anesthesia through the submandibular approach and sent for histopathologic examination. the tumor was found to be a well circumscribed, solid mass that shelled out easily and completely [figure 4 ]. tumor mass with extracted teeth microscopic examination revealed a tumor consisting of fibrous tissue with myxoid areas along with islands and strands of inactive odontogenic epithelium. based on clinical, radiographic, and histological findings, a diagnosis of cof was made and the patient was advised a long - term follow - up. cof is a rare and benign neoplasm that could appear very similar to the endodontic lesions and/or to the other odontogenic tumors. shafer. in 1983 considered odontogenic fibroma a distinct neoplasm with its own histopathologic and clinical features separating it from other odontogenic tumors. wesley. in 1975 suggested a set of criteria for diagnosing odontogenic fibroma as follows. clinically, the lesion is central in the jaws and has a slow persistent growth that results in painless cortical expansion.radiologically, its appearance varies, but like the ameloblastoma and odontogenic myxoma, most examples are multilocular radiolucent lesions that involve relatively large portions of the jaws in the later stages. in some instances, they may be associated with unerupted and/or displaced teeth.histopathologically, the most consistent feature is a tumor composed predominantly of mature collagen fibers with numerous interspersed fibroblasts. the presence of small nests and/or strands of inactive odontogenic epithelium is a variable feature.the lesion is benign and responds well to surgical enucleation with no tendency to undergo malignant transformation. clinically, the lesion is central in the jaws and has a slow persistent growth that results in painless cortical expansion. radiologically, its appearance varies, but like the ameloblastoma and odontogenic myxoma, most examples are multilocular radiolucent lesions that involve relatively large portions of the jaws in the later stages. in some instances histopathologically, the most consistent feature is a tumor composed predominantly of mature collagen fibers with numerous interspersed fibroblasts. the presence of small nests and/or strands of inactive odontogenic epithelium is a variable feature. the lesion is benign and responds well to surgical enucleation with no tendency to undergo malignant transformation. using these criteria, they diagnosed only seven cases of true odontogenic fibroma and added one new case, all in the mandible. gardner in 1980 attempted further clarification of lesions previously described as odontogenic fibroma and classified them into three different, yet probably related, lesions : first, the hyperplastic dental follicle.second, a fibrous neoplasm with varying collagenous fibrous connective tissue containing nests of odontogenic epithelium simple type.third, a more complicated lesion with features of dysplastic dentine or cementum - like tissue and varying amount of odontogenic epithelium who type. because this group was similar to the calcifying odontogenic tumor described by pindborg in who publication in 1971, gardner designated it as odontogenic fibroma (who type). the distinguishing features between the two lesions are that in the calcifying odontogenic tumor but not in the odontogenic fibroma (who type), this lesion stains positive with amyloid stains. second, a fibrous neoplasm with varying collagenous fibrous connective tissue containing nests of odontogenic epithelium simple type. third, a more complicated lesion with features of dysplastic dentine or cementum - like tissue and varying amount of odontogenic epithelium who type. because this group was similar to the calcifying odontogenic tumor described by pindborg in who publication in 1971, gardner designated it as odontogenic fibroma (who type). the distinguishing features between the two lesions are that in the calcifying odontogenic tumor but not in the odontogenic fibroma (who type), this lesion stains positive with amyloid stains. till now, approximately 80 patients have been reported as single cases or case series in english literature.[91115 ] gardner has referred the tumor made up of connective tissue and odontogenic islands resembling dental follicle as the simple cof and to the tumor described by the who as the who - type cof. histologically, the simple type is characterized by a tumor mass made up of mature collagen fibers interspersed usually by many plump fibrobalsts that are very uniform in their placement and tend to be equidistant from each other. small nests or islands of odontogenic epithelium that appear entirely inactive are present in variable but usually in quite minimal amounts. the who type also consists of relatively mature but quite cellular fibrous connective tissue with few to many islands of odontogenic epithelium. osteoid, dysplastic dentin or cementum - like material is also variably present. according to marx, most cofs require an incisional biopsy because their presentation suggests more aggressive disease, and once the diagnosis is established, a panoramic radiograph is sufficient for treatment planning. the resultant bony cavity is closed at the mucosal level without the need for drains or packing. dunlap and barker presented two cases of maxillary odontogenic fibroma treated by curettage with follow - up of 9 years and 10 years with no evidence of recurrence. since then, svirsky. have reported a 13% (2 out of 15 cases) rate of recurrence. jones. reported a case which recurred 16 months after surgery. according to marx, if recurrence is observed, the original pathological specimen as well as biopsy specimen should be reviewed. it is possible that an odontogenic myxoma with fibrous features often termed as fibromyxoma was interpreted as a cof. it is also possible that an ossifying fibroma with few bony or cementum - like components was interpreted as the type of cof that has its usual mature fibrous connective tissue and its own calcific deposits which are believed to be either cementum or dentin. | central odontogenic fibroma (cof) is an extremely rare benign tumor that accounts for 0.1% of all odontogenic tumors. it appears as an asymptomatic expansion of the cortical plate of the mandible or maxilla. radiologically it presents as a unilocular or multilocular radiolucency. it responds well to surgical enucleation with no tendency for recurrence. we describe a case of cof in mandibular right posterior region in a 16-year - old female. the lesion was surgically removed and analyzed histopathologically. |
atrial fibrillation (af) is the most common cardiac arrhythmia in clinical practice, and contributes to a high prevalence of mortality and morbidity. factors such as aging, obesity, diabetes, hypertension, and cardiovascular diseases are considered to increase the risk of developing af. omentin, a novel adipokine, is produced and secreted mainly by visceral adipose tissue. omentin is codified by two genes named omentin-1 and omentin-2, and the former is the major circulating form. recombinant omentin-1 results in increased insulin - stimulated glucose uptake and akt phosphorylation in human adipocytes. recently, omentin-1 has been shown to be correlated with obesity, hypertension, diabetes, and coronary artery disease (cad). the aim of this study was to determine the correlation between serum omentin-1 concentrations and the presence of af and atrial remolding. a cross - sectional study was performed in a consecutive population of 220 patients who were diagnosed with af. the criteria for a diagnosis of af were in accordance with the guidelines established by american heart association. patients were excluded from the study if they had valvular heart disease, hyperthyroidism, acute coronary syndrome, previous cardiac surgery, or systemic disease. af patients were divided into three groups according to american heart association guidelines : paroxysmal af (n=70), persistent af (n=78), and permanent af (n=72). the study plan was approved by the research ethics committee of our hospital (no. anthropometric (height, weight, and blood pressure), clinical, and laboratory analysis were performed. venous blood was collected after a minimum of 10 hours of fasting for further examination. an enzyme - linked immunosorbent assay kit (cusabio biotech corporation, usa) was utilized to evaluate serum omentin-1 concentrations. transthoracic echocardiography was performed by experienced echocardiologists on all patients to evaluate the characteristics of their left atrial diameter (lad). the unpaired t - test, chi - square tests, or the mann - whitney u test was utilized to determine the parameter differences between af patients and control patients. comparison of the characteristics between the three af subgroups was performed by chi - square tests, one - way anova, or the kruskal - wallis test. a cross - sectional study was performed in a consecutive population of 220 patients who were diagnosed with af. the criteria for a diagnosis of af were in accordance with the guidelines established by american heart association. patients were excluded from the study if they had valvular heart disease, hyperthyroidism, acute coronary syndrome, previous cardiac surgery, or systemic disease. af patients were divided into three groups according to american heart association guidelines : paroxysmal af (n=70), persistent af (n=78), and permanent af (n=72). the study plan was approved by the research ethics committee of our hospital (no. anthropometric (height, weight, and blood pressure), clinical, and laboratory analysis were performed. venous blood was collected after a minimum of 10 hours of fasting for further examination. an enzyme - linked immunosorbent assay kit (cusabio biotech corporation, usa) was utilized to evaluate serum omentin-1 concentrations. transthoracic echocardiography was performed by experienced echocardiologists on all patients to evaluate the characteristics of their left atrial diameter (lad). the unpaired t - test, chi - square tests, or the mann - whitney u test was utilized to determine the parameter differences between af patients and control patients. comparison of the characteristics between the three af subgroups was performed by chi - square tests, one - way anova, or the kruskal - wallis test. af patients showed higher levels of sbp, dbp, ldl - c, and lad, as well as statin, aspirin, and warfarin treatment, compared to healthy controls (table 1). af patients showed significantly reduced serum omentin-1 concentrations compared to healthy controls (table 1). permanent af patients had lower serum omentin-1 concentrations compared to paroxysmal af and persistent af patients (figure 1). furthermore, significant decreased serum omentin-1 concentrations were observed in persistent af patients compared to paroxysmal af patients (figure 1). simple and multiple logistic regressions both showed a significant association of serum omentin-1 with a decreased risk of developing af (table 3). the roc curve of serum omentin-1 concentrations determining af development are shown in figure 2. pearson correlation analysis revealed a negative relationship of serum omentin-1 concentrations with bmi (r=0.283, p<0.001), tg (r=0.256, p<0.001), and lad (r=0.332, p<0.001). af patients showed higher levels of sbp, dbp, ldl - c, and lad, as well as statin, aspirin, and warfarin treatment, compared to healthy controls (table 1). af patients showed significantly reduced serum omentin-1 concentrations compared to healthy controls (table 1). permanent af patients had lower serum omentin-1 concentrations compared to paroxysmal af and persistent af patients (figure 1). furthermore, significant decreased serum omentin-1 concentrations were observed in persistent af patients compared to paroxysmal af patients (figure 1). simple and multiple logistic regressions both showed a significant association of serum omentin-1 with a decreased risk of developing af (table 3). the roc curve of serum omentin-1 concentrations determining af development are shown in figure 2. pearson correlation analysis revealed a negative relationship of serum omentin-1 concentrations with bmi (r=0.283, p<0.001), tg (r=0.256, p<0.001), and lad (r=0.332, p<0.001). the present study indicated that af patients had reduced serum omentin-1 concentrations compared to the healthy controls. serum omentin-1 concentrations were negatively correlated with left atrial diameter in af patients. in short, this is the first study that has demonstrated the association of serum omentin-1 and af. some adipokines, such as adiponectin and resistin were reported to be associated with the development of af. our results also showed a correlation between omentin-1, another adipokine, and af development. these results point to an important role for adipose tissue and adipokine in the pathophysiology of af. therefore, serum omentin-1 may be utilized as a new biomarker to predict and assess the risk of developing af. large body size, assessed using body surface area in youth, and weight gain from age 20 to midlife, have both been independently correlated to af development. during a median time of 12.9 years follow - up, bmi was associated with an increased risk of 4.7% with each kilogram per square meter of developing af. in addition, overweight and obesity were associated with adjusted short - term increased risk of af development. plasma omentin-1 concentrations, as well as omentin-1 gene expression levels, were significantly decreased in obese and overweight subjects compared to lean subjects. therefore, omentin-1 may have a protective effect in af development and persistence of af partly by inhibiting obesity or regulating lipid metabolism. omentin inhibited platelet - derived growth factor bb - induced vascular smooth muscle cell migration by reducing oxidative stress. this indicates that omentin-1 may serve as a target for treating hypertension by inhibiting vascular structural remodeling. in addition, omentin treatment inhibited pulmonary arterial hypertension in rats by inhibiting vascular structural remodeling and abnormal contractile reactivity. furthermore, omentin treatment inhibited contractile dysfunction and insulin resistance in cardiomyocytes. decreased serum omentin-1 concentrations were found in patients with cad. the aforementioned results indicate a close correlation between omentin-1 and diabetes, hypertension, and cad. furthermore, a negative correlation was found between serum omentin-1 and inflammatory mediators such as tnf-, interleukin-6 (il-6), and c - reactive protein. in conclusion, serum omentin-1 concentrations were inversely correlated with the development of af and atrial remolding. | backgroundomentin-1 is one of the adipokines associated with obesity, diabetes, and coronary heart disease development. we determined to investigate whether serum omentin-1 concentrations were correlated with the presence of atrial fibrillation (af).material / methodsserum omentin-1 concentrations were examined in a cross - sectional population that included 220 patients with af (70 with paroxysmal af, 78 with persistent af, and 72 with permanent af) and 115 healthy controls.resultsreduced serum omentin-1 concentrations were found in af patients compared to the controls. in addition, patients with permanent af had lower serum omentin-1 concentrations compared to patients with persistent af and patients with paroxysmal af. significantly decreased serum omentin-1 concentrations were observed in persistent af patients compared to paroxysmal af patients. spearman correlation analysis suggested that serum omentin-1 concentrations were negatively correlated with left atrial diameter in af patients.conclusionsserum omentin-1 concentrations were correlated with the presence of af and atrial remolding. |
initially introduced in the mid-1960 's, with the adoption of radioactive i into clinical practice, prostate brachytherapy remains a highly effective treatment modality for organ confined prostate cancer. with further development of the transperineal approach in the 1980 's tumor registry studies initially revealed an increase utilization of prostate brachytherapy as a primary treatment modality in the late 1990 's and early 2000 's, as targeted radiotherapy approaches were limited during that time period. however, this trend has reported to have recently reversed as newer technologies have gained acceptance, such as intensity modulated radiotherapy (imrt), proton therapy, as well as improvement of surgical options, such as robotic assisted prostatectomy [4, 5 ]. others have suggested that outside issues, such as financial reimbursement, lack of physician skill proficiency in brachytherapy, as well as stage migration towards more intermediate and high grade disease, which may be less likely to include prostate brachytherapy, have played a role in this trend. these issues have led to several provocative studies suggesting that prostate brachytherapy is on a steep decline. most of these studies reporting on the decline of prostate brachytherapy have analyzed its usage in comparison to all available treatment options. in this study we sought to utilize the national cancer database (ncdb) in order to evaluate the temporal trends of prostate brachytherapy in the setting of men with low risk disease who have elected to undergo treatment with radiotherapy. by limiting our cohort to those who are most likely to select brachytherapy (patients with low risk disease) and have selected radiation therapy over observation or surgery, we sought to obtain a more nuanced view into whether prostate brachytherapy is really on the decline compared to external beam radiation. the ncdb is a hospital - based registry that is the joint project of the american cancer society and the commission on cancer of the american college of surgeons. it is estimated that 70% of all diagnosed malignancies in the united states are captured by facilities participating in this registry and reported to the ncdb. the commission on cancer 's ncdb and the hospitals participating in the ncdb are the source of the de - identified data used in this study. however, they have not verified and are not responsible for the statistical validity or conclusions derived by the authors of this study. exemption was obtained from the new york harbor veterans affairs committee for research and development prior to the initiation of this study. men with prostate cancer diagnosed between 2004 - 2012 and treated with either external beam radiation and/or prostate brachytherapy were abstracted from the ncdb. in order to be included, men had to be clinically staged as t1c - t2anx-0mx-0 adenocarcinoma of the prostate, gleason score 6, and prostate specific antigen 10.0 ng / ml, who were treated with radiation therapy were included in this analysis. this limited inclusion to men with low risk prostate cancer as classified by the national comprehensive cancer network. in order to accurately assess treatment selection by facility type, we further limited analysis to those who received all of their therapy in one center, which is suggested by the ncdb for analyses that include facility type as one of the covariables. descriptive statistics were used to analyze brachytherapy utilization over time, as well as by other factors such as facility type, insurance status, and race. comparisons were made via, fisher 's exact test, and mann whitney test where appropriate. the variables analyzed in the multivariate model included age grouping (70 years), year of diagnosis (2004 through 2012 in single year increments), race (white, black, other), distance from treatment center (divided into 4 quartiles), facility type (academic, non - academic), and insurance type (none, private insurance, medicaid, medicare, other government, unknown). significant values were defined as those with a p - value 50% of men undergoing radiation therapy choosing brachytherapy in 2012. however, given the current trend, one would expect this to continue to decline into the next decade. the utilization rates in our study are higher than in other reports, which is likely due to different methodologies. the seer database study looked at global declining rates of brachytherapy amongst all risk groups, but did not detail the relative usage of brachytherapy compared to ebrt by risk group. the aforementioned ncdb study compared brachytherapy usage to all other modalities, including surgery, hormones, and no treatment. in addition, they excluded several brachytherapy variables due to questions regarding the fidelity of those variables at the time of their analysis, which may have led to some degree of underestimation of brachytherapy usage. in fact, a prior seer based study using more similar methodology to ours reported that prostate brachytherapy utilization was 56.1% for low risk patients, which is consistent with our findings. regardless of the precise comparative methodologies used, the consistent findings from all of these studies are that prostate brachytherapy appears to be declining. this is somewhat surprising, as many studies to date demonstrate an increase in prostate - specific antigen (psa) progression free survival with brachytherapy versus other modalities. the prostate cancer results study group recently published a study, which included almost 850 different publications and found that compared to ebrt or radical prostatectomy, brachytherapy was associated with the most sustained psa progression free survival over 12 years. another retrospective analysis looked at almost 8,000 low and intermediate risk patients treated in canada with radiotherapy, and found that the use of brachytherapy led to statistically significant improvement in psa progression free survival, with a hazard ratio of 4.58 for intermediate risk patients and 2.90 for low risk patients. the recently reported ascende - rt trial showed an increased survival with the addition of brachytherapy to ebrt in intermediate and high risk patients. several possibilities have been suggested to explain this trend, including increased utilization of robotic assisted surgery, fewer practitioners being trained, and possibly financial incentives. with imrt paying almost $ 15,000 per patient, prostate cancer is among the top five most expensive cancers in the united states, with approximately $ 12 billion spent annually. it is estimated that approximately 70,110 low risk prostate cancers will be diagnosed in the united states within the next ten years, and the cost to treat them with low - dose - rate (ldr) brachytherapy would be $ 18.3 billion, $ 32.2 billion for high - dose - rate (hdr) brachytherapy and $ 54 billion for imrt. in the current era of increased emphasis on cost containment, these financial issues will likely become an urgent healthcare issue in the united states. although financial incentive seems like a strong driver for the decrease in brachytherapy utilization, our study demonstrated a much larger decline of brachytherapy utilization in academic centers, where one would expect financial incentive to play less of a role. this finding suggests that there may be other factors driving these changes and is of particular concern as academic centers provide most of resident training throughout the county. recent association of residents in radiation oncology (arro) survey reported an enormous decrease in resident reported clinical experience in brachytherapy from 88% in the 2005 - 2008 survey to 15 - 30% in the 2013 - 2015 survey. another recently published study determined that the number of academic centers performing 1 brachytherapy procedure a month has increased from 56.4% to 73.7%. this translates into practicing physicians who are uncomfortable with performing the procedure, limiting treatment options offered to patients, and will likely lead to further decline through the years. it is incumbent on academic centers to take an increased responsibility for this issue, in order to prevent a nationwide drought of brachytherapy efficient physicians. we also noted that distance clearly plays a role in brachytherapy utilization, as there was an increase in utilization in patients who lived further from treatment facilities. this was most pronounced in those that lived furthest from treatment facilities, where the brachytherapy peaked to 70% in those patients. as typical ebrt treatment require 8 - 9 weeks of therapy, it would be taxing for one that lives far away from the treatment facilities to undergo daily treatment for that time period, further favoring brachytherapy in that group. as with all tumor registry studies, limitations to this study include selection bias, incomplete data, and coding errors. the ncdb in particular is a hospital based database, and therefore if patients either received external beam at an outpatient facility and received brachytherapy at an ncdb hospital or vice versa, it may lead to an over or underestimation of the findings. for example, if a patient received external beam at a hospital but brachytherapy at a private institution, this may have underestimated true brachytherapy utilization. in attempt to account for that potential bias, only patients who received all of their treatment at the same facility were included in this analysis. in conclusion, prostate brachytherapy remains for now the most commonly selected radiation treatment for low risk disease. however, there has been a consistent trend of reduced brachytherapy usage over the last decade, which if it continues may eventually lead to marginalization of prostate brachytherapy over time. given its favorable clinical and cost effectiveness, further studies should be directed to determine barriers to brachytherapy, particularly at academic centers, in order to reduce this trend. | purposeseveral prior studies have suggested that brachytherapy utilization has markedly decreased, coinciding with the recent increased utilization of intensity modulated radiation therapy, as well as an increase in urologist - owned centers. we sought to investigate the brachytherapy utilization in a large, hospital - based registry.material and methodsmen with prostate cancer diagnosed between 2004 - 2012 and treated with either external beam radiation and/or prostate brachytherapy were abstracted from the national cancer database. in order to be included, men had to be clinically staged as t1c - t2anx-0mx-0, gleason 6, psa 10.0 ng / ml. descriptive statistics were used to analyze brachytherapy utilization over time and were compared via 2. multivariate logistic regression was used to assess for covariables associated with increased brachytherapy usage.resultsthere were 89,413 men included in this study, of which 37,054 (41.6%) received only external beam radiation, and 52,089 (58.4%) received prostate brachytherapy. the use of brachytherapy declined over time from 62.9% in 2004 to 51.3% in 2012 (p < 0.001). this decline was noted in both academic facilities (60.8% in 2004 to 47.0% in 2012, p < 0.001) as well as in non - academic facilities (63.7% in 2004 to 53.0% in 2012, p < 0.001). the decline was more pronounced in patients who lived closer to treatment facilities than those who lived further. the use of intensity modulated radiation therapy increased during this same time period from 18.4% in 2004 to 38.2% in 2012 (p < 0.001). on multivariate analysis, treatment at an academic center, increasing age, decreasing distance from the treatment center, and years of diagnosis from 2006 - 2012 were significantly associated with reduced brachytherapy usage.conclusionsin this hospital - based registry, prostate brachytherapy usage has declined for low risk prostate cancer as intensity modulated radiation therapy usage has increased. however, it still remains the treatment of choice for 51.3% of patients as of 2012. |
malignancy - producing granulocyte colony - stimulating factor (g - csf) is rare. robinson first described a g - csf - producing tumor (gcsf - pt) in 1974, and it has since been reported in various sites of cancer. g - csf is a glycoprotein that stimulates the proliferation and maturation of precursor cells in the bone marrow into fully differentiated neutrophils [2, 3 ]. carcinoma of unknown primary site (cup) is also a rare tumor, accounting for approximately 3% of all cancer diagnoses. cup often has a poor outcome due to its early dissemination, aggressive behavior and unpredictable nature as well as its metastatic potential and pattern. although platinum / taxane combination (e.g., cbdca + ptx) chemotherapy is widely used for patients suffering from cup, its response rate is only about 3040%, with a median overall survival of merely 9 months. among new regimens, the combination of carboplatin, paclitaxel, bevacizumab and erlotinib is thought to be highly desirable. so far, cup which produces g - csf (gcsf - cup) is too uncommon to be treated with a precise chemotherapy regimen. we herein report a challenging case of gcsf - cup with aggressive growth, treated with combination chemotherapy of carboplatin, paclitaxel, bevacizumab and erlotinib. a 75-year - old man presented to his primary care clinic due to a left neck lump and was referred to our hospital. he had broken his left ankle in a traffic accident, and he had been smoking half a pack of cigarettes per day for 55 years. his complete blood count and blood chemistry were as follows : white blood cell (wbc) count 25,120/l (normal range 3,5909,640/l) with 82% neutrophils, 3% monocytes and 8% lymphocytes ; no immature granulocytes were observed ; platelet count 21.8 10/l (normal range 14.833.9 10/l), and c - reactive protein level 4.45 mg / dl (normal level < 0.3 mg / dl). various tumor markers were elevated, and scc, cyfra and ca19 - 9 were extremely increased (table 1). his wbc count increased gradually up to 44,680/l, with a differential count of 94% neutrophils during a 6-week period after his first visit, while the serum g - csf concentration (normal range 3.732.3 pg / ml) was increased as high as 283 pg / ml during the same period, suggesting that the tumor grew rapidly. a contrast - enhanced ct scan showed massive lymphadenopathy of the left cervix, left subclavian artery, left mediastinum, mesenteric and para - aortic lymph nodes, with peripheral rim enhancement, consolidation in the right lower lung and a 1-cm low - density liver lesion (fig. f - fdg pet / ct demonstrated a high fdg accumulation in the lymph nodes, pulmonary consolidation and bone marrow (fig. a bone marrow smear showed that the rate of myelopoietic cells was increased but that of erythropoietic cells and megakaryocytes decreased, with normal cellularity. the rate of eosinophils was not increased, and therefore, chronic myelocytic leukemia was not suspected (fig. 1c, d). a needle biopsy of the cervical lymph node and endobronchial ultrasound - guided transbronchial needle aspiration were performed, and cytological examinations of the cervical lymph node and bronchial lavage fluid indicated adenocarcinoma. immunohistochemical (ihc) examination showed cytokeratin (ck)7(+), ck20(+), muc1(+), muc2(), cea(+), p63(+), cd5(/+), cdx2(), human gastric mucin(/+), n - cam(), thrombomodulin(), sp - a(), ttf-1(), alk(), psa(), er(), pgr(), her2() and egfr() expression (fig. since the clinical and pathological investigations could not detect the primary site, the patient was diagnosed with gcsf - cup. after the approval of the intramural ethics committee and after having received the written informed consent from the patient, he was treated with carboplatin (auc 6), paclitaxel (175 mg / m), bevacizumab (15 mg / kg) and erlotinib 150 mg / day. after the start of chemotherapy, g - csf slightly decreased, but no change was seen on the ct scan (fig. 3). thirteen days later, intestinal perforation occurred. with the surgeon 's consultation, the patient was given palliative therapies and died 23 days after the start of chemotherapy. treatment - related toxicity during chemotherapy was grade 4 anemia, grade 3 platelet count decrease, grade 1 aspartate aminotransferase elevation and grade 5 intestinal perforation. his autopsy confirmed bulky lymph nodes of the neck, showing a remarkably shrunken appearance compared to before the therapy (fig. 1b), mediastinal and para - aortic lymphadenopathy and massive thickening of the mesentery. histopathological assessment proved poorly differentiated adenocarcinoma with no specific morphologic features, invasion to the right lung and intestine as well as lymph node metastasis, and a well - differentiated hepatocellular carcinoma without any metastasis or invasion in the liver. ihc examination of the mesentery showed the same results as those from the needle biopsy, and the tumor cells were positive for g - csf with immunohistochemistry (fig. although several reports have reviewed each site of gcsf - pt (e.g., g - csf - producing gastric tumor), the frequency and histology are still unclear. g - csf, producing tumor and case reports ; 176 cases are reported and 171 described in japanese. additionally, we explored the japan medical abstracts society database looking for the key words producing and case report in japanese ; 354 cases, including 102 cases from pubmed, have been described since june 2014. hence, we reviewed 420 cases of gcsf - pt, with the exception of 4 cases that did not refer to the histopathology due to a lack of specimens. gcsf - pt cases are classified according to the primary site and pathologically (table 2). the most common lesions of gcsf - pt are found in the lung, urinary tract (renal pelvis, ureter, urethra and bladder), stomach and duodenum, and the esophagus. others included 2 malignant fibrous histiocytomas, 1 epithelioid sarcoma and 1 undifferentiated sarcoma in the soft tissue and bone ; 1 squamous cell carcinoma in the middle ear ; 1 squamous cell carcinoma in the head and neck ; 1 squamous cell carcinoma of the urachus, and 1 prostate adenocarcinoma. gcsf - pt sometimes secretes other hormones simultaneously, such as parathyroid hormone - related protein c in 21 cases, -fetoprotein in 3 cases, -human chorionic gonadotropin in 2 cases, antidiuretic hormone in 1 case and erythropoietin in 1 case, respectively. our case did not have any clinical feature of abnormal hormone secretion other than g - csf. diagnostic criteria for gcsf - pt are the following : (1) extreme leukocytosis, (2) elevated g - csf activity, (3) a decrease in the wbc count after tumor resection, or (4) proof of g - csf production in the tumor. our case matched criteria 1, 2 and 4 and was therefore diagnosed with gcsf - pt. pet - ct scans show an abnormally high f - fdg uptake, not only by the tumor itself but also diffusely throughout the bone marrow. this finding could be explained by the elevated bone marrow metabolism associated with the excessively active production of granulocytes under g - csf stimulation and is considered to be useful for the diagnosis of gcsf - pt. moreover, we confirmed no malignancy with bone marrow puncture, and the increase in only myelopoietic cells is compatible with excessive g - csf secretion. meanwhile, cup is relatively rare, and standard clinical evaluation, unlike in most cancers, does not identify the anatomic primary site because of its extremely small size or possibly local regression due to antitumor immune defenses as well as its protracted clinical latency. since it is difficult to detect the primary organs, investigations with imaging, tumor and ihc markers are performed to at least classify the type of carcinoma for the purpose of a therapy guideline. however, 20% of cup patients show clinical and/or pathologic features that fit into one or several defined treatable subsets (e.g., isolated axillary lymph node tumors of female patients are treated as stage ii breast cancer). these patients have clinical features that strongly suggest a specific diagnosis, even though an anatomic primary site can not be identified, and the prognosis is similar to these cancers. the remaining 80% of patients with cup do not fall into any of these favorable subsets and have been treated with empiric chemotherapy. in our case, ct, mri and pet images as well as endoscopy did not detect the primary site. ihc examinations showed adenocarcinoma and ck7/ck20 positivity, generally suggesting transient cell carcinoma, pancreatic ductal carcinoma, cholangiocellular carcinoma and gastric adenocarcinoma. the results of muc1, muc2, cea and cdx2 pointed to a possible pancreatic ductal carcinoma, and p63 positivity indicates ureteral carcinoma ; however, thrombomodulin negativity suggests the opposite. a slight positivity of human gastric mucin implies gastric carcinoma, a negativity of n - cam denies neurogenic tumor, and a cd5 result may show lymphocyte infiltration ; other ihc stains are not helpful in predicting a likely primary tumor. according to these clinical and histopathological results, the primary site could not be detected ; therefore, our case was diagnosed with cup. the primary organ has been identified in 70% of cup patients by autopsy, but our case has also failed in this matter. so far, a combination of both gcsf - pt and cup does not seem to have been reported, and this study is likely the first case. although, because of the rarity of the disease, there is a lack of data from randomized phase iii trials which would be helpful for the treatment of patients with cup, the regimens with newer chemotherapeutic agents (e.g., taxanes, gemcitabine, irinotecan) show modest improvement ; most of these newer regimens report response rates of 3050%, and the median survival time was 810 months. a phase ii trial of the paclitaxel - carboplatin plus bevacizumab - erotinib regimen for cup patients was reported in 2009. this regimen produced a response rate of 53% ; the median overall survival time was 12.6 months, the median progression - free survival 8 months, and the 1- and 2-year survival rates 51 and 27%, respectively. both the median progression - free survival and 2-year survival rate are the best among previously reported empiric chemotherapy regimens. similar to the reported effectiveness, this new regimen was also presumed to be efficacious in our case because a decrease in g - csf and wbc was observed after the treatment and because the appearance of the bulky neck lymph node remarkably shrank. if leukocytosis is detected in relation to a nonlymphoid hematopoietic malignant tumor, gcsf - pt should be reckoned. careful observation is paramount in cup treatment due to the unpredictable nature of its metastatic potential and pattern. | granulocyte colony - stimulating factor (g - csf)-producing nonhematopoietic malignancies have been reported in various organs and are associated with a poor clinical outcome. moreover, carcinoma of unknown primary site (cup) is an uncommon malignancy that occurs in about 26% of cancer patients. cup also has a poor prognosis due to its missing profile. since both g - csf - producing carcinoma and cup are rare, g - csf - producing cup (gcsf - cup) is considered to have an even poorer prognosis and is seldom encountered. herein, we report the case of a gcsf - cup patient. a 75-year - old man was admitted to our hospital complaining of cervical lymphadenopathy. multiple bulky lymph nodes without a primary site were revealed by image analysis. his complete blood count showed leukocytosis, and his blood chemistry panel indicated highly elevated levels of g - csf. although the patient was treated with combination chemotherapy of carboplatin, paclitaxel, bevacizumab and erlotinib, he died of intestinal perforation due to tumor invasion 23 days after the start of the therapy. an autopsy confirmed that the tumor was positive for anti - g - csf antibody, but the primary site was still not detected. |
emphysematous cholecystitis (ec) is an uncommon variant of acute cholecystitis, which is caused by secondary infection of the gallbladder wall with gas - forming organisms [15 ]. the mortality rate of ec is as high as 25% because of its high complication rate [2, 5 ] due to gangrene, gallbladder perforation, pericholecystic abscess and bile peritonitis. emergency surgical intervention is necessary for ec treatment for which open cholecystectomy has traditionally been performed [1, 35 ]. here a 78-year - old male was admitted to our hospital for an investigation of vertigo. after admission, he developed fever and abdominal distention. a chest x - ray in the up - right position and abdominal computed tomography (ct) the chest x - ray showed the presence of gas within the gallbladder and in its wall (fig. 1). the ct scan revealed emphysematous changes in the gallbladder wall and air within the gallbladder (fig. the patient was referred to the department of surgery and emergent surgery was performed. figure 1:up - right chest x - ray demonstrating air in the gallbladder (arrow) and in its wall (arrowheads). figure 2:(a and b) abdominal computed tomographic scans of axial and coronal views revealing emphysematous changes in the gallbladder wall and air in the gallbladder. up - right chest x - ray demonstrating air in the gallbladder (arrow) and in its wall (arrowheads). (a and b) abdominal computed tomographic scans of axial and coronal views revealing emphysematous changes in the gallbladder wall and air in the gallbladder. based on the hemodynamically stable status of the patient, we selected a laparoscopic approach. exploration of the abdominal cavity revealed gangrene and necrosis of the gallbladder. to facilitate the resection, we initially punctured the gallbladder using a percutaneous needle to extract bile. although there was significant adhesion, we were able to safely dissect the calot 's triangle and detected a cystic duct and artery. his postoperative course was uneventful. a bile culture obtained during surgery revealed clostridium perfringens, enterobacter cloacae and enterococcus faecalis as the causative organisms. ec is an uncommon variant of acute cholecystitis characterized by potentially high mortality and morbidity because of a high incidence of gangrene or perforation. garcia. reviewed 20 cases of ec and reported that the mortality rate was as high as 25% and the morbidity rate as high as 50%. emergent surgical intervention for ec is indicated with cholecystectomy as the definitive treatment, although percutaneous cholecystostomy for critically ill patients presents another option [1, 4, 5 ]. bouras. reported that the laparoscopic approach for ec can be considered a safe procedure. owing to the advances of laparoscopic surgery, it has allowed to perform emergency cholecystectomy. our patient was hemodynamically stable ; therefore, we selected the laparoscopic approach, which was successfully and safely completed. the causative organisms of ec include c. perfringens, escherichia coli and species of the genera staphylococcus, streptococcus, pseudomonas and klebsiella [15 ]. ec is associated with diabetes mellitus and primarily affects males, as in the present case. the symptoms of ec are almost the same as those of acute cholecystitis, including right upper quadrant pain, nausea, vomiting and low - grade fever [2, 4 ]. a prompt diagnosis of ec is essential and plain abdominal x - rays or ultrasound can be diagnostically useful with a sensitivity of up to 95%. however, contrast - enhanced abdominal ct scans may offer more information. in our patient, a chest x - ray in the up - right position detected air in the gallbladder wall and an abdominal ct revealed typical presentation of ec. emergency intervention is indicated and the laparoscopic approach can be considered a safe and effective method in hemodynamically stable patients. | emphysematous cholecystitis (ec) is an uncommon variant of acute cholecystitis, which is caused by secondary infection of the gallbladder wall with gas - forming organisms. the mortality rate of ec is still as high as 25%. emergency surgical intervention is indicated. open cholecystectomy has been traditionally accepted as a standard treatment for ec. we present a case of ec successfully treated by laparoscopic surgery. laparoscopic cholecystectomy for ec is considered to be safe and effective when indicated. |
cervical cancer is the third most common cancer and the fourth cause of cancer related mortality among women worldwide.1 in developing countries where access to screening is absent or limited, cervical cancer is the third cause of cancer mortality among women and is therefore much higher than in developed countries where this pathology is the seventh leading cause of cancer death.1 in morocco, cervical cancer is a major public health problem.2,3 fortunately, the cervical cancer has a long precancerous period that provides an opportunity for the screening and treatment before it progresses to cervical cancer. therefore, improving screening tests is a priority goal for the early diagnosis of cervical cancer. p16 is a cyclin - dependent kinase inhibitor that regulates the activity of cyclin - dependent kinases 4 and 6. when the high risk (hr)-human papillomavirus (hpv) e7 oncoprotein inactivates retinoblastoma, p16 expression increases dramatically.47 moreover, overexpression of p16 protein in cervical cancer is used as a diagnostic tool and has been directly associated with infection by hr - hpv genotypes.8,9 the aim of this study was to evaluate the combination of p16 protein expression, hpv typing, and histopathology for the early detection of cervical cancer among moroccan women. the study was conducted in the laboratory of histo - cytopathology of institut pasteur du maroc from january 2014 to october 2014 in casablanca, morocco under an approved human use protocol (institutional review board, ref : 013/14) to enroll and obtain tissue from the cervix of females to test for abnormal or precancerous conditions, or cervical cancer. the tissues were obtained by performing biopsies during routine physical examination to determine the health status of females ranging from 38 to 69 years of age. the biopsies were transferred to specimen collecting tubes containing 10% formalin and transported to the laboratory for processing and screening of precancerous or cancerous cells. tissue sections of 3 to 4 microns were prepared in paraffin and then placed on silanized slides. p16 protein expression was performed by immunohistochemistry using a cintec histology kit (biogen / roche, germany) according to the manufacturer s instructions. signal amplification in situ hybridization (csa - ish, genpoint, dako, glostrup, denmark) with biotinylated probes for hpv-6/11, 16/18, and 31/33/51 (enzo, new york, ny, usa) was used according to manufacturer protocols. after dewaxing and hydration, the 96 tissue sections were treated with proteinase k solution for 5 minutes at room temperature. the denaturation of target dna was carried out on a hotplate at 95c and transferred to 37c for hybridization with probes. the revelation of reaction product the slides were counterstained with mayer s hematoxylin, rinsed in ethanol and xylene, mounted by the permanent mounting medium (dako), and then examined by light microscope. data were entered into a database using ibm spss ver. 20.0 for windows (ibm co., armonk associations between hpv status, histopathological diagnosis, and p16 expression were analyzed using test or fisher s exact test. spearman s rank correlation coefficients were analyzed to investigate the possible correlations between hpv status, histopathological diagnosis, and p16 expression. all statistical tests were two - sided and results with values of p 0.05) nor between hsil and invasive cancer (p > 0.05) were observed. of the seventy - one hpv positive cases, we detected 67.6% (48/71) of hr - hpv (hpv 16 and 18), 24% of low risk - hpv (hpv 6 and 11), 1.4% intermediate risk - hpv (hpv 31, 33, and 35), and 7% coinfections (hpv 6/11 and 16/18). moreover, correlation between p16 expression and histopathological grade showed a positive correlation (correlation coefficient = 0.470, p < 0.05). furthermore, the hr - hpv status was significantly correlated with histopathological grade (correlation coefficient = 0.364, p < 0.05) (fig. the current screening tests for cervical cancer (histology and cytology) are unable to distinguish between the lesions which progress to cancer and those that do not.10 to overcome these limitations, an additional test is required to determine if oncogenic hpv virus has already increased genetic instability and made the infected cells sensitive to processing, leading eventually to the development of cancer. this test relies on the hr - hpv detection that exerts its oncogenic potential in infected woman.10 in this study, the hpv dna was detected in 74% of the biopsies. this prevalence was very high compared to that previously reported among moroccan women with normal and abnormal cytology (15.7%).3 however, another study conducted by khair.11 on cancer biopsies of the cervix found a higher incidence rate 92% close to our study. worldwide, hpv prevalence was very variable : in africa 22.1%, central america and mexico 20.4%, northern america 11.3%, europe 8.1%, and asia 8.0%.12 in our study, we found some convergence of hr - hpv in hsil and invasive cancer. this result is consistent with those reported in nigeria that showed the presence of 100% of hr - hpv in all samples with abnormal cytology.13 in addition, hr - hpv detection was related to hsil and invasive cancer among chinese and hungarian populations.14,15 in this report, hpv 16 and 18 were the most detected. this finding seems to be in line with previous data and highlighted that hpv16 and hpv18 are the most common types associated with cervical cancer worldwide.1622 the p16 protein overexpression was detected in 72.9%. in previous moroccan study, the overexpression of p16 was detected in 92.45% of cervical cancer specimens.23 our results were similar to those reported in many studies, where nearly 100% of high grade lesions and invasive cancers showed a strong overexpression of p16 protein, while non - dysplastic lesions remained negative.24,25 interestingly, our finding highlighted a trend positive correlation between cervical lesions severity, p16 expression, and hr - hpv prevalence. so these two markers are likely indicators of the severity of cervical lesions, and may be used in case of diagnostic s doubt particularly between lsil and hsil. in fact, many reports revealed p16 overexpression by immunohistochemistry in a very high proportion of neoplasia intraepithelial high grade, 80% to 100% of cervical intraepithelial neoplasia (cin)2 and almost all cin3, and in nearly all invasive cervical cancers, with diffuse expression throughout the height of squamous epithelium.2629 thus, these data indicate that p16 protein expression can be used to improve the histopathological diagnosis of precancerous cervical lesions.30 our findings highlighted a strong link between hr - hpv and hsil and/or invasive cancers. moreover, we found that p16 protein is a promising marker for the early diagnosis of precancerous and cancerous lesions of the cervix. the results of this study provide an improved screening approach for the diagnosis of the risk of lesions progressing to cervical cancer. | cervical cancer is a major public health problem in morocco. the cervical cancer has a long precancerous period that provides an opportunity for the screening and treatment. improving screening tests is a priority goal for the early diagnosis of cervical cancer. this study was conducted to evaluate the combination of p16ink4a protein expression, human papillomavirus (hpv) typing, and histopathology for the identification of cervical lesions with high risk to progress to cervical cancer among moroccan women. a total of 96 cervical biopsies were included in this study. signal amplification in situ hybridization with biotinylated probes was used to detect hpv. immunohistochemistry was used to evaluate the expression of p16ink4a protein. hpv dna was detected in 74.0% of the biopsies (71/96). of the seventy - one positive hpv cases, we detected 67.6% (48/71) of high risk (hr)-hpv (hpv 16 and 18), 24% of low risk - hpv (hpv 6 and 11), 1.4% intermediate risk - hpv (hpv 31, 33, and 35), and 7% coinfections (hpv 6/11 and 16/18). overexpression of p16ink4a protein was observed in 72.9% (70/96) of the biopsies. in addition, p16ink4a protein detection was closely correlated with recovery of hr hpv. our result showed that p16ink4a expression level is correlated with hr - hpv status. |
mobile applications (commonly referred to as apps) are designed for mobile devices such as smartphones and tablets. today, devices with faster processors, smaller batteries, improved memory, and highly efficient operating systems are capable to process many different advanced functions, especially apps, which currently affects our personal and work environments. in medicine, from patient monitoring and diagnostics to more efficient medical education and communication, smartphones serve a pivotal role in the practice of medicine today. furthermore, the food and drug administration estimates 500 million smartphone users worldwide will use a health care app by 2015. medical apps usually include education, consultation, and reference tools and can be focused in many different medical specialties such as family medicine, dermatology, cardiology, and surgery. pathology education has been traditionally done with stored glass slide sets, textbooks, and journals. however, increasing the number of specific online material such as video lectures, pathology journals, and reference textbooks, all together with the always difficult to use glass slide teaching sets because of some usual problems such as haphazardly organized, occupy workspace, faded stains, and broken slides makes apps a very straightforward way for learning and being up to date in pathology. the main benefits of developing a smartphone application that organize pathology online information is to offer an interactive reference resource with the potential to be viewed anywhere at any time. in this light, apps are by definition a reference resource that includes practical portability, widespread accessibility, and useful organization. until date, there are few number of smartphone pathology - related applications available, and most of them are focused on dermatopathology. the goal of this article is to relay the benefits of general pathologist - helper (gp - helper), in creating a reference tool for general pathologists and associate members. the aim of developing the application was to organize online pathology material in order to be user - friendly, comprehensive, timesaver and an interactive resource for pathologists, a pathologist in training, technicians, and medical students. the app was designed for android and ios devices (a superior bar was included with a specific navigation system). in addition, app appearance was customized using pink and violet colors as h & e staining. the app 's data required an active, hard - working online search looking forward to finding the best pathology information available. credibility and the accuracy of contents from every website included in the app support information such as contact details of the creator and manager director are shown when touching logotype bars. in summary, the app has permanent information (offline data) about different pathology protocols (tnm latest edition, protocols regarding management of tumors of unknown primary origin, and flowcharts for some of the most difficult tumors to diagnose) and a database with more than 5000 immunohistochemistry results from different tumors. online material shows more than 1100 links to free available pathology videos from youtube website ; about 250 links regarding information about the most important antibodies managed in pathology departments ; more than 70 websites about pathology and cytology societies ; 46 websites about different pathology providers ; more than 75 links to the most outstanding international journals of pathology ; more than 60 links to online reference textbooks in pathology and cytology ; 17 links regarding interesting pathology websites ; more than 10 links to websites containing information about how to obtain a certification in pathology ; 9 links to websites with pathology unknown cases and quiz questions ; and one link to http://www.news-medical.net/tag/feed/pathology.aspx, a website that keep us up to date in scientific upcoming news. expert consult section is also available and provides e - mails and websites from more than two experts in 18 pathology fields from around the world. that is if the list box is not sufficient to show the whole links in every section. beta versions were distributed to colleagues for feedback using google play developer console. after incorporating their feedback, the final master version of the app eventually, gp - helper integrates two external interactive places such as forum gp - helper and community gp - helper, with complex development. the aim of developing the application was to organize online pathology material in order to be user - friendly, comprehensive, timesaver and an interactive resource for pathologists, a pathologist in training, technicians, and medical students. the app was designed for android and ios devices (a superior bar was included with a specific navigation system). in addition, app appearance was customized using pink and violet colors as h & e staining. the app 's data required an active, hard - working online search looking forward to finding the best pathology information available. credibility and the accuracy of contents from every website included in the app support information such as contact details of the creator and manager director are shown when touching logotype bars. in summary, the app has permanent information (offline data) about different pathology protocols (tnm latest edition, protocols regarding management of tumors of unknown primary origin, and flowcharts for some of the most difficult tumors to diagnose) and a database with more than 5000 immunohistochemistry results from different tumors. online material shows more than 1100 links to free available pathology videos from youtube website ; about 250 links regarding information about the most important antibodies managed in pathology departments ; more than 70 websites about pathology and cytology societies ; 46 websites about different pathology providers ; more than 75 links to the most outstanding international journals of pathology ; more than 60 links to online reference textbooks in pathology and cytology ; 17 links regarding interesting pathology websites ; more than 10 links to websites containing information about how to obtain a certification in pathology ; 9 links to websites with pathology unknown cases and quiz questions ; and one link to http://www.news-medical.net/tag/feed/pathology.aspx, a website that keep us up to date in scientific upcoming news. expert consult section is also available and provides e - mails and websites from more than two experts in 18 pathology fields from around the world. that is if the list box is not sufficient to show the whole links in every section. beta versions were distributed to colleagues for feedback using google play developer console. after incorporating their feedback, the final master version of the app eventually, gp - helper integrates two external interactive places such as forum gp - helper and community gp - helper, with complex development. one specific issue worth discussing was the creation of the multichannel chat called community gp - helper. furthermore, chat moderators are supported by the host page in collaboration with gp - helper administrator. discussions were organized into 13 rooms taking into account different user 's interests : chats for the general pathologist, a pathologist in training, technicians, and students are in english but also in spanish language, however, chats for associate members, dermatopathologist, hematopathologist, molecular pathologist, and neuropathologist are only in english. guest access is available in order to allow users to quickly start chatting without creating an account first. however, users can create an account with a username and password within your chat room and edit profile. the chat room allows the user to instant messaging and upload, or exchanged data pictures and videos either taken by phone 's camera, or from phone 's gallery. emoticons are also available to bring your chat room to life with graphical smilies that users can insert into their messages. the database must be managed by chatango corporation. one specific issue worth discussing was the creation of the multichannel chat called community gp - helper. furthermore, chat moderators are supported by the host page in collaboration with gp - helper administrator. discussions were organized into 13 rooms taking into account different user 's interests : chats for the general pathologist, a pathologist in training, technicians, and students are in english but also in spanish language, however, chats for associate members, dermatopathologist, hematopathologist, molecular pathologist, and neuropathologist are only in english. guest access is available in order to allow users to quickly start chatting without creating an account first. however, users can create an account with a username and password within your chat room and edit profile. the chat room allows the user to instant messaging and upload, or exchanged data pictures and videos either taken by phone 's camera, or from phone 's gallery. emoticons are also available to bring your chat room to life with graphical smilies that users can insert into their messages. the database must be managed by chatango corporation. the forum (http://forum-gp-helper.mboards.com) was developed to offer interactive and permanent information about pathology and closely related themes for users. the website was developed using miarroba, a popular open source content management system platform that host the information on mysql. the forum (http://forum-gp-helper.mboards.com) was developed to offer interactive and permanent information about pathology and closely related themes for users. the website was developed using miarroba, a popular open source content management system platform that host the information on mysql. the application was accepted by google play app store on july 2015, and it is freely available. the hospital universitario de araba altogether with the spanish society of pathology supported this app. logotype of the app shows a background composed by squamous cell carcinoma of the lung stained with h & e [figure 1 ]. the home screen has the star button that links the user to the main index, and the exit button as a shortcut to way out [figure 2 ]. majority of information in the app is written in capital letters in order to facilitate reading. home screen displaying the start button the application was designed to be user - friendly with all accessible pages available on the main index [figure 3 ]. the main index includes many buttons such as protocols, tumors database, societies, journals, providers, antibodies, videos, bibliography, interesting websites, practicing websites, certification, forum, expert consult, breaking news, and community (wi - fi connection required). main index layout showing some accessible user buttons many different pathology lecture videos and websites with pathology quiz features are shown after linking on the specific button displayed on every list. for instance, clicking on neoplasia (gross specimens) in gross examination of human specimen 's page, the user will be able to watch this free available video on youtube website. this enables the user to learn and evaluate their ability to render a diagnosis on macro and micro photographies. furthermore, offline content such as in tumors database section, you will be able to introduce either a partial or full name of a tumor and the app will show you its immunophenotype [figure 4 ]. pathology protocols (tnm latest edition, protocols regarding management of tumors of unknown primary origin, and flowcharts for some of the most difficult tumors to diagnose) are also included [figure 5 ]. hematopoietic neoplasms gp - helper was intended to be a comprehensive pathology platform for pathologists, a pathologist in training, technicians, medical students, and associate members. easy to handle, the main goal of the app is to organize the pathology information that is sometimes randomly shown on the internet. forum gp - helper and community gp - helper will be able to show us much different information, not only to get a feedback opinion about the app but also to enhance professional connections, a better way of working in the 21 century. the home screen has the star button that links the user to the main index, and the exit button as a shortcut to way out [figure 2 ]. majority of information in the app is written in capital letters in order to facilitate reading. the application was designed to be user - friendly with all accessible pages available on the main index [figure 3 ]. the main index includes many buttons such as protocols, tumors database, societies, journals, providers, antibodies, videos, bibliography, interesting websites, practicing websites, certification, forum, expert consult, breaking news, and community (wi - fi connection required). many different pathology lecture videos and websites with pathology quiz features are shown after linking on the specific button displayed on every list. for instance, clicking on neoplasia (gross specimens) in gross examination of human specimen 's page, the user will be able to watch this free available video on youtube website. this enables the user to learn and evaluate their ability to render a diagnosis on macro and micro photographies. furthermore, offline content such as in tumors database section, you will be able to introduce either a partial or full name of a tumor and the app will show you its immunophenotype [figure 4 ]. pathology protocols (tnm latest edition, protocols regarding management of tumors of unknown primary origin, and flowcharts for some of the most difficult tumors to diagnose) are also included [figure 5 ]. tumors database section. gp - helper was intended to be a comprehensive pathology platform for pathologists, a pathologist in training, technicians, medical students, and associate members. easy to handle, the main goal of the app is to organize the pathology information that is sometimes randomly shown on the internet. forum gp - helper and community gp - helper will be able to show us much different information, not only to get a feedback opinion about the app but also to enhance professional connections, a better way of working in the 21 century. apps are software programs that have been developed to run on a computer or mobile device to accomplish a specific purpose. the introduction of mobile computing devices (personal digital assistants, followed by smartphones, and tablet computers) with more advanced features, such as global positioning systems, sound recorders, web searching, high - quality cameras, as well as faster processors and improved memories, smaller batteries, better operating systems, and high - resolution screens, mobile devices have essentially become handheld computers. in this light, mobile computing devices are able to facilitate the development of huge amounts of mobile device apps for both personal and professional use, with greatly impacted in many fields, including medicine. while recent studies have demonstrated their efficacy, more studies are needed to validate formally these apps in this emerging mobile health (mhealth) era. apps are being increasingly used in many medical specialties such as dermatology, family medicine, surgery, anesthesiology, radiology, in pathology, most of the apps are focus on dematopathology. due to visual pattern recognition is of vital importance for the pathologist, video and image - based apps are of great benefit. for instance, they can be used for educational, with the integration of quiz features and eventually, focusing on clinical purposes. however, one of the main barriers to use apps in pathology is more related with a properly pathology department location, due to they usually are in lower floors of the hospital, without internet connectivity, where web - hosted data in the application may not be available. regarding this affirmation, i describe hereby gp - helper app, one of the few apps developed thinking for every professional worker from every department of pathology. besides, i anticipate that in the future many more smartphone apps will become available for the pathology community. finally, this technical note highlights the importance of technological developments in healthcare, and it is an example of how pathologists can also contribute in this matter. | introduction : smartphone applications (apps) have become increasingly prevalent in medicine. due to most pathologists, pathology trainees, technicians, and medical students use smartphones ; apps can be a different way for general pathology education. general pathologist - helper (gp - helper) is a novel app developed as a reference tool in general pathology and especially for general pathologists, developed for android and ios platforms.materials and methods:gp - helper, was created using mobincube website platform. this tool also integrates forum gp - helper, an external website created using miarroba website (http://forum-gp-helper.mboards.com) and community gp - helper a multichannel chat created using chatango website platform.results:the application was released in july 2015, and it is been periodically updated since then. the app has permanent information (offline data) about different pathology protocols (tnm latest edition, protocols regarding management of tumors of unknown primary origin, and flowcharts for some of the most difficult tumors to diagnose) and a database with more than 5000 immunohistochemistry results from different tumors. online data have links to more than 1100 reference pathology video lectures, 250 antibodies information, more than 70 pathology association websites, 46 pathology providers, and 78 outstanding pathology journal websites. besides this information, the app has two interactive places such as forum gp - helper and community gp - helper that let users to stay in touch everywhere and every time. expert consult section is also available.conclusions:gp-helper pretends to integrate offline and online data about pathology with two interactive external places in order to represent a reference tool for general pathologists and associate members. |
the causes of pulmonary embolism can be venous thromboembolism, and nonthrombotic embolism like septic, fat, air, amniotic fluid and tumour embolism. tumour embolism to lungs can arise from cancers of breast, stomach, liver, kidney and rarely from choriocarcinoma. choriocarcinoma is a malignant, trophoblastic cancer, belonging to the malignant end of the spectrum in gestational trophoblastic disease, which can occur following molar pregnancy, ectopic pregnancy, abortion and even normal pregnancy [2, 3 ]. choriocarcinomas spread via blood and lymphatics with early haematogenous spread to lungs, resulting in pulmonary embolism, pulmonary oedema, pulmonary hypertension or acute respiratory distress syndrome. in a young female presenting with persistent shortness of breath, cough and chest pain, the possibility of metastatic pulmonary embolism should be considered as surgery and chemotherapy can cure the choriocarcinoma metastasizing to the lungs. a 25-year - old lady was admitted in the pulmonology ward with the diagnosis of pneumonia. the patient had presented with a history of progressive shortness of breath, chest pain and persistent cough with occasional haemoptysis. she had a history of being treated with antibiotics and anti - tb drugs in outpatient basis. since her symptoms were persistent and her general condition was deteriorating, she was admitted to the ward. on examination, she had crepitations in bilateral chest and an oxygen saturation of only 80%. cect chest was done, which revealed pulmonary embolus occluding the main pulmonary artery, and right and left pulmonary arteries (fig. 1). serum beta - human chorionic gonadotrophin (hcg) level was found to be significantly high. she was managed further with chemotherapy (emaco regimen) with excellent response to the treatment. figure 3:histopathological image showing the trophoblastic elements. a ct scan image showing thrombus in the pulmonary artery. women in the reproductive age group with lung metastasis present with dyspnoea, chest pain, cough and haemoptysis. as the diagnosis can always be misleading and patient might be treated in the line of pneumonia or tuberculosis, it is very essential to have a high index of suspicion. surgery is indicated in patients with haemodynamic instability and those with massive tumour burden occluding main pulmonary and branch pulmonary arteries. so, chemotherapy should be initiated as soon as the diagnosis is strongly suspected or confirmed. pulmonary embolism due to choriocarcinoma should always be suspected in a reproductive age woman presenting with intractable shortness of breath. | pulmonary embolism carries a significant morbidity and mortality. metastatic choriocarcinoma presenting as pulmonary embolism is a rare event. here, we report a case of a 25-year - lady with a history of worsening shortness of breath for 4 months who was treated as a case of pneumonia and tuberculosis. owing to the worsening condition, she had a contrast enhanced computed tomography (cect) chest done and was diagnosed to have pulmonary embolism. she underwent pulmonary embolectomy. the histopathological examination of the embolus revealed it to be metastatic choriocarcinoma. she showed a good response to chemotherapy. metastatic choriocarcinoma should be considered as a differential diagnosis in females presenting with pulmonary embolism. |
between 70 and 75% of all ovarian tumors are of epithelial origin, with borderline ovarian tumors (bots) accounting for 10% of these neoplasms. bots are defined as atypical proliferative tumors or tumors of low malignant potential with microinvasion into the stroma. there are seven types of bots : serous, mucinous, endometrioid, clear cell, brenner, undifferentiated and mixed tumors. the incidence of bots among cysts without obvious signs of malignancy is low about 0.6%. although the diagnosis of bot is based on histopathological examination, the preoperative evaluation of clinical, radiological and laboratory picture of the patient is of great importance. accurate radiological findings related to the character of the lesion may play a crucial role in planning surgical treatment. this is also important due to the fact that the disease usually affects young women who wish to maintain their reproductive potential by undergoing a possibly less radical surgery. the aim of this article was to present a case of a patient with ovarian tumor in order to show the underestimated role of imaging techniques in the diagnostic process. a 27-year - old woman with pelvic pain and abdominal discomfort presented to the department of gynecology, gynecologic oncology and gynecologic endocrinology in the medical university of gdask. no changes were observed in the morphology of the external genitalia. the body of the uterus was movable but shifted posteriorly by a mass adherent to the right ovary. the ca 125 was over 600 u / ml. transvaginal ultrasonography (tvu) of the lower pelvis showed an encapsulated, well - delineated structure mostly formed by a liquid content, measuring 12.2 3.3 5.2 cm, with a solid fraction present in its internal wall in the form of intraluminal exophytic papillary projections (fig. 2). ultrasound examination of mostly hypoechogenic multilocular cystic tumor with well - delineated smooth borders and a diameter of 12 cm, with small solid echogenic papillary projections (multi - locular solid type, however only one loculus is seen) ultrasound examination : a. the same hypoechoic cystic tumor ; b. there is a satellite tumor with a diameter of 3 cm of the same echostructure on the right cystoscopy revealed no lesions in the urinary bladder. computed tomography (ct) confirmed the presence of cystic lesion with a solid fraction in the right ovary (fig. 3). a. a contrast - enhanced ct scan showing no enhancement of the cystic portion of the tumor and mild wall enhancement with clearly visible large amount of exophytic papillary projections. b. non - contrast - enhanced ct scan showing a cystic tumor with smooth margins and a diameter of 11 cm (30 h.u.) lying anteriorly to and above the bladder, slightly on the right laparotomy was selected as the surgical procedure due to the tumor size and the criteria for low risk malignancy. a cystic, multilocular tumor with a diameter of 11 cm that was adherent the omentum, peritoneum, urinary bladder and vesicouterine pouch was found. the wall of the cyst was up to 1.5 cm thick, the internal surface was covered with wide intraluminal papillae up to 1.5 cm in height. mucinous cystadenoma with limited malignancy of right ovary was diagnosed and staged ia / pt1an0mx according to the international federation of gynecology and obstetrics (figo) and the american joint committee on cancer / union for international cancer control (ajcc / uicc) staging system. ovarian cancer is the second most common gynecologic malignancy and the leading cause of death among women with gynecological cancers. imaging modalities play an important role in the diagnosis, staging and treatment in this subgroup of neoplasms, however, it is difficult to suggest a simple algorithm. ultrasound seems to be the first choice from the technical and economical point of view. there are no specific radiological features selectively describing bots detectable by ultrasound or other, more advanced modalities. us examination plays an important role in detecting and describing adnexal masses, with the tvu being of greater importance. it is not sufficient to evaluate tumor masses as precisely as recommended by the iota (international ovarian tumor analysis). the questions that often arise are as follows : a) which algorithms to follow (images are only supportive) b) which course of action to take operate or observe ? detailed evaluation provides different approaches : like iota s terms or simple tools proposed by mcdonald. who based their research on 3 types of adnexal masses plus / minus tumor marker elevation (solid vs. cystic vs. complex + ca 125). they found the combination of solid / complex mass with ca 125 elevation to be responsible for the identification of 77.3% of eoc cases. however, unilocular solid cysts which most commonly contained papillary projections predominated among all groups. ct is not a routine procedure for the evaluation of adnexal masses. in our case, ct was performed for preoperative confirmation of bot, however, it probably could have been avoided. bent. described 4 categories of bot appearances on mri scans : 1) unilocular cysts ; 2) minimally septated cysts with papillary projections ; 3) markedly septated lesions with plaque - like excrescences and 4) predominantly solid lesions with exophytic papillary projections. this research also described the most important clinical and imaging characteristics that could indicate a suspicion of bot : in young woman with normal or mildly elevated ca 125 levels, showing predominantly cystic lesion at tvu examination with regular thin wall and the presence of normal ipsilateral ovarian stroma. although the mri findings did not differ from those derived from ultrasound studies, it allowed for a more objective and comprehensive evaluation of organs and disorders. radiological assessment plays an important role in establishing the final diagnosis and treatment planning in patients with bot. the role of a gynecologist is to make the final decision regarding further observation or surgery. the role of radiologist is to provide clinicians with assistance during the diagnostic progress, using available imaging modalities with ultrasound as the first choice. | borderline ovarian tumors represent about 10% of all epithelial ovarian cancers, but in contrast to epithelial ovarian cancers, they constitute a group of tumors with a much better prognosis. an assessment of clinical presentation, physical examination, radiological and biochemical findings is necessary to tailor management strategies for patients with ovarian tumors. the article, which is based on a case report, describes different approaches for preoperative diagnosis as well as discusses approaches that might bring some insights on tumor histology. furthermore, it raises a question about which imaging techniques should be proposed for a reliable diagnosis of borderline ovarian tumors to ensure safe surgery planning. |
the avon longitudinal study of parents and children (alspac) is a prospective cohort study investigating the health and development of children in the south - west of england,17 the full details of which, including details of representativeness of the sample, are available on the study website (http://www.bristol.ac.uk/alspac). pregnant women resident in one of three bristol - based health districts with an expected date of delivery between 1 april 1991 and 31 december 1992 were invited to take part in the study. of these women, 14 541 were recruited. from these pregnancies, there were 14 062 live born children, 13 988 of whom were alive at 1 year. follow - up has included parent and child - completed questionnaires, links to routine data and clinic attendance. a random subsample of children from the last 6 months of recruitment (children in focus group approximately 10% of the total cohort) were invited to clinics between the ages of 4 months and 5 years ; all children were invited to clinics from the age of 7 years onwards. ethics approval for the study was obtained from the alspac law and ethics committee and the local research ethics committees. a questionnaire at 32 weeks ' gestation asked mothers to report their educational attainment, which was categorised as below o - level (ordinary level ; exams taken in different subjects usually at age 1516 years at the completion of legally required school attendance, equivalent to the present uk general certificate of secondary education), o - level only, a - level (advanced level ; exams taken in different subjects usually at age 18 years), or university degree or above. we also examined socioeconomic differentials with head of household occupational social class and father 's education (categorised as above), but since results and conclusions were the same as those presented here for mother 's education these are not presented or discussed further. birth length (crown heel) was measured for almost the whole cohort by alspac staff who visited newborns soon after birth (median 1 day, range 114 days), using a harpenden neonatometer (holtain ltd, crosswell, crymych, uk). from birth to 5 years, measurements are also available for the majority of the cohort from health visitor records, which form part of standard childcare in the uk. in this cohort we had up to four measurements taken on average at 2, 10, 21 and 48 months of age, which we have demonstrated in previous work to have good accuracy.18 for a random 10% of the cohort, we also have measurements from children in focus clinics, held between the ages of 4 months and 5 years. at these clinics, crown heel length for children aged 425 months was measured using a harpenden neonatometer (holtain ltd), and from 25 months onwards standing height was measured using a leicester height measure (seca, hamburg, germany). from age 7 years upwards, all children were invited to annual clinics, at which standing height was measured (without shoes) to the last complete millimetre using the harpenden stadiometer (holtain ltd). across all ages, implausible measurements (> 4 sd from the mean for gender and age - specific category) were re - coded as missing (101 measurements from 96 individuals, approximately 0.1% of all available measurements). all other available measures were used in analyses. to account for the likely reduced accuracy of parent - reported measurements,19 a binary indicator of measurement source (research clinic or health records versus parent reports from questionnaires) was included in all models. we estimated individual growth trajectories using a linear spline mixed - effects model (two levels measurement occasion and individual), fitted using the statistical package mlwin version 2.10 (http://www.cmm.bristol.ac.uk/mlwin/index.shtml). such models allow for the change in scale and variance of height over time and use all available data from all eligible children under a missing at random assumption. they also allow for individual variation in growth trajectories, as random effects allow each individual to have different intercepts and slopes. models for growth between birth and 10 years were constructed separately for boys and girls, for all individuals with data on maternal education and at least two length / height measurements (n=12 366). the methodology identified spline points that defined periods of approximately linear growth based on the data. the modelling approach confirmed that there were three spline points (four periods of linear growth) between birth and 10 years : 03 months, 310 months, 1029 months, 29120 months for boys, and 02 months, 211 months, 1132 months and 32120 months for girls. five coefficients thus describe average growth in the cohort birth length (ie, the baseline measurement) and mean linear growth for the four periods described above. full details of the statistical methodology are presented in the supplementary material, available online only. socioeconomic inequality in the growth trajectories was estimated by fitting interaction terms in the random effects model between maternal education and the constant term (representing birth length) and each of the slopes for the growth periods. the parameters for these interaction terms demonstrate whether there are differences in birth length or growth in each period between socioeconomic groups (ie, the associated p values test the null hypothesis of no difference in birth length or growth in each period by socioeconomic group). the only other covariate included in models was a binary indicator of source of height measurement (questionnaire vs measured by alspac clinic staff or health visitors). the avon longitudinal study of parents and children (alspac) is a prospective cohort study investigating the health and development of children in the south - west of england,17 the full details of which, including details of representativeness of the sample, are available on the study website (http://www.bristol.ac.uk/alspac). pregnant women resident in one of three bristol - based health districts with an expected date of delivery between 1 april 1991 and 31 december 1992 were invited to take part in the study. of these women, 14 541 were recruited. from these pregnancies, there were 14 062 live born children, 13 988 of whom were alive at 1 year. follow - up has included parent and child - completed questionnaires, links to routine data and clinic attendance. a random subsample of children from the last 6 months of recruitment (children in focus group approximately 10% of the total cohort) were invited to clinics between the ages of 4 months and 5 years ; all children were invited to clinics from the age of 7 years onwards. ethics approval for the study was obtained from the alspac law and ethics committee and the local research ethics committees. a questionnaire at 32 weeks ' gestation asked mothers to report their educational attainment, which was categorised as below o - level (ordinary level ; exams taken in different subjects usually at age 1516 years at the completion of legally required school attendance, equivalent to the present uk general certificate of secondary education), o - level only, a - level (advanced level ; exams taken in different subjects usually at age 18 years), or university degree or above. we also examined socioeconomic differentials with head of household occupational social class and father 's education (categorised as above), but since results and conclusions were the same as those presented here for mother 's education these are not presented or discussed further. birth length (crown heel) was measured for almost the whole cohort by alspac staff who visited newborns soon after birth (median 1 day, range 114 days), using a harpenden neonatometer (holtain ltd, crosswell, crymych, uk). from birth to 5 years, measurements are also available for the majority of the cohort from health visitor records, which form part of standard childcare in the uk. in this cohort we had up to four measurements taken on average at 2, 10, 21 and 48 months of age, which we have demonstrated in previous work to have good accuracy.18 for a random 10% of the cohort, we also have measurements from children in focus clinics, held between the ages of 4 months and 5 years. at these clinics, crown heel length for children aged 425 months was measured using a harpenden neonatometer (holtain ltd), and from 25 months onwards standing height was measured using a leicester height measure (seca, hamburg, germany). from age 7 years upwards, all children were invited to annual clinics, at which standing height was measured (without shoes) to the last complete millimetre using the harpenden stadiometer (holtain ltd). across all ages, implausible measurements (> 4 sd from the mean for gender and age - specific category) were re - coded as missing (101 measurements from 96 individuals, approximately 0.1% of all available measurements). all other available measures were used in analyses. to account for the likely reduced accuracy of parent - reported measurements,19 a binary indicator of measurement source (research clinic or health records versus parent reports from questionnaires) was included in all models. we estimated individual growth trajectories using a linear spline mixed - effects model (two levels measurement occasion and individual), fitted using the statistical package mlwin version 2.10 (http://www.cmm.bristol.ac.uk/mlwin/index.shtml). such models allow for the change in scale and variance of height over time and use all available data from all eligible children under a missing at random assumption. they also allow for individual variation in growth trajectories, as random effects allow each individual to have different intercepts and slopes. models for growth between birth and 10 years were constructed separately for boys and girls, for all individuals with data on maternal education and at least two length / height measurements (n=12 366). the methodology identified spline points that defined periods of approximately linear growth based on the data. the modelling approach confirmed that there were three spline points (four periods of linear growth) between birth and 10 years : 03 months, 310 months, 1029 months, 29120 months for boys, and 02 months, 211 months, 1132 months and 32120 months for girls. five coefficients thus describe average growth in the cohort birth length (ie, the baseline measurement) and mean linear growth for the four periods described above. full details of the statistical methodology are presented in the supplementary material, available online only. socioeconomic inequality in the growth trajectories was estimated by fitting interaction terms in the random effects model between maternal education and the constant term (representing birth length) and each of the slopes for the growth periods. the parameters for these interaction terms demonstrate whether there are differences in birth length or growth in each period between socioeconomic groups (ie, the associated p values test the null hypothesis of no difference in birth length or growth in each period by socioeconomic group). the only other covariate included in models was a binary indicator of source of height measurement (questionnaire vs measured by alspac clinic staff or health visitors). data on growth and maternal education are available for 6380 boys and 5986 girls ; 88% of the alspac cohort. approximately 30% of children included had mothers in the lowest education category, and approximately 13% were in the highest category (table 1). there was a median of seven measurements per child (interquartile range five to 10 measurements), with most children having at least one measurement in each growth period (table 2). the number of measurements was greater in higher socioeconomic groups, for example the median was six measures in the lowest maternal education category and eight in the highest maternal education category. at least two - thirds of height measurements after birth were measured in alspac clinics or by health visitors (table 2), and are therefore likely to have good accuracy. in the multilevel models, the indicator for questionnaire measurements tended to have a negative coefficient, that is parents tended to overreport their child 's height. only a small percentage of alspac participants are of non - white ethnicity (5.5%) ; models excluding non - white children did not differ. summary of measurements included in the growth model for 12 366 individuals included in models that is excluding parent - reported measurements from questionnaires. all birth length measurements either extracted from health records or measured by avon longitudinal study of parents and children (alspac) staff. 03 1029 months for boys, 1132 months for girls 29120 months for boys, 32120 months for girls. figure 1 shows the average predicted growth trajectories across childhood from our models ; differences between actual and predicted measurements are shown in supplementary table 1 (available online only) ; these were very small in each period indicating good model fit. these values indicate weak levels of autocorrelation and given the goodness of fit of our model as demonstrated in supplementary table 1 (available online only), together with computational difficulties in incorporating autocorrelation into growth models in mlwin, we did not account for autocorrelation in further analyses. average height (cm) trajectories for boys and girls predicted by the multilevel models. there is a clear gradient in birth length across categories of maternal education for both boys and girls, with babies born to mothers in the lowest education categories being shortest and each sequential increase in category of maternal education being associated with an increase in average birth length (p values for interactions between birth length and maternal education 0.0003 for boys, < 0.0001 for girls). for boys there is a mean difference in birth length of 0.41 cm between the lowest and highest maternal education categories ; representing 0.9% of the average birth length of a son of a degree - educated mother. for girls the mean difference between the lowest and highest maternal education categories is 0.65 cm, representing 1.3% of the average birth length of a daughter of a degree - educated mother (table 3). predicted lengths / heights across categories of maternal education there is statistical evidence of socioeconomic differences in growth for some, but not all, growth periods (table 4). for growth in the first few months of life, there is no statistical evidence of a difference between maternal education categories, although there is some indication that growth rates tend to be higher in the lower socioeconomic groups. for growth later in childhood growth rates tend to be higher in the higher maternal education categories, with most growth periods demonstrating a linear pattern across all four categories of maternal education. the differences in growth rates across maternal education categories are small, but result in a 1 cm widening of the absolute difference between the highest and lowest maternal education categories, while the relative socioeconomic differential in height remains similar from birth to 10 years (tables 3 and 4). figures 2 and 3 show the curves for the highest and lowest categories of maternal education for boys and girls, respectively. these indicate the small absolute difference in height between these extreme groups with the more detailed analysis across all categories shown in tables 3 and 4. mean growth rates across categories of maternal education average predicted growth trajectories for boys in the lowest and highest maternal education categories. average predicted growth trajectories for girls in the lowest and highest maternal education categories. by the age of 10 years, the mean difference between children in the lowest and highest maternal education categories is 1.4 cm (1.0% of average height for the son of degree - educated mothers) for boys and 1.7 cm (1.2% of average height for the daughter of degree - educated mothers) for girls. this means that at 10 years old, despite boys being on average taller than girls, girls in the highest maternal education category are on average 0.8 cm taller than boys in the lowest maternal education category (table 3). data on growth and maternal education are available for 6380 boys and 5986 girls ; 88% of the alspac cohort. approximately 30% of children included had mothers in the lowest education category, and approximately 13% were in the highest category (table 1). there was a median of seven measurements per child (interquartile range five to 10 measurements), with most children having at least one measurement in each growth period (table 2). the number of measurements was greater in higher socioeconomic groups, for example the median was six measures in the lowest maternal education category and eight in the highest maternal education category. at least two - thirds of height measurements after birth were measured in alspac clinics or by health visitors (table 2), and are therefore likely to have good accuracy. in the multilevel models, the indicator for questionnaire measurements tended to have a negative coefficient, that is parents tended to overreport their child 's height. only a small percentage of alspac participants are of non - white ethnicity (5.5%) ; models excluding non - white children did not differ. summary of measurements included in the growth model for 12 366 individuals included in models that is excluding parent - reported measurements from questionnaires. all birth length measurements either extracted from health records or measured by avon longitudinal study of parents and children (alspac) staff. 03 1029 months for boys, 1132 months for girls 29120 months for boys, 32120 months for girls. figure 1 shows the average predicted growth trajectories across childhood from our models ; differences between actual and predicted measurements are shown in supplementary table 1 (available online only) ; these were very small in each period indicating good model fit. these values indicate weak levels of autocorrelation and given the goodness of fit of our model as demonstrated in supplementary table 1 (available online only), together with computational difficulties in incorporating autocorrelation into growth models in mlwin, we did not account for autocorrelation in further analyses. average height (cm) trajectories for boys and girls predicted by the multilevel models. there is a clear gradient in birth length across categories of maternal education for both boys and girls, with babies born to mothers in the lowest education categories being shortest and each sequential increase in category of maternal education being associated with an increase in average birth length (p values for interactions between birth length and maternal education 0.0003 for boys, < 0.0001 for girls). for boys there is a mean difference in birth length of 0.41 cm between the lowest and highest maternal education categories ; representing 0.9% of the average birth length of a son of a degree - educated mother. for girls the mean difference between the lowest and highest maternal education categories is 0.65 cm, representing 1.3% of the average birth length of a daughter of a degree - educated mother (table 3). predicted lengths / heights across categories of maternal education there is statistical evidence of socioeconomic differences in growth for some, but not all, growth periods (table 4). for growth in the first few months of life, there is no statistical evidence of a difference between maternal education categories, although there is some indication that growth rates tend to be higher in the lower socioeconomic groups. for growth later in childhood growth rates tend to be higher in the higher maternal education categories, with most growth periods demonstrating a linear pattern across all four categories of maternal education. the differences in growth rates across maternal education categories are small, but result in a 1 cm widening of the absolute difference between the highest and lowest maternal education categories, while the relative socioeconomic differential in height remains similar from birth to 10 years (tables 3 and 4). figures 2 and 3 show the curves for the highest and lowest categories of maternal education for boys and girls, respectively. these indicate the small absolute difference in height between these extreme groups with the more detailed analysis across all categories shown in tables 3 and 4. mean growth rates across categories of maternal education average predicted growth trajectories for boys in the lowest and highest maternal education categories. average predicted growth trajectories for girls in the lowest and highest maternal education categories. by the age of 10 years, the mean difference between children in the lowest and highest maternal education categories is 1.4 cm (1.0% of average height for the son of degree - educated mothers) for boys and 1.7 cm (1.2% of average height for the daughter of degree - educated mothers) for girls. this means that at 10 years old, despite boys being on average taller than girls, girls in the highest maternal education category are on average 0.8 cm taller than boys in the lowest maternal education category (table 3). even in a highly developed country such as the uk, socioeconomic differentials in infant and child height persist, with linear gradients in birth length and growth rates across four categories of maternal education. we have shown, in a cohort of children born in the uk in the early 1990s, that most of the socioeconomic inequality in height during childhood is driven by differences in birth length, with only small additional widening of height inequality due to differences in growth rates after infancy. as the scale of height increases over childhood, there is an increase in absolute inequality in height between birth and 10 years. for a scaled variable such as height, however, a more meaningful way of examining patterns of inequality over childhood is relative inequality. these data demonstrate little meaningful increase in the relative inequality in height after birth, indicating that height inequality is primarily present at birth and does not widen considerably through childhood growth. there is considerable evidence from cross - sectional studies demonstrating the existence of height inequalities at young ages. for example, social gradients in height were demonstrated in british 2-year - olds from the carnegie (boyd orr) survey, a cross - sectional survey conducted between 1937 and 1939,20 in british 7-year - olds in the 1960s,21 and in 511-year - olds in england and scotland in 1972.22 one study of 616-year - olds from the usa, and a further study of 1016-year - olds from sweden, however, found no evidence of height inequalities.23 24 few studies have investigated the patterns of height inequality across childhood using longitudinal data. one study compared two measurements of 7569 children aged 510 years from england and scotland. the children were measured in 1972 and again in 1973 ; the study found no change in the socioeconomic differential in height, leading the authors to conclude that height inequalities are established before the age of 5 years.25 our findings that differences in height are due primarily to birth length rather than childhood growth may well differ in other settings, particularly low and middle - income countries and previous generations in high - income countries, where there may be important socioeconomic patterning of infant growth due to illness and nutrition.26 as, in this population at least, the socioeconomic differential in height is present at birth and does not widen much across childhood, socioeconomic inequalities in height reflect socioeconomic conditions experienced before birth. the underlying reasons for the observed socioeconomic differential in birth length are likely to be multifactorial, and may include maternal behaviours such as smoking during pregnancy, which has been shown in the alspac cohort to have a dose response relationship with offspring height at age 7.5 years.27 furthermore, fetal and maternal genetics might result in socioeconomic differentials in birth length, because height is highly heritable28 and there is evidence for assortative mating by height29 and by socioeconomic position (sep).30 finally, epigenetic effects (different expression of genetic variation determined by environmental factors related to sep) could explain our findings. further studies are required to determine the underlying mechanisms for the associations that we have found. for example, one study compared height inequality between members of the 1958 birth cohort and their offspring. inequalities in height narrowed between the two generations from 2 to 3 cm at the age of 7 years in cohort members to less than 1 cm among their children (born approximately 26 years later).16 it would be interesting in further analyses to explore the role of grandparental education in the socioeconomic patterning of the growth trajectories in alspac, and as the data become available to explore how socioeconomic patterning of height trajectories differs between the alspac participants and their parents and offspring. the absolute difference we observe in the alspac participants is relatively small, and is smaller than the differences observed in previous generations in the uk, including the 1958 birth cohort.16 it is possible, therefore, that socioeconomic differentials in height will decrease further over time as living conditions improve, although a review utilising data from 10 european countries showed persisting height inequalities, with no narrowing of the height differential between educational groups in younger cohorts compared with previous generations.1 height differences, probably at least partly through their associations with sep, have been shown to result in disparities in many other aspects of life, with taller people experiencing, for example, increased job success and reporting greater life satisfaction.31 32 the main strengths of this study are its uniqueness in examining longitudinal height trajectories from birth to age 10 years in a large sample size with a large number of repeat measurements. our modelling approach has allowed us to examine trajectories of height across childhood, taking account of the different timings and numbers of measurements between children. by using a linear spline model, we have been able to simplify the trajectories, while retaining good fit between actual and predicted values. however, we have restricted our analyses to growth up to 10 years of age. at this age very few children will have gone through puberty. among those with relevant data (information provided by parents using tanner 's puberty questions) the vast majority of the cohort were prepubertal at age 10 years (82% at tanner stages 1 or 2 ; 96% at stages 13). when further data are available, it will be interesting to see whether and how height inequalities alter as the whole cohort passes through puberty. we were able to include the vast majority of participants in our study and those included did not differ with respect to sep from those excluded due to missing data. however, in our data we have fewer measurements of height for lower sep children. when analyses were restricted to only participants who had at least nine measurements the findings were essentially identical to those presented here. our study has focused on height, but previous research has suggested that leg length is the component of total height that is most socially patterned.20 we do not have serial measurements of leg length in this study and are unaware of any other studies that have such data. to conclude, our results suggest that socioeconomic differentials in length / height in contemporary high - income populations are already present at birth, suggesting that genetic, epigenetic or intrauterine characteristics influenced by maternal behaviours may be important. there are no socioeconomic differences in growth in early infancy, but slightly faster growth in later childhood results in minimal widening of the height inequality. further research would be valuable to explore the mechanisms that underlie the differentials we have identified. despite improvements in living standards and increasing secular trends in height, socioeconomic inequalities in height persist, with individuals from more deprived backgrounds tending to be shorter. what is not known is at what age such socioeconomic inequalities in height emerge, and whether and how they change across childhood. our results suggest that socioeconomic differentials in length / height in contemporary high - income populations are present at birth, with some small widening of inequality in later childhood, suggesting that they are driven by genetic, epigenetic or maternal intrauterine characteristics. even in a highly developed country such as the uk, socioeconomic differentials in infant and child height persist, with linear gradients in birth length and growth rates across four categories of maternal education. we have shown, in a cohort of children born in the uk in the early 1990s, that most of the socioeconomic inequality in height during childhood is driven by differences in birth length, with only small additional widening of height inequality due to differences in growth rates after infancy. as the scale of height increases over childhood, there is an increase in absolute inequality in height between birth and 10 years. for a scaled variable such as height, however, a more meaningful way of examining patterns of inequality over childhood is relative inequality. these data demonstrate little meaningful increase in the relative inequality in height after birth, indicating that height inequality is primarily present at birth and does not widen considerably through childhood growth. there is considerable evidence from cross - sectional studies demonstrating the existence of height inequalities at young ages. for example, social gradients in height were demonstrated in british 2-year - olds from the carnegie (boyd orr) survey, a cross - sectional survey conducted between 1937 and 1939,20 in british 7-year - olds in the 1960s,21 and in 511-year - olds in england and scotland in 1972.22 one study of 616-year - olds from the usa, and a further study of 1016-year - olds from sweden, however, found no evidence of height inequalities.23 24 few studies have investigated the patterns of height inequality across childhood using longitudinal data. one study compared two measurements of 7569 children aged 510 years from england and scotland. the children were measured in 1972 and again in 1973 ; the study found no change in the socioeconomic differential in height, leading the authors to conclude that height inequalities are established before the age of 5 years.25 our findings that differences in height are due primarily to birth length rather than childhood growth may well differ in other settings, particularly low and middle - income countries and previous generations in high - income countries, where there may be important socioeconomic patterning of infant growth due to illness and nutrition.26 as, in this population at least, the socioeconomic differential in height is present at birth and does not widen much across childhood, socioeconomic inequalities in height reflect socioeconomic conditions experienced before birth. the underlying reasons for the observed socioeconomic differential in birth length are likely to be multifactorial, and may include maternal behaviours such as smoking during pregnancy, which has been shown in the alspac cohort to have a dose response relationship with offspring height at age 7.5 years.27 furthermore, fetal and maternal genetics might result in socioeconomic differentials in birth length, because height is highly heritable28 and there is evidence for assortative mating by height29 and by socioeconomic position (sep).30 finally, epigenetic effects (different expression of genetic variation determined by environmental factors related to sep) could explain our findings. further studies are required to determine the underlying mechanisms for the associations that we have found. for example, one study compared height inequality between members of the 1958 birth cohort and their offspring. inequalities in height narrowed between the two generations from 2 to 3 cm at the age of 7 years in cohort members to less than 1 cm among their children (born approximately 26 years later).16 it would be interesting in further analyses to explore the role of grandparental education in the socioeconomic patterning of the growth trajectories in alspac, and as the data become available to explore how socioeconomic patterning of height trajectories differs between the alspac participants and their parents and offspring. the absolute difference we observe in the alspac participants is relatively small, and is smaller than the differences observed in previous generations in the uk, including the 1958 birth cohort.16 it is possible, therefore, that socioeconomic differentials in height will decrease further over time as living conditions improve, although a review utilising data from 10 european countries showed persisting height inequalities, with no narrowing of the height differential between educational groups in younger cohorts compared with previous generations.1 height differences, probably at least partly through their associations with sep, have been shown to result in disparities in many other aspects of life, with taller people experiencing, for example, increased job success and reporting greater life satisfaction.31 32 the main strengths of this study are its uniqueness in examining longitudinal height trajectories from birth to age 10 years in a large sample size with a large number of repeat measurements. our modelling approach has allowed us to examine trajectories of height across childhood, taking account of the different timings and numbers of measurements between children. by using a linear spline model, we have been able to simplify the trajectories, while retaining good fit between actual and predicted values. however, we have restricted our analyses to growth up to 10 years of age. at this age very few children will have gone through puberty. among those with relevant data (information provided by parents using tanner 's puberty questions) the vast majority of the cohort were prepubertal at age 10 years (82% at tanner stages 1 or 2 ; 96% at stages 13). when further data are available, it will be interesting to see whether and how height inequalities alter as the whole cohort passes through puberty. we were able to include the vast majority of participants in our study and those included did not differ with respect to sep from those excluded due to missing data. however, in our data we have fewer measurements of height for lower sep children. when analyses were restricted to only participants who had at least nine measurements the findings were essentially identical to those presented here. our study has focused on height, but previous research has suggested that leg length is the component of total height that is most socially patterned.20 we do not have serial measurements of leg length in this study and are unaware of any other studies that have such data. to conclude, our results suggest that socioeconomic differentials in length / height in contemporary high - income populations are already present at birth, suggesting that genetic, epigenetic or intrauterine characteristics influenced by maternal behaviours may be important. there are no socioeconomic differences in growth in early infancy, but slightly faster growth in later childhood results in minimal widening of the height inequality. further research would be valuable to explore the mechanisms that underlie the differentials we have identified. despite improvements in living standards and increasing secular trends in height, socioeconomic inequalities in height persist, with individuals from more deprived backgrounds tending to be shorter. what is not known is at what age such socioeconomic inequalities in height emerge, and whether and how they change across childhood. our results suggest that socioeconomic differentials in length / height in contemporary high - income populations are present at birth, with some small widening of inequality in later childhood, suggesting that they are driven by genetic, epigenetic or maternal intrauterine characteristics. | backgroundsocioeconomic differentials in adult height are frequently observed, but the age at which these inequalities emerge and the patterns they follow through childhood are unknown.subjects and methodsusing data from the avon longitudinal study of parents and children (alspac), height trajectories from birth to 10 years (n=12366) were modelled. individual trajectories were estimated using mixed - effects models. differences in trajectories by socioeconomic position (sep) were investigated.resultsthere was a clear gradient in birth length across categories of maternal education ; average birth length in boys was 0.41 cm lower in the lowest maternal education category compared with the highest, which is 0.9% of the average birth length for the highest sep category (equivalent results for girls 0.65 cm, 1.3%). socioeconomic differences in childhood growth were small, and only resulted in minimal widening of the height inequality with increasing age. by the age of 10 years, the mean difference between children in the lowest and highest maternal education categories was 1.4 cm for boys and 1.7 cm for girls ; similar proportionate differences to those seen at birth (1.0% for boys and 1.2% for girls). patterns were the same when father 's education or household occupational social class were used to measure sep.conclusionsthe socioeconomic differential in height during childhood in this cohort of children born in the uk in the 1990s arises largely through inequalities in birth length, with small increases in the inequality from differences in growth in later childhood. |
lichen sclerosus (ls) is a chronic inflammatory skin disease of unknown etiology. at present, the typical first line of treatment for ls includes various topical steroids to control inflammation. its epidemiology is probably underestimated, but it has been calculated to have a prevalence of 0.10.3% among all patients referred to a community - based dermatology department. although the underlying cause is unknown, researchers have linked the disease to autoimmune mechanisms. a recent study analyzed serum autoantibody profiles in patients with ls versus healthy controls. this study found the presence of circulating autoantibodies to a specific skin protein, extracellular matrix protein 1 (ecm1), in most patients with ls. they concluded that ecm1 is a plausible target antigen for autoimmune mechanisms observed in ls pathology. this is the main rationale for the off - label use of ketamine infusions for the treatment of neuropathic pain. however, ketamine exhibits extensive polypharmacological effects, which has led it to be regarded as it also exhibits robust immunoinhibitory effects that have been studied mainly in the context of developing a novel antidepressant. entourage effect that could be uniquely beneficial for the treatment of painful inflammatory conditions with a possible autoimmune component such as ls. a 66-year - old female patient presented to the florida spine institute (fsi) as a referral from her dermatologist for the treatment of chronic pain in her bilateral lower limbs. she had begun having pain in her lower limbs approximately 8 years prior to her first visit to the fsi. the patient had developed a rash that encompassed large portions of her lower limbs and anus. the patient had been diagnosed with ls et atrophicus at the department of dermatology and cutaneous surgery of the university of south florida (tampa, fla., there, the patient had been treated with steroids, with little improvement. over time, the patient had developed significant skin scarring, skin atrophy, and edema. her pain was treated with a common neuropathic pain agent (gabapentin) and an opiate (hydrocodone). this pain regimen gave the patient mild relief, but her ability to perform physical activities was only minimally affected. the patient stated that pain, weakness, and edema in her lower limbs were her main complaints and the reason for her intolerance of physical activity. the pain was described as constant burning with deep aching pain and sporadic sharp pains. on a visual analog scale (vas), she stated that if her pain were controlled, she would be able to increase her physical activity and performance. on physical exam, the patient 's skin revealed skin atrophy, scarring, and plaques consistent with ls et atrophicus located in both legs, thighs, buttocks, and the anus (fig. her lower limbs also demonstrated edema, color changes, and temperature changes in the distal parts of the legs (when compared to the hands and upper thighs). the major muscle groups in her lower limbs were considerably weak, but she had a manual muscle strength grade of at least 4 out of 5. thus, the patient was treated as a case of possible neuropathic pain, which was likely a complication of the patient 's ls. the patient 's pain started after months of having her rash and escalated to a severity at which her ambulation and ability to perform her activities of daily living were significantly affected. in addition, the patient 's sensory, vasomotor, and motor dysfunction as well as edema started months after the rash had started. at the time of her first visit, methadone was chosen because, in addition to being an opioid analgesic, it acts as a mild nmda receptor antagonist, a novel target for neuropathic pain. the patient 's pain improved over the next month (to vas 5/10). despite the improvement in the patient 's pain, the main goal for the patient and treating physician was ultimately to discontinue opiate medications while maintaining adequate pain control it has the ability to provide conscious sedation, but, more importantly, it is a potent nmda receptor antagonist. in may 2014, the patient was started on the ketamine infusion protocol set forth by the fsi. on day 1, the patient was infused for approximately 4 h with 200 mg ketamine, 200 mg lidocaine, and 6 mg midazolam in a 100-ml bag of saline. the ketamine dosage was increased by 200 mg each day until 800 mg was reached. the overall clinical course of the patient in this case study is plotted in fig. 2. four days following the last ketamine infusion during a follow - up visit, the patient reported improvement in her pain levels (to vas 4/10). one month after the ketamine infusions, the patient was seen for another follow - up. areas that had previously been causing much discomfort due to sclerotic plaques and lesions showed drastic improvement. in addition, the patient reported decreased allodynia, hyperpathia, and hypersensitivity in her lower limbs. the patient volunteered to undergo 2 ketamine booster infusions over the course of 2 days. in july 2014, her pain had significantly reduced (to vas 1/10) from her pre - ketamine infusion state, and her ls rash and edema continued to be significantly reduced (fig. the patient 's ability to perform activities of daily living and ambulation were greatly improved. over the course of 8 months, she received ketamine booster infusions every 23 months. her pain continues to be controlled without the use of opiates (vas 1/10). the patient 's rashes, edema, allodynia, and hypersensitivity continue to be significantly improved compared to the pre - ketamine infusion state. she has undergone physical therapy with significant improvement in ambulation ability and exercise / physical activity tolerance. the patient claims to be at a level of pain control and physical activity similar to her premorbid state. a 66-year - old female patient presented to the florida spine institute (fsi) as a referral from her dermatologist for the treatment of chronic pain in her bilateral lower limbs. she had begun having pain in her lower limbs approximately 8 years prior to her first visit to the fsi. the patient had developed a rash that encompassed large portions of her lower limbs and anus. the patient had been diagnosed with ls et atrophicus at the department of dermatology and cutaneous surgery of the university of south florida (tampa, fla., there, the patient had been treated with steroids, with little improvement. over time, the patient had developed significant skin scarring, skin atrophy, and edema. her pain was treated with a common neuropathic pain agent (gabapentin) and an opiate (hydrocodone). this pain regimen gave the patient mild relief, but her ability to perform physical activities was only minimally affected. the patient stated that pain, weakness, and edema in her lower limbs were her main complaints and the reason for her intolerance of physical activity. the pain was described as constant burning with deep aching pain and sporadic sharp pains. on a visual analog scale (vas), she stated that if her pain were controlled, she would be able to increase her physical activity and performance. on physical exam, the patient 's skin revealed skin atrophy, scarring, and plaques consistent with ls et atrophicus located in both legs, thighs, buttocks, and the anus (fig. her lower limbs also demonstrated edema, color changes, and temperature changes in the distal parts of the legs (when compared to the hands and upper thighs). the major muscle groups in her lower limbs were considerably weak, but she had a manual muscle strength grade of at least 4 out of 5. thus, the patient was treated as a case of possible neuropathic pain, which was likely a complication of the patient 's ls. the patient 's pain started after months of having her rash and escalated to a severity at which her ambulation and ability to perform her activities of daily living were significantly affected. in addition, the patient 's sensory, vasomotor, and motor dysfunction as well as edema started months after the rash had started. at the time of her first visit, methadone was chosen because, in addition to being an opioid analgesic, it acts as a mild nmda receptor antagonist, a novel target for neuropathic pain. the patient 's pain improved over the next month (to vas 5/10). despite the improvement in the patient 's pain, the main goal for the patient and treating physician was ultimately to discontinue opiate medications while maintaining adequate pain control it has the ability to provide conscious sedation, but, more importantly, it is a potent nmda receptor antagonist. in may 2014, the patient was started on the ketamine infusion protocol set forth by the fsi. on day 1, the patient was infused for approximately 4 h with 200 mg ketamine, 200 mg lidocaine, and 6 mg midazolam in a 100-ml bag of saline. the ketamine dosage was increased by 200 mg each day until 800 mg was reached. the overall clinical course of the patient in this case study is plotted in fig. 2. four days following the last ketamine infusion during a follow - up visit, the patient reported improvement in her pain levels (to vas 4/10). one month after the ketamine infusions, the patient was seen for another follow - up. areas that had previously been causing much discomfort due to sclerotic plaques and lesions showed drastic improvement. in addition, the patient reported decreased allodynia, hyperpathia, and hypersensitivity in her lower limbs. the patient volunteered to undergo 2 ketamine booster infusions over the course of 2 days. in july 2014, her pain had significantly reduced (to vas 1/10) from her pre - ketamine infusion state, and her ls rash and edema continued to be significantly reduced (fig. the patient 's ability to perform activities of daily living and ambulation were greatly improved. over the course of 8 months, she received ketamine booster infusions every 23 months. her pain continues to be controlled without the use of opiates (vas 1/10). the patient 's rashes, edema, allodynia, and hypersensitivity continue to be significantly improved compared to the pre - ketamine infusion state. she has undergone physical therapy with significant improvement in ambulation ability and exercise / physical activity tolerance. the patient claims to be at a level of pain control and physical activity similar to her premorbid state. a recent case - control study suggested that ls is characterized by aberrant autoimmune function. that study compared a total of 190 women with adult - onset ls with healthy controls. the authors found that patients with ls more frequently had autoimmune disorders (28 vs. 9% ; p < 0.001), supporting an autoimmune association with ls. thus, pharmacotherapies that address inflammation and autoimmune responses should be particularly useful for the treatment of ls. to our knowledge, there have been no other reports on the effects of ketamine in patients with ls until now. in our case study, we report that the pain and dermatological pathology associated with ls in one patient were significantly reduced following ketamine infusion therapy that was employed for neuropathic pain. even though this is an extremely rare case, we felt that these serendipitous observations related to the effects of ketamine in ls might spark further research to better understand its etiology. moreover, this case study provides evidence that supports intravenous ketamine as an alternative treatment option for patients who can not manage their ls with topical steroids. that intravenous ketamine infusions drastically improved symptoms of ls in one patient is scientifically interesting, in part due to its debatable etiology. ketamine is known to exert analgesic, anesthetic, bronchodilatory, immunomodulatory, neuroprotective, and antidepressant effects, probably via multiple pharmacological modalities. although extensive efforts are already underway to repurpose ketamine for medical use beyond anesthesia, no other studies have looked at ls. as described in this case study, intravenous ketamine infusion not only provided rapid and sufficient analgesia but it also produced drastic and unexpected improvements in lesions associated with severe ls. we believe that these therapeutic effects could be due to not yet fully understood immunomodulatory properties of ketamine. nonetheless, the results achieved for this patient merit further investigation. moreover, we hope that future studies reveal the importance of ketamine 's polypharmacological effects in the context of various disease states, rather than fearing the drug 's nonspecificity. | a patient reported to the florida spine institute (clearwater, fla., usa) with severe lichen sclerosus of the anogenital region and legs. the patient 's pain presentation was neuropathic with hypersensitivity, allodynia, swelling, and weakness. the patient had failed multiple pain management modalities including opioid therapy, anticonvulsants, and antidepressants. the patient completed a standard intravenous ketamine infusion regimen developed at the florida spine institute and reported complete abolishment of her pain syndrome. for the first time, we report that ketamine infusions also dramatically improved a patient 's lichen sclerosus. that ketamine is known to have immunomodulatory properties, and given the clinical observations described in this case report, suggests that ketamine should be explored as a possible new therapeutic option for managing lichen sclerosus, especially in cases that are refractory to conventional therapies. |
a 19-year - old male motorcycle rider was hit by a car and complained of pain in the left wrist. a physical examination revealed tenderness of the left wrist and hypesthesia of the 4th and 5th finger. the plain radiographs revealed an intraarticular comminuted fracture of the distal radius with posterolateral displacement of the distal fragment and an ulnar styloid process fracture (fig. a closed reduction and percutaneous k - wires fixation with an external fixator were performed on the 2nd day after the trauma. postoperatively, the patient complained of a continuous tingling sensation at the medial 1/2 of the 4th and 5th fingers and the progressively development of a clawing deformity. at 6 weeks after surgery, the external fixator was removed and the range of motion exercise was started. at 8 weeks postoperatively, ultrasonography and electrophysiologic study were performed due to the continuous ulnar nerve palsy. the ultrasonographic findings showed that the continuity of the ulnar nerve was maintained but there was swelling of the ulnar nerve in the guyon 's canal and fibrosis of the tissues around the nerve. the electrophysiologic study revealed a decrease in the sensory and motor conduction velocity of the ulnar nerve and denervation potential in the intrinsic muscle of the hand. at 12 weeks postoperatively, ulnar nerve exploration, decompression and neurolysis was performed due to the lack of improvement in the neurological symptoms. the intraoperative findings revealed swelling of the ulnar nerve and compression by the adjacent fibrous tissues and adhesion in the guyon 's canal (fig. a 27-year - old male complained of pain in his left wrist after a fall from a height. a physical examination revealed severe swelling and tenderness in the left wrist and hypesthesia in the 4th and 5th finger were observed. the plain radiographs revealed a severe intraarticular comminuted fracture of the left distal radius with an ulnar styloid process fracture (fig. an open reduction was performed on the day of the trauma. a small 2 cm - incision the bone deficiency in the metaphysis was treated with an allogeneous cancellous bone graft, and an external fixator was applied. postoperatively, the patient complained of a tingling sensation in the medial 1/2 of the 4th and 5th finger and clawhand deformity. the ulnar nerve palsy was still present 6 weeks after surgery, which required ultrasonography and an electrophysiologic study. the ultrasonographic findings showed that the continuity of the ulnar nerve was maintained but there was swelling of the nerve in the guyon 's canal. the electrophysiologic study revealed a decrease in the sensory and motor conduction velocity of the ulnar nerve and the denervation potential in the intrinsic muscle of the hand. at 8 weeks postoperatively, ulner nerve exploration, decompression and neurolysis was performed due to the lack of improvement in the neurological symptoms. the intraoperative findings revealed swelling of the ulnar nerve and adhesion by the adjacent fibrous tissues (fig. 4). the neurological symptoms began to improve at the 4th week since the nerve exploration. at 1 year postoperatively, minor numbness was felt in the 4th finger and 5th finger but the clawhand deformity had disappeared. a 19-year - old male motorcycle rider was hit by a car and complained of pain in the left wrist. a physical examination revealed tenderness of the left wrist and hypesthesia of the 4th and 5th finger. the plain radiographs revealed an intraarticular comminuted fracture of the distal radius with posterolateral displacement of the distal fragment and an ulnar styloid process fracture (fig. a closed reduction and percutaneous k - wires fixation with an external fixator were performed on the 2nd day after the trauma. postoperatively, the patient complained of a continuous tingling sensation at the medial 1/2 of the 4th and 5th fingers and the progressively development of a clawing deformity. at 6 weeks after surgery, the external fixator was removed and the range of motion exercise was started. at 8 weeks postoperatively, ultrasonography and electrophysiologic study were performed due to the continuous ulnar nerve palsy. the ultrasonographic findings showed that the continuity of the ulnar nerve was maintained but there was swelling of the ulnar nerve in the guyon 's canal and fibrosis of the tissues around the nerve. the electrophysiologic study revealed a decrease in the sensory and motor conduction velocity of the ulnar nerve and denervation potential in the intrinsic muscle of the hand. at 12 weeks postoperatively, ulnar nerve exploration, decompression and neurolysis was performed due to the lack of improvement in the neurological symptoms. the intraoperative findings revealed swelling of the ulnar nerve and compression by the adjacent fibrous tissues and adhesion in the guyon 's canal (fig. a 27-year - old male complained of pain in his left wrist after a fall from a height. a physical examination revealed severe swelling and tenderness in the left wrist and hypesthesia in the 4th and 5th finger were observed. the plain radiographs revealed a severe intraarticular comminuted fracture of the left distal radius with an ulnar styloid process fracture (fig. an open reduction was performed on the day of the trauma. a small 2 cm - incision the bone deficiency in the metaphysis was treated with an allogeneous cancellous bone graft, and an external fixator was applied. postoperatively, the patient complained of a tingling sensation in the medial 1/2 of the 4th and 5th finger and clawhand deformity. the ulnar nerve palsy was still present 6 weeks after surgery, which required ultrasonography and an electrophysiologic study. the ultrasonographic findings showed that the continuity of the ulnar nerve was maintained but there was swelling of the nerve in the guyon 's canal. the electrophysiologic study revealed a decrease in the sensory and motor conduction velocity of the ulnar nerve and the denervation potential in the intrinsic muscle of the hand. at 8 weeks postoperatively, ulner nerve exploration, decompression and neurolysis was performed due to the lack of improvement in the neurological symptoms. the intraoperative findings revealed swelling of the ulnar nerve and adhesion by the adjacent fibrous tissues (fig. 4). the neurological symptoms began to improve at the 4th week since the nerve exploration. at 1 year postoperatively, minor numbness was felt in the 4th finger and 5th finger but the clawhand deformity had disappeared. a median nerve injury following a fracture of the distal radius is a relatively common neurological complication that is found in 2 - 7% of cases and is associated with high energy trauma. carpal tunnel syndrome after a fracture of the distal radius is caused by an increase in pressure on the carpal tunnel due to swelling or bleeding into the carpal tunnel in most cases.1 - 3) in contrast, ulnar nerve injury is an extremely rare event with only 30 cases reported worldwide.2 - 10) according to bacorn and kurtzke4) an ulnar nerve injury was found in only 1 out of 2,000 patients (0.05%) with a fracture of the distal radius. however, soong and ring3) suggested a higher frequency of ulnar nerve injuries, showing that 5 of their study population treated for a distal radial fracture within a 2 year period presented with an ulnar nerve injury. in our experience, the incidence of ulnar nerve injury was similar to that reported by soong and ring3). we encountered 4 cases of ulnar nerve injury subsequent to a fracture of the distal radius. two of them were combined with a closed fracture and 2 of them were accompanied with open fracture involving severe soft tissue damage, such as a degloving injury. we postulate that the higher incidence of severe displacement, comminuted fractures, intra - articular fractures and open fractures are due to an increase in the number of high - energy injuries caused by motor vehicle accidents and industrial accidents. the cases reported thus far have also shown that an ulnar nerve injury mostly affects young people as a result of a high - energy injury caused by traffic accidents, falls from a height and sports injuries, and is common in patients with severe displacement and comminution, combined a distal ulnar fracture and open fracture. the 2 young patients in this study resulted from a high - energy injury caused by a motorcycle accident and a fall from a height. in them, the displacement or intra - articular comminution was severe and combined with an ulnar styloid process fracture. an ulnar nerve injury is caused primarily by direct contusion, traction and nerve compression due to fibrosis of the adjacent tissues or swelling, intraneural fibrosis, and rarely by laceration in a distal radial fracture.3,5 - 7) zoega7) reported from their intraoperative findings and a cadaver study that an ulnar nerve injury can occur as a result of contusion caused by a posterior and radial displacement of the distal radius fragment. clark and spencer5) reported that compression by thick fibrous tissues around the ulnar nerve resulted in progressive ulnar nerve palsy and demonstrated that a permanent ulnar nerve injury could be avoided even when it was displaced or extended in a fracture of the distal radius because it has a higher mobility and extensibility than the median nerve. according to soong and ring3) the ulnar nerve is more vulnerable to traction and contusion than to compression because the ulnar nerve is located outside the carpal tunnel and is fixed in the guyon 's canal. considering that our cases presented with swelling as a result of a contusion of the ulnar nerve as well as compression and adhesion by the adjacent tissues, it is believed that traction, contusion and compression by the adjacent fibrous tissues are the main causes of an ulnar nerve injury in a closed fracture with laceration being a rare cause. however, it is not believed that an ulnar nerve injury is caused by an open fracture involving extensive damage to the soft tissues. with regard to the treatment of an ulnar nerve injury after a distal radial fracture, there is no disagreement regarding the need for early nerve exploration when combined with an open fracture or acute carpal tunnel syndrome. however, there is some controversy regarding the treatment of an ulnar nerve injury in a closed fracture. some authors reported that careful observation can lead to recovery,3,7) while vance and gelberman6) reported that rapid recovery could be obtained with early decompression. in addition, bourrel and ferro1) suggested that neurolysis should be performed when there are no signs of neural recovery in the 6 months after the trauma. in our cases, the neurological symptoms did not disappear after reduction and fixation of the fracture, and no recovery was observed after 2 - 3 months of trauma. ultrasonography and an electrophysiologic study were performed, and the location and pattern of the ulnar nerve injury were observed. however, median and ulnar nerve injuries should be considered when a high - energy injury, wide displacement, or comminution is accompanied. considering that an ulnar nerve injury combined with a closed fracture of the distal radius results from a contusion or compression by the adjacent tissues in most cases, the treatment options should be chosen depending on the degree of recovery after an anatomical reduction and fixation of the fracture. when signs of recovery are not observed during the observations, nerve exploration after ultrasonography and an electrophysiologic study is recommended to identify the pattern, location and cause of the injury as well as the possibility of regeneration. | ulnar nerve palsy subsequent to a fracture of the distal radius is extremely rare compared to a median nerve injury. the lesion tends to occur in younger patents with a high - energy mechanism of injury and a severe injury pattern consisting of wide displacement, comminution, combined distal ulnar fracture and open fracture. the mechanism of injury can contribute to a direct contusion and traction, compression secondary to prolonged edema and tissue fibrosis, intraneural fibrosis and laceration. we report 2 cases of progressive ulnar nerve palsy subsequent to closed fractures of the distal radius. the neurological symptoms recovered in all cases who underwent nerve decompression and neurolysis at 2 or 3 months after the trauma. it is recommended that cases with high - energy, widely displaced or comminuted fractures of the distal radius be evaluated carefully for ulnar nerve as well as median nerve injury. |
systems biology is a discipline that examines the organizational relationships between biological structures in an organism, and thus it covers a very broad scope of biology from the macroscopic to the microscopic biology of organisms, ranging from mankind and other animals, plants, and microorganisms to their organs, tissues, cells, and subcellular organelles and structures through to molecular structures and the interactions between different molecular structures and systems. this broad scope of systems biology demands many different types of instruments for different aspects of imaging and signal detection. instruments and devices such as cameras and x - ray imaging systems are used for whole organism imaging in man and larger animals [1, 2 ], ivis imaging system has been developed for small animal imaging in vivo, ultrasonic devices are applied for the organ and tissue imaging, the ultraview living cell imager has been developed for cells and subcellular imaging, and various types of biochip have been designed for the analysis of the tissue, cells, and molecular structures. except for ultrasonic devices, all of the above detection - analysis systems require excellent optical design for the best performance of their particular objectives. one important type of optical system useful for the analysis of subcellular and molecular structures is the confocal chip scanner with a low background noise. advanced biochip analysis platforms [7, 8 ] analyze the content of a particular microarray slide chip, including gene chips, protein chips, cell chips, tissue chips, and others. many researchers work with small molecule biochips, used for the detection of dna variations by dna hybridization, of proteins by the immunoreaction of proteins, and other more specialist analyses such as for dna sequencing, ligase chain reaction (lcr), and others. some important biochip detection systems [9, 10 ] have been developed, such as the fluorescence microscope imaging system and the laser confocal scanner. in all these detection systems, optical design has played a keyrole in obtaining high - clarity images of microscopic objects, and the optical objective is crucial to the detection sensitivity, the resolving power, and the working distance of the detection system. usually, the bigger the numerical aperture of the objective, the higher its resolving power, and the higher the power of collecting the fluorescence signals bound on the tested object, however to achieve this structure, the working distance of the objective must also be shorter. for example, when the conventional microscope objective has an na > 0.6, then its working distance wd is usually smaller than 1 mm. for example, the zeiss plan objective 440050, magnification 40, na=0.65, wd=0.6 mm, and the nikon plan objective model 40, na=0.65, and wd=0.57 mm. in this paper, a novel confocal optical system design and a dual laser confocal scanner are described in which the materials and lenses employed are as small as practicable ; the optical design and the potential pitfalls of an objective with a long working distance and a high numerical aperture have been considered and resolved for application to biochips. the collecting fluorescence has very high efficiency, and generally the instrument has excellent resolving power and an excellent signal - to - noise ratio. the design of a confocal optical system with high - performance detection of biological sample slides and biochips centers around the image quality, including the consideration of the resolution, zoom, aberration, and optical transfer functions, each of which can be optimized by using rays tracing calculations. in the process of optical design, all parameters of the optical system structure, including the surface curvature radius of each lens, the thickness of the lenses, the transmitted materials used, the separation distances, and the surface apertures can be varied to obtain an excellent optical specificity and image quality. the functional relationships between the specificity, image quality, and structural parameters of the system can be described as follows : (1)1(1,,i)=1k(1,,i)=k, where i and k are two natural numbers, 1k are various aberrations of the optical specificity and the image quality, 1k are functions of the specificity, the image quality, and the structural parameters of system, 1i correspond to all the structural parameters of system. based on the polynomial expansion and the minimum binary iterative method, when all structural parameters 1i are modified repeatedly, an approximate minimum aberration will be obtained in (1). applying the above optical design method, an optimum confocal optical system structure for the detection of biochips the objective system is a combination of seven lenses including two doublets and uses only three glass materials, zk7, zk11, and zf2. the objective system has a high numerical aperture of 0.72 for collecting the signal from the object, a focal length of 13.06 mm, and a front focal length of 3.22 mm to provide a working distance of approximately 3.0 mm. there is a parallel ray path between the objective and the magnifying lenses, to which it is convenient to also add the filters and the dichroic mirror for the incident laser when building the confocal scanning system. the optical structure parameters of confocal scanning system were optimized in figure 1, where there are an objective and a magnifying lenses, the objective consists of seven lenses, the magnifying lenses consists of five lenses, there is a parallel beam between the objective and magnifying lenses. a configuration of the objective collecting fluorescence is shown in figure 2, where the distance from the focal plane to the front surface of first lens of objective is z, r is the radius of effective aperture of the objective, is the half of aperture angle. the emission fluorescence (em - fluor) of molecule bound on biochips is ideally a spherical wave, and the fluorescence bound on the biochip collected by the objective as shown in figure 2 is described approximately by the formula (2)i(z)k02z2r2, where k0 is a constant, z is the distance from the center of source in the focal plane to the first surface of objective, r is the radius of effective aperture of the objective. when biochips are placed at the focal plane of the objective, and the objective has a numerical apture na = nsin, where n is the refractive index, then the formula (2) can be simplified to formula (3)i(z)1n2k02na2(1+na2+na4+). formula (3) indicates that the intensity of the collecting fluorescence of molecules on biochips has a direct ratio to the square of the higher power of the na. by developing a structure form using formula (3), we created a confocal optical system with a numerical aperture of 0.72, and an efficiency of collected fluorescence of more than 2-fold better than that of other commercial confocal biochip scanners [9, 10 ] whose objective has a smaller numerical aperture than 0.5. in a confocal scanning system the smaller the laser scanning spot of optical system, the higher the scanning resolution power of the confocal system. the scanning spot diagrams are referenced to the real chief ray as shown in figure 3. this option allows selection of two other reference points, the centroid and the middle. the middle is defined so that the maximum ray errors are equal in both the x- and the y - directions. when a laser with a real beam diameter of about 1 mm is imaged by the objective shown above in figure 1, then the spot diagram on the focal plane is as shown in figure 3, where the scale is 0.4 m, the spot diameter on the focal plane is smaller than 0.4 m, which is an ideal scanning probe beam with a more high resolving power of < 0.5 m. the optical speciality and the aberration of the imaging system of our novel optical structure can be analyzed using the optical design software zemax - ee. for the spot diagram shown in figure 3, the root mean square (rms) radius on the focal plane is 0.105 m, and the geometric radius on the focal plane is 0.183 m, which corresponds to a confocal optical system with a scanning resolution power smaller than 0.4 m. the optical path difference (opd) is a scalar quantity and it is identical to those for ray aberration fans at the tangential and sagittal directions px and py, respectively. the data plotted in figure 4(a) is the optical path difference of the system in figure 1, which is the difference between the optical path length of the ray and the optical path length of the chief ray. the vertical axis scale of graph is one wave, while the opd maximum of system in figure 1 is smaller than 5 waves. in figure 4(a), there is a small optical path difference among 3 wavelengths 570 nm, 620 nm, and 670 nm, but the maximum optical path difference is smaller than 3 waves, which is lower than a normal visible light imaging system of 5 waves and can be used for confocal scanning system very well. the encircled energy diagram is the percentage of total energy enclosed as a function of distance from either the chief ray or the centroid at the image of a point object, while the diffraction limit curve is for the aberration - free encircled energy computed on - axis. the encircled energy diagram of the system is shown in figure 4(b), where the horizontal coordinate is the radius, and the vertical coordinate is the normalized fraction of the enclosed energy. the encircled energy diagram shows a diffused intensity spot in focal plane of system, the smaller the radius of encircled energy diagram is, the more the fluorescence energy is collected by the pinhole in the focal plane. in figure 4(b), the radius of encircled energy diagram is smaller than 10 m, where the efficiency of fluorescence collected is near to 100%, when a pinhole with a radius of 10 m is set at the focal plane of the magnifying lenses, then the efficiency of collection of the fluorescence of an object is near to the diffraction limit. geometric image analysis is used to model extended sources, to analyze useful resolution, to represent the appearance of imaged objects, and to provide intuition as to image rotation. the diffraction image analysis accounts for the finite pass band and other diffraction - related effects of real optical systems based upon fourier optics. the geometric image analysis of the optical system is shown in figure 5(a), while the diffraction image analysis of the optical system in the object area illuminated by the scanning spot is shown in figure 5(b), both of which have a high efficiency of 100%. in figure 5(a), the geometric image analysis shows a nice roundness of model extended sources, there is good image rotation invariability of system. in figure 5(b), the diffraction image analysis shows a nice uniformity and clear outline of the letter f as an object, there is good image quality of system to the object area illuminated. applying the above optical design parameters, we constructed a new confocal optical scanning system which has been further developed into the advanced confocal scanner specially for biochip application, as illustrated in figure 6, where the objective and magnifying lenses are designed in figure 1, two filters for the dye cy3 and the dye cy5 are bought from chroma corporation, pmt (photomultiplier tube) is bought from hamamatsu corporation, laser 1 is a solid laser with wavelength 532 nm and power 25 mw, laser 2 is a semiconductor laser with wavelength 635 nm and power 25 mw, splitter is a dichroic mirror from chroma corporation, the mirror is machined into an elliptical mirror with a small hole 1 mm in center, xy scanning platform is designed with 2 m moving control precision. pinhole is a small hole with diameter 20 m, a / d electronic card is designed with precision 16 bit and frequency 1 mhz. computer is chosen with piii cpu or higher cpu speed, biochip is developed by capitalbio corporation. it is characterized by an objective with a large numeral aperture of na=0.72, a long working distance of 3.0 mm, and a sensitivity of fluorescence detection of about 0.1 fluors/m. compared to other similar commercial scanners, it has a higher - resolution power and an excellent signal - to - noise ratio. in figure 6, when the laser beam irradiates the biochip on the xy scanning platform from the laser 1 with wavelength 532 nm, or from the laser 2 with wavelength 635 nm, the fluorescence of biological sample on the biochip is induced and collected by pmt. after a / d (analogue / digital) transfer is complete, the fluorescence signal from the biochip is loaded into the computer for digital image processing. when pairs of identical gradient signal biochips from full moon (full moon biosystems, sunnyvale, calif, usa) were labeled with either cy3-tagged or cy5-tagged probes, and dual color fluorescence was detected by using our newly developed confocal scanner and by another commercial confocal scanner (us popular brand s), the features of the scanning image analyses are shown in figures 7(a)7(d), where the right small spot array image is a local area magnifying view for left scanning images. our newly developed confocal scanner shows a high scanning resolution, an excellent contrast, and a signal - to - noise ratio seen in figures 7(a) and 7(c). the detection sensitivity of the new confocal scanner was further illustrated in figure 7(e), where a full moon (full moon biosystems, sunnyvale, calif, usa) normal molecular density biochip was used and the unit of density is the molecule number per the square micron, and the signal is the relative intensity in the range of 0 to 65 535. in figure 7(e), a molecule with a density of 0.071 fluors/m was detected with a signal of 547, where snr (signal - to - noise ratio) is greater than 2, which indicates that the new confocal scanner is with a sensitivity of fluorescence about 0.1 fluors/m. the gene expression analysis is an important method to study the different gene functions of organisms, which is usually performed on high - density biochips with several tens of thousands of probes in a small 20 mm 60 mm area, where dual fluorescent color labels with the dyes cy3 and cy5 can be used to produce a gene expression spectrum. in order to analyze the gene expression of biochips, it is important for the scanning system to possess high scanning resolution, an excellent contrast, and signal - to - noise ratio, over a wide range of signal intensities. the newly developed confocal optical scanning system can be readily used for the gene expression analysis of biochips. the gene expression spectrum for a high - yield variety of cotton as shown in figure 8 was obtained by using the developed confocal scanner. in figure 8, the dual fluorescent color - labeled biochips were hybridized with 15 reference probes at the beginning of first row and with 687 gene reporter probes representing expressed genes from the two cottons. messanger rna from common cotton was labeled with cy3, and mrna from a high - yield cotton was labeled with cy5. the dual fluorescent color - labeled biochips were scanned twice using the new confocal scanner, where one scan was illuminated by the green laser with a wavelength 532 nm to induce fluorescence of cy3, and the second scan was illuminated by the red laser with a wavelength 635 nm to induce the fluorescence of cy5. figure 8(a) is the image of the gene expression spectrum of the common cotton labeled with cy3, and figure 8(b) is the image of the gene expression spectrum of the high - yield cotton labeled with cy5. figure 8(c) is the combined images of the gene expression spectrums from both the high - yield cotton and the common cotton, where there is an obvious color change if there is a difference between the level of gene expression of mrnas labeled by cy3 and by cy5. figure 8(d) is the scatter plot of gene expression to analyze the differences between the gene message labeled with cy3 to message labeled with cy5, where the identical level of expression of the same genes in the two plant varieties distributes along the line direction of 45 degrees with increasing relative intensity of signals. the larger the difference of gene expression between the two varieties, the farther the position of gene in the scatter plot is away from the 45-degree line. in figure 8(d), except for 15-reference probes at row 1, there are 36 genes with a 2-fold signal difference between the common cotton and the high - yield cotton, distributed over a broad range of relative signal intensities, which shows that there are 36 important genes to improve the yield of cotton. the new confocal scanner is also useful for fluorescence detection of the immunoreactions of proteins on chips. we describe here a protein microarray chip for the parallel detection of autoantibodies in the serum of patients with autoimmune diseases, including systemic lupus erythmatosus (sle), mixed connective tissue disease (mctd), sjgren 's syndrome (ss), sjgren 's syndrome a (ssa), sjgren 's syndrome b(ssb), smith (sm), ribonucleoprotein (rnp), scleroderma (scl), systemic sclerosis (ssc), dermotomyositis (dm), double - stranded dna(dsdna), phosphate - buffered saline tween-20(pbst), and polymyositis (pm). purified autoantigens (ssa-52, ssa-60, ssb, sm, rnp-68, scl-70, jo-1, dsdna, centromere b, ribosomal p0, and extracts of hep-2 cells) are immobilized on the gel chip as shown in figure 9(a), where qc quality control, bc blank control, rc reaction control, nc negative control, 1-jo-1, 2-sm, 3-scl-70, 4-cenp - b, 5-dsdna, 6-ssb, 7-ssa-52, 8-extracts of hep-2 cells, 9-ssa-60, 10-ribosomal p0, 11-rnp-68. the protein microarray chip was incubated with 30 l of a five diseases - mixed serum (diluted 1 : 100 with pbst) for 30 minutes at room temperature. after being rinsed and washed one time for 5 minutes with pbst, the chip was incubated with 30 l of cy3-labeled goat antihuman igg antibody for 30 minutes at room temperature. after another rinse and 5-minutes pbst wash, the chip was briefly centrifuged to dry it. the mixed serum of sle, ss, ssc, mctd, dm, and pm positive sera patient was tested on the protein microarray chip, which was then scanned using our confocal scanner. the result of scanning image in figure 9(b) clearly showed that this mixed sera contained anti - jo-1 at row 2 from column 1 to column 3, anti - sm at row 2 from column 4 to column 6, anti - scl-70 at row 2 from column 7 to column 9, anticentromere b at row 3 from column 1 to column 3, anti - dsdna at row 3 from column 4 to column 6, anti - ssb at row 3 from column 7 to column 9, anti - ssa-52 at row 4 from column 1 to column 3, antinuclear antibodies at row 4 from column 7 to column 9, anti - ssa-60 at row 5 from column 1 to column 3, anti - ribosomal p0 at row 5 from column 4 to column 6, and anti - rnp-68 at row 5 from column 7 to column 9. this developed confocal scanner is good for some applications of biochips, such as dna hybridization and immuno - reaction of proteins. the long working distance of the newly developed confocal scanner has a particular advantage for work with biochips enclosed with a hybridization gasket, or conventional microscope slides with thick covers, or even with an uncovered liquid surface. this feature is necessary for observation of real - time (rt) events on the chip surfaces, such as monitoring rt - fluorescence pcr. the high scanning resolution power available with a scanning beam of 0.4 m and with a sensitivity of detected fluorescence of 0.1 fluors/m are both important for obtaining an excellent images of small objects with quite clear definition, high contrast, and with a high signal - to - noise ratio. this improved clarity of the images could be seen when comparing scans of a full moon normal molecular density biochip by using our developed confocal scanner with that of using another commercial confocal scanner (see figure 7). when compared to other common microscope objective designs where the objective of a typical microscope has a numerical aperture of na=0.65 and a working distance smaller than 1 mm (such as the zeiss plan objective 440050, magnification 40, na=0.65, wd=0.6, wd=0.6 mm, and the nikon plan objective model 40, na=0.65, wd=0.6, wd=0.57 mm), it is obvious that the optical design of the newly developed confocal scanning system is highly advanced, with an optimum combination of lenses, including a high numerical aperture of 0.72, a long working distance of 3.0 mm. the use of only 7 lenses and only 3 different kinds of optical glasses of zk7, zk11, and zf2 is also beneficial. the limited number of glasses and lenses reduces the compound aberration in the use of large numbers of lenses and multiple glasses. in addition, these glass materials favor manufacture with small material error and small machining error. furthermore, the developed confocal scanner can be used for scanning cells and tissues slide, and because the high numerical aperture optical design has a very short focal depth 0. 7 m, it is also good for the tomography imaging of a cubic object. | a novel confocal optical system design and a dual laser confocal scanner have been developed to meet the requirements of highly sensitive detection of biomolecules on microarray chips, which is characterized by a long working distance (wd>3.0 mm), high numerical aperture (na=0.72), and only 3 materials and 7 lenses used. this confocal optical system has a high scanning resolution, an excellent contrast and signal - to - noise ratio, and an efficiency of collected fluorescence of more than 2-fold better than that of other commercial confocal biochip scanners. the scanner is as equally good for the molecular imaging detection of enclosed biochips as for the detection of biological samples on a slide surface covered with a cover - slip glass. some applications of gene and protein imagings using the dual laser confocal scanner are described. |
the double - stranded conformation of genomic dna must be unwound to provide single - stranded dna (ssdna) intermediates required for dna replication, recombination, and repair. the ssdna intermediates can adopt various structures like junctions, g - quadruplex, and intramolecular triplex [13 ]. in cells, the unwinding of double - stranded dna (dsdna) is catalyzed by a class of ubiquitous enzymes termed dna helicases. helicases disrupt one or more base pairs within the duplex dna and then translocate vectorially to the next duplex region to repeat the process [59 ]. the helicase activity is cycled by the binding and hydrolysis of an ntp through a number of energetic (conformational) states that have different affinities for ssdna and dsdna. previously, it was reported that almost all helicases appear to function as oligomers (usually dimers or hexamers). oligomerization provides multiple binding sites necessary for dna or rna target recognition, interaction with accessory proteins, and atp binding [5, 6 ]. recg is a well - characterized helicase from escherichia coli that unwinds dna junctions in vitro. it catalyzes the interconversion of forks and junctions [1, 12, 13 ]. it is necessary in cellular processes such as dna replication, recombination, and repair [5, 6 ]. the conversion of a replication fork into a holliday junction requires the simultaneous unwinding of the leading and lagging strands followed by the reannealing of the two parental strands and the annealing of the two nascent strands. there is no information available on the relationship between the structural states of recg and its function. any study to determine the mechanism of recg action must, therefore, be addressing the differences between structural states of inactive and active forms of recg. nucleic acids possess an intrinsic structural polymorphism critical in nucleic acid - nucleic acid, nucleic acid - protein, and nucleic acid - drug interactions. the structures adopted are especially influenced by ionic properties like ion concentration, charge, and size [1416 ]. moreover, living cells contain soluble and insoluble molecules such as proteins, nucleic acids, saccharides, lipids, and metabolites that can alter the stabilities of canonical and noncanonical nucleic acid structures. the total concentration of biomolecules reaches 400 g l in cells, leading to what is referred to as molecular crowding [17, 18 ]. in the crowded intracellular environment, these crowded conditions stabilize the noncanonical dna structures such as triplexes and g - quadruplexes, whereas they destabilize the duplex form [19, 20 ]. elucidation of the interactions between helicases and dna substrates with various structures is a very important step in understanding the mechanism through which helicases bind and unwind dsdna. in this study, the structure and function of recg were investigated under diluted and crowded conditions shedding light on how the structural properties of recg correlate with activity. dna oligonucleotides of high performance liquid chromatography (hplc) purification grade were purchased from hokkaido system science (see table s1 in supplementary materials available online at doi:10.1155/2012/392039). single - strand concentrations of the dna sequences were determined by measuring absorbance at 260 nm at a high temperature using a shimadzu 1700 spectrophotometer connected to a thermoprogrammer. single - strand extinction coefficients were calculated from mononucleotide and dinucleotide data using the nearest neighbor approximation [21, 22 ]. the gene encoding recg was amplified using kod - plus dna polymerase (toyobo) with e. coli bl 21 genomic dna as the template and the following primers : primer - s (5-ggaattccatatgaaaggtcgcctgttagatg-3) and primer - as (5-cccgctcgagtcatgcgttggagtaacgttc-3). the pcr products were inserted into the ndei and xhoi sites of the pet-26b vector (merck). recg was expressed in e. coli strain rosetta2 (de3) at 28c in lb medium supplemented with 30 mg l kanamycin and 34 mg l chloramphenicol. to induce recg expression, isopropyl -d - thiogalactopyranoside (iptg) was added to a final concentration of 1 mm when the optical density of the cells reached approximately 0.6 at 600 nm. the procedure for purification recg was as follows : cells were suspended in 20 mm tris - hcl (ph 8.5), 100 mm nacl, and membranes were disrupted with a sonicator. recg was purified over a histrap hp column using buffer a (20 mm tris (ph 8.0) containing 100 mm nacl) and buffer b (20 mm tris (ph 8.0) containing 2 m nacl) and then a hiload 26/60 superdex 200-pg column (ge healthcare) using buffer a (20 mm tris (ph 8.0) containing 100 mm nacl and 5 mm imidazole) and buffer b (20 mm tris (ph 8.0) containing 500 mm nacl and 1 m imidazole). circular dichroism (cd) experiments were performed on a j-820 spectropolarimeter (jasco) at 4c and 37c in a 0.1-cm path length cuvette. samples of 5 m recg were prepared in 30 mm mes (ph 7.0) and 0.5 mm na2edta containing 100 mm nacl or 100 mm nacl and 1 mm mgcl2 or 5 mm mgcl2, with and without 1 mm atp and 0 wt% or 40 wt% poly(ethylene glycol) with an average molecular mass of 200 (peg 200). the cd spectra shown are the average of at least three scans from 200 to 350 nm. the temperature of the cell holder was regulated by a temperature controller (ptc-348, jasco), and the cuvette - holding chamber was flushed with a constant stream of dry n2 gas to avoid condensation of water on the cuvette exterior. cd melting curves of recg were recorded at 222 nm in the presence of 30 mm mes (ph 7.0) and 0.5 mm na2edta containing 100 mm nacl or 100 mm nacl and 1 mm mgcl2 or 5 mm mgcl2 and 0 wt% or 40 wt% peg 200. to analyze the functional activity of recg, 1 m frayed duplex dna was titrated with successive additions of 50 nm recg and 0.1 mm atp. samples were prepared in the presence of 30 mm mes (ph 7.0) and 0.5 mm na2edta containing 100 mm nacl or 100 mm nacl and 1 mm mgcl2 or 5 mm mgcl2. before measurement, the frayed duplex dna was heated to 95c, gently cooled at a rate of 0.5c min, and incubated at 4c overnight. melting curves of dna structures were obtained by measuring the uv absorbance at 260 nm. samples were prepared in 30 mm mes (ph 7.0) and 0.5 mm na2edta containing 100 mm nacl or 100 mm nacl and 1 mm mgcl2 or 5 mm mgcl2. before measurement, the samples were heated to 95c, gently cooled at a rate of 0.5c min, and incubated at 4c overnight. the melting temperature (tm) values for dna structures were obtained from the uv melting curves as described previously [21, 22 ]. firstly, we used cd spectroscopy to study the structural states of recg with and without atp in the presence of different cations (na, mg, or both na and mg) at 0 wt% and 40 wt% peg 200, a neutral cosolute to study systematically the effects of molecular crowding. recg was expressed in e. coli and purified by using ion exchange chromatography and affinity chromatography followed by dialysis. figure 1(a) shows cd spectra at 4c or 37c of 5 m recg in the presence of 100 mm na with or without atp at 0 wt% peg 200. the cd spectra without atp displayed a positive peak at 198 nm and negative peaks at 208 nm and 220 nm, which are characteristic of an -helix. in the presence of 1 mm atp, the cd spectra of recg had a positive peak at 195 nm and a negative peak at 225 nm. the crystal structure of thermotoga maritime recg reveals three structural domains (figure s1). the largest domain (domain 1) is at n - terminus, which forms a long -helix and a -strand. the c - terminal residues of the protein extend from the end of domain 3 and cross - back to contact domain 1, forming a hook that wraps around the extended -helix, which provides a nucleotide binding site. recently, the structure of e. coli recg was modeled based on the coordinates of thermotoga maritime recg usingswiss - pdb viewer. apart from the missing n - terminal sequences that form a separate fold in the thermotoga maritime recg, the e. coli recg structure is essentially identical. morikawa suggested that the insertion before the helicase core in the recg sequence compared to the thermotoga maritime recg contributes to the specific recognition of the branched dna structure by e. coli recg. the -strand in the insertion domain recognizes the junction through a stacking interaction with several aromatic residues in recg (figure s1). thus, the -helix and the -strand structures observed by the cd measurements likely correspond to the atp binding domain and the dna junction recognition structure, respectively. we also measured the cd spectra of recg as a function of atp concentration from 0 mm to 1 mm under physiologically relevant ionic conditions (100 mm nacl and 1 mm mgcl2) at 37c (figure s2a). the cd spectra of recg without atp displayed a positive peak at 196 nm and negative peaks at 210 nm and 222 nm that remained unchanged at atp concentrations below 0.17 mm. at atp concentrations from 0.34 mm to 1 mm, the cd spectra gradually shifted to a positive peak at 216 nm and a negative peak at 228 nm. figures s2b and s1c show the plots of molar ellipticity at 210 nm and 225 nm, respectively, obtained from cd spectra versus increasing atp concentration. the appearance of biphasic curves at 210 nm and 225 nm further confirms the structural transition. the difference of the transition points at 210 nm and at 225 nm might indicate that the dependency on atp is different between the -helix and the -strand conformations. these cd results clearly indicate that the -helix to -strand transition in recg depends on the atp concentration. we also explored the effect of atp binding on recg under similar ionic conditions and a molecular crowding condition of 40 wt% peg 200 (figure 1(b)). cd spectra displayed positive peaks at 190 nm and 198 nm and negative peaks at 208 nm and 220 nm in the absence of atp. in contrast to the spectra under the diluted conditions, no significant differences were observed in the cd spectra in the presence of 40% peg 200 even after the addition of atp, indicating that -helix structure was dominant. in 100 mm nacl and 1 mm mgcl2 (figures s3a and s3b) or 5 mm mgcl2 (figures s3c and s3d) at 0 wt% or 40 wt% peg 200, we observed that recg without atp folded into -helical conformation, whereas it was converted into the -strand conformation after the addition of atp. this result is identical to the result obtained with 100 mm na only, indicating that under dilute conditions the binding of atp to recg regulates the structural transition from an -helix to -strand independent on the nature of cation. on the other hand, the cell - mimicking molecular crowding condition favored the folding of recg into the -helical conformation even in the presence of atp. previous studies have shown that molecular crowders can stabilize the native state of a protein [29, 30 ], promote a oligomerization [31, 32 ], shift an open - closed equilibrium toward a closed form as a substrate - binding state, and affect a folding rate of protein [3335 ]. protein - folding variants are proposed to have key roles in a number of pathophysiological processes [3640 ]. in view of published results that support the hypothesis that molecular crowding conditions are more relevant to the conditions in cells than dilute conditions, the atp - independence of the recg folding may have biological significance. to investigate in detail the structural changes induced in recg upon atp binding, we explored the thermal stability of recg using cd melting at 222 nm. normalized cd melting curves of 5 m recg in the presence of 100 mm na (figure s4a), 100 mm na and 1 mm mg (figure s4b), or 5 mm mg (figure s4c) in the absence and presence of 1 mm atp were recorded. the estimated values of melting temperature t1/2 (the temperature at which 50% of a protein sample is denatured) are given in table s2. addition of atp stabilized the recg by 3c, 3.5c and 1c in the presence of 100 mm na, 100 mm na and 1 mm mg, or 5 mm mg, respectively. close internal packing of the backbone atoms in -strand structures of recg optimizes van der waals interactions and minimizes energetically unfavorable hydrophobic interactions between nonpolar protein groups and water molecules in the environment. collectively, these factors help to reduce the net free energy of the -strand and thereby increase its stability. our results suggest that recg adopts its active functional structure after atp binding to facilitate the process of unwinding of nucleic acid substrate, because atp bindings play an important role in the functional activity of various proteins [4245 ]. in the absence of atp, recg was stabilized by 2c, 1.0c, and 2.5c in the presence of 100 mm na, 100 mm na and 1 mm mg, or 5 mm mg respectively at 40 wt% peg 200 in comparison to 0 wt% peg 200. furthermore, addition of atp thermally stabilized the recg at 40 wt% peg 200, and recg was maximally stable (t1/2 = 50c) at 40 wt% peg 200 in the presence of atp. folded proteins usually have hydrophobic cores and charged or polar side - chains occupy the solvent - exposed surface. minimizing the number of hydrophobic side chains exposed to water is an important driving force behind the folding process. the strength of hydrogen bonds depends on their environment, thus hydrogen bonds enveloped in a hydrophobic core contribute more than hydrogen - bonds exposed to the aqueous environment. under molecular crowding conditions, low water activity may induce the interdomain rearrangement in recg to allow formation of the -helix containing a hydrophobic core. the -helix form was stabilized by 2c (t1/2 = 50c) at 40 wt% peg 200 than at 0 wt% peg 200 (t1/2 = 48c) in the presence of 5 mm mg. we designed a 62-mer dna oligonucleotide to form a frayed dna duplex containing noncomplementary arms and a stem region (figure 2). the enzyme should then translocate to the dsdna to allow the strand separation with a 35 polarity. the ssdna extension was introduced at the 5 end to stimulate the activity of recg on the strand opposite to that used for the primary contact of recg. we first measured the stability of frayed duplex by recording the uv melting of 1 m dna duplex in the presence of 100 mm na (figure s5a), 100 mm na and 1 mm mg (figure s5b), and 5 mm mg (figure s5c) at 0 wt% or 40 wt% peg 200. the lower temperature transition could be due to the intramolecular interaction of the bases constituting the arm region of the frayed duplex. the higher - temperature transition could be due to the melting of the stem region. in the absence of peg 200, the mid - points of the higher - temperature transitions were 70c, 71c, and 72c in the presence of 100 mm na, 100 mm na and 1 mm mg, and 5 mm mg, respectively. in 40 wt% peg 200, the higher - temperature transitions in the presence of 100 mm na, 100 mm na and 1 mm mg, and 5 mm mg were 65c, 66c, and 66c, respectively. to confirm the higher melting transition due to stem region, we have also recorded the stability of stem region of frayed duplex by recording the uv melting of 1 m dna duplex in the presence of 100 mm na (figure s5a), 100 mm na and 1 mm mg (figure s5b), and 5 mm mg (figure s5c) at 0 wt% or 40 wt% peg 200. we observed single transition for the thermal melting of the stem region of frayed duplex which confirms that upper transition in frayed duplex is due to the intermolecular structure (figure s6). in the absence of peg 200, the mid - points of the higher - temperature transitions were 74c, 74.5c, and 74.5c in the presence of 100 mm na, 100 mm na and 1 mm mg, and 5 mm mg, respectively. in 40 wt% peg 200, the higher - temperature transitions in the presence of 100 mm na, 100 mm na and 1 mm mg, and 5 mm mg were 74c, 67c and 66c, respectively. the decrease in the t1/2 values under molecular crowding conditions is in good agreement with previous reports of dna duplex stabilities in molecular crowders. the cd spectrum of the dna in the presence of 100 mm na without peg 200 was characterized by a positive peak at 275 nm and negative peaks at 211 nm and 247 nm, a spectrum typical of the b - form conformation (figure 3). we then added recg and atp to the dna. at 50 nm recg and 0.1 mm atp, we observed a decrease in intensity of the positive peak at 275 nm and a red shift such that negative peaks were located at 215 nm and 250 nm (figure 3(a)). spectra were recorded after successive additions of recg and atp, and after each addition we observed a decrease in intensity of the positive peak. at 250 nm recg and 0.5 mm atp, there was complete loss in original b - form conformation of the dna, indicating unwinding of the duplex. previous report described the role of recg in the processing of stalled replication forks, and acted by reversing the fork past the damage to create a four - way junction that allows template switching and lesion bypass. it has also been reported that recg unwinds both the leading and lagging strand duplex arms of a three - way junction and the unwinding of these arms was found to be coordinated. in our study, as frayed duplex contains only single - stranded long overhangs at both the terminus, therefore, we propose that recg should bind to the frayed duplex (as it mimics the replication fork) and should convert the duplex into single strands. therefore, to confirm the same we have recorded the cd spectra of single stands constituting the frayed duplex under dilute and molecular crowding conditions (figure s7). the cd spectrum of each single strand of frayed duplex in the presence of 100 mm na, 100 mm na and 1 mm mg, and 5 mm mg with and without peg 200 was characterized by a positive peak at 274 nm and negative peaks at 244 nm. these cd signatures indicate that each single strand of the frayed duplex folds into typical of the b - form conformation due to the formation of intramolecular structure. to better understand the structures of frayed duplex after unwinding by recg, secondary structures of single strands were predicted using m - fold [51, 52 ]. the monomers of frayed duplex showed 3 short complementary stems with a varying number of bases in loops along with short dangling ends at opposite terminus in each strand (figure s8). when cd spectra were recorded after successive additions of recg and atp, and after each addition, we observed a decrease in intensity of the positive peak (figure 3 and figure s9). at 250 nm recg and 0.5 mm atp, there was complete loss in original b - form conformation of the dna, indicating unwinding of the duplex into single strands solely due to the enzymatic activity of recg in the presence of atp. earlier studies indicated that the substrate specificity of recg was critically dependent on the concentrations of atp and mgcl2, and under certain conditions, recg preferentially unwound three strand junctions of dna [1, 11 ]. here, we observed functional activity of recg in the presence of na and absence of mg. next, we recorded the cd spectra in the presence of 100 mm na and 1 mm mg (figure s9a) and in 100 mm na and 5 mm mg (figure s9b) without peg 200. we observed an overall decrease in cd ellipticity at 273 nm that shifted to 283 nm after the successive additions of recg and atp, although the extent of decrease of the positive peak was observed to be less than that observed without mg. these results clearly indicate that the functional activity of recg was not dependent on the nature of the cations. a recent paper showed that the conformational change of the dna - binding domain of hel308 helicase, which is a member of the same superfamily-2 helicase family as recg, is coordinated by the atp binding. as we observed a structural transition from an -helix to -strand upon atp binding under dilute conditions (figure 1(a)) and functional activity under the same conditions, we propose that the -strand structure is the conformation of recg that unwinds our dna substrate under dilute conditions. the -strand structure could function as a helix opener by actively disrupting base pairs of a frayed dna duplex, and this may be the basis of recognition of dna junctions. to evaluate the effect of molecular crowding, we repeated our analysis of the activity of recg in solution containing 40 wt% peg 200. the cd spectra of the dna were measured in 100 mm na (figure 3(b)), 100 mm na and 1 mm mg (figure s9c), and 5 mm mg (figure s9d) in 40 wt% peg 200. of note, the conformation of the frayed duplex is different in 40 wt% peg 200 than in 0 wt% peg 200, as mentioned above stability of the frayed duplex was found to be lower under molecular crowding conditions than under dilute conditions. in 100 mm na in the presence of the crowding agent, the positive peak at 275 nm shifted to 288 nm after the successive addition of 50 nm recg and 0.1 mm atp. this indicates the catalytic activity of recg under molecular crowding conditions is different from that under the dilute conditions. similar decreases in intensities of the positive peak at 275 nm and the negative peaks at 221 nm and 247 nm were observed when recg and atp were added to the frayed duplex in the presence of 100 mm na and either 1 mm mg or 5 mm mg and 40 wt% peg 200 (figures s9c and s9d). these cd results clearly indicate that recg unwound the frayed duplex both under dilute and molecular crowding condition independent on the nature of cations, although it is quite possible that the extent of unwinding of frayed duplex and catalytic efficiency of recg may depend on the nature of cation. in the absence of crowder and in the presence of atp, recg was converted from an -helix form to a -strand form. under the crowding conditions, on the other hand, recg remained in -helix form even when atp was added. the atp binding site of recg is in domains 2 and 3 which both have -helical conformations. it has been reported that the seca helicase, which has homologous structure and function to recg undergoes a transition to an -helix form upon atp binding. the conformational change to -strand form observed in this study may suggest a transition to the active dna - binding form of recg. based on our results, we propose that the -strand form observed under diluted conditions is the active structure for binding and unwinding of dna, whereas the -helix form observed under the crowded conditions is the preactive structure that stably binds atp. the different unwinding activities observed may be due to the interdomain flexibility of recg, which allows substantial conformational changes imparting the ability to recognize more than one dna structure. furthermore, our results can provide significant information to explore and design the small ligands regulating the recg helicase activity with a molecular crowding condition. firstly, the secondary structures of recg were different with and without a molecular crowding agent, peg 200. the structural transition from -helix to -strand observed under the dilute conditions depended solely on the binding of atp and not on the nature of cations present in solution. the molecular crowding conditions favored the folding of recg bound to atp into an -helical structure. secondly, the frayed duplex was unwound by recg under dilute and molecular crowding conditions, indicating that the -strand conformation may be the active structure of recg, with the -helix form the pre - active structure of recg. molecular crowding conditions played a critical role in the functional structure of recg, and our data suggest the reason for the observed differences in the unwinding activity in vivo and in vitro. we are currently working to determine the binding stoichiometry of atp with recg and to quantify functional activity of recg under dilute and molecular crowding conditions on various dna substrates. | in an atp - dependent reaction, the escherichia coli recg helicase unwinds dna junctions in vitro. we present evidence of a unique protein conformational change in the recg helicase from an -helix to a -strand upon an atp binding under dilute conditions using circular dichroism (cd) spectroscopy. in contrast, under molecular crowding conditions, the -helical conformation was stable even upon an atp binding. these distinct conformational behaviors were observed to be independent of na+ and mg2 +. interestingly, cd measurements demonstrated that the spectra of a frayed duplex decreased with increasing of the recg concentration both under dilute and molecular crowding conditions in the presence of atp, suggesting that recg unwound the frayed duplex. our findings raise the possibility that the -helix and -strand forms of recg are a preactive and an active structure with the helicase activity, respectively. |
during disease monitoring, a stem with small, irregular, brown lesions was sampled at a farm in cheonan, chungnam province, in september of 2011. the infected twig was rotted, and sunken cankers and reddish - brown needles were seen on the infected twig (fig. 1). after incubation in a humid condition, white mycelia, having dark brown necrosis, developed around the twig cavity. prior to further analysis the ducc505 isolate was grown on pda and maintained at 25. a 5-mm diameter mycelial plug was cut from the margin of a 5-day - old culture of the isolate, and was placed centrally in an 85-mm petri dish containing pda. the isolate was cultured at 25, and colony characteristics such as color, shape and size were recorded. the colony diameter was measured daily by scoring the average length for a period of 7 days. colonies were whitish, having crenated edges, aerial mycelium on the surface, and with black gregarious fruiting bodies (fig. conidia were 17~22 3.5~4.2 m, fusiform, 4-septate, and straight to slightly curved. the basal cell was of conical shape with an obtuse end, and pale brown in color. the three median cells were brown : the second cell from base was pale brown, the third cell was a darker brown, and the fourth cell was brown. there were 2~3 tubular, apical appendages arising from the apex of the apical cell. the ducc505 isolate grew better on pda than oat meal agar and malt extract agar (fig. the optimum temperature for mycelial growth of the isolate ducc505 on pda was 25 (fig. the isolate ducc505 grew well in a broad range of ph, from 5 to 10 (fig. 3c). these growth properties could be attributed to overcome the ph and low temperature stress in environment. so far, none of fungicides have been registered for the disease control of acanthopanax sp. in the agrochemical use guide book in korea. also, no fungicide has ever been tested for p. ellipsospora isolated from acanthopanax sp. we therefore tested five kinds of fungicides for this study that are commercially available for ascomycete plant pathogens in korea. to understand the agrochemical sensitivity of the ducc505 isolate, we grew it with different concentration of fungicides, and the results are summarized in fig. 3d. in the benomyl and tebuconazol supplemented media, the mycelial growth was completely inhibited at 10 g / ml. this result is similar to pestalotiopsis microspora that is sensitive to tebuconazole. however, the isolate showed relative resistance in all media containing azoxystrobin, dimethomorph and triflumizole. overall, it is suggested that among the five fungicides, benomyl and tebuconazol are the appropriate choice for the control of p. ellipsospora. the ducc505 fungal isolate was grown on pda plates for 5 days at 25. mycelia were harvested by scraping the fungal colonies with a sterile blade. genomic dna was extracted as described by kim., with modifications. from the extracted genomic dna, partial translation elongation factor 1 polymerase chain reaction (pcr) was performed as described previously using the universal primers tef728 (5'-cat cga gaa gtt cga gaa gg-3 ') and tef1 (5'-gcc atc ctt gga gat acc agc-3 '). the pcr products were purified with a high pure pcr purification kit (roche, basel, swiss) and sequencing was carried out by macrogen inc. the nucleotide sequence of partial tef1- gene of the fungal isolate shares 99% (531/536) sequence identity with that of known pestalotiopsis ellipsospora. the tef1- gene sequence of the ducc505 was deposited in genbank dna database under accession number kc534872. 4). thus, based on the molecular and morphological data generated, the isolate was identified as p. ellipsospora. the young twigs were inoculated with a droplet of the ducc505 conidia suspension (1.3 10 conidia / ml), which was prepared from the fungal cultures grown on pda. the inoculated young twigs were incubated in a humid chamber at 25 for 7 days. as rotting progressed, superficial white mycelium and small black acervuli were observed (fig. 5). p. ellipsospora was reisolated from the rotted twig lesion, thus fulfilling koch 's postulates. these results demonstrated that p. ellipsospora ducc505 was able to infect a twig of a. divaricatus. this is the first detailed report describing p. ellipsospora isolated from acanthopanax in korea, and it appears to be the first confirmation proving its pathogenicity on acanthopanax twigs. | acanthopanax divaricatus, a member of the araliaceae family, has been used as an invigorant in traditional korean medicine. during disease monitoring, a stem with small, irregular, brown lesions was sampled at a farm in cheonan in 2011. the symptoms seen were sunken cankers and reddish - brown needles on the infected twig. the isolated fungal colonies were whitish, having crenated edges and aerial mycelium on the surface, and with black gregarious fruiting bodies. the reverse plate was creamy white. conidia were 17~22 3.5~4.2 m, fusiform, 4-septate, and straight to slightly curved. the nucleotide sequence of the partial translation elongation factor 1 alpha gene of the fungal isolate, shares 99% sequence identity with that of known pestalotiopsis ellipsospora. based on the results of the morphological and molecular analyses, the fungal isolate was identified as p. ellipsospora. in korea, this is the first report of canker on a. divaricatus. |
an aromatase inhibitor (ai) showed efficacy that was superior to that of tamoxifen (tam) in large - scale randomized clinical trials in breast cancer patients, and ais are now the most extensively used drugs for postoperative adjuvant therapy for hormone - dependent postmenopausal breast cancer [1, 2 ]. however, on the minus side, manifestation of joint symptoms (joint pain and stiffness) as drug - related adverse reactions of ais has become an important safety issue. joint symptoms have been variously reported to occur at an incidence of 20% to 36% in patients administered ais, and discontinuation of administration of ais is sometimes unavoidable [3, 4 ]. it has been said that a decrease in serum estrogen (e2) is associated with the causation of joint symptoms due to ai administration [510 ]. however, there have been no reports of stringent studies of such an association, and the detailed mechanisms involved in any such association thus remain unclear. in recent years, there have been reports that ais are poorly metabolized in some patients, while in other patients ais do not show efficacy because of genomic polymorphism of the metabolizing enzymes. since march of 2008 we have been monitoring the e2 levels in breast cancer patients who were started on ai therapy, and we reported that some patients show renewed elevation of e2 in spite of being thought to be clinically postmenopausal. the present prospective study was designed with the objective of elucidating whether or not there truly is an association between manifestation of joint symptoms in response to ai administration and a decrease in serum e2. accordingly, patients who were administered an ai were monitored with regard to drug - related adverse reactions, including joint symptoms, their clinical findings, and the e2 level. a total of 159 hormone - dependent postmenopausal breast cancer patients were started on administration of an ai at tokushima breast care clinic during the period from march 2008 through october 2009. the ai treatment consisted of anastrozole, in an oral dose of 1 mg / day. menopause was defined as the state of having undergone bilateral oophorectomy, age of 60 years or older, or an age of under 60 years with amenorrhea for at least 12 months, and serum e2, lh, and fsh levels that satisfy the diagnostic criteria for postmenopause. for each enrolled patient, the following information was elucidated and recorded : the patient 's age at enrollment, age at menarche, number of childbirths, bmi, age at menopause, time interval from menopause until ai administration, presence / absence of therapy prior to ai administration, and clinicopathological findings. in addition, one adverse reaction of ai inhibitors is elevation of serum cholesterol, a lipid metabolism abnormality. for this reason, we carried out measurement of both e2 and total cholesterol in this study. the serum levels of total cholesterol (tc) and e2 were assayed prior to administration of the ai and then at 3, 6, 9, and 12 months after starting the ai therapy. (kyoto, japan), and the serum e2 level was measured by eclia (electrochemiluminescence immunoassay) in this laboratory. the laboratory 's standard values for the postmenopausal levels of those hormones are 130219 mg / dl for tc and 1040 pg / ml for e2 (lower limit of detection : 5 pg / ml). patients who maintained e2 at 5 pg / ml for more than 6 consecutive months following the start of ai administration were classified as rebound cases. patients who showed a transient increase or did not show an initial decrease in e2 after the start of ai administration but who then showed a decrease within 6 months following ai administration were defined as decreased cases. similarly, patients whose serum tc level was 10 iu / ml or more for at least 6 months following the start of ai administration were classified as elevated in addition, the bone density was determined prior to the start of ai administration and then every 4 months thereafter. at each scheduled time of bone density testing the patients were asked whether they experienced drug - related adverse reactions, that is, joint symptoms and hot flashes, which are known to occur at high incidences with ais. the bone density was measured on the basis of the speed of sound (sos) for propagation of an ultrasound pulse through the calcaneal bone. the bone density was expressed as the percent of the sos relative to that of the young adult mean value (% yam) and the percent of the sos relative to that of the age - matched mean value (% age). patients showing a decrease in the bone density of 10% or more that persisted for at least 4 months were classified as decreased cases, while all other patients were classified as drug - related adverse reactions were evaluated in accordance with the common terminology criteria for adverse events (ctcaes) version 4.0. the analyzed population of this study consisted of 104 patients who had not experienced joint symptoms or hot flashes prior to ai administration and were observed and subjected to the above clinical testing for at least 6 months subsequent to the start of ai administration. in addition, patients who had ingested a concomitant medication that could be thought to be related to an adverse reaction were excluded from the analysis of that drug - related adverse reaction. statistical analyses were performed using the chi - square test and trend analysis by logistics regression analysis. a p value of <.05 was defined as representing a significant difference. the design of this study was approved by the ethics committee of the institute of medical science, the university of tokyo, and the university of tokushima (protocol no. it is necessary to acknowledge that a limitation of this study is that the study population was small. the disease stage was stage 0 in 11 patients, stage i in 58 patients, and stage ii in 35 patients, and 25 patients had axillary lymph node metastases. sixty - six patients had not received any treatment prior to ai administration, while 21 patients had been administered tam monotherapy and 10 patients had received chemotherapy alone (table 1). the mean follow - up period for the 104 patients was 14.6 months, and 8 (7.7%) of the patients were classified as e2 rebound cases. in this study, patients who showed continuous e2 elevation even after the start of ai administration and patients who showed no decrease in e2 after the start of ai administration were classified as rebound cases. however, because the sample size of this study was a bit small, these two types of patients were pooled and analyzed as rebound cases. in addition, because the lower limit of detection for e2 was 5 pg / ml, it was impossible to identify patients whose e2 level did not decrease among the patients (32/104 patients ; 30.8%) with a baseline e2 level below that lower limit of detection. the incidences of ai - related adverse reactions were 31.7% (33 patients) for joint symptoms and 18.3% (19 patients) for hot flashes. the severity of the joint symptoms was grade 1 in 22 patients, grade 2 in 7 patients, and grade 3 in 4 patients. chi - square testing for possible correlations between joint symptoms and various background factors found that the incidence of joint symptoms was significantly higher in the patient group aged less than 55 years (p <.05). in addition, the patient group with less than a 5-year interval between menopause and the start of ai therapy showed a tendency to experience more joint symptoms than the patient group with an interval of 10 or more years (p =.056). moreover, none of the patients in the e2 rebound patient group experienced joint symptoms, and the incidence of joint symptoms was significantly lower than in the e2 decreased patient group (p <.05). the tc - elevated patient group showed a significantly higher incidence of joint symptoms compared with the tc - nonelevated patient group (p <.05). in univariate analysis, joint symptoms were found to manifest at a significantly higher incidence in the following patient strata : a preai % yam of 80% or more (p <.05), e2 decreased (p <.05), and tc elevated (p <.01). the time interval between menopause and the start of ai therapy showed a tendency to influence the manifestation of joint symptoms (p =.059). multivariate analysis did not find any significant correlations between the manifestation of joint symptoms and the analyzed patient factors (table 2). chi - square testing and univariate analysis for possible correlations between hot flashes and various background factors found that the incidence of hot flashes was significantly higher in the following patient strata : age of less than 55 years (p <.05, p <.05), menarche at an age of less than 12 years (p <.05, p <.05), no prior therapy (chisquare testing only : p <.01), less than a 5-year interval between menopause and the start of ai therapy (p <.01, p <.01), a preai % yam of 80% or more (p <.05, p <.01), and tc elevated (p <.01, p <.05). multivariate analysis did not find any significant correlations between the manifestation of hot flashes and the analyzed patient factors (table 3). aromatase inhibitors (ais) are currently the drugs of choice for postoperative adjuvant therapy for hormone - dependent postmenopausal breast cancer [1, 2 ]. however, manifestation of joint symptoms (joint pain and stiffness) as drug - related adverse reactions of ais has become an important safety issue [3, 4 ]. a decrease in e2 has been said to be one cause of the joint symptoms associated with ai administration [510 ]. however, the possible mechanism of such causation remains unclear, and there have been no stringent studies of such an association between a decrease in e2 and manifestation of joint symptoms. accordingly, with the aim of elucidating the detailed mechanisms involved in ai - related adverse reactions, the present study was designed and executed by prospectively surveying adverse reactions and measuring the e2 level in postoperative postmenopausal breast cancer patients who were administered an ai (anastrozole). joint symptoms manifested at an incidence of 31.7% in these ai - treated patients, and they interfered with the activities of daily life of approximately 4% of the patients. it has been said that joint symptoms in ai - treated patients tend to occur more readily if the interval between menopause and the start of ai therapy is within 5 years and if the patient 's age is 5059 years. our present study results also show that joint symptoms tended to occur at a higher incidence in younger patients, when the interval between menopause and the start of ai therapy was shorter and when the pre - ai % yam was higher. it can thus be surmised that factors associated with younger patients are more likely to lead to manifestation of joint symptoms. other factors that have been suggested to be involved in the manifestation of joint symptoms include the presence of prior therapy and obesity, but the published literature does not show any consensus in this regard [3, 5 ]. the incidence of hot flashes in this cohort was 18.3%, which was lower than the incidence of joint symptoms. however, hot flashes also tended to manifest more frequently in the same younger patient population as joint symptoms. tc elevation was associated with both joint symptoms and hot flashes, but a decrease in bone density did not show any association with either adverse reaction. these differences might be attributable to the fact that tc elevation is a change that manifests in a short time - frame, whereas a decrease in bone density is a change that occurs over a longer period of time. the e2 level was also analyzed for possible association with the manifestation of ai - related adverse reactions. hot flashes are considered to be a functional change that occurs under sympathetic nerve stimulation due to a decrease in e2, but it was reported that they do not correlate with the e2 value. thus, it has been surmised that hot flashes do not occur as a result of a simple decrease in e2. on the other hand, with regard to the possibility of association of joint symptoms with e2, joint symptoms did not occur in patients with e2 rebound, whereas their manifestation was associated with a decrease in e2. however, multivariate analysis did not show a clear significant difference in relation to a decrease in e2. accordingly, the results indicated the possibility that a decrease in e2 is not directly associated with manifestation of joint symptoms and instead suggested the possibility that a decrease in e2 causes some other secondary change(s) in joints, which is followed by manifestation of joint symptoms. aromatase is present in osteoblasts, synovial cells, and chondrocytes of articular cartilage, and one report surmised that a local decrease in the e2 level in bones and joints is involved in joint symptoms. some studies used sonography and magnetic resonance imaging to objectively investigate the local findings in patients with joint symptoms due to ai therapy [18, 19 ]. it was reported that carpal tunnel syndrome, wrist effusion, tendon sheath enhancement and thickening, and tenosynovial changes were seen in patients with joint symptoms due to ai therapy, findings that suggest the possibility that joint symptoms are caused by secondary organic changes that occur in joints. for the present study, we used the eclia method to measure the e2 levels ; this method has a lower limit of detection of 5 pg / ml. this level of sensitivity can be considered a drawback of this study, since some of the enrolled patients had an e2 level which was too low to detect even before the start of ai administration. a liquid chromatography - tandem mass spectrometry (lc - ms / ms) method has become available in recent years, which permits more sensitive assay of e2 [11, 21 ]. in the future, it will be necessary to prospectively investigate organic changes in joints during ai therapy by using this highly sensitive assay technique to monitor e2 levels and also applying imaging modalities. an additional drawback of our current study is the small number of analyzed cases, and there is a need to perform a future study in a larger cohort. ai - related adverse reactions occurred more readily in younger patients, and the chi - square test and univariate analysis both showed that a decrease in e2 was associated with manifestation of joint symptoms. these results indicate that joint symptoms due to ai therapy are affected by a decrease in e2. however, multivariate analysis did not show any clear association of a decrease in e2 with joint symptoms, suggesting the possibility that a decrease in e2 is indirectly involved in the manifestation of those symptoms. | background. joint symptoms (jss) are problematic adverse drug reactions (adrs) of aromatase inhibitors (ais). involvement of decreased serum estradiol (se) has been suggested. patients and methods. 104 postmenopausal breast cancer patients administered an ai were prospectively investigated regarding various clinical parameters, js and hot flashes as adrs, and the se level. results. js manifested in 31.7% of patients and hot flashes in 18.3%. chi - square testing showed a significantly higher incidence of js in several patient strata : 6 consecutive months, with no js. in chi - square testing, hot flashes showed a significantly higher incidence in patients < 55 years old. conclusion. ai - adrs occurred more readily in younger patients. decreased se may be indirectly involved in js. |
approximately 30% of acute strokes occur in those older than 80 years of age.6)9)24) however, elderly patients tend to have high rates of poor outcomes and in - hospital mortality.1)4)8)13)14)16)17)23)25)26)30) furthermore, family members of elderly patients are often reluctant to accept aggressive treatment. recently, prospective trials have shown the benefit of endovascular treatment (evt) for acute ischemic stroke.5)12)20)22)32) however, some trials included only patients aged 80 or less,20)32) and although trial inclusion criteria had no age limitation, a small proportion of elderly patients over 80 years were included.5)12)22) therefore, the evidence supporting that endovascular treatment can be actively recommended for elderly patients as for younger patients is not sufficient. to determine whether evt for acute ischemic stroke is also beneficial in acute ischemic stroke patients over 80 years of age who are candidates for evt, outcomes were compared between patients who underwent evt and those who did not, in terms of functional outcomes and rates of symptomatic hemorrhage, in - hospital morbidities, and mortality. this retrospective study was approved by our institutional review board. from a stroke registry (january 2009-march 2014) at our institution, data was collected on patients matching the evt criteria for acute ischemic stroke of our institution : (1) time from onset 120% difference between diffusion weighted imaging and time to peak map), (4) an initial national institute of health stroke scale [nihss ] score of 5, and (5) within time from onset of 4.5 hours, no clinical response to intravenous tpa, as described in previous studies.21)28) patients who had intracranial hemorrhage, large infarctions (infarction size on diffusion mri 1/3 on middle cerebral artery territory), bleeding diathesis, or serious illnesses affecting life expectancy including malignancy were excluded. for 478 patients matching the evt criteria retrieved from the registry, additional criteria were applied : age > 80 years and a prestroke modified rankin scale (mrs) score of 2. a total of 156 patients were selected as study subjects. to ensure agreement with the above mentioned criteria, the clinical and radiologic data of the patients were re - reviewed by two investigators (k.h. and c.h.k.). the stroke mri protocol and the method of measuring diffusion - perfusion mismatch used have been previously described.18) at our institution, intravenous (iv) administration of tissue plasminogen activator (tpa) is considered, regardless of patient age, when indicated. accordingly, of the 156 study subjects, tpa was administered in 95 patients matching iv tpa indication, but none of them showed a clinical response. in the remaining 61 patients, iv tpa was not used, from passing its time window in 58 patients or from a previous history of intracranial hemorrhage in 3 patients. unlike iv tpa administration, evt for patients over 80 years of age was not actively recommended during the study period. information on risks and benefits of evt was provided to first - degree family members (spouse, sons, and daughters) and evt was performed when they agreed. because none of the patients were clearly conscious, they were unable to participate in this decision. of the 156 study subjects, family members of 100 (64.1%, non - evt group) patients refused evt, thus they received standard medical treatment in the stroke unit in accordance with stroke guidelines.19) the remaining 56 (35.9%, evt group) patients underwent evt according to agreement of family members. in line with our evt protocol, we primarily used clot retrieval using a penumbra reperfusion catheter (penumbra, alameda, california) or a solitaire fr stent (ev3, plymouth, minnesota). intra - arterial infusion of fibrinolytics was reserved for cases with a remaining small amount of thrombi or distal migrating emboli. technical details have been previously described.3)21)28) after evt, they were also managed according to the standard treatment guidelines in the stroke unit. basic demographic and clinical data (past history, occlusion site, trial of org 10172 acute stroke treatment [toast ] classification, time from onset, initial nihss score, iv tpa use, hemorrhage, in - hospital mortality, and mrs score [prestroke, and at 3 and 12 months after stroke ]) were retrieved from the stroke registry. according to the criteria proposed by the european cooperative acute stroke study (ecass) group,15) hemorrhage was classified as : hemorrhagic infarction type 1 (hi-1), hemorrhagic infarction type 2 (hi-2), parenchymal hematoma type 1 (ph-1), or parenchymal hematoma type 2 (ph-2). symptomatic hemorrhage was defined as one associated with a 4-point increase in nihss score or a 1-point deterioration in level of consciousness.11) the stroke registry was controlled by two stroke neurologists (h.j.b. and m.k.h.). morbidities, including the need for endotracheal intubation, gastrointestinal bleeding, pneumonia, and urinary tract infection (uti), were obtained by review of medical records (s.p.b. and b.l.). patients were considered to have a hospital - acquired infection, when they had clinical, radiological, and laboratory findings compatible with infection and the organism was confirmed by culture. the angiographic outcomes of patients who underwent evt were reviewed and graded by two neurointerventionists (g.h. and the effects of evt were determined using : (1) good functional outcome (defined as a mrs score of 0 - 2) rates at 3 and 12 months after stroke, (2) symptomatic hemorrhage rates, (3) in - hospital morbidity rates (endotracheal intubation, gastrointestinal bleeding, pneumonia, and uti), and (4) in - hospital mortality rates. statistical analysis was performed using stata (version 13, stata corp lp, college station, tx). the mann - whitney u test was used for bivariate analysis of continuous variables and the or fisher 's exact test for bivariate analysis of nominal factors. the effects of evt were compared using binary logistic regression analysis, adjusted for confounders significantly associated with outcome after ischemic stroke : age (120% difference between diffusion weighted imaging and time to peak map), (4) an initial national institute of health stroke scale [nihss ] score of 5, and (5) within time from onset of 4.5 hours, no clinical response to intravenous tpa, as described in previous studies.21)28) patients who had intracranial hemorrhage, large infarctions (infarction size on diffusion mri 1/3 on middle cerebral artery territory), bleeding diathesis, or serious illnesses affecting life expectancy including malignancy were excluded. for 478 patients matching the evt criteria retrieved from the registry, additional criteria were applied : age > 80 years and a prestroke modified rankin scale (mrs) score of 2. a total of 156 patients were selected as study subjects. to ensure agreement with the above mentioned criteria, the clinical and radiologic data of the patients were re - reviewed by two investigators (k.h. and c.h.k.). the stroke mri protocol and the method of measuring diffusion - perfusion mismatch used have been previously described.18) at our institution, intravenous (iv) administration of tissue plasminogen activator (tpa) is considered, regardless of patient age, when indicated. accordingly, of the 156 study subjects, tpa was administered in 95 patients matching iv tpa indication, but none of them showed a clinical response. in the remaining 61 patients, iv tpa was not used, from passing its time window in 58 patients or from a previous history of intracranial hemorrhage in 3 patients. unlike iv tpa administration, evt for patients over 80 years of age information on risks and benefits of evt was provided to first - degree family members (spouse, sons, and daughters) and evt was performed when they agreed. because none of the patients were clearly conscious, they were unable to participate in this decision. of the 156 study subjects, family members of 100 (64.1%, non - evt group) patients refused evt, thus they received standard medical treatment in the stroke unit in accordance with stroke guidelines.19) the remaining 56 (35.9%, evt group) patients underwent evt according to agreement of family members. in line with our evt protocol, we primarily used clot retrieval using a penumbra reperfusion catheter (penumbra, alameda, california) or a solitaire fr stent (ev3, plymouth, minnesota). intra - arterial infusion of fibrinolytics was reserved for cases with a remaining small amount of thrombi or distal migrating emboli. technical details have been previously described.3)21)28) after evt, they were also managed according to the standard treatment guidelines in the stroke unit. basic demographic and clinical data (past history, occlusion site, trial of org 10172 acute stroke treatment [toast ] classification, time from onset, initial nihss score, iv tpa use, hemorrhage, in - hospital mortality, and mrs score [prestroke, and at 3 and 12 months after stroke ]) were retrieved from the stroke registry. according to the criteria proposed by the european cooperative acute stroke study (ecass) group,15) hemorrhage was classified as : hemorrhagic infarction type 1 (hi-1), hemorrhagic infarction type 2 (hi-2), parenchymal hematoma type 1 (ph-1), or parenchymal hematoma type 2 (ph-2). symptomatic hemorrhage was defined as one associated with a 4-point increase in nihss score or a 1-point deterioration in level of consciousness.11) the stroke registry was controlled by two stroke neurologists (h.j.b. and m.k.h.). hospital morbidities, including the need for endotracheal intubation, gastrointestinal bleeding, pneumonia, and urinary tract infection (uti), were obtained by review of medical records (s.p.b. and b.l.). patients were considered to have a hospital - acquired infection, when they had clinical, radiological, and laboratory findings compatible with infection and the organism was confirmed by culture. the angiographic outcomes of patients who underwent evt were reviewed and graded by two neurointerventionists (g.h. and the effects of evt were determined using : (1) good functional outcome (defined as a mrs score of 0 - 2) rates at 3 and 12 months after stroke, (2) symptomatic hemorrhage rates, (3) in - hospital morbidity rates (endotracheal intubation, gastrointestinal bleeding, pneumonia, and uti), and (4) in - hospital mortality rates. statistical analysis was performed using stata (version 13, stata corp lp, college station, tx). the mann - whitney u test was used for bivariate analysis of continuous variables and the or fisher 's exact test for bivariate analysis of nominal factors. the effects of evt were compared using binary logistic regression analysis, adjusted for confounders significantly associated with outcome after ischemic stroke : age (< 85 or 85 years), time from onset (< 4.5 or 4.5 - 8 hours), initial nihss score (moderate, 5 - 14 or severe, 15), and prestroke mrs score (0 or 1 - 2). " tpa use " was excluded from the logistic regression models, because of a lack of clinical response after tpa use in all cases and correlation with time from onset causing a multicollinearity problem. basic demographic and clinical data were not significantly different in the evt and non - evt groups (table 1). in the evt group, successful recanalization (tici grade 2b) was achieved in 46 patients (82.1%). of these, 25 patients did not achieve good outcomes and thus, futile recanalization, defined as mrs 3 despite successful recanalization, was 54.3%. complications occurred in 3 (5.4%) patients during evt : vessel rupture in 2 patients and intracranial artery dissection in 1 patient. of these, good outcomes were achieved in 2 patients at 3 and 12 months after stroke. recanalization failed in 1 patient with vessel rupture, whose outcome was poor (mrs 5 at 3 and 12 months), although bleeding was stopped by temporary balloon occlusion. at 3 months after stroke, good functional outcomes were achieved in a greater portion of patients in the evt group than in the non - evt group (20 [35.7% ] vs. 11 [11.0% ], adjusted odds ratio [or ] 4.779, 95% confidence interval [ci ] 1.972 - 11.579, p = 0.001 ; table 2 and fig. at 12 months follow - up, this trend continued and the good functional outcome rate was significantly higher in the evt group than in the non - evt groups (20 [35.7% ] versus 14 [14.0% ], adjusted or 3.705, 95% ci 1.574 - 8.722, p = 0.003 ; fig. 1b). in the non - evt group, proportions of patients who were severely disabled or dead (mrs score of 4 - 6) were over 80% and significantly higher than in the evt group at 3 months (83 [83.0% ] vs. 28 [50.0% ], adjusted or 6.950, 95% ci 2.844 - 16.985, p < 0.001) and 12 months (82 [82% ] vs. 32 [57.1% ], adjusted or 4.180, 95% ci 1.837 - 9.510, p = 0.001) after stroke (fig. 7 [12.5% ] versus 29 [29.0% ], adjusted or 0.262, 95% ci 0.098 - 0.703, p = 0.008) and uti (9 [16.1% ] versus 34 [34.0% ], adjusted or 0.256, 95% ci 0.099 - 0.657, p = 0.005) were significantly less frequent in the evt group during admission (table 2). hemorrhage occurred in 40 (25.6%) patients, including 8 (5.1%) with symptomatic hemorrhage during admission. although hemorrhage types (hi, 6 [10.7% ] vs. 11 [11.0% ] ; ph, 9 [16.1% ] vs. 14 [14.0% ], p = 0.941) were similar in the two groups, symptomatic hemorrhage rates were significantly higher in the evt group (6 [10.7% ] vs. 2 [2.0% ], adjusted or 6.859, 95% ci 1.139 - 41.317, p = 0.036). in - hospital mortality rate did not differ significantly between the evt and non - evt groups (7 [12.5% ] vs. 8 [8.0% ], adjusted or 1.380, 95% ci 0.408 - 4.664, p = 0.604). the present study shows that evt for acute ischemic stroke was associated with good functional outcomes at 3 and 12 months after stroke in patients over 80 years of age who were candidates for evt. it also reduced rates of in - hospital morbidities, such as hospital - acquired infections. although symptomatic hemorrhage occurred more frequently after evt, overall functional outcome of elderly patients treated by evt was still better and in - hospital mortality rate was not higher than that of patients not treated with evt. our results indicate that the benefit of evt for acute ischemic stroke is maintained even in patients over 80 years of age. therefore, elderly patients should not be simply excluded from evt candidates because of their age. this is consistent with subgroup analysis of recent prospective trials reporting that no significant interaction was detected between the benefit of evt and age.5)12) in addition to functional outcomes, in - hospital morbidities were also evaluated as a primary outcome of evt, because these often lead to unfavorable or fatal outcome in elderly patients.8)13) we found that pneumonia and uti developed more frequently in the non - evt group. poor clinical condition in patients with acute ischemic stroke is associated with these in - hospital morbidities and, in turn, their developments are predictors for unfavorable outcome and mortality.2)7)10)27)29)31)33)34)35)36) therefore, these findings suggest that a better clinical condition achieved by evt would reduce in - hospital morbidities, contributing to a more favorable outcome in evt groups. especially in elderly patients with acute ischemic stroke, symptomatic hemorrhage is frequent and associated with high rates of in - hospital mortality and poor outcome.1)4)8)13)14)16)17)23)25)26)30) in the current study, however, in - hospital mortality rates were not different between the two study groups. in addition, more than 80% of patients not treated by evt were severely disabled or dead (mrs score of 4 - 6) due to acute stroke or its sequelae at 3 and 12 months after stroke, thus this proportion was found to be much higher than that of the evt group. therefore, these results indicate that the benefit of evt overweighs the risk of symptomatic hemorrhage after evt. in practice, we frequently encounter elderly patients with acute ischemic stroke, but no consensus has been reached on this issue. although our results do not provide definite evidence obtained from a well - designed randomized study, we believe that they could provide some useful information for physicians and patients, until sufficient data become available these include its retrospective nature, single center experience, and its relatively small cohort. in addition, selection bias is likely to have been introduced by involving family members in the treatment decision - making process, and the general management afforded to both evt and non - evt groups may have been less than aggressive due to considerations of patient age. nevertheless, the general management guidelines followed were identical for the evt and non - evt groups and all patients were managed without abandoning treatment, because only patients whose family - members agree on treatment for stroke are permitted to be referred to our tertiary referral hospital. the present study demonstrated that evt improved functional outcomes at 3 and 12 months after stroke in patients over 80 years of age with acute ischemic stroke who were candidates for evt. these results indicate that the benefit of evt for acute ischemic stroke is maintained in elderly patients. in these patients, symptomatic hemorrhage occurred more frequently after evt, but the benefit of evt overweighs the risk of symptomatic hemorrhage, considering the high rate of poor outcomes in patients who did not undergo evt. accordingly, our findings suggest that evt can be considered in elderly patients, if indicated. | objectivewe evaluated the effect of endovascular treatment (evt) for acute ischemic stroke in patients over 80 years of age.materials and methodsthe records of 156 acute stroke patients aged over 80 years who were considered as candidates for evt were analyzed. fifty - six patients (35.9%, evt group) underwent evt and 100 patients (64.1%, non - evt group) did not. outcomes, in terms of functional outcomes and rates of symptomatic hemorrhage, in - hospital morbidity and mortality, were compared between groups. each comparison was adjusted for age, time from onset, initial national institute of health stroke scale, and pre - stroke modified rankin scale (mrs).resultsmore patients in the evt group achieved good outcomes (mrs score of 0 - 2) at 3 months (35.7% vs. 11.0%, adjusted odds ratio [or ] 4.779 [95% confidence interval 1.972 - 11.579 ], p = 0.001) and 12 months (35.7% vs. 14.0%, adjusted or 3.705 [1.574 - 8.722 ], p = 0.003) after stroke. during admission, rates of hospital - acquired infection including pneumonia (12.5% vs. 29.0%, adjusted or 0.262 [0.098 - 0.703 ], p = 0.008) and urinary tract infection (16.0% vs. 34.0%, adjusted or 0.256 [0.099 - 0.657 ], p = 0.005) were significantly lower in the evt group. more symptomatic hemorrhages (10.7% vs. 2.0%, adjusted or 6.859 [1.139 - 41.317 ], p = 0.036) occurred in the evt group, but no significant difference was observed in in - hospital mortality rate (12.5% vs. 8.0%, adjusted or 1.380 [0.408 - 4.664 ], p = 0.604).conclusionevt improved functional outcome and reduced the risk of hospital - acquired infections in acute stroke patients over 80 years of age without increasing the risk of in - hospital mortality, although symptomatic hemorrhage occurred more frequently after evt. |
for the self - assembling process, a solution containing 10 nm scaffold strand, 50 nm of each staple strand (reverse - phase cartridge purified, bioneer inc.), 5 mm tris + 1 mm edta (ph 7.9 at 20 c), 16 mm mgcl2 was heated to 80 c for 4 min, cooled down to 60c over the course of 80 min, and cooled further down to 24 c over the course of 72 h. the folded objects were electrophoresed on a 1.5% agarose gel containing 45 mm tris borate + 1 mm edta and 11 mm mgcl2 at 70 v for 3 h. to prevent the origami structures from denaturation during electrophoresis, the gel - box was cooled in an ice - water bath. the gel - band containing the structures was physically extracted from the gel and run through spin columns (freezensqueeze spin columns, biorad) at 5000 rcf. the dna tensegrity objects were then imaged with tem after negative staining with uranyl formate on a fei tecnai t12 biotwin at 80 kv. a 12-helix bundle kite with four springs of equal length (300 nt) was designed such that the restriction site for ecori is located in one of the 300-nt spring regions. the assembled kites with a double - stranded region at the ecorri restriction site (gaattc + 10 bases or more overhang on both sides of the sequence) were incubated for one hour with the enzymes (20 l of 10 nm scaffold, 80 nm each staple, 1xneb - buffer 2, 20 units of enzyme, new england biolabs, inc.). uncut and cut tensegrity kites were electrophoresed on a 1.5% agarose gel. the gel - bands were physically extracted and imaged with tem after negative staining with uranyl formate. for the self - assembling process, a solution containing 10 nm scaffold strand, 50 nm of each staple strand (reverse - phase cartridge purified, bioneer inc.), 5 mm tris + 1 mm edta (ph 7.9 at 20 c), 16 mm mgcl2 was heated to 80 c for 4 min, cooled down to 60c over the course of 80 min, and cooled further down to 24 c over the course of 72 h. the folded objects were electrophoresed on a 1.5% agarose gel containing 45 mm tris borate + 1 mm edta and 11 mm mgcl2 at 70 v for 3 h. to prevent the origami structures from denaturation during electrophoresis, the gel - box was cooled in an ice - water bath. the gel - band containing the structures was physically extracted from the gel and run through spin columns (freezensqueeze spin columns, biorad) at 5000 rcf. the dna tensegrity objects were then imaged with tem after negative staining with uranyl formate on a fei tecnai t12 biotwin at 80 kv. a 12-helix bundle kite with four springs of equal length (300 nt) was designed such that the restriction site for ecori is located in one of the 300-nt spring regions. the assembled kites with a double - stranded region at the ecorri restriction site (gaattc + 10 bases or more overhang on both sides of the sequence) were incubated for one hour with the enzymes (20 l of 10 nm scaffold, 80 nm each staple, 1xneb - buffer 2, 20 units of enzyme, new england biolabs, inc.). the gel - bands were physically extracted and imaged with tem after negative staining with uranyl formate. | tensegrity or tensional integrity is a property of a structure that relies on a balance between components that are either in pure compression or in pure tension for its stability [1,2 ]. tensegrity structures exhibit extremely high strength - to - weight ratios and great resilience, and are therefore widely used in engineering, robotics and architecture [3,4 ]. here we report nanoscale, prestressed, three - dimensional tensegrity structures in which rigid bundles of dna double helices resist compressive forces exerted by segments of single - stranded dna that act as tension - bearing cables. our dna tensegrity structures can self - assemble against forces up to 14 pn, which is twice the stall force of powerful molecular motors such as kinesin or myosin [5,6 ]. the forces generated by this molecular prestressing mechanism can be employed to bend the dna bundles or to actuate the entire structure through enzymatic cleavage at specific sites. in addition to being building blocks for nanostructures, tensile structural elements made of single - stranded dna could be used to study molecular forces, cellular mechanotransduction, and other fundamental biological processes. |
map affects domestic and wild animals and, in cows, causes chronic enteritis, diarrhea, weight loss, and progressive emaciation that can eventually lead to death. map has also been linked to human crohn 's disease, a systemic disorder that causes mainly a chronic inflammation of the intestine. it is suggested that humans might be infected through contaminated milk, although relatively little is known about map survival during industrial milk manipulation. thus, laboratory assays were performed to evaluate map heat resistance according to differential distribution of heat treatment during pasteurization [36 ]. in contrast, other authors support the theory that map is able to resist pasteurization when it is present in raw milk [713 ]. viable map was detected in commercial pasteurized milk in the uk, the usa, the czech republic, and india [10, 11, 1416 ]. furthermore, in the czech republic, using f-57 or is900 real - time pcr, map was detected in 49% of samples of powdered infant milk, with one study yielding viable map. nevertheless, little was published regarding map in dairy products other than liquid of powdered milk and some cheese. in general, fermented milk products provide significant barriers to pathogen 's growth, starting with heat treatment during pasteurization, followed by the addition of a starter culture and the acidity environment during fermentation as well as refrigerated storage. van brandt. inoculated map into ultrahigh temperature (uht) milk and observed that initial map counts, during yogurt fermentation followed by storage at 6c, remained unchanged for over 6 weeks, regardless of the starter culture that was used or the fat content of milk. it is unknown whether the treatment to which milk is subjected to prepare yogurt is sufficient to inactivate viable map. although there are some studies on the behavior of pathogenic bacteria such as escherichia coli (e. coli) and listeria monocytogenes during the processing of yogurt, they are affected by different temperatures and low ph. e. coli is an important foodborne pathogen and dairy products may contain these bacteria, normally due to postpasteurization contamination. e. coli o157:h7, the most studied bacterium of the enterobacteriaceae family, is able to survive the acidic conditions during yogurt preparation and thus causes bacterial enteric infections to the consumers. massa. found that e. coli o157:h7 survived fermentation conditions of low ph levels of 4.5 and at temperatures of 42c up to 5 h. bachrouri. found that e. coli o157:h7 can grow during the preparation of yogurt and survive for 1021 days at different refrigerated storage temperatures. several studies have shown that the addition of probiotic cultures in yogurt shortens the survival of l. monocytogenes and e. coli o157:h7 during the storage period [2124 ]. however, not much literature is available about similar studies using salmonella enteritidis (s. enteritidis). the aim of this study was to find out the viability of map, e. coli, and s. enteritidis during the traditional processing and refrigerated storage of yogurt made from milk after experimental inoculation and determine if there are synergistic or antagonistic effects between these bacteria. bacterial strains were isolated at the bacteriology laboratory of the national institute of agricultural technology (inta), balcarce (argentina), using map strain inta sb, isolated from a commercial milk, e. coli strain inta 116/c3, obtained from bovine feces, and s. enteritidis strain inta 86/360, isolated from poultry. for the preparation of the inocula map was cultured onto solid herrold 's egg yolk medium (heym) plus 0.0002% (wt / vol) mycobactin j (allied monitor inc., fayette, mo, usa) and sodium pyruvate (sigma chemical co., st. louis, mo, usa) for 8 weeks at 37c. for the preparation of the inoculum, few colonies were suspended in 15 ml of phosphate buffered saline (pbs) (ph = 7), adjusted to turbidity of 0.5 in mcfarland scale containing 1.5 10 colony forming units (cfu) ml. e. coli was grown onto mcconkey (mc) agar and s. enteritidis was cultured onto xilosa - lisina - desoxicolato agar with the addition of 0.46% of tergitol-4 (sigma chemical co., st. afterwards, one colony of either e. coli or s. enteritidis was suspended in pbs ph 7.2 and used to seed brain - heart infusion (bhi) broth (oxoid, uk), which were further incubated overnight at 37c. for the preparation of the inocula both bhi tubes were diluted with pbs (ph = 7.2) to a final concentration of 1.5 10 colony forming units (cfu) ml. three traditional yogurts were prepared using 360 ml of commercial uht milk, 125 g of commercial yogurt containing starter culture of streptococcus thermophilus and lactobacillus bulgaricus, and 5 ml of the corresponding bacterial inoculum : yogurt 1 (y1) : map, yogurt 2 (y2) : e. coli + s. enteritidis, and yogurt 3 (y3) : map + e. coli + s. enteritidis. the fermentation was carried out in a thermostatic orbital shaker (new brunswick scientific, model e24) at 43c for 3 h. afterwards, the yogurts were stored at 4c for 20 days. samples of 50 ml were taken out before and throughout the preparation time at 20 min, 45 min, 65 min, 90 min, 115 min, 135 min, 155 min, 180 min, and 270 min. furthermore, samples were also taken during storage at 4.5 h, 24 h, 48 h, 4 days, 10 days, 15 days, and 20 days after inoculation. map. for decontamination of the samples the method proposed by tacquet. was used with some modifications : 35 ml of each yogurt was centrifuged at 6000 rpm for 20 min in a refrigerated centrifuge (sigma 16-k, germany) and the pellet was resuspended in 15 ml of oxalic acid at 5% (wt / vol). the samples were kept at 37c for 10 min and then centrifuged once more. afterwards, the pellet was resuspended in 2 ml pbs and finally 160 l was cultured in heym plus vancomycin 0.01% (wt / vol), amphotericin b 5% (wt / vol), nalidixic acid 0.3% (wt / vol), and nystatin 0.01% (wt / vol). the medium was incubated at 37c for 40 days and was weekly observed for map growth. the final cfu was calculated taking into account that each slant was inoculated with 160 l. the results are expressed as cfu ml of yogurt. 5 ml was taken from each yogurt sample and 10 decimal logarithmic dilutions were made in pbs (ph = 7.2). using the miles and misra method, an aliquot of 20 l from each dilution was plated onto triplicate mc and xldt4 for e. coli and s. enteritidis strains, respectively. the agar plates were incubated overnight at 37c and afterwards colonies were counted in the dilution that permitted the identification of 330 separate colonies. ph was measured using a ph meter (schott gerate, cg 820, usa). the linear regression curves of log counts were analyzed versus time of preparation and storage. the analysis was performed only for e. coli and s. enteritidis by comparing the behavior of both bacteria in the yogurt and of each in the two yogurt bacteria which were inoculated. at the beginning of the preparation process, counts of map in y1 and y3 were 46 and 127 cfu ml, respectively. during the preparation process (up to 135 min) map was not detected in any of the yogurts. after that, map counts in y1 were 5.6 cfu ml at 180 min, but map was not isolated from y3. after 4.5 h and during the storage at 4c, the cfu ml increased and reached a maximum of 56,5 and 62,7 cfu ml at 4 days in both yogurts, after which the viability began to fall again (figures 1 and 2). the initial counts for e. coli and s. enteritidis were 5.3 and 5.5 log of cfu ml and decreased during the first hour of preparation. both bacteria reached their maximum counts at 24 h (6.4 log of cfu the viability of both bacteria continued to slightly decline until the end of the trial (5 log for e. coli and 4.6 log for s. enteritidis) (figures 3, 4, 5, and 6)., the ph gradually decreased to 4.5. from 180 min to 4.5 h, values remained in that range. afterwards, the ph remained stable at 4.5 throughout storage until the end of the trial (figures 7 and 8). the regression analysis of the log counts of e. coli and s. enteritidis versus the preparation and storage time in y3 gave an r of 0.94 (p 0.05). on the other hand, the behavior analysis of e. coli in y3 and y2 showed no significant differences (p > 0.05). instead, s. enteritidis showed a different behavior in both yogurts (p < 0.05), which occurs in y3 inoculated with map. as milk is a nutrient medium suitable for growth of microorganisms, raw milk can be an important source of pathogens for humans. the ability of map to survive certain food processing methods and to persist unfavorable conditions, combined with its possible involvement in crohn 's disease, has raised concern with respect to human exposure to this pathogen. for the commercial production of yogurt and other fermented milk products, raw milk is usually first pasteurized. if indeed map is present in pasteurized milk, it might be transferred to humans upon consumption of by - products. therefore, it is interesting to gain insights into the behavior of map in such products as the acidic nature of yogurt (ph about 44.5) generally contributes to the inactivation of bacterial pathogens. map was isolated until the end of the present study (20 days of storage), showing its capacity to withstand the acidic conditions generated during yogurt preparation and the low temperatures of storage. in all samples, van brandt. found that initial map counts remain unchanged over the 6 weeks of refrigerated storage, regardless of starter culture used and the fat content of milk, but did not evaluate its isolation during preparation. sung and collins found that at low ph levels in vitro, map expressed two proteins related at the acid tolerance. however, they could not predict map survival curve in foods where other factors might affect its viability. in the present work, map was not isolated during the preparation process, which suggests that the sudden decrease in ph might have caused damage to the bacteria and therefore it was impossible to culture it on artificial culture media. furthermore, map was recuperated at 4.5 h after inoculation suggesting that initially it might have been in a viable noncultivable state and after an adaptation period. in a previous work, during the preparation and storage of cheese made with caprine and bovine milk, map was isolated only when nonpasteurized milk was used up to 60 days of maturation. high values of ph were observed between 30 and 45 days of storage, coinciding with the further map isolation. it has also been observed that ph, temperature, and salt concentrations can affect map viability [3133 ]. to our knowledge, there are no referential studies on the survival of s. enteritidis during the preparation of dairy products like yogurt. however, some studies have focused on e. coli, especially the pathogenic serotype o157:h7, which causes hemolytic uremic syndrome. observed that e. coli o157:h7 counts increased during fermentation, whereas dineen. therefore, in this study, high inoculation levels were chosen in order to assure recuperation of both e. coli and s. enteritidis at the entire process of production and conservation of the yogurts. hence, it was demonstrated that e. coli counts had decreased slowly, while s. enteritidis maintained stable. these differences may be explained by the fact that in these abovementioned works the fermentation conditions were different : higher fermentation temperature (467c), longer fermentation time of 24 h, and lower final ph (4.1). similarly, soudah. observed that e. coli o157:h7 did not survive fermentation in yogurt containing lactobacillus delbrueckii subsp. however, other researchers found that e. coli o157:h7 survived the fermentation of yogurt at 42c [19, 23 ]. furthermore, the exposure to acid environments for short times increased the tolerance acid of e. coli, thus permitting survival for longer periods. these results are in agreement with those of the present work, where, despite suffering a slight decline, e. coli was recovered during the fermentation process at 43c and a final ph of 4.7. these results suggest that the conditions of fermentation are important factors that may affect the survival of e. coli. observed that viability of e. coli began to decline until 7 days of storage, when no viable bacteria were detected.. found that no viable e. coli o157:h7 was detected in yogurts made with and without probiotics, at 16 and 28 days of storage, respectively. govaris. did not detect viable e. coli o157:h7 between 5 and 8 days of storage at 4c or between 4 and 6 days of storage at 12c and the ph of the yogurts was stable. however, in the present work, using a similar inoculation dose in the yogurts, e. coli and s. enteritidis were isolated at 20 days of cold storage. this reveals the capacity of these bacteria to tolerate acidity conditions when they are present in raw material. on the other hand, lactic bacteria produce compounds such as organic acids, hydrogen peroxide, diacetyl, and bacteriocins, which inhibit the growth of contaminant microflora and pathogens such as staphylococcus aureus, clostridium botulinum, and l. monocytogenes. in the present study, it is suggested that the presence of map in y3 contributed to the reduced survival of s. enteritidis in comparison with y2. there is also a lack of information about the competitive inhibition of map and other bacteria for nutrients in the yogurt during preparation. nevertheless, if such competition does occur, map would be in disadvantage as it is the cultivable mycobacterium with the slowest growth, with a slow generation time of over 20 h, under optimal growth conditions, whereas s. enteritidis has a generation time of 20 minutes. further studies should be performed to assess the competitive inhibition mechanisms of these pathogens in vitro as well as their resistance to different temperatures and ph levels in order to improve optimal conditions for the processing of dairy products and to ensure their elimination. the present study presents evidence that pathogen bacteria like map, e. coli, and s. enteritidis can survive yogurt fermentation conditions and low ph levels followed by refrigerated storage for at least 20 days. thus these pathogens are present in raw materials and they might reach consumers. the importance of implementing good manufacturing practices during the production and storage of yogurt is highlighted. therefore, similar studies should be carried out with different concentrations of bacteria, in an attempt to simulate natural contamination. | the purpose of this study was to determine the viability of mycobacterium avium subsp. paratuberculosis (map), escherichia coli (e. coli), and salmonella enteritidis (s. enteritidis) during preparation and refrigerated storage of yogurt. three yogurts were prepared using pasteurized commercial milk. each yogurt was artificially contaminated with (1) map, (2) e. coli + s. enteritidis, and (3) map + e. coli + s. enteritidis. samples were taken during and after the fermentation process until day 20 after inoculation. map was not detected during their preparation and short - term storage but was recuperated after starting at 180 min after inoculation storage. live bacterial counts of e. coli, and s. enteritidis increased during the first 24 hours, followed by a slight decrease towards the end of the study. in this study it was shown how map, e. coli, and s. enteritidis resisted the acidic conditions generated during the preparation of yogurt and low storage temperatures. this work contributes to current knowledge regarding survival of map, e. coli, and s. enteritidis during preparation and refrigerated storage of yogurt and emphasizes the need to improve hygiene measures to ensure the absence of these pathogenic microorganisms in dairy products. |
hypertension (htn) is an important public health problem in both economically developed and developing nations. as per world health organization report, studied secular trends in the age - adjusted mean systolic blood pressure (sbp) worldwide. their findings revealed mean age - adjusted sbp declined by approximately 2 mmhg from 1980 to 2008. the age - adjusted sbp was highest in low - income and middle - income countries. in the same period, mean age - adjusted sbp declined in economically developed regions such as australia, north america, and western europe and increased in economically developing regions such as oceania, east africa, and south and southeast asia. additionally, due to the growth and aging of the population around the world, the number of people with uncontrolled hypertension was reported to have increased between 1980 and 2008. worldwide, 7.6 million premature deaths (about 13.5% of the global total) were attributed to high blood pressure. about 54% of stroke and 47% of ischemic heart disease worldwide were attributable to high blood pressure. hypertension has been associated with increased risk of coronary artery disease and is an independent risk factor for cardiovascular and cerebrovascular diseases [5, 6 ]. a meta - analysis also reported that lower values of blood pressure are associated with higher risk of cardiovascular disease and also with chronic kidney diseases [7, 8 ]. the situation is critical in the southeast asian region with studies reporting htn as an important risk factor for attributable burden of disease in the region [9, 10 ]. an alarming rise in htn projected by the global burden of hypertension 2005 study and the gbd 2010 study portrays a grim picture for the indian population [2, 9 ]. beyond programs aimed at the prevention of hypertension, treatment of hypertension remains a challenge in many parts of the world. looking at the existing burden of disease, the indian government has launched the national program for prevention and control of cancer, diabetes, cardiovascular diseases and stroke for prevention and control of disease at community level. reliable information about the prevalence of hypertension is essential to the development of national and local level health policies for prevention and control of hypertension. thus, this study was conducted with the objective of finding prevalence of hypertension and its risk factors in a rural area in delhi. it was a community based cross - sectional study conducted in two rural field practice areas, covered under the department of community medicine, maulana azad medical college, new delhi. study population was constituted by all people above 18 years of age residing in two villages of delhi. the sample size was calculated on the basis of a previous study which recorded prevalence of hypertension in rural population as 15%. taking 95% confidence interval, the required sample size for an 18,800 population was 194 (derived by epi - info software version 7). a predesigned, pretested, semistructured questionnaire was used by the surveyors containing items to assess sociodemographic profile like age, sex, identification data, socioeconomic status, and so forth. the who steps approach was employed to study the profile of the hypertension in the population. information on sociodemographic variables and behavioural risk factors was collected, that is, tobacco use, alcohol use, and related factors using a questionnaire (step 1) ; clinical measurements such as weight, height, and blood pressure were obtained using standardized protocols and instruments (step 2) ; cholesterol, triglycerides, and high density lipoprotein (hdl) were measured (step 3). it was adapted by including local terms and translated into local (hindi) language and back translated into english by hindi and english expert and field tested on 20 subjects in rural community. self - reported history of use of tobacco as beedi or cigarette or any other form of tobacco and alcohol consumption as well as history of hypertension and diabetes mellitus was obtained from the respondents. blood pressure was recorded three times in sitting position, in the right arm, using a standard android dial bp apparatus (mercury type of bp apparatus is phased out from health care setting). the standard protocol was followed and the average of the last two readings was used in the analyses. hypertensive subjects were defined as those with systolic blood pressure (sbp) equal to or more than 140 mmhg and/or diastolic blood pressure (dbp) equal to or more than 90 mmhg or those being treated for hypertension [13, 14 ]. body mass index (bmi) was calculated as weight in kilograms divided by height in meters squared. blood sugar fasting and postprandial and cholesterol levels were measured among study subjects. each selected subject written informed consent was obtained and referral services were provided if required at the rural health centre. the prior ethical clearance for the study was obtained from the institutional ethics committee. table 1 shows the sociodemographic characteristics such as age, sex, and religion of nonhypertensive (n = 863) and hypertensive (n = 142) groups. the hypertensive group was significantly higher in individuals more than 35 years than those less than 35 years. the mean number of years of education for nonhypertensive cases was 8.6 years and for hypertensive cases it was 6.2 years. there was no significant difference in the two groups in monthly per capita income but significant difference was seen in occupation categories. out of these 142 hypertensive subjects, previous history of hypertension was given by 68 (6.8%) of subjects and 50 (5.0%) subjects reported history of high bp records in the past one year. out of these 68 subjects, only 29 (42.6%) reported that they were taking antihypertensive medications, 49 (72.1%) reported lesser intake of salt in diet, 25 (36.8%) were doing exercise for control of bp, and 43 (63.2%) were taking efforts for weight control for bp. yield of this study was detecting 74 new cases of hypertension in the rural population of delhi. it can be seen that there was no significant difference in tobacco intake, both present and past tobacco use in the two groups. the hypertensive group was significantly higher in those who take alcohol than in the other group when inquiring about the alcohol intake in the past one year (p value = 0.02). hypertension was found in 20.7% of subjects with raised total cholesterol level and 11.1% among those with normal values which was statistically significant (= 16.2, p value = 0.01). similarly there was significant difference in raised triglyceride levels in the two groups. a significantly higher number of study subjects were hypertensive overweight and obese group as compared to the other group (= 8.87, p value = 0.03). 12 (8.4%) were diabetic among hypertensive cases and 130 (91.5%) were nondiabetic. table 3 shows results of multivariate analysis for hypertension and its risk factors which showed that age, education, and cholesterol levels were independently associated with hypertension. multivariate analysis using logistic regression was used to find independent association of various factors. all the variables with p value less than or equal to 0.1 in univariate analysis as shown in table 3, age group less than 35 years has lesser odds of having hypertension than age group more than 35 years. in education classes, taking illiterate as baseline, those educated up to primary and high school level had significantly lesser odds of hypertension. similarly, in occupation categories, taking unemployed as baseline, those who were retired had significantly higher odds of hypertension. subjects with normal cholesterol levels had lesser odds of having hypertension than those with raised levels. the present study showed that the prevalence of hypertension was significantly higher in individuals more than 35 years as compared to those less than 35 years. wang. also found that both systolic and diastolic blood pressure were inversely associated with the level of school education independent of all other risk factors. education makes the people aware of the disease and what precautions can be undertaken by the healthy individual. no significant association was found between hypertension and monthly per capita income but significant differences were found in different occupation classes. these are consistent with the findings in study conducted by tsutsumi. which revealed that occupation and related stress were as independent risk factor of hypertension. the confounding factors were adjusted in multivariate analysis which showed that only retired subjects had significantly higher odds of hypertension. the present study showed that only 42.6% of subjects who were known hypertensives were taking antihypertensive medications. similar results were stated by kusuma. in their study carried out in delhi and found that only 59% of hypertension patients were on medication. this difference could be due to our study population that belongs to a rural area where tendency of not starting long term medication is always present. among modifiable factors, this is inconsistent with findings of other studies where tobacco use has been found to be associated with hypertension [20, 21 ]. this could be due to the low level of tobacco use in this population as compared to national average of 35% which is around 48% in males and 20% in females while in the present study it is only approximately 11%. alcohol has been reported as an independent risk factor by other authors as well [22, 23 ]. the prevalence of hypertension was found to be consistently increased with increasing bmi as revealed by other authors [16, 24 ]. in multivariate analysis, raised total cholesterol, age more than 35 years, and education were independent risk factors of hypertension as reported by other authors as well. any causal association can not be derived from the present cross - sectional study design. more research with appropriate study design is needed to find if any causal association exists between hypertension and the discussed variables. it can be concluded that there is significant burden of hypertension in rural areas in delhi. age, education, and cholesterol levels were independent risk factors of hypertension in the present study. cholesterol levels should be cut down using approaches of behaviour change communication (bcc) in community. | introduction. hypertension is an important public health challenge in both economically developing and developed countries. it is one of the risk factors for cardiovascular mortality. data is available on hypertension in urban population but few studies are reported in rural areas. materials and methods. it was a community based cross - sectional study conducted in two rural areas in delhi among 1005 subjects selected using systematic random sampling method. who steps approach was used to collect data. blood pressure, body mass index, and blood sugar were measured. data analysis was done using spss version 16. odds of hypertension among subjects with risk factors were calculated. p value less than 0.05 was considered significant. results. the prevalence of hypertension was 14.1% among study subjects. hypertension was significantly higher in individuals more than 35 years than those less than 35 years. hypertension was significantly higher in those who take alcohol and in subjects with raised total cholesterol level but in multivariate analysis only age, education, and cholesterol levels were independently associated with hypertension. conclusion. there is significant burden of hypertension in rural areas in delhi. age, education, and cholesterol levels were independent risk factors of hypertension. |
percutaneous interventions have paved the way for new therapeutic areas on heart with a minimally invasive approach competing heart surgery. consequently, indications of percutaneous interventions have grown over the last decade, particularly for elderly patients, who are frequently challenged for surgical approaches. although percutaneous interventions have been well validated in most of the patients, their efficacy and safety in elderly patients continue to be debated. the use of interventional cardiology in continually aging population is known to carry a higher risk of complications, and because elderly patients are usually excluded from clinical studies, the performance of percutaneous intervention on elderly patients is still to be confirmed. this is particularly true for alcohol septal ablation (asa) in symptomatic patients with drug - refractory obstructive hypertrophic cardiomyopathy (hcm). although the comparison of myomectomy and asa by randomized clinical trial seems impossible to perform,1 asa is widely used in europe. it is nowadays admitted that short- and long - term outcomes of asa are mostly favorable.2,3 this is well validated for young- and middle - age patients.4 although few studies suggest that the age is an independent predictor of mortality after asa,5 other studies suggest that the hemodynamic benefit is distinct according to the age of patients.6 efficacy and safety of asa in elderly patients remain to be confirmed. the objective of this study was to compare the efficacy and safety of asa in patients under and over 65 years old. fifty - one consecutive patients with obstructive hcm who underwent asa in the university hospital of rangueil, toulouse, france, between january 2010 and july 2015 were retrospectively included. the clinical diagnosis of obstructive hcm was based on the demonstration by bidimensional echocardiography of a nondilated and hypertrophied left ventricle (maximum left wall thickness 15 mm) in the absence of another cardiac or systemic disease that could produce a similar degree of hypertrophy.79 indication of asa was retained according to the european society of cardiology guidelines in patients with symptoms and left ventricular (lv) outflow tract obstruction 50 mmhg at rest or after exercise despite an optimal medical treatment. demographic data, cardiovascular risk factors, electrocardiography, biochemistry, medication and procedural characteristics were abstracted from medical records. the study was approved by our institutional review board (research ethics committee of the university hospital of toulouse), and all patients were informed of the study design. due to the retrospective design of the study the research ethics committee of the university hospital of toulouse deemed written informed consent not necessary. all patients underwent a transthoracic echocardiography examination at baseline, during the procedure of asa and during the follow - up with a commercially available ultrasound vivid e9 system (ge vingmed ultrasound as, horten, norway) or philips ie33 (philips medical systems, the netherlands) using a 2.5 mhz transducer. a complete m - mode and two - dimensional gray scale echocardiography including the three standard apical views (four, three and two chambers) using high frame rates (> 60 frames / sec) all echocardiographic data were synchronized to the electrocardiogram, and acquisition was performed during breath hold. the peak instantaneous lv outflow tract gradient was measured with continuous - wave doppler in the apical five - chamber view with the simplified bernoulli equation. patients with lv outflow tract obstruction 60 frames / sec) all echocardiographic data were synchronized to the electrocardiogram, and acquisition was performed during breath hold. the peak instantaneous lv outflow tract gradient was measured with continuous - wave doppler in the apical five - chamber view with the simplified bernoulli equation. patients with lv outflow tract obstruction < 50 mmhg at rest were tested by exercise echocardiography. lv end - diastolic, end - systolic volumes and ejection fractions were measured using simpson s biplane method. cardiac magnetic resonance (cmr) was performed on a 1.5-t mr commercial scanners, either avanto, siemens medical solutions (erlangen, germany) or intera, philips medical systems (best, the netherlands) using a 12-element phased - array cardiac coil with vector - cardiac gating in all patients. following scout imaging, balanced steady - state free precession breath - hold images were acquired : slice thickness 6 mm (long - axis and four - chamber views) or 810 mm (contiguous short - axis views [no gap between slices, from the atrioventricular ring to the apex ]). subsequently, delayed contrast - enhanced cmr images were obtained in the same long - axis, four - chamber and short - axis orientations 10 minutes after injection of 0.2 mmol / kg of gadolinium dimenglumine (magnevist, berlex imaging, wayne, nj, usa) using a phase - sensitive inversion recovery spoiled gradient echo sequence. lv ejection fraction and mass were calculated using clinically available software (argus [siemens medical solutions ] and viewforum [philips medical systems ]). left atrial diameter was measured in the long - axis and four - chamber view. a temporary pacemaker was placed in all patients except those who already had a permanent pacemaker. a 6f guiding catheter was engaged in the ostium of the left main coronary artery, and a 10151.53-mm over - the - wire balloon catheter was advanced over a 0.014-inch wire into the target septal perforator artery. for two patients, the target septal artery was reached through the right coronary artery. myocardial contrast echocardiography was used through the balloon lumen to delineate the culprit septal segments. ethanol 15 ml was injected into the artery supplying the culprit septal segments and left in place for 5 minutes. if complete heart block was present at 48 hours after the procedure, a permanent dual - chamber pacemaker was implanted. in - hospital major acute cardiac events (mace) were defined by death and/or cardiac tamponade and/or third - degree atrioventricular block requiring permanent stimulation and/or hemorrhage from the vascular path requiring transfusion. follow - up consisted of a functional and echocardiographical assessment and was performed by review of the patient s last hospital consultation. univariate analyses were performed using the mann whitney rank sum test for continuous variables and the fisher s exact test for categorical variables for intergroup comparisons. comparisons of continuous variables were performed by a mann whitney u test or spearman s correlation when appropriate. then, two models of multivariate analysis were performed using a multiple regression model including, among baseline characteristics, all univariate parameters with a p - value < 0.10 and either age in years (model 1) or age over 65 years (model 2). all statistical analyses were performed using spss version 20.0 (spss inc., chicago, il, usa) and p<0.05 defined statistical significance. all patients had symptoms with a balanced distribution of poor dyspnea (new york heart association [nyha ] class ii) and severe dyspnea (nyha class iii and iv), without any difference between patients under and over 65 years old. the demographic, echocardiographic and cmr characteristics of patients are listed in table 1. comparing to patients under 65 years old, there were more females in the group over 65 years old, with a smaller weight and a trend to a higher rate of arterial hypertension. lv outflow tract obstruction at rest, use of diuretic and average dose of diuretic were higher in patients over 65 years old. most of the patients (88%) had radial access, without any difference between young and elderly patients (p=0.242). there was no difference between young and elderly patients in terms of the morphine administration during the procedure with doses of 8.33.6 and 7.22.1 mg (p=0.081), respectively. as shown in table 1, the target septal artery was the first one for most of the patients over 65 years old, whereas 35% of the patients under 65 years old required the ablation of another septal artery : most of the time the second one, except for two patients who required the ablation of the septal artery from the posterior descending artery. the average hospital stay was 83 and 94 days for patients under and over 65 years old, respectively (p=0.633). table 2 shows in - hospital cardiac events after asa among patients under and over 65 years old. among patients over 65 years old, 2 (7%) died before being discharged. mace, defined by death and/or cardiac tamponade and/or third - degree atrioventricular block requiring permanent stimulation and/or hemorrhage from the vascular path requiring transfusion, were more frequent in patients over 65 years old in comparison with younger patients (12 [43% ] versus 2 [9% ], respectively, p=0.007). as shown in table 3, by univariate analysis, age, age over 65 years old, female and hypercholesterolemia were associated with a risk of increase for mace after asa. after multivariate analysis, there was still a trend of increased risk for mace with age, and particularly age over 65 years, but the association was no longer statistically significant. the average duration of follow - up was 2219 and 1615 months for patients under and over 65 years old, respectively (p=0.354). there was no difference between patients under and over 65 years old regarding the efficacy of the procedure with a decrease of the nyha class of 1.30.6 and 1.40.7 (p=0.510) and the maximum lv outflow tract gradient of 8657 and 8136 mmhg (p=0.733), respectively. the average nyha class at the time of follow - up was 1.30.4 and 1.20.4 (p=0.359) for patients under and over 65 years old, respectively. the impact of the procedure on the nyha class for both groups is illustrated in figure 1. all patients had symptoms with a balanced distribution of poor dyspnea (new york heart association [nyha ] class ii) and severe dyspnea (nyha class iii and iv), without any difference between patients under and over 65 years old. the demographic, echocardiographic and cmr characteristics of patients are listed in table 1. comparing to patients under 65 years old, there were more females in the group over 65 years old, with a smaller weight and a trend to a higher rate of arterial hypertension. lv outflow tract obstruction at rest, use of diuretic and average dose of diuretic were higher in patients over 65 years old. most of the patients (88%) had radial access, without any difference between young and elderly patients (p=0.242). there was no difference between young and elderly patients in terms of the morphine administration during the procedure with doses of 8.33.6 and 7.22.1 mg (p=0.081), respectively. as shown in table 1, the target septal artery was the first one for most of the patients over 65 years old, whereas 35% of the patients under 65 years old required the ablation of another septal artery : most of the time the second one, except for two patients who required the ablation of the septal artery from the posterior descending artery. the average hospital stay was 83 and 94 days for patients under and over 65 years old, respectively (p=0.633). table 2 shows in - hospital cardiac events after asa among patients under and over 65 years old. among patients over 65 years old, 2 (7%) died before being discharged. mace, defined by death and/or cardiac tamponade and/or third - degree atrioventricular block requiring permanent stimulation and/or hemorrhage from the vascular path requiring transfusion, were more frequent in patients over 65 years old in comparison with younger patients (12 [43% ] versus 2 [9% ], respectively, p=0.007). as shown in table 3, by univariate analysis, age, age over 65 years old, female and hypercholesterolemia were associated with a risk of increase for mace after asa. after multivariate analysis, there was still a trend of increased risk for mace with age, and particularly age over 65 years, but the association was no longer statistically significant. the average duration of follow - up was 2219 and 1615 months for patients under and over 65 years old, respectively (p=0.354). there was no difference between patients under and over 65 years old regarding the efficacy of the procedure with a decrease of the nyha class of 1.30.6 and 1.40.7 (p=0.510) and the maximum lv outflow tract gradient of 8657 and 8136 mmhg (p=0.733), respectively. the average nyha class at the time of follow - up was 1.30.4 and 1.20.4 (p=0.359) for patients under and over 65 years old, respectively. the impact of the procedure on the nyha class for both groups is illustrated in figure 1. the incidence of cardiovascular disease rises with age, and age itself is considered as a risk factor for the development of cardiovascular diseases. clinical and experimental evidence proves that the aging process promotes structural and functional remodeling of the heart, which occurs even in healthy older adults in the absence of an overt cardiovascular disease. it is legitimate to suppose that age - related effects on heart can modify the natural time course of heart disease. hcm may develop in various age groups as a consequence of different pathological processes10 and may be a different disease in young and elderly patients. whether the disease takes the same form in the elderly than in the young or middle - aged patients has not been clarified.11 consequently, the benefits and the risks of asa may change with age. our study shows that 1) asa has the same efficacy among patients under and over 65 years old ; and 2) patients over 65 years old have more in - hospital complications of the procedure. regarding the benefits of asa in elderly when compared with younger patients, our results are consistent with the previous ample studies where asa produced significant and similar improvements in lv outflow tract obstruction and symptoms, regardless of age, and despite baseline differences.6,12,13 faber provided conflicting results finding that younger age was associated with a less favorable hemodynamic outcome.14 this could be partly explained by a higher basal interventricular septal thickness in younger patients at baseline, as reported by gietzen.6,13 anyway, all these studies confirm that the results of asa on the elderly are at least as good as on younger patients up to 12 months.6,12 our study confirms these results with an average follow - up of 1615 months. we found a higher rate of major acute in - hospital cardiac events following asa in elderly patients compared with younger patients. these findings are consistent with the previous study of leonardi, who have reported higher procedural complication rates for elderly patients in comparison to younger patients.12 a recent study among 1,275 highly symptomatic patients treated with asa demonstrated that age was an independent predictor of all - cause mortality over a follow - up of 5.7 years.15 if the influence of age on long - term all - cause mortality seems logical, its impact on the procedural complication rate is still to confirm. in our cohort, by univariate analysis, absolute age and age over 65 years old are associated with an increased risk for acute procedural complication, but no longer after multivariate analysis. whether the sample of our study is too small for allowing enough power to differentiate risk factors for procedural complications remains to be clarified. we can not overlook that there were two deaths among elderly patients, whereas there was none among younger patients. however, the highest rate of complication among elderly in previous studies was wildly influenced by the difference of access site bleeding : all the procedures were performed with transfemoral arterial access.12 in our cohort, most of the procedures were performed through radial access, which had a protective effect by univariate analysis. although the impact of age on postprocedural complications was no longer significant by multivariate analysis, our study is probably underpowered to discriminate independent predictor of mace. the trend of increased risk associated with age after multivariate analysis, in agreement with previous studies ; suggests that asa is associated with an increased rate of complications among elderly patients. first, our study is a retrospective observational study conducted at a single center, and, therefore, unmeasured confounding factors may influence the observed associations. second, as aforesaid, the sample of our study is probably too small for allowing enough power to discriminate age as a risk factor for adverse cardiac events following asa. however, the lower impact of age after multivariate analysis highlights the weight of comorbidities and confounding factors in the age - related procedural complications. finally, our study focused on asa and did not integrate a control group with optimal medical treatment alone or myomectomy. first, our study is a retrospective observational study conducted at a single center, and, therefore, unmeasured confounding factors may influence the observed associations. second, as aforesaid, the sample of our study is probably too small for allowing enough power to discriminate age as a risk factor for adverse cardiac events following asa. however, the lower impact of age after multivariate analysis highlights the weight of comorbidities and confounding factors in the age - related procedural complications. finally, our study focused on asa and did not integrate a control group with optimal medical treatment alone or myomectomy. although it is considered as disease of the young age and because of its age - related penetrance, obstructive hcm is commonly diagnosed in the elderly, which are frequently challenged for surgical management. consequently, symptomatic patients despite optimal medical treatment referred for asa have increased. our study shows that elderly patients referred for asa have the same benefits as younger patients through an average follow - up of 1615 months | backgroundthe performance of alcohol septal ablation (asa) in elderly symptomatic patients with drug - refractory obstructive hypertrophic cardiomyopathy is still to be confirmed. the objective of this study was to compare the efficacy and safety of asa in patients under and over 65 years old.methods and resultsfifty - one consecutive patients with obstructive hypertrophic cardio - myopathy who underwent asa were retrospectively included and reviewed for in - hospital major acute cardiac events and follow - up. twenty - eight patients were over 65 years old. left ventricular outflow tract obstruction at rest, use of diuretic and average dose of diuretic were higher in patients over 65 years old. there was no difference in hospital stay between patients under and over 65 years old. among patients over 65 years old, 2 (7%) died before being discharged. major acute cardiac events were more frequent in patients over 65 years old in comparison with younger patients (43% versus 9%, respectively, p=0.007). the average follow - up duration was 1615 months. there was no difference between patients under and over 65 years old regarding the efficacy of the procedure with a decrease of the new york heart association class of 1.30.6 and 1.40.7 (p=0.510) and the maximum left ventricular outflow tract gradient of 8657 and 8136 mmhg (p=0.733), respectively.conclusionelderly patients have the same benefits as younger patients after asa but have more complications including mortality events. |
apart from thorough detection and diagnosis of dental caries, compiling a treatment plan is another important task of a dentist. the initial assessment of hard tooth tissue is normally visual and, depending on indication and availability, x - rays are used for further detection and treatment planning. the visual classification system international caries detection and assessment system (icdas - ii) was developed to provide clinicians, epidemiologists, and researchers with an evidence - based method for standardized data collection in different settings and better comparison between studies. reproducibility and accuracy of icdas - ii have already shown to be promising for occlusal caries detection. icdas criteria have the potential to aid treatment planning. depending on the visual assessment, activity of a lesion, and patient 's risk status, apart from purely visual and visual - tactile caries diagnosis, there are several other methods for the detection of dental caries on occlusal surfaces. this includes radiography, laser or light fluorescence - based methods, and electrical impedance measurements. it is well known that fluorescence - based methods make use of the phenomenon that carious lesions fluoresce more strongly than sound tissues when excited by light at specific wavelengths. the devices diagnodent and diagnodent pen (kavo, biberach, germany) function on the same principle : they emit red light at 655 nm that causes fluorescence of bacterial metabolites in infected dentine. the fluorescence emitted from the tooth is measured and translated into a numerical scale from 0 to 99. the vistaproof fluorescence camera and the recently devised vistacam ix (both : drr dental, bietigheim - bissingen, germany) have leds that emit high - energy blue - violet light at 405 nm onto the tooth surface. this wavelength stimulates porphyrins produced by caries - related bacteria to emit red light, containing less energy. this fluorescence is recorded by the camera, transferred to a computer, and processed with special software (dbswin, drr). the result is a digital image that shows lesions in different colors with respective numerical values between 0 and 4, predicting the extent and depth of caries [5, 79 ]. radiographic examination is quite commonly used in caries detection and generally it is possible to detect approximal lesions earlier than with visual diagnosis alone. however, the validity of detecting enamel caries is low for the occlusal surfaces. to date, only a few studies exist which look at the impact of using visual inspection, radiographic examination, or laser fluorescence devices in treatment decision [12, 13 ]. no study to date has looked at the impact of the use of the new vistacam ix fluorescence device in treatment decisions. thus the aim of this study was to evaluate the performance of visual icdas - ii, laser fluorescence, fluorescence - based camera, and radiographic (bw) examinations for occlusal caries detection and their ability to make treatment decisions when used as a single method or as a combination of two methods. the teeth were collected in a dental practice and informed consent was obtained for the use of teeth for scientific purposes. the teeth were stored in a thymol - water solution immediately after extraction. within 24 h, they were cleaned thoroughly and then stored in water. the occlusal surfaces were photographed digitally (leica zoomsystem z6 apo / qwin standard v 3.4.0 software, leica microsystems, wetzlar, germany). one site within the pit and fissure system of each tooth was marked on black and white images of the tooth surface for ease of relocation. all sites were visually examined by two investigators (doctoral student calibrated by an experienced investigator) using the international caries detection and assessment system (icdas - ii) and a consensus score for each site was achieved. the chosen sites were recorded as : 0 = sound (n = 13) ; 1 = first visible sign of noncavitated lesion seen only when the tooth is dried ; 2 = visible noncavitated lesion seen when wet and dry ; 3 = microcavitation in enamel ; 4 = noncavitated lesion extending into dentine seen as an undermining shadow ; 5 = small cavitated lesion with visible dentine : less than 50% of surface ; 6 = large cavitated lesions with visible dentine in more than 50% of the surface. 1 = first visible sign of noncavitated lesion seen only when the tooth is dried ; 2 = visible noncavitated lesion seen when wet and dry ; 3 = microcavitation in enamel ; 4 = noncavitated lesion extending into dentine seen as an undermining shadow ; 5 = small cavitated lesion with visible dentine : less than 50% of surface ; 6 = large cavitated lesions with visible dentine in more than 50% of the surface. prior to the evaluations, the examiners were trained in using the devices according to the manufacturer 's recommendations. the diagnodent laser fluorescence device (lf) prior to the laser fluorescence readings, the device was zeroed using an obviously sound enamel spot on the tooth (zero value)., the laser fluorescence device was moved along the surface of the investigation site and the peak value was recorded (possible reading values 099). using the vistacam ix fluorescence - based camera (fc), images of all occlusal tooth surfaces were taken.for each tooth, the distance spacer was used in order to achieve optimum results with the caries filter. the optical head of the camera was placed on the occlusal surface of the tooth and the control ring was pressed in order to freeze the image taken by the camera. the saved images were analyzed by the camera software (dbswin, drr dental) with possible reading values from 04. detailed information on the scales of both lf and fc is given in tables 1 and 4. intraexaminer reproducibility for both devices was assessed by repeating the measurements on 1/3 of the investigation sites (n = 28) within 1 day. digital bw radiographs were taken of all teeth using the gendex dental x - ray machine (soredex, helsinki, finland) at 65 kv, 6.5 ma, and exposure time of 0.12 seconds. all radiographs were processed by the digora optime image scanner (soredex, helsinki, finland). the teeth were placed in rows (3 teeth in each row) in suitable molds (a - r x - ray model, frasaco gmbh, tettnang, germany). tft (thin - film transistor) color monitor (flexscan l 768, eizo, avnet technology solutions gmbh, nettetal, germany) by the investigators and a consensus score for each site was achieved. the sites were recorded using the following scores : 0 = no radiolucency, 1 = radiolucency in the outer half of enamel, 2 = radiolucency in the inner half of enamel, up to the enamel - dentine junction, 3 = radiolucency in the outer half of dentine, 4 = radiolucency in the inner half of dentine. 1 = radiolucency in the outer half of enamel, 2 = radiolucency in the inner half of enamel, up to the enamel - dentine junction, 3 = radiolucency in the outer half of dentine, 4 = radiolucency in the inner half of dentine. for all sites a consensus treatment decision was made by the examiners using the following scores : 0 = no treatment / use of fluorides, 1 = sealant application, 2 = round bur and sealant application, and 3 = restoration (e.g., composite, amalgam, and glass ionomer). for further analysis the treatment decisions were collapsed into preventive care (pc : score 0 - 1) and operative care (oc : score 2 - 3). to determine the depth of the lesions, all tooth surfaces were opened with rotating instruments. the end point of the excavation was reached when there was no longer any sign of caries. in the process, the lesions ' depths were divided up into the following categories : sound tooth surface (score 0), caries in the enamel (clinical score 1), caries down to the enamel - dentine junction (clinical score 2), and caries in the first third of dentine (clinical score 3), caries down to the second third of dentine or near pulp, or pulp already effected (clinical score 4). all the sites were investigated by two examiners and a consensus clinical score was derived for each investigation site. all findings were recorded on data collection forms and later transferred to an excel table. the consensus clinical scores were used to calculate sensitivity and specificity at the d1 and d3 diagnostic threshold. at the d1 diagnostic threshold all clinical scores 14 were classed as caries, for the d3 diagnostic threshold clinical scores 3 and 4 were classed as caries only. using these sensitivity and specificity values roc (receiver operating characteristic) analyses sensitivity and specificity values for the lf measurements were obtained at the cut - off values according to the literature. for the fc, sensitivity and specificity values were used firstly at the cut - off values according to the manufacturer and secondly at the cut - off values which were determined according to the literature. the performance of each method (auc) was interpreted by using the following classification : 0.500.60 fail, 0.600.70 poor, 0.700.80 fair, 0.800.90 good, and 0.901.0 excellent [16, 17 ]. a nonparametric test (the mcnemar test) was used to show whether different methods or combinations of methods lead to significant changes in treatment decisions (= 0.05). a total of 84 occlusal sites on posterior teeth were examined (52 molar and 32 premolar teeth). the distribution of the sites according the icdas - ii criteria was code 0 = 13, code 1 = 15, code 2 = 26, code 3 = 12, code 4 = 2, code 5 = 12, and code 6 = 4. while using the fc device, intraclass correlation coefficients (icc) for intraexaminer reproducibility were 0.98 for the lf device and 0.97 for the fc. the distribution of the sites according to the different methods cross - tabulated with the gold standard scores is presented in table 1. all methods assessed nearly all sound tooth surfaces correctly, except for fc when the cut - off values according to the manufacturer were used. all correlations were significant at the 0.01 level or at the 0.05 level, respectively (two tailed). the highest correlation was found between icdas - ii and fc measurements followed by the correlation between icdas - ii and the gold standard. the lowest correlation was found between the findings of bitewing radiography and the gold standard. sensitivity, specificity, and the areas under the roc curves (auc) at d1 and d3 diagnostic thresholds are presented in table 3. the use of icdas - ii, lf, and fc demonstrated good to excellent diagnostic performances (auc between 0.88 and 1.0), while the performance of bitewing radiography for detection of occlusal lesions was shown to be weak (auc 0.56 and 0.59, resp.). at the d1 diagnostic level specificity for the same findings were observed for the sensitivity of the fc at d3 diagnostic level. table 4 presents the distribution of the sites according to the different methods cross - tabulated with the treatment decision. table 5 shows whether the treatment decisions changed significantly from preventive care to operative care when different methods were used for treatment planning. for example, when treatment decisions with icdas - ii were cross - tabulated with treatment decisions after bitewing assessment, operative rather than preventive treatment was chosen for significantly more sites (n = 19 and p = 0.003). similar findings were observed for treatment decisions involving icdas - ii versus fc with regular cutoffs (p 1.5 can be expected to indicate dentine caries. a comparable cut - off point (> 1.4) for the determination of dentine caries was found in other studies using the vistaproof [9, 24, 25 ]. in the present study, treatment decisions were determined using the cut - off points according to the manufacturer and according to the literature, respectively. it could be seen that sensitivity values at the d1 and d3-level were higher when cutoffs according to the literature were used (table 3). when these cutoffs were used for treatment planning, almost all of the clinically dentine lesions were planned to be treated operatively (34/36, table 7). however, it should be taken into account that 4 investigation sites could not be assessed due to technical problems while using the fc for caries detection. the use of bw for occlusal caries detection showed low sensitivity (table 3). other authors had reported higher values of sensitivity but similar specificity values when bw radiographs were evaluated for occlusal caries detection [26, 27 ]. looking at the correlation coefficients found in our study (table 2) it can be observed that bw had the weakest correlation compared to all other methods and to the gold standard, respectively. when bw was used for treatment decisions, more than 70% (29/40, table 7) of clinically dentine lesions usually studies on using radiography and treatment decisions tend to use bw for approximal caries detection rather than for occlusal lesions.. suggested in a recently published study that the icdas examination shows better performance than radiographic examination for occlusal caries detection. usually establish the validity of a detection system by using a gold standard against which the sensitivity and specificity of the diagnostic method can be calculated. a common gold standard used for occlusal caries lesions is the histological evaluation of hard tissue sections, with large variations in the methodology. teeth are hemisected at the place to be examined, or hard tissue sections are prepared whose thickness can vary considerably. in order to be close to clinical conditions we determined the depth of the lesions by opening the tooth surfaces with rotating instruments. this method might show some shortcomings, and in order to validate whether the caries was removed in total sectioning of the excavated teeth could probably have been an additional option. the results of our study show that preventive care for sound surfaces was suggested with each detection method, indicating high specificity of the treatment decision (table 7). almost all enamel lesions which were found clinically were planned to be treated using preventive treatment options. the greatest difference was found for treatment planning of dentine lesions, where the use of fc (with cut - offs according to the literature) had the greatest agreement between operative treatment and dentine lesions, followed by use of icdas - ii. this information may suggest that the use of fc is the primary criterion for evaluating the condition of the dental surface before the establishment of a treatment plan, followed by icdas - ii. but it should be taken into account that use of detection tools should always be seen as an adjunct to a primary visual detection of dental caries and thus it should rather be the other way round : that a tooth surface is first observed by a visual detection method and then as a second step, another device can be used for further detection. ie and verdonschot observed that although carious lesions can be observed by visual examination, caries preventive strategies will not be initiated until very late. a solution to this problem would be to detect caries lesions at an early stage of development, for example, by using systems like icdas - ii. in the study by diniz. icdas has proved to be reliable for early caries detection, but there was no strong correlation with histology. in the present study the findings (correlation to gold standard, performance, sensitivity, and specificity values) indicate that icdas - ii may have a high potential for detection and treatment planning, and that other devices, especially the fluorescence camera, could add substantial information to the visual examination, enabling examiners to plan treatment more accurately. of course, issues like caries activity and patient - based information can not be taken into account for treatment decisions within the limitations of an in vitro study. | aim. to use visual inspection (icdas - ii), laser fluorescence (lf), fluorescence based camera (fc) and radiographic examination (bw) for detection of caries and for treatment decision. methods. the occlusal sites of 84 extracted permanent teeth were examined using all methods and treatment decisions (preventive or operative care) were recorded based on each method independently. for validation of the findings, fissures were opened with rotating instruments and clinical depth was determined as gold standard. correlations (rs), sensitivity, specificity and auc were calculated. mcnemar test was used to show whether different methods led to significant changes in treatment decisions. results. highest correlation was found between icdas - ii and fc (rs 0.84), icdas - ii and gold standard (0.82) and fc and gold standard (0.81). icdas - ii provided the highest performance (auc 1.0), followed by fc (0.95) and lf (0.88). the greatest difference was found for treatment planning of dentine lesions, where the use of fc (cut - offs according to the literature) had the greatest agreement between operative treatment and dentine lesions, followed by use of icdas - ii. conclusion. icdas - ii may have high potential for detection and treatment planning, and other devices, especially the fluorescence camera, can add substantial information to the visual examination, enabling examiners plan treatment more accurately. |
the preoperative evaluation included assessment of the position of the limb on the side of the fused hip. the optimal position for hip fusion is 15-30 of hip flexion, 5-10 of adduction, and 0-10 external rotation.7) in addition to general and more specific examinations of the spine and lower extremities, the ipsilateral knee on the side of the hip fusion was examined with the knee off the end of the examination table to check the degree of flexion, stability, and patellar tracking. the preoperative radiographic evaluation included long leg standing views to assess the overall alignment of the limb and knee, and ensure optimal position of the fused hip.2) for the surgical procedure, the patient was placed supine on the operating table and the knee was placed at the level of the break in the table. throughout the procedure, the table was elevated and lowered episodically, and the foot of the table was flexed and flattened as needed, keeping the area sterile, using multiple sheets at the distal end when the table was lowered or flexed. after inflation of the tourniquet, a midline skin incision was made with a medial parapatellar capsular approach. the patella was everted 90 and a resection of 8-mm thickness was made initially. to facilitate exposure, both of the patients ' knees were very stiff preoperatively, making initial femoral guide placement difficult. after excising the menisci and cruciate ligaments, a tibial resection was first made using an extramedullary guide system to make gentle flexion of the knee possible for distal femoral resection (fig. we resected 10 mm of bone from the less worn plateau to achieve a tibial cut surface that was perpendicular to the shaft of the tibia in the coronal plane. the posterior tibial slope was set to 7 in the sagittal plane, as recommended by the manufacturer (nexgen, zimmer, warsaw, in, usa). a femoral intramedullary guide rod was placed, and an anterior provisional resection was made using the reference to the transepicondylar axis (fig. subsequently, the distal femoral resection was made at 5 of valgus, per the preoperative templating, considering the angle between the anatomical and mechanical axes of the femur. the size for the femoral component was selected, and the final anterior and posterior femoral cuts were then made. the flexion and extension gaps and limb alignment osteophytes were excised and contracted soft tissues were released to balance the knee. finally, tibial, femoral, and patellar preparation were performed and trial components placed. an 87-year - old man had his left hip fused surgically 52 years earlier due to previous femoral head and acetabular fractures. severe knee pain and limitation of activities of daily living due to ipsilateral knee osteoarthritis resulted in the need for tka (table 1, fig. 2a). his chronological age, medical condition, and satisfactory position of the fused hip made tha prior to the tka unnecessary. the position of components9) was satisfactory in radiographs at 4.7 years postoperative (table 1, fig. 2b). a 63-year - old man had a left hip fracture and left upper extremity amputation due to war injuries. he was hospitalized intermittently and in and out of a spica cast for several years. the skin and soft tissues around the hip were so atrophic and scarred that conversion of the hip fusion to tha was not considered a safe option. the clinical and radiographic results were satisfactory at the 7.8 years postoperative, although severe quadriceps scarring limited flexion (table 1, fig. an 87-year - old man had his left hip fused surgically 52 years earlier due to previous femoral head and acetabular fractures. severe knee pain and limitation of activities of daily living due to ipsilateral knee osteoarthritis resulted in the need for tka (table 1, fig. 2a). his chronological age, medical condition, and satisfactory position of the fused hip made tha prior to the tka unnecessary. the position of components9) was satisfactory in radiographs at 4.7 years postoperative (table 1, fig. a 63-year - old man had a left hip fracture and left upper extremity amputation due to war injuries. he was hospitalized intermittently and in and out of a spica cast for several years. the skin and soft tissues around the hip were so atrophic and scarred that conversion of the hip fusion to tha was not considered a safe option. the clinical and radiographic results were satisfactory at the 7.8 years postoperative, although severe quadriceps scarring limited flexion (table 1, fig. occasionally, performing tha first prior to tka is not the best option in patients with severe knee osteoarthritis and ipsilateral hip fusion because of the high complication rate of the conversion tha and the already satisfactory function of patients with a well - aligned fused hip. we report a surgical technique and results of two patients where we performed tka without a conversion tha. a major limitation of this study is a report of the surgical technique with the results of only two patients. however, patients with this specific condition are extremely uncommon. in this report, the preoperative and intraoperative challenges and technical considerations, as well as the clinical and radiographic results are addressed.2,3,8) one previous report suggested that tka alone is unlikely to provide a satisfactory result.3) another study reported an acceptable clinical result with a postoperative range of motion (rom) of 84,2) and a third study reported that postoperative stiffness was encountered and manipulation of the knee under anesthesia was frequently necessary.8) reasons for the postoperative limited rom might be due to quadriceps scarring, the use of older implants, and suboptimal positioning of knee component due to surgical difficulties encountered. however, adjustment of the operating table position and modification of the surgical steps facilitated initial exposure and the bone cuts. we episodically flexed the foot of the table 90 to allow maximum assistance for flexion of the knee. we opted to resect the patella and tibia before performing any femoral cuts, given the stiffness of the knee and difficulties accessing the femur. tka alone below a fused hip should be reserved for patients in whom the hip was fused in an optimum position with satisfactory alignment.2) malpositioning of a fused hip in excessive internal or external rotation prohibits accurate positioning of a tka.3) inaccurate axial alignment of components will lead to functional limitation and mechanical failure. our preoperative physical examination included an assessment of the position of the limb on the side of the fused hip. tka without takedown of the fused hip and conversion tha should be considered only in patients with optimum positioning of the fused hip. in conclusion, tka can be performed successfully with adjustments of the table position and modification of the surgical steps in patients in whom the hip is arthrodesed in an already acceptable position. this also avoids the unnecessary conversion of a well - aligned fused hip and avoids the complications of a conversion tha. | we report the surgical technique used to perform posterior - stabilized total knee arthroplasty (tka) in two patients with a well positioned and functional hip arthrodesis. intraoperatively, the operating table was placed in an increased trendelenburg position. episodically, we flexed the foot of the table by 90 to allow maximal knee flexion to facilitate exposure and bone cuts. we opted to resect the patella and tibia first to enable exposure, given the stiffness of the arthritic knee. one patient 's medical condition prohibited complex conversion total hip arthroplasty (tha) prior to the tka. the other patient 's scarred soft tissues around the hip, due to chronic infection and multiple operations, made tha risky. the final outcome provided satisfactory results at a minimum of 2 years postoperatively. tka can be successfully performed with adjustments of table position and modification of the sequence of surgical steps in patients with ipsilateral hip fusion. |
we have not yet fully realized how covertly they have entered our environment and what problems they bring. next to alcohol and tobacco, other drugs are forcefully entering out lives ; they are abused often by teenagers who wish to intensify the current state of joy and happiness. the drug will, at least for a while, allow users feel strong and to forget about disappointments, probably because the reality of life affected them more severely than expected and they have no other means of dealing with it. some authors claim that the most probable causes of illegal drug use among young people may be : low self - esteem, peer pressure, examples of adults, psychological problems, boredom and curiosity, alienation, and biological predisposition. use of illegal drugs is the number one public health problem for university students because it is the leading cause of preventable death and injury among students age 1825 years. during the past decade the use of marijuana has increased considerably among college and university students, whereas alcohol consumption has held relatively steady during the same period. by means of this study the study was carried out at the constantine the philosopher university in nitra, the fifth biggest city in slovakia. the research group consisted of females studying teaching, because females are predominant this field of the study. we studied this group from 2 points of view : the group of students as a whole and 2 sub - groups. as a criterion for division into sub - groups, we used their answers to the questions asked in the questionnaire in which they confirmed use of any illegal drug. the students admitting use of illegal drugs were marked as group e (experimental, n=72 ; 33.48%), the students, who have never used an illegal drug were marked as group c (control, n=143 ; 66.51%). to obtain responses from students, we used anonymous questionnaires. we tried to avoid possible interference from irrelevant factors, for example, before an exam there is a bias because the students do not pay much attention to a questionnaire. the questionnaire contained 24 items, each of which offered multiple choice answers, out of which only 1 could be selected, which allowed expressing of ratio of agreement, or disagreement with the specified statement, or to present a neutral attitude. the first area was aimed at obtaining basic information on students and select characteristics of their family environment (age, location of parental home, parents education, communication with parents, understanding and support from parents, parents awareness about their children s free - time activities, and consumption of alcohol and tobacco by parents). in the second area, we determined the extent of sports activities of female students. if your male friend used drugs, would you have abandoned him ? will you have committed treason or spying if you alert his parents or teacher to the illegal drug use ? would you be able to refuse an illegal drug if somebody (for instance some of your friends) offered it to you ? the fourth area gathered information on opinions and attitudes of female about issues related to use of illegal drugs, their own illegal drug use experience (collecting information about drugs, places of most frequent contact of young people with drugs, time of first use, opinion on anti - drug activities, extent of consuming of alcohol and smoking, reasons for drug abuse). for evaluation of answers we used pearson s chi - square () test of independence by which we measured statistical significance of differences between students who use and do not use illegal drugs. it means that knowledge of 1 of the variables can not help to improve estimation of the value of another variable. if p - value is lower than the chosen level of significance (p<0.05 ; p<0.001) then the null hypothesis is denied ; the measured difference is too high for being only the consequence of random selection and it is therefore statistically significant. if a p - value is equal to or higher than the chosen level of significance, the null hypothesis can not be denied, meaning that the measured difference may be the consequence of random selection and it is not statistically significant. for evaluation, for evaluation of answers we used pearson s chi - square () test of independence by which we measured statistical significance of differences between students who use and do not use illegal drugs. it means that knowledge of 1 of the variables can not help to improve estimation of the value of another variable. if p - value is lower than the chosen level of significance (p<0.05 ; p<0.001) then the null hypothesis is denied ; the measured difference is too high for being only the consequence of random selection and it is therefore statistically significant. if a p - value is equal to or higher than the chosen level of significance, the null hypothesis can not be denied, meaning that the measured difference may be the consequence of random selection and it is not statistically significant. for evaluation, we confirmed a significant relationship between using illegal drugs and the place of residence (=7.585 ; p<0.05). we found that education of parents has statistical significance for their children using illegal drugs (=10.14 ; p<0.05). a very high percentage of parents whose daughters used illegal drugs had a university degree (n=26 ; 31.32%). communication between parents and their children and parents paying attention to their children plays an important role in risk - behavior (use of illegal drugs) of their children. most of the university women surveyed said they had excellent support, help, and understanding from their parents (n=60 ; 83.33%). we found a statistically significant dependence between use of illegal drugs and understanding of parents in groups e and c (=5.643, p<0.05). parents of students who have never used illegal drugs were interested in how their children are spending their free time (n=98 ; 68.53%). in this parameter we confirmed significant dependence between the experimental and control groups (=18.667 ; p<0.001). the most common response of both the experimental group and control group mothers was that they drink alcohol only occasionally (n=39 ; 28.46%) or students from both groups most frequently answered that their fathers drank only occasionally. a statistical significance was not been detected between using illegal drugs by students and alcohol drinking of their parents (mothers : =0.110 ; p<0.05, fathers : =5.869 ; p<0.05). the smoking of parents and using illegal drugs by their children has not show any statistically significant dependence (mothers : =2.461 ; p<0.05, fathers : =3.676 ; p<0.05). female students using illegal drugs had grown in families where parents used to smoke daily (mothers : n=12 ; 17.39%, fathers : n=25 ; 35.21%). results of the second range of issues are shown in table 2 and figure 1. there was no significant association between sports leisure activities and taking illegal drugs (=5.768 ; p<0.05). analysis results of the third range of issues are shown in table 3. in this part we found out whether taking illegal drugs impacts relationships among friends. the student answers to the question about leaving their boyfriend who is taking drugs were equal. yes (n=22 ; 30.55%) and no (n=28 ; 38.88%) have higher percentage in the e group of students. the response i do not know was frequent in the c group of students (n=67 ; 44.37%). students in the e group considered that telling the parents of their boyfriend that he is using illegal drugs is a form of betrayal (n=20 ; 27.77%). on the other hand, in the c group of students this response was about 16% less frequent. both study groups indicated that illegal drugs were most frequently present at discos and at gatherings of friends. a significant association was detected between taking illegal drugs and alcoholism (=16.645 ; p<0.001), as well as with smoking (=6.226 ; p<0.05). students had first used illegal drugs in high school (n=49 ; 70.40%) and 19.70% (n=14) of students reported that their first contact with illegal drugs was in elementary school. the highest number of participants prefer this drug (n=72 ; 77.17%, figure 2). a significant association (=39.456 ; p<0.001) was also detected between taking illegal drugs and source of information about drugs. the students who do not us illegal drugs have learned about illegal drugs at school (n=89 ; 44.95%), and the students who are taking illegal drugs learned about them largely from their friends (n=47 ; 43.92%). few students in either group mentioned parents as a source of illegal drug information (e=4.16%, c=6.56%). we found differences in illegal drug awareness in slovakia. while more than half students in the c group consider their drug awareness as adequate (n=74 ; 52.48%), the students in the e group consider that as an inadequate (n=34, 47.22%) and this was statistically significant (=16.897 ; p<0.001). most young women declare curiosity as a reason for trying illegal drugs (n=59 ; 67.81%). agreement with legalization of marijuana was reported by 41.66% of drug students using illegal drugs and 8.39% of students who do use illegal drugs and this difference was statistically significant (=33.83 ; p<0.001). most students (n=71 ; 80.19%) reported that someone had offered them an illegal drug. we confirmed a significant relationship between using illegal drugs and the place of residence (=7.585 ; p<0.05). we found that education of parents has statistical significance for their children using illegal drugs (=10.14 ; p<0.05). a very high percentage of parents whose daughters used illegal drugs had a university degree (n=26 ; 31.32%). communication between parents and their children and parents paying attention to their children plays an important role in risk - behavior (use of illegal drugs) of their children. most of the university women surveyed said they had excellent support, help, and understanding from their parents (n=60 ; 83.33%). we found a statistically significant dependence between use of illegal drugs and understanding of parents in groups e and c (=5.643, p<0.05). parents of students who have never used illegal drugs were interested in how their children are spending their free time (n=98 ; 68.53%). in this parameter we confirmed significant dependence between the experimental and control groups (=18.667 ; p<0.001). the most common response of both the experimental group and control group mothers was that they drink alcohol only occasionally (n=39 ; 28.46%) or students from both groups most frequently answered that their fathers drank only occasionally. a statistical significance was not been detected between using illegal drugs by students and alcohol drinking of their parents (mothers : =0.110 ; p<0.05, fathers : =5.869 ; p<0.05). the smoking of parents and using illegal drugs by their children has not show any statistically significant dependence (mothers : =2.461 ; p<0.05, fathers : =3.676 ; p<0.05). female students using illegal drugs had grown in families where parents used to smoke daily (mothers : n=12 ; 17.39%, fathers : n=25 ; 35.21%). results of the second range of issues are shown in table 2 and figure 1. there was no significant association between sports leisure activities and taking illegal drugs (=5.768 ; p<0.05). analysis results of the third range of issues are shown in table 3. in this part we found out whether taking illegal drugs impacts relationships among friends. the student answers to the question about leaving their boyfriend who is taking drugs were equal. yes (n=22 ; 30.55%) and no (n=28 ; 38.88%) have higher percentage in the e group of students. the response i do not know was frequent in the c group of students (n=67 ; 44.37%). students in the e group considered that telling the parents of their boyfriend that he is using illegal drugs is a form of betrayal (n=20 ; 27.77%). on the other hand, both study groups indicated that illegal drugs were most frequently present at discos and at gatherings of friends. a significant association was detected between taking illegal drugs and alcoholism (=16.645 ; p<0.001), as well as with smoking (=6.226 ; p<0.05). students had first used illegal drugs in high school (n=49 ; 70.40%) and 19.70% (n=14) of students reported that their first contact with illegal drugs was in elementary school. the highest number of participants prefer this drug (n=72 ; 77.17%, figure 2). a significant association (=39.456 ; p<0.001) was also detected between taking illegal drugs and source of information about drugs. the students who do not us illegal drugs have learned about illegal drugs at school (n=89 ; 44.95%), and the students who are taking illegal drugs learned about them largely from their friends (n=47 ; 43.92%). few students in either group mentioned parents as a source of illegal drug information (e=4.16%, c=6.56%). we found differences in illegal drug awareness in slovakia. while more than half students in the c group consider their drug awareness as adequate (n=74 ; 52.48%), the students in the e group consider that as an inadequate (n=34, 47.22%) and this was statistically significant (=16.897 ; p<0.001). most young women declare curiosity as a reason for trying illegal drugs (n=59 ; 67.81%). agreement with legalization of marijuana was reported by 41.66% of drug students using illegal drugs and 8.39% of students who do use illegal drugs and this difference was statistically significant (=33.83 ; p<0.001). most students (n=71 ; 80.19%) reported that someone had offered them an illegal drug. our study deals with use of illegal drugs by female university students, and discusses some selected aspects (e.g., family background, sport activities, and relationships with friends) to ascertain their views, attitudes and behavior in relation to illegal drug use. as subjects we chose female university students because after the graduation they will work at primary and secondary schools as teachers and they will shape the younger generation with their attitudes and beliefs in the future. adolescent substance abuse potentially holds a number of negative implications for the health and well - being of the individual, including increased risk for injury and death from interpersonal violence, motor vehicle injury, drowning, increased probability of engaging in high - risk sexual behaviors ; and increased risk for suicidal ideation and behaviors. some other studies indicated that there is a link between substance abuse and getting involved in crime, and a high prevalence of substance abuse among juvenile offenders. not having friends was positively associated with use of tobacco and illicit drugs and negatively associated with alcohol use. in our group, 72 (33.48%) young women admitted taking illegal drugs and 143 (66.51%) had never tried illegal drugs. in some studies it has been shown that there is a strong association between adolescent age and substance abuse. logistic regression analysis indicated that the age of students had a relationship with lifetime alcohol use, but results showed that there is no significant association between age and lifetime drug abuse. analysis of issues related to family background of respondents confirmed the relationship between drug use, place of residence, and education of parents. students of both studied groups considered the communication with their parents was satisfactory and also positively evaluated their parents in providing assistance and support. their parents are also interested how they are spending their leisure time and which activities they prefer. also negatively associated with such risk behaviors were characteristics of the family context represented by : living with parents, having meals together, and parental supervision (when parents know what the child does in their free time). these factors of family background showed a statistically significant relationship between experimental and control group. parental consumption of legal drugs (alcohol and cigarettes) did not show statistical significance. we did not confirm a statistically significant relationship between leisure time sport activity and drug use. it is interesting that the students of both groups did not strictly express an unfavorable opinion about abandonment of a friend who would take drugs. the most frequent place of contact with illegal drugs is during meeting with friends and at the disco. consumption of legal drugs (alcohol and cigarettes) in the studied population was at a satisfactory level, and statistical analysis did not confirm the association between experience with drug and alcohol consumption and smoking. the findings of this study, like those in other studies, showed that lifetime alcohol use and lifetime drug abuse relates to the smoking status of friends. in spite of these findings, we can not determine whether having smokers as close friends are a risk factor for substance abuse or if students who used illegal drugs choose smoker as their friends. on the other hand, the adolescents who become friends may have common characteristics that may have association with substance abuse. some authors have argued that the use of alcohol and tobacco tends to precede and to increase the risk of initiating illegal drug use. similarly, other authors found that regular smokers are more likely to drink and are 1030 times more likely to use illegal drugs than non - smokers. the results of a study conducted in 10th grade students showed that 16.9% of students were experimenters and 2.5% of students were regular smokers. furthermore, the results of a recent study showed that 32% of students had experienced alcohol consumption and 2.1% of them had lifetime drug abuse. during the past decade, the use of marijuana and other illicit drugs has increased considerably among college students, whereas alcohol use has held relatively steady during the same period. first experiences with illegal drug use tended to be in high school, followed by elementary school and university. marijuana is the most consumed illegal drug in our studied population of students, followed by hashish. epidemiological research indicates young americans 1829 years old are the most likely to use marijuana or other illicit drugs relative to other age groups. most students, who are not using drugs, learned about illegal drugs from school, while the students who already have experience with drug use learned about drugs from their friends. most students (in both studied groups) considered they had adequate knowledge about drugs. it is a striking finding, because having negative information about drugs did not hamper them from taking drugs. student may believe they have adequate knowledge about drugs, but deeper analysis of their knowledge proves that this knowledge is drawn from friends, media, and magazines, and is only superficial. insufficient information from parents about negative effects of drugs on the human body can be considered as a negative phenomenon. while more than half of students not using drugs considered awareness of this issue in slovakia to be sufficient, drug users consider it to be inadequate. both groups of young women considered that the most effective form of prevention by is discussion with the reformed male drug user. curiosity is the most common cause that led to the first contact with any drug in our group of young women. we assume that the main reason is to fit in with a group of friends, but this was not confirmed in our study. slovak research conducted at the university shows that curiosity is on the leading reason, but that other reasons include friends, false heroism, personal problems, family problems, incidental contact with a dug dealer, problems at school, and boredom. most drug users agree with legalizing marijuana in slovakia, as opposed to young women, who have negative attitude towards drugs. we have determined that education of parents has a statistically significant influence on drug abuse of their children. communication between parents and children and parents paying attention to their children have significant roles in risky behavior (drug abuse). parents of students not abusing drugs were interested in how their children spend their free time. we have not confirmed a statistically relevant correlation between spending of free time engaged in sports and drug abuse. in the area evaluating the attitudes towards friends, we have not detected any statistically relevant differences between the groups. we have confirmed the relationship between consumption of alcohol and drug abuse and smoking. the most consumed, so - called soft drug, in our group of female university students is marijuana. we have recorded statistically significant dependence between drug abuse and source of information on drugs and also between drug abuse and attitude towards legalization of marijuana. a relative majority of young woman stated curiosity as a reason for use of drugs. our study has revealed some context, pointing to the need of more prevention at primary school. because the issue of drug use by university students is still studied, our findings are important for comparison and generalization of causes of the steady increase in the number of young people using illegal drugs. these findings suggest that institutions of higher education may need to provide opportunities for screening to detect students at risk for drug abuse. more importantly, additional efforts are needed to ensure that adequate assessment, monitoring, and treatment options are available for those individuals at risk for drug abuse. past college - based longitudinal studies have shown that many forms of drug use tend to increase during the transition from high school to college and then decline as students graduate and assume post - college responsibilities. however, more prospective longitudinal studies are needed to determine whether these findings also apply to the course of drug use disorders among university students. | backgroundthis study is focused on the issue of illegal drug use among female university students preparing to become teachers. the main aim was to determine the frequency of drug abuse in a group of young women (n=215, mean age 20.44 years).material / methodsusing survey methods, we determined that 33.48% of female university students in slovakia use illegal drugs and 66.51% of students have never used illegal drugs. differences between these groups were determined using statistical analysis, mostly in 4 areas of survey questions.resultswe determined that education of parents has a statistically significant influence on use of illegal drugs by their children (2=10.14 ; p<0.05). communication between parents and children and parental attention to children have a significant role in determining risky behavior (illegal drug use, 2=8.698, p<0.05). parents of students not using illegal drugs were interested in how their children spend their free time (68.53%). we confirmed the relationship between consumption of alcohol and illegal drug use (2=16.645 ; p<0.001) and smoking (2=6.226 ; p<0.05). the first contact with drugs occurs most frequently at high school age. the most consumed soft drug in our group of female university students is marijuana.conclusionsour findings are relevant for comparison and generalization regarding causes of the steady increase in number of young people using illegal drugs. |
nowadays nonalcoholic fatty liver disease (nafld) known as the most common chronic liver disease worldwide and its prevalence continue to rise. prevalence of nafld has been reported between 2.8% and 24% in the general population in different countries. nafld is reported as the most common reason for elevated alanine aminotransferase (alt) levels in iranian population and its prevalence is estimated 7% for children and 35% for adults. due to the lack of enough scientific evidence, the optimum approach for nafld treatment is unclear. there are different therapeutic methods which all of them are based on the modification of underlying etiologic factors. increasing evidence shows the influence of the intestinal flora on liver pathology. in the other, nafld is associated with increased intestinal permeability that is related to severity of hepatic steatosis and intestinal bacterial overgrowth has been reported in 50% of these patients. in addition, changes in intestinal bacterial flora due to stress or improper eating habits can play an important role in the pathogenesis or progression of nafld. fructooligosaccharids such as inulin, other oligosaccharides, lactulose, resistant starch and dietary fiber (prebiotics), and boost probiotics response. they can enhance the growth of bifidobacteria or lactobacilli and may be helpful in controlling or reduction of harmful bacteria growth. in several clinical trials, beneficial effects of probiotics it seems that liver fat metabolism can be affected by bacteria and potentially by probiotics. in addition, there is some evidence that shows probiotics have a protective effects on acute liver injury and symbiotic can leading to improvement in both liver inflammation and fibrosis in animal model., there is no study, which investigated the effect of symbiotic in nafld patients. hence with respect to the high prevalence of the fatty liver disease in our country and lack of accurate data on this patient 's condition in iran and neighboring countries, more researches on treatment of this patients seem necessary. therefore, in present clinical trial we evaluated the effect of short - term symbiotic supplementation, as a simple, low cost and without side effect treatment component on liver enzymes, c - reactive protein (crp) and steatosis grade in nafld patients. the protocol of this randomized, double - blind, placebo - controlled clinical trial was approved by the research ethics committee of isfahan university of medical sciences and registered in iranian registry of clinical trial (irct2013122811763n15). nafld volunteers (by ultrasound) were referred to isfahan endocrine and metabolism research center and upon meeting the study criteria were enrolled into this study (from march to july 2014). the inclusion criteria included age 1860 years, no other liver disease (such as hepatitis c, hepatitis b and autoimmune hepatitis ; wilson 's disease), no organ transplantation, no inflammatory bowel disease, no self - reported specific disease and malignancies, no pregnancy and lactation, no corticosteroids, amiodarone, tamoxifen, cyclines, perhexiline, methotrexate, hydralazine, laxatives, and oral contraceptive pill medication, no vitamin - mineral, antioxidant, and omega-3 supplementation. study volunteers were excluded for failure to follow the study 's guidelines (< 90% compliance, subject 's compliance was evaluated by counting the remaining capsules at the end of the 4 and 8 week), and antibiotics therapy during the study. based on aspartate transaminase (ast) and alt as main variables of the study, 34 patients were required in each group (power 80% and = 5%). considering 20% sample loss, 80 patients were enrolled. participants were randomly allocated to two numerically equal groups from a double - blind, 80-person list, using a table of random digits and given either symbiotic in form of a 500 mg capsule (familact, produced by zisttakhmir company) containing seven species of probiotic bacteria (lactobacillus casei, lactobacillus acidophilus, lactobacillus rhamnosus, lactobacillus bulgaricus, bifidobacterium breve, bifidobacterium longum, streptococcus thermophilus) and fructooligosaccharidesor a placebo capsule (containing 120 mg starch). faculty of pharmacy, isfahan university of medical sciences, prepared placebo capsules, similar in shape and appearance as symbiotic capsules. all study participants ingested the capsules (symbiotic or placebo) once daily for 8 weeks. six participants were excluded during the study (because of unwillingness or failure to follow the study 's guidelines), which left 38 volunteers in the symbiotic group and 36 in the placebo group [figure 1 ]. a flow chart of patients who entered the study informed consent was obtained from all participants, at the beginning of the study. all data collection and measurements general information including age, sex, smoking, menopausal status, medical history, and medication were collected using interview. body weight was measured to the nearest 0.1 kg with minimal clothing by means of a digital seca balance. height was measured to the nearest 0.5 cm without shoes by means of a seca stadiometer. body mass index (bmi) was calculated for each patient (bmi = weight in kg / height 2 in m). we metered waist and hip circumference on a horizontal plane at the level of the iliac crest by an ergonomic circumference measuring tape (model 201 ; seca gmbh and co, kg, hamburg, germany). alt and ast were measured with colorimetric method (using kits from pars azmoon company, tehran, iran). laboratory reference ranges of alt, ast, and crp were considered 531 (iu / l), 531 (iu / l) and up to 1.0 (mg / dl), respectively. ultrasound grading to state liver steatosis was determined by means of an east medical sonographic scanner equipped with a convex 3.5 mhz browser by a skilled radiologist. nonalcoholic steatohepatitis (nash) grade was assessed using the national health and nutrition examination survey iii criteria : grade 0 normal, grade 1 mild, grade 2 moderate, grade 3 severe. the compendium of physical activities was used to standardize the assignment of metabolic equivalent of task intensities. dietary intake was collected by food record (3-day food record in gram before, and the same after intervention). dietary data were analyzed using nutritionist iv software adjusted for iranian foods (version 4.1, first databank division, the hearst corporation, san bruno, ca, usa). data were assessed for normality by the shapiro wilk test. in case of normal distribution, paired t - test and in case of nonnormal distribution, wilcoxon signed rank test were used for comparing data within groups. independent t - test (in case of normal distribution) or nonparametric statistical test, mann whitney u - test (in case of nonnormal distribution), was used for comparing data between two groups. analysis of covariance (ancova) was used for evaluating between group differences based on quantitative data ; adjustment was made for differences in baseline covariates. within and between groups differences based on qualitative variables all tests were two - sided, and p < 0.05 were considered statistically significant. the protocol of this randomized, double - blind, placebo - controlled clinical trial was approved by the research ethics committee of isfahan university of medical sciences and registered in iranian registry of clinical trial (irct2013122811763n15). nafld volunteers (by ultrasound) were referred to isfahan endocrine and metabolism research center and upon meeting the study criteria were enrolled into this study (from march to july 2014). the inclusion criteria included age 1860 years, no other liver disease (such as hepatitis c, hepatitis b and autoimmune hepatitis ; wilson 's disease), no organ transplantation, no inflammatory bowel disease, no self - reported specific disease and malignancies, no pregnancy and lactation, no corticosteroids, amiodarone, tamoxifen, cyclines, perhexiline, methotrexate, hydralazine, laxatives, and oral contraceptive pill medication, no vitamin - mineral, antioxidant, and omega-3 supplementation. study volunteers were excluded for failure to follow the study 's guidelines (< 90% compliance, subject 's compliance was evaluated by counting the remaining capsules at the end of the 4 and 8 week), and antibiotics therapy during the study. based on aspartate transaminase (ast) and alt as main variables of the study, 34 patients were required in each group (power 80% and = 5%). considering 20% sample loss, 80 patients were enrolled. participants were randomly allocated to two numerically equal groups from a double - blind, 80-person list, using a table of random digits and given either symbiotic in form of a 500 mg capsule (familact, produced by zisttakhmir company) containing seven species of probiotic bacteria (lactobacillus casei, lactobacillus acidophilus, lactobacillus rhamnosus, lactobacillus bulgaricus, bifidobacterium breve, bifidobacterium longum, streptococcus thermophilus) and fructooligosaccharidesor a placebo capsule (containing 120 mg starch). faculty of pharmacy, isfahan university of medical sciences, prepared placebo capsules, similar in shape and appearance as symbiotic capsules. all study participants ingested the capsules (symbiotic or placebo) once daily for 8 weeks. six participants were excluded during the study (because of unwillingness or failure to follow the study 's guidelines), which left 38 volunteers in the symbiotic group and 36 in the placebo group [figure 1 ]. a flow chart of patients who entered the study informed consent was obtained from all participants, at the beginning of the study. all data collection and measurements general information including age, sex, smoking, menopausal status, medical history, and medication were collected using interview. body weight was measured to the nearest 0.1 kg with minimal clothing by means of a digital seca balance. height was measured to the nearest 0.5 cm without shoes by means of a seca stadiometer. body mass index (bmi) was calculated for each patient (bmi = weight in kg / height 2 in m). we metered waist and hip circumference on a horizontal plane at the level of the iliac crest by an ergonomic circumference measuring tape (model 201 ; seca gmbh and co, kg, hamburg, germany). alt and ast were measured with colorimetric method (using kits from pars azmoon company, tehran, iran). laboratory reference ranges of alt, ast, and crp were considered 531 (iu / l), 531 (iu / l) and up to 1.0 (mg / dl), respectively. ultrasound grading to state liver steatosis was determined by means of an east medical sonographic scanner equipped with a convex 3.5 mhz browser by a skilled radiologist. nonalcoholic steatohepatitis (nash) grade was assessed using the national health and nutrition examination survey iii criteria : grade 0 normal, grade 1 mild, grade 2 moderate, grade 3 severe. the compendium of physical activities was used to standardize the assignment of metabolic equivalent of task intensities. dietary intake was collected by food record (3-day food record in gram before, and the same after intervention). dietary data were analyzed using nutritionist iv software adjusted for iranian foods (version 4.1, first databank division, the hearst corporation, san bruno, ca, usa). data were assessed for normality by the shapiro wilk test. in case of normal distribution, paired t - test and in case of nonnormal distribution, wilcoxon signed rank test were used for comparing data within groups. independent t - test (in case of normal distribution) or nonparametric statistical test, mann whitney u - test (in case of nonnormal distribution), was used for comparing data between two groups. analysis of covariance (ancova) was used for evaluating between group differences based on quantitative data ; adjustment was made for differences in baseline covariates. within and between groups differences based on qualitative variables all tests were two - sided, and p < 0.05 were considered statistically significant. there were no differences in baseline characteristics between symbiotic and placebo groups [table 1 ]. we observed no significant differences in age, anthropometric measures, crp level, liver enzymes, dietary intake, and physical activity status between two groups. baseline characteristics of the study subjects who received symbiotic or placebo none of the participants had normal grade steatosis at baseline and comparison of severity of hepatic steatosis between two groups before (p = 0.59) and after the intervention (p = 0.18) through k test showed no significant difference. in symbiotic group within group changes assessment through mcnemar test showed that 50% and 25% of patients with mild (grade 1) and moderate (grade 2) nafld became normal, respectively. in 43.8% of patients with moderate nafld, severity of hepatic steatosis reduced to grade 1and in the rest of subjects remained unchanged. only in 9.1% of patients with mild nafld in symbiotic group steatosisseverity increased to grade 2. in the placebo group, 23.5% of patients with mild (grade 1) nafld recovered and in 11.8% and 5.9%, steatosis severity increased to grade 2 and 3, respectively. severity of steatosis in a patient with grade 3 nafld in placebo group fell to grade 2. in general, changes in symbiotic group were significant and in placebo group were not significant [table 2 ]. comparison of steatosis grade before and after the intervention between symbiotic and placebo groups crp and liver enzymes levels at baseline and after intervention period were reported in table 3. alt and ast levels did not change within the intervention group but were significantly increased in the placebo group. ancova detected no differences in inflammatory markers and hepatic function tests levels between two groups after adjustment for energy intake and baseline values. crp, liver enzymes and anthropometric measures of participants before and after the intervention comparison of two groups at base line and the end of study showed no significant differences in dietary intake and physical activity level. paired t - test and wilcoxon signed ranks test also showed no significant within group changes in these variables [table 4 ]. there were no differences in baseline characteristics between symbiotic and placebo groups [table 1 ]. we observed no significant differences in age, anthropometric measures, crp level, liver enzymes, dietary intake, and physical activity status between two groups. none of the participants had normal grade steatosis at baseline and comparison of severity of hepatic steatosis between two groups before (p = 0.59) and after the intervention (p = 0.18) through k test showed no significant difference. in symbiotic group within group changes assessment through mcnemar test showed that 50% and 25% of patients with mild (grade 1) and moderate (grade 2) nafld became normal, respectively. in 43.8% of patients with moderate nafld, severity of hepatic steatosis reduced to grade 1and in the rest of subjects remained unchanged. only in 9.1% of patients with mild nafld in symbiotic group steatosisseverity increased to grade 2. in the placebo group, 23.5% of patients with mild (grade 1) nafld recovered and in 11.8% and 5.9%, steatosis severity increased to grade 2 and 3, respectively. severity of steatosis in a patient with grade 3 nafld in placebo group fell to grade 2. in general, changes in symbiotic group were significant and in placebo group were not significant [table 2 ]. comparison of steatosis grade before and after the intervention between symbiotic and placebo groups crp and liver enzymes levels at baseline and after intervention period were reported in table 3. alt and ast levels did not change within the intervention group but ancova detected no differences in inflammatory markers and hepatic function tests levels between two groups after adjustment for energy intake and baseline values. comparison of two groups at base line and the end of study showed no significant differences in dietary intake and physical activity level. paired t - test and wilcoxon signed ranks test also showed no significant within group changes in these variables [table 4 ]. the present study shows that an 8-week supplementation with a 500 mg symbiotic capsule (familact, produced by zisttakhmir company) containing seven species of probiotic bacteria (l. casei, l. acidophilus, l. rhamnosus, l. bulgaricus, b. breve, b. longum, s. thermophilus) and fructooligosaccharides in nafld patients does not change high - sensitivity - crp, alt and ast levels in comparison between two groups but is associated with steatosis grade improvement. based on our knowledge, this is the first study that investigates the effect of symbiotic supplementation on liver enzymes, crp and ultrasound findings in nafld patients. nowadays, there is no registered drug for the treatment of nafld and new data are coming. our results showed that in symbiotic group aminotransferase levels remained static as a result of symbiotic supplementation while they were increased in placebo group., demonstrated that supplementation with vsl#3 (s. thermophilus and several species of lactobacillus and bifidobacteria) in rats for 9 weeks had no effect on alt. in xu. study on sprague - dawley rats, probiotic supplementation for 12 weeks also showed no effect on alt but attenuated hepatic fat accumulation. review of animal studies showed that the levels of liver enzymes reduced in 22% of cases as a result of treatment with probiotics. a double - blind clinical trial by aller., in 2011 on 30 patients with nafld showed that 6 months treatment with probiotics reduced alt and ast levels., in 2013 conducted a meta - analysis to investigate results of four randomized trials involving 134 nafld / nash patients and concluded that probiotic therapy decreased alt and ast, significantly. most experimental and human studies in this field, in contrast with our results, had led to a reduction in liver enzymes. despite observing some degrees of hepatic steatosis at baseline, but mean liver enzymes level was normal in the present study. hence, detecting no change in alt and ast levels seems reasonable. on the other hand, liver enzymes increase in the placebo group noted that a symbiotic can at least prevents progression of the disease and a longer period of intervention with a higher dosage of supplement in patients with elevated levels of liver enzymes might show significant effects on these markers. in our study previous studies in animals with alcoholic and nafld suggest that treatment with probiotics can reduce liver damage. according to wong., study in 2013, intra hepatic triglyceride content in patients with nash was reduced as a result of 6 months intervention with probiotics., study showed no effect on inflammation and liver steatosis but their study duration was less than ours was and our supplements were containing probiotics and prebiotics that can amplify the effects. moreover, although weight in the symbiotic group significantly decreased, dietary intakes and physical activity levels (as the main factors contributing to the prevention and treatment of nafld patients) showed no significant differences at the baseline and the end of study between two groups. therefore, it can be concluded that the observed changes in weight, liver enzymes and steatosis were not related to physical activity and dietary status. moreover, as other studies showed, symbiotic supplementation can cause weight loss. in addition, the observed results in liver enzymes and the degree of steatosis after adjustment for energy intake and baseline values remained unchanged, so we can conclude that the steatosis improvement and stability of transaminases levels in the symbiotic group (against their increase in the placebo group) might be a result of symbiotic supplementation. it seems the beneficial effects of symbiotic can be related to reducing the impact of pathogenic bacteria on nafld development by exclusion or inhibition of invading bacteria, as well as by producing antimicrobial factors such as scfa. control of bacterial flora can lead to a reduction of endotoxins and other toxic compounds derived from bacteria, such as ethanol, phenol, and indole, which cause liver damage. on the other side, they can inhibit urease activity of gut microflora bacteria, their ammonia production, and its diffusion into the portal system. it is said that crp levels are significantly associated with fatty liver histological features (steatosis grade, necroinflammation, and fibrosis) ; this supports the earlier hypothesis that nafld is associated with low - grade systemic inflammation. crp in our study remain unchanged as results of loguercio., study which showed 3 months of treatment with probiotic vsl#3, study in 2013 also showed no effect for 12 weeks probiotic supplementation on crp in bio f1b golden syrian hamsters but decreased liver triglyceride and cholesterol concentrations. malaguarnera in a double - blind, placebo - controlled randomized control trial in 2011 on 66 nafld patient showed that probiotic with fos and lifestyle modification (i.e. diet and exercise) for 24 weeks significantly reduced serum ast levels, tumor necrosis factor - alpha, and crp. intervention duration of these studies was longer than present investigation and this can be cause of differences. symbiotic capsules in our study were containing fructooligosaccharides, which are now considered because of their prebiotic properties. fructooligosaccharides can make bifidobacteria the dominant species in the colon and may help to control or reduce the growth of harmful bacteria. in animal models, oligofructose increased the gut bifidobacterial content of high - fat diet fed mice which reduced oxidative stress and adipose tissue inflammation and resulted in an improvement of glucose tolerance. in addition daubioul. in a placebo - controlled pilot study evaluated the effect of oligofructose on nafld patients and observed oligo - fructose consumption resulted in reduced insulin and aminotransferase levels. to our search this is the first double - blind placebo controlled clinical trial that evaluated the effect of symbiotic as a low cost without side effect therapeutic component on steatosis grade and liver enzymes in iranian nafld patients. nevertheless, several limitations must be considered in the interpretation of our findings, including limited duration of the clinical trial and the sample size. furthermore, it is well known that liver histology is the gold standard for nafld / nash. although ultrasonography is reasonably accurate, it can not identify fatty infiltration of the liver below a threshold of 30% but because of budget limitation we were not able to do the best. based on the present study results, it can be concluded that symbiotic supplementation for 8-week can improve steatosis in nafld patients and might be useful in management of nafld or be protective again progression of the disease. further clinical trials with longer intervention period and higher doses of symbiotic may need for showing more clear results in this regard. food security research centre, school of nutrition and food science, isfahan university of medical sciences, isfahan, iran. food security research centre, school of nutrition and food science, isfahan university of medical sciences, isfahan, iran. | background : regarding to the growing prevalence of nonalcoholic fatty liver disease (nafld), concentrating on various strategies to its prevention and management seems necessary. the aim of this study was to determine the effects of symbiotic on c - reactive protein (crp), liver enzymes, and ultrasound findings in patients with nafld.methods:eighty nafld patients were enrolled in this randomized, double - blind, placebo - controlled clinical trial. participants received symbiotic in form of a 500 mg capsule (containing seven species of probiotic bacteria and fructooligosaccharides) or a placebo capsule daily for 8 weeks. ultrasound grading, crp, and liver enzymes were evaluated at the baseline and the end of the study.results:in the symbiotic group, ultrasound grade decreased significantly compared to baseline (p < 0.005) but symbiotic supplementation was not associated with changes in alanine aminotransferase (alt) and aspartate transaminase (ast) levels. in the placebo group, there was no significant change in steatosis grade whereas alt and ast levels were significantly increased (p = 0.002, p = 0.02, respectively). crp values remained static in either group.conclusions:symbiotic supplementation improved steatosis in nafld patients and might be useful in the management of nafld or protective against its progression. |
during obstructive jaundice, the impairment of intestinal mucosal barrier and the translocation of endotoxins and intestinal bacteria can cause intestinal infections and secondary multiple organ injury, which are pivotal for the deterioration of the disease [16 ]. astragalus injection, as an extract from radix astragali, is characterized by low cost, extensive pharmacological effects and few side effects and therefore has unique advantages and prospects in the treatment of oj. although some studies [8, 9 ] showed that astragalus could significantly improve the oxygen free radical - scavenging abilities, inhibit excessive inflammatory response, and thereby mitigate hepatic and renal injury in oj rats, no study on the protective effects of astragalus on intestinal mucosa was found in domestic and foreign literature. in this study, we investigated the protective effects and mechanisms of astragalus injection on small intestinal mucosa of oj rats to provide a theoretical basis for clinical application of this injection. the healthy male sd rats of clean grade, weighing between 270 and 330 g, were provided by the laboratory animal research center of the zhejiang university of traditional chinese medicine (hangzhou, china). radix astragali injection (each 10 ml vial contains active components equivalent to 20 g of the original medicine) was purchased from chiatai qingchunbao pharmaceutical co., ltd (hangzhou, china). the serum nitrogen monoxidum (no), malonaldehyde (mda), and superoxide dismutase (sod) kits were all purchased from nanjing jiancheng bioengineering research institute (nanjing, china), and the calculation units for content are respectively mol / l, nmol / ml, and u / ml. the anti - nf-b p65 and anti - bax antibody were purchased from santa cruz biotechnology, inc. (santa cruz, california, usa) tunel assay kit was purchased from takara bio inc. 180 rats were utilized for oj - associated experiments and randomly divided into sham - operated, model control, and treated group (n = 60), which were further randomly subdivided into 7d, 14d, 21d, and 28d groups (n = 15) according to time duration after operation. after rats were anesthetized with an intraperitoneal injection of 2.5% sodium pentobarbital (0.2 ml/100 g), the abdominal cavity was opened to identify and dissociate common bile duct along the hepatoduodenal ligament. for rats in the model control group and the treated group, the proximal end of common bile duct was double ligated with surgical threads, common bile duct was cut off, and a layered suture of the abdominal wall was performed to close the abdominal cavity. for rats in the sham - operated groups, common bile duct was only dissociated but not ligated, and a layered suture of the abdominal wall was also performed to close the abdominal cavity. an intraperitoneal injection of radix astragali injection at a dose of 0.75 ml/100 g / d was given to rats in the treated groups while equal volume of physiological saline solution was used in the sham - operated and the model control group. different injection was maintained until the end of the 7-day, 14-day, 21-day, and 28-day observation period in the corresponding groups. after mercy killing rats anesthetized by sodium pentobarbital in batches, we collected the serum of rats to detect no, mda and sod contents and observed the pathological changes of intestinal muscoa. the determination of these serum parameters was conducted according to the instructions provided by the kits. the pathological severity score of intestinal muscoa was conducted according to our report for related standard. and then we prepared the tissue microarrys section for intestine tissue with diameter of 1.5 mm, stained them, observed the changes in the expression levels of bax and nf-b p65 proteins as well as the apoptosis index of intestinal muscoa. envision two - step method was used to detect the expression levels of bax and nf-b p65 protein in the intestinal muscoa. the evaluation standard was as follows : (1) the staining intensity was evaluated according to the extent of cell coloration : represented negative staining ; + represented mild staining, positively stained cells showed a yellow pigment ; + + represented moderate staining, positively stained cells showed a brown pigment ; + + + represented intense staining, positively stained cells showed a dark brown pigment, each of which was scored as 0, 1, 2, and 3 points, respectively, during statistical analysis ; (2). the evaluation standard of the positive rate : there was no positive cells () ; the percentage of positive cells was less than 25% (+) ; the percentage of positive cells ranged between 26% and 50% (+ +) ; the percentage of positive cells was more than 50% (+ + +), each of which was scored as 0, 1, 2, and 3 points, respectively, during statistical analysis. in tissue microarrays sections, dna nick in situ end - labeling (tunel) staining steps were performed as follows : baking sections under 60c for 30 minutes, routine deparaffinage, and milli - q wash for 5 minutes. processing tissue with protease k (10 ug / ul) under room temperature for 15 minutes, pbs washes for 5 minutes. using 3% h2o2 solution to block endogenous peroxydase for 5 minutes, pbs wash for 5 minutestwice. adding 30 ul reaction solution in freezing condition (tdt enzyme : labeling safe buffer = 1 : 10), 37c incubation for 90 minutes, pbs wash for 5 minutes twice. adding 50 ul anti - fitc hrp conjugate, 37c incubation for 30 minutes, pbs wash for 5 minutes twice. hematoxylin counterstain, water wash and wash fully with water after differentiation till return blue ; routine dehydration and transparence ; neutral gum mounting. apoptotic index = apoptotic cell count / total cell count 100%. after input into the excel sheets, normal data was expressed as means (standard deviation) while nonnormal data were expressed as medians (interquartile range). analysis of variance and pairwise comparisons were used in normal data ; whereas nonnormal data were subjected to nonparametric test, among which kruskal - wallis h test was used for pairwise comparisons and mann - whitney u test for multiple comparisons. yates ' chi - square test () was used for intergroup comparisons of mortality rates. all rats were alive in the sham - operated groups on all time points after operation. 2 and 1 rats died in the model control and treated group on 7d, respectively ; 4 and 3 rats died in the model control and treated group on 14d, respectively ; 4 rats died in both model control and treated group on 21d ; 7 and 6 rats died in the model control and treated group on 28d, respectively. the total mortality rate of the model control and treated group on 28d were significantly higher than those in the sham - operated group (p.05). pathological changes under light microscopyno obvious difference in pathological changes was observed among each time point after operation. the intestinal mucosal epithelium was not intact and inflammatory cell infiltration of proper layer was seen in very few rats (see figure 1). no obvious difference in pathological changes the intestinal mucosal epithelium was not intact and inflammatory cell infiltration of proper layer was seen in very few rats (see figure 1). gross pathological changeson 7d after operation, intestinal wall and peritoneum became jaundice in the majority of rats. on 14d after operation, varying degrees of yellow staining of the intestinal wall and peritoneum were seen in the majority of rats. the intestinal canal was enlarged and showed fluid retention. on 21 and 28d after operation, on 7d after operation, intestinal wall and peritoneum became jaundice in the majority of rats. on 14d after operation, varying degrees of yellow staining of the intestinal wall and peritoneum were seen in the majority of rats. the intestinal canal was enlarged and showed fluid retention. on 21 and 28d after operation, pathological changes under light microscopyno obvious difference in pathological changes was observed among each time point after operation. on 7d after operation, intestinal mucosa was normal in the majority of rats, and the edema of submucous layer were present in very few rats. on 14d after operation, intestinal mucosa was normal in the majority of rats but not intact in some rats, and the edema of proper layer, submucous layer, and serosal layer were seen in some rats. on 21d after operation, intestinal mucosa was not intact in the majority of rats, the edema of proper layer, submucous layer, and serosal layer were seen in the majority of rats, and very few rats showed no abnormality of the intestinal mucosa. on 28d after operation, focal necrosis in intestinal mucosal epithelium as well as the edema of proper layer, submucous layer and serosal layer were seen in the majority of rats (see figures 2 and 3). on 7d after operation, intestinal mucosa was normal in the majority of rats, and the edema of submucous layer were present in very few rats. on 14d after operation, intestinal mucosa was normal in the majority of rats but not intact in some rats, and the edema of proper layer, submucous layer, and serosal layer were seen in some rats. on 21d after operation, intestinal mucosa was not intact in the majority of rats, the edema of proper layer, submucous layer, and serosal layer were seen in the majority of rats, and very few rats showed no abnormality of the intestinal mucosa. on 28d after operation, focal necrosis in intestinal mucosal epithelium as well as the edema of proper layer, submucous layer and serosal layer were seen in the majority of rats (see figures 2 and 3). gross pathological changeson 7d after operation, no obvious difference was observed when compared to that in model control group. on 14d after operation, intestinal wall became jaundice in half of the rats, but intestinal canal was not enlarged and showed no fluid retention. on 21, and 28d after operation, no obvious difference was observed when compared to those in model control group. on 7d after operation, no obvious difference was observed when compared to that in model control group. on 14d after operation, intestinal wall became jaundice in half of the rats, but intestinal canal was not enlarged and showed no fluid retention. on 21, and 28d after operation, no obvious difference was observed when compared to those in model control group. pathological changes under light microscopythe pathological changes in the small intestine of rats at various time points showed varying degrees of mitigation. on day 7 in treated group, the small intestine of the majority of rats showed no abnormality while the small intestine of extremely few rats showed inflammatory cell infiltration in proper layer, submucous layer, and serosal layer. on day 14 in treated group, the small intestine of some rats showed no abnormality, the small intestine of some rats showed inflammatory cell infiltration in proper layer, submucous layer, and serosal layer, and the small intestine of extremely few rats showed focal necrosis in the mucosa. on day 21 in treated group, the small intestine of some rats showed no abnormality while the other rats showed inflammatory cell infiltration in proper layer, submucous layer and serosal layer. on day 28 in treated group, the small intestine of some rats showed inflammatory cell infiltration in proper layer, submucous layer and serosal layer while the extremely few rats showed no abnormality (see figure 4). the pathological changes in the small intestine of rats at various time points showed varying degrees of mitigation. on day 7 in treated group, the small intestine of the majority of rats showed no abnormality while the small intestine of extremely few rats showed inflammatory cell infiltration in proper layer, submucous layer, and serosal layer. on day 14 in treated group, the small intestine of some rats showed no abnormality, the small intestine of some rats showed inflammatory cell infiltration in proper layer, submucous layer, and serosal layer, and the small intestine of extremely few rats showed focal necrosis in the mucosa. on day 21 in treated group, the small intestine of some rats showed no abnormality while the other rats showed inflammatory cell infiltration in proper layer, submucous layer and serosal layer. on day 28 in treated group, the small intestine of some rats showed inflammatory cell infiltration in proper layer, submucous layer and serosal layer while the extremely few rats showed no abnormality (see figure 4). pathological severity scores of the sham - operated group (on 7, 14, and 21d) was significantly lower than model control group (p.05) ; see table 1, figures 5 and 6. the sham - operated group (on 21d) was significantly lower than model control group (p.05). so we surmise that nf-b protein has no significant impact on the pathological injury of the small intestine mucosa of oj rats. apoptosis is a kind of gene - controlled physiological cell death to maintain the body 's normal physiological functions and stability. the stability of small intestinal mucosa also depends on the equilibrium between the proliferation and apoptosis of epithelial cells. some factors such as ischemia / reperfusion injury, bacterial infections and nutritional deficiencies may induce the apoptosis of intestinal epithelial cells [2227 ] and cause intestinal dysfunction [28, 29 ]. yang. found that the total extract of astragalus had inhibitory effects on both in vivo and in vitro hepatic cell injury and apoptosis. in the present study, we found that, on day 14, the apoptosis index in small intestinal mucosa in astragalus treated group was significantly lower than that in model control group, and small intestinal mucosal inflammation and necrosis and microvilli defects in treated group were milder than those in model control group, indicating that astragalus can exert protective effects on the small intestinal mucosa of oj rats through inhibiting the apoptosis of small intestinal mucosal epithelial cells. bax, as an apoptosis - inducing gene [3135 ], can promote bcl-2 [3638 ] or bcl - x dimer formation and thus induce cell apoptosis. in this study, no statistical difference existed in the products of the staining intensity and the positive staining rate of bax protein at all time points was noted between treated group and model control group, suggesting that bax protein may have no impact on oj - induced intestinal mucosa injury and other unknown factors are involved in this process. in summary, astragalus injection can increase the content of serum sod, decrease the content of mda, and inhibit the apoptosis of small intestinal mucosa cells, thereby mitigate the pathological injury of the small intestinal mucosa and exerting protective effects on the small intestinal mucosa of oj rats. however, it is worth mentioning that the therapeutic effects of astragalus are limited and auxiliary. therefore, combined use of other drugs that can protect the small intestinal mucosa is needed during the course of treatment. | objective. to research the protective effects and mechanisms of radix astragali injection on the intestinal mucosa of rats with obstructive jaundice (oj). methods. the rats were randomly divided into sham - operated, model control and radix astragali treated group. we observed the pathological changes of intestinal mucosa, expression levels of bax and nf-b proteins, and apoptosis indexes in intestinal mucosa as well as serum no, mda and sod contents, respectively, on 7d, 14d, 21d and 28d after operation. results. the pathological severity score (on 7d and 14d), apoptotic indexes (on 14d) of the intestinal mucosa and serum mda content (on 14d) of treated group were significantly lower than those in the model control group (p <.05). the serum sod contents (on all time points) of treated group were significantly higher than those in the model control group (p <.05). the sham - operated group (on 21d) of the product of staining intensity and positive rate of bax protein was significantly lower than model control group (p <.05). conclusion. radix astragali injection could protect the intestinal mucosa of oj rats by increasing the content of sod, reducing the content of mda, inhibiting the apoptosis and relieving the pathological changes of intestinal mucosa. |
hydatid disease is a parasitic disease caused by the larvae of a tapeworm belonging to the genus echinococcus. it can affect any part of the human body, the liver and the lung being the most frequently involved organs. an intra - thoracic but extra - pulmonary location is very rare, representing less than 0.1% of all cases. mediastinal localization is exceptional with less than 150 cases reported in the literature so far. we report an even rarer presentation of hydatid cyst in the posterior mediastinum causing erosion of the overlying ribs and adjoining vertebra with extension into the spinal canal, assuming a dumbbell shape, and simulating an aggressive malignancy. there have been less than 10 cases of hydatid disease with mediastinal localization and intraspinal extension reported until date. this case report emphasizes that a differential diagnosis of hydatid should be considered for a mediastinal cystic mass, however, aggressive it may appear, clinically or radiologically. a 22-year - old female, farmer by occupation, presented with complaints of chest pain and progressive weakness of left lower limb that had persisted for 3 months. respiratory system examination revealed dullness on percussion and tenderness in left infraclavicular, suprascapular, and interscapular regions. radiographs of chest, performed in posterior - anterior (pa) and lateral projections, demonstrated well - defined homogenous soft tissue opacity in the posterior mediastinum. there was erosion of the 4 rib posteriorly, scalloping of superior margin of 5 rib, and widened fourth intercostal space [figure 1a and b ]. contrast enhanced computed tomography (ct) of the thorax revealed a well - defined, multiloculated cystic mass in the left paravertebral space, extending into the spinal canal by causing erosion of the adjoining pedicle and lamina of fourth thoracic vertebra. there was erosion of the overlying 4 rib with scalloping of the 5 rib and widened intervening intercostal space [figures 2 and 3 ]. chest x - ray (a) posterior anterior (pa) and (b) lateral views show well - defined homogeneous soft tissue opacity within posterior mediastinum, causing erosion of the 4 rib posteriorly, scalloping of the superior margin of the 5 rib and widening of the 4 intercostal space (arrow). contrast enhanced computed tomography (ct) thorax in (a) mediastinal and (b) lung windows show multiloculated cystic lesion in left paravertebral space, causing erosion of pedicle and lamina of the 4 dorsal vertebra, with intraspinal extension (blue arrow). (a) three dimensional sagittal multiplanar reformation shows posterior mediastinal mass causing destruction of adjoining vertebra (arrow) with intraspinal extension. (b) volume rendered coronal image shows erosion of the 4 rib (straight arrow) and scalloping of the 5 rib (curved arrow) with widened 4 intercostal space. based on the imaging findings, the possible differential diagnosis included neurogenic tumors, aggressive malignant masses like round cell tumors (ewing 's sarcoma, primitive neuroectodermal tumors, and others), lymphoma, or metastasis. since the mass was predominantly cystic with multiloculated appearance and considering the occupation of the patient, hydatid cyst was also considered a possible differential diagnosis. the serological test with enzyme linked immunosorbant assay (elisa) was positive for echinococcosis, thus suggesting hydatid cyst as a possible etiology. the patient was operated and the cystic mass along with the involved 4 rib were completely excised. postoperative recovery was uneventful and the patient was discharged and put on adjuvant chemotherapy with mebendazole for 2 years. the patient was monitored with routine follow - ups for 5 years with no evidence of recurrence. hydatid disease continues to be a major health problem in underdeveloped areas where animal husbandry is common and no veterinary control exists. it is endemic in parts of south america, mediterranean regions, the middle east, africa, and australia. humans are secondary hosts and are infected by the ingestion of food or water contaminated by eggs of these parasites. in humans, other sites in decreasing frequency include peritoneum, kidney, brain, mediastinum, heart, bone, soft tissues, spinal cord, spleen, pleura, adrenal glands, bladder, ovary, scrotum, and thyroid gland. mediastinal hydatid cysts are very rare, accounting for less than 0.1% of all cases and less than 1% of thoracic involvement. the patients may remain asymptomatic, the lesion being discovered incidentally on a routine chest radiograph. the patients may present with non - specific symptoms of chest pain, cough, and dyspnoea. these symptoms may be produced as a result of involvement of neighboring structures and other etiologies like hemoptysis, superior vena cava syndrome, phrenic or recurrent laryngeal nerve compression, vertebral destruction with intraspinal extension and neurological symptoms, and bernard horner syndrome. the complications of mediastinal hydatid cyst can be significant and include rupture into the mediastinum, pleural cavity and right ventricle, cysto - aortic fistula with possible embolization, and infection and compression of vital structures. posterior mediastinal hydatid cyst with intraspinal extension is an extremely rare entity, with less than 10 similar cases reported in the literature till date. though the most common cause of dumbbell masses is neurogenic tumor, a large variety of unusual lesions may produce similar picture. these include neoplastic lesions such as benign / malignant peripheral nerve sheath tumors, plasmacytoma, chondrosarcoma, chondroid chordoma, superior sulcus tumor, metastasis, and nonneoplastic lesions such as infectious process (tuberculosis, hydatid cyst), aneurysmal bone cyst, synovial cyst, traumatic pseudomeningocele, arachnoid cyst, vertebral artery tortuosity, and hemangiomas. paravertebral masses with vertebral destruction and intraspinal extension have been described as spinal hydatids by some authors, and belong to group 5, paravertebral hydatid disease, according to the braithwaite and lee 's classification. the differential diagnosis of posterior mediastinal cystic lesions includes bronchogenic cysts, cystic lymphangioma, enteric cyst, and neurogenic tumors. presence of a posterior mediastinal mass with rib and vertebral destruction and intraspinal extension can raise the possibility of aggressive malignant masses like round cell tumor (ewing 's sarcoma, primitive neuroectodermal tumor, etc.), lymphoma, metastasis, or neurogenic tumor. serological tests can often be negative, especially when the cyst is intact and uncomplicated. in suspected cases, aspiration cytology under imaging guidance ct defines the cystic nature of the mass and delineates its exact anatomical relations. though presence of multiloculated appearance and calcification may suggest the diagnosis, there is no pathognomonic sign of hydatid cyst on ct. magnetic resonance imaging is indicated in defining the intraspinal extension, especially when patients present with neurological signs. the gold standard for treatment of mediastinal hydatid cyst is radical removal of the germinative membrane and pericyst. if total excision is not possible due to invasion of vital structures, partial pericystectomy after removal of germinative membrane can be performed. it has been proposed that better results are obtained by combining surgical procedure with antihelminthic therapy postoperatively. to conclude, hydatid cyst should always be considered in the differential diagnosis of mediastinal cystic lesions, especially in endemic areas, even when the lesion appears aggressive clinically and on imaging. | hydatid cyst is known to affect all possible anatomical locations of the human body. however, the mediastinal localization is extremely rare. this benign, commonly asymptomatic and incidentally detected disease, at times may simulate an aggressive malignancy by its potential to cause osseous destruction and intraspinal extension. a young female, farmer by occupation, presented with complaints of left chest pain and monoparesis of the left lower limb. radiograph followed by computed tomography (ct) of the chest demonstrated a cystic mass within the posterior mediastinum, eroding and scalloping overlying ribs and extending into the spinal canal by causing destruction of adjoining vertebra, and assuming a dumbbell shape. the serology was positive for echinococcosis. the patient underwent surgery and the postoperative histopathology confirmed the diagnosis of hydatid cyst. the patient recovered with no complications or recurrence. hydatid cyst should always be considered in the differential diagnosis of mediastinal cystic lesions, however aggressive the lessions may appear. |
it reveals and defines both the scientific and artistic dimensions of nursing. in spite of the validity of historical research in addition, there are no classic references for the novice about how to analyze a huge amount of data. if this is true, how do novice researchers become familiar with robust techniques and processes of inquiry required in research ? hamilton stated that novice historians can understand the importance of historical techniques by immersing themselves in good historiography. but this strategy seems too difficult to follow by novice researchers. history is not merely a list of events, but an impartial evaluation of the entirety of human interrelationships in time and space. historical research stages consist of the following : identifying the area of interest, raising questions, formulating a title, reviewing the literature, data gathering and analysis, interpreting data, and writing the narrative. due to the importance of the data gathering and analysis stage in qualitative historical studies, and the interconnection between these two stages, it is vital that the researcher selects a proper research method at this point. one of the methods used in a qualitative historical studies approach is the oral history method, and this was applied in a project related to the nature of nursing practice during the iran since oral history lacks a clear and well - developed analysis method, the authors decided to develop a method based on the experiences they have d during their research, and also considering the strengths and weaknesses of the methods that have been used in the other oral history project. the aim of this study is focused on the development of an analysis method for oral history. in this methodological article, a method for data analysis suitable for oral history researches has been suggested based on qualitative research tradition and experiences of the authors. in order to clarify the data obtained by the researcher through oral history interviews, a historical research methodology is required to produce a good narrative, along with the application of a proper analysis. for this purpose, a four - stage method is introduced and adopted. each stage is connected and related to the previous one, while the final stage connects to the first and closes the circuit, which means that all data analysis stages, in a sense, are complementary to one another. these analysis stages are : data gathering through interviews with the oral witness and first - level codingsecond - level coding and determining the sub - categoriesthird - level coding and determining the main categoriesconnecting the main categories to each other and writing the narrative. data gathering through interviews with the oral witness and first - level coding second - level coding and determining the sub - categories third - level coding and determining the main categories connecting the main categories to each other and writing the narrative. the authors hope that by applying this method, researchers in the field of nursing would be able to analyze oral history data in a new form. the authors are of the opinion that this method is applicable in all disciplines which are concerned with oral history. this method has been practically applied in two articles titled nurses experiences in chemical emergency departments : iran iraq war, 19801988 and the wartime experience of civilian nurses in the iran it should be noted that most of the information in recent decades concerning the digital oral history paradigm have applied the related computer software. at this stage, the data collected through interviews, oral witnesses, and first - level coding evolve into the following two facts : familiarity with the data and its organizationextraction of the initial codes. familiarity begins as the interviews proceed ; therefore, the proper selection of participants is essential. in reference to the digital oral history paradigm, the interviews are recorded with a voice recorder, and other primary sources like personal notes, photos, etc. since there is a close correlation between the data, well - conducted interviews contribute to the next stage 's analysis. the interview must be objective and proper arrangement of the questions is essential in order to obtain good results. the interviews consist of four sets of questions : warm - up, memory, judgment, and follow - up. the researcher, through an in - depth interview, encourages the participants to recall the events and related details and to relate them clearly in the interview. according to thomson, a voice - recorded interview not only allows the participant to verbally express their views, but also administers and illustrates the spoken words. the second interview represents the experience in detail and the context in which it happened, while the third interview explores the meaning that their experience holds for them. after each interview, the researcher listens to the responses many times, in order to determine the applicable portions for planning and addressing the next interview to achieve the final objective. the recorded interviews are transcribed by hand or using a computer programs and then, the data may be transferred to a computer assisted qualitative data analysis software (caqdas) for textual analysis. these notes help the researcher to recall the expressions / emotions of the interviewee when writing the narrative. the codes used are labels that contain the interview briefing or the researcher 's impressions regarding the interview. coding takes place parallel to data compilation, by constant comparison of the codes against one another and by putting them next to other codes. symbols or abbreviations categorize words or expressions of a given data, and coding facilitates data recovery. an example of coding at this stage : in some military operations like kheibar, 1985, the conditions were such that we worked for three days nonstop due to the excessive number of wounded. i remember that the young doctors and nurses could nt resist and just lay on the floor next to the patient 's bed. code assigned to speech participant 2 : (p2i1c156) nonstop nursing activities according to speziale (2010), the researcher should begin coding and analyzing data as he compiles it. the first stage of coding summarizes the data along with organizing it to facilitate labeling of the categories. coding is considered as the first interpretation stage and is applied to obtain the participants live experiences. the obtained codes are a practical ring in the chain of events in historical research. lived experiences are what the participant has been through so far. at this stage, the following two things evolve : the data with close conceptions are extracted from and studied on the initial codes and set next to one another to form the sub - categories. here it should be mentioned that a sub - category may contain many codes, which are determined at stage one of the coding. at this point, the emphasis here is put on the key concepts that are going to be applied in the fourth stage, where the study starts to take shape. in fact, at this stage, the pieces of the puzzle are arranged in a manner in which the whole picture is revealed. the credibility of the oral history method and this critical stage is determined by analyzing the approved documentation. to assure coding validity at different stages, the codes are sent to the participants. stage three shows the focus of the study regarding data correlation with the study, and at this stage the main categories are formed by similar sub - categories and the inter - connections appear. the narrative is the outcome of the words spoken by the participants, and packed in correlation with the main categories. in the narrative, the historical picture of the participants perspective on the historical events can be depicted in the research. to write the narrative, the following are used : books, primary and secondary sources, newspaper articles, poems, songs, and other items that have played a role in the reconstruction of historical events such as wars. such historical narratives are of special importance : revealing the events, projecting the findings, answering questions addressed in the study, exposing categories, clarifying ambiguities, and preventing bias in researcher 's accounts. historic narration attracts readers attention to the event by revealing a complete perception of the experience. here, the authors respond to the questions raised in the process of the study, determine the primary and secondary sources, combine the ideas, and share the views of the participants. all these enable the reader to have a meaningful and inspiring assessment of the presented issue through this model [figure 1 ]. analyzing oral history judging the study and its success is related to joining the categories to one another and the acceptance of the work for publication as a final product. in order to clarify the data obtained by the researcher through oral history interviews, a historical research methodology is required to produce a good narrative, along with the application of a proper analysis. for this purpose, a four - stage method is introduced and adopted. each stage is connected and related to the previous one, while the final stage connects to the first and closes the circuit, which means that all data analysis stages, in a sense, are complementary to one another. these analysis stages are : data gathering through interviews with the oral witness and first - level codingsecond - level coding and determining the sub - categoriesthird - level coding and determining the main categoriesconnecting the main categories to each other and writing the narrative. data gathering through interviews with the oral witness and first - level coding second - level coding and determining the sub - categories third - level coding and determining the main categories connecting the main categories to each other and writing the narrative. the authors hope that by applying this method, researchers in the field of nursing would be able to analyze oral history data in a new form. the authors are of the opinion that this method is applicable in all disciplines which are concerned with oral history. this method has been practically applied in two articles titled nurses experiences in chemical emergency departments : iran iraq war, 19801988 and the wartime experience of civilian nurses in the iran it should be noted that most of the information in recent decades concerning the digital oral history paradigm have applied the related computer software. at this stage, the data collected through interviews, oral witnesses, and first - level coding evolve into the following two facts : familiarity with the data and its organizationextraction of the initial codes. familiarity begins as the interviews proceed ; therefore, the proper selection of participants is essential. in reference to the digital oral history paradigm, the interviews are recorded with a voice recorder, and other primary sources like personal notes, photos, etc. since there is a close correlation between the data, well - conducted interviews contribute to the next stage 's analysis. the interview must be objective and proper arrangement of the questions is essential in order to obtain good results. the interviews consist of four sets of questions : warm - up, memory, judgment, and follow - up. the researcher, through an in - depth interview, encourages the participants to recall the events and related details and to relate them clearly in the interview. according to thomson, a voice - recorded interview not only allows the participant to verbally express their views, but also administers and illustrates the spoken words. the second interview represents the experience in detail and the context in which it happened, while the third interview explores the meaning that their experience holds for them. after each interview, the researcher listens to the responses many times, in order to determine the applicable portions for planning and addressing the next interview to achieve the final objective. the recorded interviews are transcribed by hand or using a computer programs and then, the data may be transferred to a computer assisted qualitative data analysis software (caqdas) for textual analysis. these notes help the researcher to recall the expressions / emotions of the interviewee when writing the narrative. the codes used are labels that contain the interview briefing or the researcher 's impressions regarding the interview. coding takes place parallel to data compilation, by constant comparison of the codes against one another and by putting them next to other codes. symbols or abbreviations categorize words or expressions of a given data, and coding facilitates data recovery. an example of coding at this stage : in some military operations like kheibar, 1985, the conditions were such that we worked for three days nonstop due to the excessive number of wounded. i remember that the young doctors and nurses could nt resist and just lay on the floor next to the patient 's bed. code assigned to speech participant 2 : (p2i1c156) nonstop nursing activities according to speziale (2010), the researcher should begin coding and analyzing data as he compiles it. the first stage of coding summarizes the data along with organizing it to facilitate labeling of the categories. coding is considered as the first interpretation stage and is applied to obtain the participants live experiences. the obtained codes are a practical ring in the chain of events in historical research. lived experiences are what the participant has been through so far. at this stage, the following two things evolve : the data with close conceptions are extracted from and studied on the initial codes and set next to one another to form the sub - categories. here it should be mentioned that a sub - category may contain many codes, which are determined at stage one of the coding. the data with close conceptions are extracted from and studied on the initial codes and set next to one another to form the sub - categories. it should be mentioned that a sub - category may contain many codes, which are determined at stage one of the coding. the emphasis here is put on the key concepts that are going to be applied in the fourth stage, where the study starts to take shape. in fact, at this stage, the pieces of the puzzle are arranged in a manner in which the whole picture is revealed. the credibility of the oral history method and this critical stage is determined by analyzing the approved documentation. to assure coding validity at different stages, stage three shows the focus of the study regarding data correlation with the study, and at this stage the main categories are formed by similar sub - categories and the inter - connections appear. the narrative is the outcome of the words spoken by the participants, and packed in correlation with the main categories. in the narrative, the historical picture of the participants perspective on the historical events can be depicted in the research. to write the narrative, the following are used : books, primary and secondary sources, newspaper articles, poems, songs, and other items that have played a role in the reconstruction of historical events such as wars. such historical narratives are of special importance : revealing the events, projecting the findings, answering questions addressed in the study, exposing categories, clarifying ambiguities, and preventing bias in researcher 's accounts. historic narration attracts readers attention to the event by revealing a complete perception of the experience. here, the authors respond to the questions raised in the process of the study, determine the primary and secondary sources, combine the ideas, and share the views of the participants. all these enable the reader to have a meaningful and inspiring assessment of the presented issue through this model [figure 1 ]. analyzing oral history judging the study and its success is related to joining the categories to one another and the acceptance of the work for publication as a final product. historical research, as a research methodology, is supported by oral history (research method), which is a valuable tool for researchers in the nursing field. during the passage of time, a lack of an objective method to assist the analysis of the data obtained through oral evidence interviews limits the researcher 's efforts. here, an attempt has been made to introduce a model for better data analysis with respect to oral history that would facilitate better research approaches where oral history is the main source. through systematic application of this method, the nursing researcher would be able to produce a correct and reliable narrative that would illustrate nursing discipline abilities and assist in the production of knowledge in this realm. | background : historical research has limitations in applying proper and credit - worthy chronology to clarify the data. in this methodology, the application of oral history is one of the ways in which answers to questions addressed by the research theme are elicited. oral history, as a clear and transparent tool, needs to be applied with guidelines for qualitative researchers regarding data analysis limitations from oral evidence and face - to - face contact. therefore, the development of a systematic method for data analysis is needed to obtain accurate answers, based on which a credit - worthy narration can be produced. the aim of this study was to introduce an ethical and objective approach for the analysis of data obtained from oral history.materials and methods : this is a methodological article that suggests an analysis method based on qualitative approach and experiences of the authors.results:a systematic method of data analysis for oral history research, based on common qualitative data analysis methods, has been suggested as the result of this article.conclusions:this new technique is equipped with measures that would assist qualitative researchers in the nursing field and other disciplines regarding analysis of qualitative data resulting from oral history studies. |
the mortality rate is one of the key indicators of an intensive care unit (icu) quality. however, the severity of patients ' illnesses, comorbidities, and demographics all strongly affect the mortality rate (1). thus a comparison of unadjusted mortality rate among icus without consideration of severity or other case - mix variables might provide an incorrect estimate of icu quality. in a multicenter study, the crude mortality rate of tertiary pediatric icus was four times higher than that of non - tertiary pediatric icus (2). the latter does not suggest that the performance of the tertiary pediatric icu was poor, as tertiary hospitals are usually referral hospitals to which more severe patients are referred. therefore, when we compare mortality data of certain units or patients ' groups for the purpose of quality care or research, the mortality data must be adjusted. mortality data on icu patients is usually adjusted using mortality prediction models. in general pediatric icus, the pediatric index of mortality (pim) 3, pediatric risk of mortality (prism) iii, are commonly used mortality prediction models (345), whereas the acute physiology and chronic health evaluation (apache), simplified acute physiology score and mortality probability model are commonly used in adult icus (6). regional variations, such as different national health systems, different populations, or disease prevalence, might affect the performance of these scoring systems. thus, some validation studies of the applicability of these scoring systems to korean adult patients have been conducted in korea (789). to the best of our knowledge, there has been no validation study of pim3 scoring system for pediatric patients in korea. another consideration when applying pim score in korean icus is the age of the patients. the developmental data of pim score were validated for children aged 3 mm and both fixed = 1 ; other or unknown = 0) ; 3) ([fio2 100]/pao2). pao2 mmhg, fio2 at the time of pao2 if oxygen via endotracheal tube or headbox (fio2 or pao2 unknown, [{ fio2 100}/pao2 ] = 0.23) ; 4) base excess in arterial or capillary blood, mmol / l (unknown = 0) ; 5) mechanical ventilation at any time during the first hour in icu (no = 0 ; yes = 1) ; 6) elective admission to icu (no = 0 ; yes = 1) ; 7) recovery from surgery or a procedure is the main reason for icu admission (no = 0 ; yes, recovery from a bypass cardiac procedure = 1 ; yes, recovery from a non - bypass cardiac procedure = 2 ; yes, recovery from a noncardiac procedure = 3) ; 8) low - risk diagnosis (no or in doubt = 0 ; yes = 1) ; 9) high - risk diagnosis (no or in doubt = 0 ; yes = 1) ; and 10) very high - risk diagnosis (no or in doubt = 0 ; yes = 1). low - risk diagnosis consists of asthma, bronchiolitis, croup, obstructive sleep apnea, diabetic ketoacidosis, and seizure disorder. high - risk diagnosis consists of spontaneous cerebral hemorrhage, cardiomyopathy or myocarditis, hypoplastic left heart syndrome, neurodegenerative disorder, and necrotizing enterocolitis. very high - risk diagnosis consists of cardiac arrest preceding icu admission, severe combined immune deficiency, leukemia or lymphoma after first induction, bone marrow transplant recipient, and liver failure. patients with multiple diagnoses were assigned to only 1 risk - diagnosis group, which has the highest risk score. pim3 score and probability of death were calculated by formulas presented in the development study (4). pim3 score = (3.8233 pupillary reaction) + (0.5378 elective admission) + (0.9763 mechanical ventilation) + (0.0671 [absolute { base excess } ]) + (0.0431 sbp) + (0.1716 [sbp/1,000 ]) + (0.4214 [{ fio2 100}/pao2 ]) (1.2246 bypass cardiac procedure) (0.8762 non - bypass cardiac procedure) (1.5164 noncardiac procedure) + (1.6225 very high - risk diagnosis) + (1.0725 high - risk diagnosis) (2.1766 low - risk diagnosis) 1.7928. probability of death = exp (pim3 score)/[1 + exp (pim3 score) ]. data on parameters that differed in the pim2 version, including the main reason for icu admission, cardiac bypass surgery, diagnosis category (low-, high - risk), were collected as described in the developmental study of pim2 (11). physiological variables from the time of first icu contact to 1 hour after arrival in the icu were recorded. the formulas and coefficients presented in previous pim2 and pim3 studies were used to calculate the predicted mortality of each patient (411). the sas program (ver. 9.3 ; sas institute, cary, nc, usa) was used for all the statistical analyses. all tests were 2-tailed, and a value of p 0.70 was regarded as acceptable, and a value of > 0.8 was regarded as good discrimination (13). the present study protocol was reviewed and approved by the institutional review board of samsung medical center (irb no. this was a retrospective cohort study of patients admitted to the pediatric intensive care unit (picu) of a single tertiary institute, from 1st january 2010 to 31st december 2014. immediate postoperative cardiac patients are usually admitted to another dedicated pediatric cardiac surgical unit, but can be admitted to this picu when medical support is more emphasized than postoperative care. many surgical patients, especially in a post organ transplantation state, are admitted to the specialty icu shared by adult patients. the inclusion and exclusion criteria were similar to those of previous studies of the pim and revised versions (pim2 and pim3) (41011), except for the age of the patients. in contrast to the criteria in previous studies, which included only patients aged 3 mm and both fixed = 1 ; other or unknown = 0) ; 3) ([fio2 100]/pao2). pao2 mmhg, fio2 at the time of pao2 if oxygen via endotracheal tube or headbox (fio2 or pao2 unknown, [{ fio2 100}/pao2 ] = 0.23) ; 4) base excess in arterial or capillary blood, mmol / l (unknown = 0) ; 5) mechanical ventilation at any time during the first hour in icu (no = 0 ; yes = 1) ; 6) elective admission to icu (no = 0 ; yes = 1) ; 7) recovery from surgery or a procedure is the main reason for icu admission (no = 0 ; yes, recovery from a bypass cardiac procedure = 1 ; yes, recovery from a non - bypass cardiac procedure = 2 ; yes, recovery from a noncardiac procedure = 3) ; 8) low - risk diagnosis (no or in doubt = 0 ; yes = 1) ; 9) high - risk diagnosis (no or in doubt = 0 ; yes = 1) ; and 10) very high - risk diagnosis (no or in doubt = 0 ; yes = 1). low - risk diagnosis consists of asthma, bronchiolitis, croup, obstructive sleep apnea, diabetic ketoacidosis, and seizure disorder. high - risk diagnosis consists of spontaneous cerebral hemorrhage, cardiomyopathy or myocarditis, hypoplastic left heart syndrome, neurodegenerative disorder, and necrotizing enterocolitis. very high - risk diagnosis consists of cardiac arrest preceding icu admission, severe combined immune deficiency, leukemia or lymphoma after first induction, bone marrow transplant recipient, and liver failure. patients with multiple diagnoses were assigned to only 1 risk - diagnosis group, which has the highest risk score. pim3 score and probability of death were calculated by formulas presented in the development study (4). pim3 score = (3.8233 pupillary reaction) + (0.5378 elective admission) + (0.9763 mechanical ventilation) + (0.0671 [absolute { base excess } ]) + (0.0431 sbp) + (0.1716 [sbp/1,000 ]) + (0.4214 [{ fio2 100}/pao2 ]) (1.2246 bypass cardiac procedure) (0.8762 non - bypass cardiac procedure) (1.5164 noncardiac procedure) + (1.6225 very high - risk diagnosis) + (1.0725 high - risk diagnosis) (2.1766 low - risk diagnosis) 1.7928. probability of death = exp (pim3 score)/[1 + exp (pim3 score) ]. data on parameters that differed in the pim2 version, including the main reason for icu admission, cardiac bypass surgery, diagnosis category (low-, high - risk), were collected as described in the developmental study of pim2 (11). physiological variables from the time of first icu contact to 1 hour after arrival in the icu were recorded. the formulas and coefficients presented in previous pim2 and pim3 studies were used to calculate the predicted mortality of each patient (411). 9.3 ; sas institute, cary, nc, usa) was used for all the statistical analyses. all tests were 2-tailed, and a value of p 0.70 was regarded as acceptable, and a value of > 0.8 was regarded as good discrimination (13). the present study protocol was reviewed and approved by the institutional review board of samsung medical center (irb no. of those, 93 patients were excluded because they were 18 years or older at the time of icu admission, and one patient was excluded because of an incomplete medical record. the final study consisted of 1,710 patients, with approximately 340 patients included annually in the study. in this study group, 208 cases (12.1%) were readmitted to the picu during the hospital stay (median interval of readmission : 9 days). as for the age, 1,656 patients were younger than 16 years of age, and the median age was 1.58 years (interquartile range [iqr ] : 0.337.85 years). the most common diagnostic category was cardiovascular disease, followed by hemato - oncological disease and respiratory disease. the other diagnostic category included hepatic dysfunction or gastrointestinal disease (57 case, 3.3%), immunodeficiency disease (52 case, 3.0%), renal disease (44 case, 2.6%), metabolic disease (39 case, 2.3%), and unclassified disease (12 case, 0.7%). the observed hospital mortality rate was 8.47%, which was higher than the predicted mortalities calculated by both the pim2 and pim3 formulas (table 1). iqr = interquartile range, icu = intensive care unit, iqr = interquartile range, pim = pediatric index of mortality. the unadjusted mortality rates and predicted mortality rates of both age groups (0.70) and better than that of the pim2 model (c - index = 0.70). 1, the calibration of both the pim2 and pim3 models was good for both age groups (p > 0.05), but the value for the pim2 model was higher than the value for the pim3 model. in addition, the expected mortality rates were closer to the observed mortality rates in pim2 model (table 2). when we divided the patients into 10 similar sized groups according to their predictive mortality risk, both the average predicted mortality and observed mortality of approximately 90% of the patients was lower than 20% (fig. 1). there were some differences in the predicted and observed mortality in the subgroups, but these were acceptable according to the hosmer - lemeshow gof test. gof = goodness - of - fit, roc = receiver - operating characteristic, pim = pediatric index of mortality, ci = confidence interval. expected hospital mortality (%) calculated by the pim3 model and observed hospital mortality (%), together with the corresponding patient number, are presented. the patients were divided into 10 similar sized groups based on predicted mortality. in the diagnostic subgroups, the mortality rate of the hemato - oncological patients was 2 to 5 times higher (18.73%) compared to that of the other subgroups (table 3). the calibration was acceptable for all the subgroups, including the cardiac, respiratory, and hemato - oncological groups. however, as shown in table 3, the discriminatory power of the c - index for the hemato - oncological patients was lower than acceptable (0.666). gof = goodness - of - fit, roc = receiver - operating characteristic, pim = pediatric index of mortality, ci = confidence interval. this is the first study to validate the pim3 scoring system in a pediatric icu in korea. we validated the pim3 score in a korean pediatric icu population using the formulas and coefficients presented in the developmental study of pim3 score (4). the present study showed that the pim3 had acceptable discrimination ability (c - index = 0.76) and good calibration, with a value of 9.4 in those aged 0.7). it was similar to the value in an italian validation study, which reported that pim3 more accurately predicted the mortality risk than pim2 (21). therefore, the pim3 score may be a reasonable choice for pediatric icus in korea, despite its additional complexity. in previous validation studies, the area under the roc curve (c - index) was acceptable, but the values varied between 0.79 and 0.92. the c - indices in those studies were higher than the value reported in this study, which means the indices had better discriminatory power. the variation may be explained regional differences in study populations. in the development study of pim2 and pim3, cardiac, respiratory, and non - cardiac postoperative groups were most common diagnostic groups (411). in contrast, in the present study, cardiac, hemato - oncological, and respiratory groups were the most prevalent diagnostic groups. the hemato - oncological subgroup was the second most common subgroup in the present study, and it had the lowest c - index (0.66) compared to the other subgroups (0.740.83). the poor performance of pim and prism score in predicting mortality of oncological patients was reported in several studies (22232425). one study reported that both prism and pim score underestimated the mortality of oncological patients (24). another study suggested poor performance of prism3 in predicting mortality of pediatric stem cell transplantation patients (25). in a study of adults, the apache ii scoring system also underestimated the mortality of bone marrow transplant recipients when the apache ii score was low (26). the underestimation of mortality rate might be caused by the innate limitation of the general mortality prediction score, which has one - size - fits - all property. for example, the predicted mortality of a respiratory failure patient, which is measured by pao2, fio2 and the usage of mechanical ventilation (in pim3 score), pao2, and pco2 (in prism3 score), can be calculated in the same way for a previously healthy child with viral pneumonia and an oncological patient with cytomegalovirus / fungal pneumonia, pulmonary veno - occlusive disease, or pulmonary graft - versus - host disease at the time of admission. since the latter patient usually has very poor prognosis (24), the observed mortality rate might be very different between the 2 patients. a general mortality prediction model might be inappropriate for use with patients in specialized oncological icus. instead, a disease - specific scoring system, reflecting specific prognosis, might be more useful (27). the observed mortality was 8.47%, and it was higher than the reported observed mortality. high unadjusted mortality may be explained by the severity of illness of patients in the icu or the poor performance of the icu team. to clarify the actual cause, the standard mortality ratio (smr), which is the observed mortality rate divided by the average predicted mortality, can be calculated (1). the smr is the most commonly used parameter of icu quality in western europe and is mandated by some countries (28). it can also be used to compare mortality data, follow changes over time, and evaluate the effect of interventions or events (2930731). to calculate the smr, the predicted mortality must first be determined. one advantage of the pim3 is that the formula and coefficients of predicted mortality is presented in the article and freely available (4), compared to the mortality prediction formula of prism3 is commercially patented and only incomplete prism score is documented in the study report. in this study, the observed mortality was higher than the predicted mortality. however, the smr was higher than 1, suggesting that the performance of the icu was low. the time patterns of admission (i.e., early admission before full disease progression or late admission when the disease is severe) can affect the physiological parameters recorded at the time of icu admission and eventually the smr. variation in the predicted mortality according to the scoring system can also influence the smr (32). in latin america, some countries have tried to develop a calibrated mortality prediction model because of regional differences among icus (734). despite its defects for example, serial measurements of icu smrs can be used to monitor internal quality improvements in hospitals. at the national level, the smr of pediatric icus can be measured every 510 years to evaluate the improvement of the quality of korean pediatric icus. first, this was a single center study conducted at a tertiary hospital. thus the findings may not be generalizable to the entire korean pediatric population. however, there are only a few pediatric icus in korea, all of which are located in tertiary hospitals, so our study population might be similar to other pediatric icu populations in korea. another problem is that many pediatric patients in korea are admitted to adult icus, but patients aged < 18 years are usually excluded in research of adult patients. to evaluate all the pediatric patients in icus, including pediatric patients in adult icus, multicenter studies, including primary and secondary hospitals, due to the diversity of the patients, a one - size - fits - all scoring system was used in the evaluation of the icu. in reality, this type of system is not suited to all patients, especially small specific subgroups. the third limitation is the retrospective data collection, although every effort was made to validate the data thoroughly. in conclusion, the performance of the pim3 scoring system in korean patients aged < 18 years was good. however, the pim3 may not be an appropriate quality measure for subgroups with hemato - oncological disease or oncology - specific icus because of its low discriminatory power. a multicenter study that includes pediatric patients in both adult and pediatric icus | to compare mortality rate, the adjustment of case - mix variables is needed. the pediatric index of mortality (pim) 3 score is a widely used case - mix adjustment system of a pediatric intensive care unit (icu), but there has been no validation study of it in korea. we aim to validate the pim3 in a korean pediatric icu, and extend the validation of the score from those aged 016 to 018 years, as patients aged 1618 years are admitted to pediatric icu in korea. a retrospective cohort study of 1,710 patients was conducted in a tertiary pediatric icu. to validate the score, the discriminatory power was assessed by calculating the area under the receiver - operating characteristic (roc) curve, and calibration was evaluated by the hosmer - lemeshow goodness - of - fit (gof) test. the observed mortality rate was 8.47%, and the predicted mortality rate was 6.57%. for patients aged < 18 years, the discrimination was acceptable (c - index = 0.76) and the calibration was good, with a 2 of 9.4 in the gof test (p = 0.313). the observed mortality rate in the hemato - oncological subgroup was high (18.73%), as compared to the predicted mortality rate (7.13%), and the discrimination was unacceptable (c - index = 0.66). in conclusion, the pim3 performed well in a korean pediatric icu. however, the application of the pim3 to a hemato - oncological subgroup needs to be cautioned. further studies on the performance of pim3 in pediatric patients in adult icus and pediatric icus of primary and secondary hospitals are needed. |
the clinical features, laboratory data, and patient outcomes are presented in table 1. among the 24 patients, 11 were treated with intermittent hemodialysis (ihd), whereas 13 were treated with a continuous modality (eight with continuous veno - venous hemodialysis (cvvhd) and five with continuous veno - venous hemodiafiltration (cvvhdf)). in two patients (patients nos. 8 and 11, table 1), ethanol infusion was started before the initial blood sample was drawn, explaining why they were acidotic in spite of their serum ethanol concentration. the two groups of patients treated with different modes of enhanced elimination were comparable by age, time to diagnosis / treatment, and number of patients. the mean time to diagnosis and treatment after the toxic alcohol consumption was 37 h in the cvvhd / hdf group and 48 h in the ihd group. all collected data were normally distributed with two exceptions : (a) serum ethanol in both groups of patients, and (b) serum methanol in the cvvhd / hdf group. there were no statistical differences between the two groups in admission data with respect to serum methanol, formate, pco2, hco3, base deficit, anion gap, and osmolal gap (all p>0.05). the patients treated with cvvhd / hdf were more acidotic than those treated with ihd (mean ph 6.90.1 vs. 7.10.1, respectively), with higher lactate levels (both p=0.04). data on formate and methanol elimination in 11 patients treated with ihd and 13 patients treated with cvvhd / hdf are presented in tables 2 and 3, respectively. the mean methanol elimination half - life in our study was 3.71.4 h for ihd and 8.11.2 h for cvvhd / hdf. the mean formate elimination half - life was 1.60.4 h during ihd and 3.61.0 h during cvvhd / hdf. the elimination half - lives of both formate and methanol on ihd were significantly shorter than on cvvhd / hdf (both p0.05). however, a significant correlation was present between the elimination half - life of formate and arterial blood ph ; the longer half - life of formate was present in more acidotic patients (p=0.038). no significant correlations of methanol and formate elimination half - lives were found with the folate substitution (treatment with either folic or folinic acid, or without folates) and with the specific antidote (ethanol or fomepizole) administration (all p>0.05). as regards the outcome, the patients with visual and cns sequelae of methanol poisoning had significantly longer elimination half - lives of formate than those without sequelae (p=0.005). the differences in mortality (p=0.36) and morbidity (survivals with sequelae, p=0.19) between ihd and cvvhd / hdf groups were not significant. the results of the multivariate regression analysis of variables influencing the elimination half - lives of formate and methanol based on all 24 cases are shown in tables 4 and 5. five independent variables included in the model explained 82.8% of the formate elimination half - life variation. the dialysate flow rate (p=0.013) was based on the cvvhd / hdf cases only ; however, it was still contributing significantly to the model. dialyzer membrane surface ' was grouped together as interchangeable with dialysate temperature ', explaining why other dialyzer properties ' is given as the term in the model, in spite of the linear correlation between both the methanol and the formate elimination half - lives. the variable clinical features ' consists of glasgow coma scale, mean arterial pressure, and pulse rate. the variable metabolic acidosis ' includes ph, pco2, hco3, base deficit, and the anion gap (ag). the two latter variables thus reflect the severity of the poisoning. for the methanol elimination half - life, the model consisting of four variables explained 81.5% of the variation (table 5). in this model, the dialysate flow rate and partially the blood flow rate were grouped together within the discrete factor dialyzer properties ' characterizing the mode of hemodialysis, whereas attempting to eliminate this factor led to the devaluation of the model. thus, this factor represented synthetically the difference in technical parameters of two modes of hemodialysis. the model for methanol was less sensitive to the factors characterizing the clinical state of the patient and more sensitive to the technical parameters of hemodialysis as compared with the model for formate. metabolic acidosis ' was the factor grouping together ph, hco3, bd, and the anion gap, reflecting the severity of metabolic acidosis, and thus describing the severity of poisoning in this model. methanol poisoning is one of the few conditions in clinical toxicology where dialysis has an essential role. both methanol and formate are small molecules (molecular weights 32 and 46 g / mol, respectively) with a volume of distribution 0.60.7 l / kg for methanol and 0.5 l / kg for formate, and no protein binding. extensive toxicokinetic data exist on the capacity of ihd to remove methanol, although the evidence is scarce for formate removal. there are no studies evaluating both the effects on methanol and formate kinetics with continuous modes of elimination (cvvhd / hdf) and only one that compared the efficacy of continuous and intermittent modes of dialysis. in that particular study, there were only three patients, and no formate kinetics was determined. the elimination of methanol without antidote therapy is of zero order with a rate of 85 mg / l / h that is i.e., about half of that of ethanol. if methanol metabolism is blocked by ethanol or fomepizole, methanol elimination is very slow (elimination half - lives of about 50 h in both cases), predominantly by pulmonary (and some renal) excretion. the kinetics of methanol depends upon metabolism, and thus the rate kinetics may vary not only depending on the amount of toxic spirits ingested but also on the time from ingestion to treatment, concomitant ingestion of different amounts of ethanol in other alcoholic beverages and foods, capability of respiratory and renal systems, as well as body weight, muscle / adipose tissue ratio, possible folate, and protein deficiency in chronic alcohol abusers, and other factors. both half - lives found during extracorporeal elimination in our study are markedly shorter than the reported endogenous elimination half - life of methanol (mean 43.152 h) during treatment with antidotes only. our results further confirm the superiority of ihd over the cvvhd / hdf in methanol elimination from serum, supporting findings from a previous study. nevertheless, the circulatory status is a concern when choosing dialysis modality in conditions where fluid removal is necessary ; this is why the continuous modalities are often preferred for hypotensive patients with acute kidney injury. in methanol poisoning, however, fluid removal is usually not necessary ; furthermore, the cases with the mean arterial blood pressure lower than 70 mmhg are rare (only one patient in our study), and therefore ihd is not expected to be riskier than cvvhd / hdf in most cases. one complication due to bleeding and one case of circuit clotting were reported in 13 cases of treatment with cvvhd / hdf. the rebound effect is present when the toxic agent continues to be transferred into the arterial circulation from peripheral body compartments, which is not the case with methanol and formate. the other possibility of methanol rebound is in the cases of delayed absorption of methanol from a full stomach. in our study, the mean time to diagnosis was 48 h in the ihd group, and thus the methanol was completely absorbed before the onset of dialysis. the possibility of formate rebound exists if incomplete adh blocking is present (due to insufficient serum ethanol levels) and continuation of the metabolic pathway of methanol elimination in addition to the elimination through hemodialysis. in all our cases, adh was completely blocked either by ethanol or by fomepizole, which was proved by first - order kinetics of methanol elimination. in contrast to the study by kan., the mean methanol elimination half - life in our study was only 54% shorter during ihd as compared with cvvhd / hdf. the smaller difference between the two modes of dialysis in our study can be explained by the higher dialysate flow in the cvvhd / hdf group. the dialysate flow rate in two patients on cvvhdf in the cited study was 1.0 l / h only, compared with a mean of 2.5 l / h in our study. the results for formate elimination during ihd substantially differed from the endogenous elimination half - life of formate reported from 2.3, 3.4, to 56 h, and in two case reports 20 (ref. 29) and 77 h. it was also 65% shorter than in a previous study by kerns. during dialysis. this study, however, possesses some weaknesses : only six patients were dialyzed, and two of them had less than three data points to calculate the half - life of formate (of whom one had an extremely short half - life before initiation of dialysis : 79 min). further, there was variability in the blood flow in two patients (out of five) during dialysis, which means that half - life can not be calculated., who calculated the mean formate elimination half - life during hemodialysis in 18 patients to be 1.810.78 h, which was statistically different from the values observed without dialysis. furthermore, the formate elimination half - life before or in the absence of dialysis calculated in the cited study (6.13.3 h) was substantially longer than in the former studies based on fewer cases. the mean elimination half - life of formate on ihd in our study was 56% shorter as compared with cvvhd / hdf (1.6 vs. 3.6 h, p 18 years of age, and met the following criteria : confirmed methanol poisoning (serum methanol > 6.3 mmol / l), documented circumstances of methanol ingestion and time to diagnosis, and sufficient laboratory data, including serum methanol and formate level on admission and at the initiation of hemodialysis. twenty - four methanol - poisoned patients were treated in 10 hospitals between september and december 2012. the patients were treated with alkalization, ethanol or fomepizole, or a combination of both antidotes (for example, start of therapy with fomepizole followed by ethanol administration). folates (folic or folinic acid) were given to 17/24 patients according to the standard protocols. patient nos 111 were treated with ihd, and patient nos 1224 were treated with continuous methods of enhanced elimination (nos 1219 with cvvhd and nos 2024 with cvvhdf). most of the patients treated with ihd (9/11 patients) were admitted to the intensive care units of internal medicine departments, and the patients treated with cvvhd / hdf were predominantly admitted to the departments of anesthesiology and resuscitation (12/13 patients). patients underwent hemodialysis if they exhibited any of the following : serum methanol higher than 15.8 mmol / l, acidemia, or visual toxicity. the choice of the method of enhanced elimination in each case was based on several factors, such as the hemodynamic stability of the patient on admission (all the patients treated with ihd had mean arterial pressure > 70 mm hg, 4/13 patients treated with cvvhd had mean arterial pressure 70 mm hg), and severity of clinical symptoms of poisoning (8/13 patients treated with cvvhd were comatose on admission (gcs 18 years of age, and met the following criteria : confirmed methanol poisoning (serum methanol > 6.3 mmol / l), documented circumstances of methanol ingestion and time to diagnosis, and sufficient laboratory data, including serum methanol and formate level on admission and at the initiation of hemodialysis. twenty - four methanol - poisoned patients were treated in 10 hospitals between september and december 2012. the patients were treated with alkalization, ethanol or fomepizole, or a combination of both antidotes (for example, start of therapy with fomepizole followed by ethanol administration). folates (folic or folinic acid) were given to 17/24 patients according to the standard protocols. patient nos 111 were treated with ihd, and patient nos 1224 were treated with continuous methods of enhanced elimination (nos 1219 with cvvhd and nos 2024 with cvvhdf). most of the patients treated with ihd (9/11 patients) were admitted to the intensive care units of internal medicine departments, and the patients treated with cvvhd / hdf were predominantly admitted to the departments of anesthesiology and resuscitation (12/13 patients). patients underwent hemodialysis if they exhibited any of the following : serum methanol higher than 15.8 mmol / l, acidemia, or visual toxicity. the choice of the method of enhanced elimination in each case was based on several factors, such as the hemodynamic stability of the patient on admission (all the patients treated with ihd had mean arterial pressure > 70 mm hg, 4/13 patients treated with cvvhd had mean arterial pressure 70 mm hg), and severity of clinical symptoms of poisoning (8/13 patients treated with cvvhd were comatose on admission (gcs < 8), whereas only 2/11 treated with ihd. another significant factor was the type of dialyzing equipment available in a medical facility : some smaller hospitals had the dialyzing equipment in the anesthesiology departments only, making only the continuous mode of hemodialysis available. ethanol was given to seven patients treated with ihd and nine patients were treated with cvvhd / hdf, whereas fomepizole (alone or followed by ethanol administration) was administered to four patients in each group. six patients treated with ihd and 11 patients treated with cvvhd / hdf received folates. ihd was initiated 2 h after admission to the hospital (range 12 h). it was performed for a median duration of 8 h. cvvhd / hdf was started 3.5 h after admission to the hospital (range 0.512 h) and lasted for a median duration of 44 h. venous blood for methanol and formate analysis was obtained on admission, at the start of hemodialysis, every 24 h during the dialysis session, and at the end of dialysis. the blood samples for the osmolality measurements were not necessarily taken at the same time as the samples taken for the methanol and formate analysis in this study, and correlation studies between osmolality and methanol could not be performed. the stated parameters of cvvhd / hdf were maintained during the observation time. in most cases, the duration of cvvhd / hdf was longer than the observation time ; however, it was not practical for the study purpose and ethical issues to continue blood sampling. blood samples were spun, serum was separated, and it was frozen until analyses. methanol was measured by a gas chromatographic method with flame ionization detection and a direct injection with internal standard (gas chromatograph chrom 5, laboratory instruments prague, czech republic), limit of detection 1.9 mmol / l, and day - to - day coefficient of variation 2.55.4%. formate was measured enzymatically on a hitachi analyzer (hitachi 912, hitachi science systems, tokyo, japan) using formate dehydrogenase (roche, meylan, france) and nad (roche), according to a previously published method. pure sodium formate (sigma - aldrich, st louis, mo) was used to prepare a standard of 1.1 mmol / l phosphate buffer and two control sera. day - to - serum ethanol was analyzed by gas chromatography with flame ionization detection and a direct injection with internal standard (gas chromatograph chrom 5, laboratory instruments). mmol / l, and the day - to - day coefficient of variation was 3.87.1%. osmolality was measured by freezing point depression method on a fiske one - ten osmometer. the reference range for the osmolal gap was 919 mosm per kg h2o. the osmolal contribution from ethanol (two patients) elimination half - life was calculated from the relationship of methanol and formate versus time : linear regression analysis determined the elimination constant (ke), from the slope of the natural logarithm of methanol or formate versus time. first - order ' intradialytic elimination half - life was then calculated from the relationship t1/2=0.693/ke. the ag and the osmolal gap were calculated by using the following formulae : ag=(na+k) (hco3+cl), and og = mo (1.86 na+glucose+urea)/0.93, where mo represents the measured osmolality. pearson correlation analysis was performed for characterization of logarithmic dependence concentration of methanol and formate on time, respectively. median, modus, arithmetic mean, and confidence intervals were applied for the calculation of average values of different groups of patients. the normality of data was verified using tests based on sample skewness and kurtosis ; the normality of one sample was based on the kolmogorov we used the independent t - tests for comparison of independent parameters between two groups of patients, and the multivariate regression models (forward, backward, and stepwise methods) for the determination of optimal sets of explanatory variables for the methanol and formate elimination half - lives. we have used also the exploratory factor analysis on pearson correlations among the monitored input variables as an auxiliary tool of explanatory regression models. statistical documentation was performed in excel (microsoft, redmond, wa), and the formal calculations were produced in qc expert software 3.1 (trilobyte, pardubice, czech republic) and in ibm spss ver. 17.0 and statistica sf ver. 10.0 (both licensed to first faculty of medicine of the charles university in prague). the study was approved by the general university hospital ethics committee in prague, czech republic. | during an outbreak of methanol poisonings in the czech republic in 2012, we were able to study methanol and formate elimination half - lives during intermittent hemodialysis (ihd) and continuous veno - venous hemodialysis / hemodiafiltration (cvvhd / hdf) and the relative impact of dialysate and blood flow rates on elimination. data were obtained from 11 ihd and 13 cvvhd / hdf patients. serum methanol and formate concentrations were measured by gas chromatography and an enzymatic method. the groups were relatively comparable, but the cvvhd / hdf group was significantly more acidotic (mean ph 6.9 vs. 7.1 ihd). the mean elimination half - life of methanol was 3.7 and formate 1.6 h with ihd, versus 8.1 and 3.6 h, respectively, with cvvhd / hdf (both significant). the 54% greater reduction in methanol and 56% reduction in formate elimination half - life during ihd resulted from the higher blood and dialysate flow rates. increased blood and dialysate flow on the cvvhd / hdf also increased elimination significantly. thus, ihd is superior to cvvhd / hdf for more rapid methanol and formate elimination, and if cvvhd / hdf is the only treatment available then elimination is greater with greater blood and dialysate flow rates. |
the natural history of acute cervical internal carotid artery (ica) occlusion with serious neurological symptom is unfavorable1). treatment for acute occlusion of the cervical ica includes intravenous administration of tissue plasminogen activator (tpa), percutaneous transluminal angioplasty, carotid endarterectomy (cea), and extracranial - to - intracranial (ec - ic) artery bypass. according to the current literature, the recanalization rate for carotid occlusion with systemic (intravenous) tpa alone ranges from 15% to 35%. a good outcome has been documented for 20% to 44% of patients treated with intravenous tpa alone for acutely symptomatic ica occlusion4,17). despite the proven benefit of cea in prevention of stroke recurrence in patients with a high - grade stenosis of the ica2,25), few studies of emergency cea for improvement of their neurologic symptoms and no rigorous clinical trials regarding the efficacy of cea in an acute stroke stage have been reported3,18). a few studies have attempted to determine whether emergent ec - ic bypass surgery is an effective treatment for main trunk stenosis or occlusion in acute stage12). previous studies have supported carotid angioplasty and stenting (cas) as a technique for secondary prevention in the chronic stroke stage8,29). however, cas in the acute stage remains challenging because of the limited therapeutic window and risk of hyperperfusion syndrome after revascularization. nevertheless, there have been some reports on the safety and effectiveness of emergency cas, even in the acute stage of ischemic stroke7,14,15,19,21,27,28,30). the purpose of this study is to demonstrate the technical feasibility and clinical efficacy of emergent cas for acute stroke due to athero - thrombotic occlusion of the cervical ica. medical records identified 17 patients who underwent emergent cas for treatment of acute stroke related to an athero - thrombotic cervical ica occlusion between 2009 and 2013. all patients underwent examination by computed tomographic (ct) perfusion imaging, followed by magnetic resonance (mr) imaging before conventional angiography. the indication for emergent cas was determined by mismatch between the diffusion - weighted mr imaging finding of localized high intensity areas and the ct perfusion finding of decreased cbf related to the occluded ica. patients who present within 4.5 hours of symptom onset and without contraindication to systemic thrombolysis are treated immediately with intravenous tpa (0.9 mg / kg) and, unless immediate neurological improvement is observed, are then transferred rapidly to the angiography suite. in contrast, patients who present between 4.5 and 6 hours after symptom onset do not undergo systemic thrombolysis, but are transferred immediately to the neurointerventional suite. patients matching the following criteria were included in our analysis : 1) onset of acute stroke symptom and arrival at the hospital within 6 hours, 2) national institutes of health stroke scale (nihss) score on admission 5, 3) athero - thrombotic occlusion of the cervical ica confirmed by catheter - based angiography : as the balloon configuration during inflation and features of residual stenosis after predilatation or the stent configuration before balloon angioplasty, and 4) negative findings on brain ct or limited hypodensities involving less than one - third of the middle cerebral artery (mca) territory. we excluded patients in whom a dissection of the ica was diagnosed due to the different nature of the disease. tia was defined as an acute transient focal neurological deficit caused by vascular disease, which showed a complete reversal within 24 hours, and caused no acute lesion, as confirmed by diffusion - weighted mr imaging. interventional procedures were performed in the neuroangiography room equipped with a digital subtraction angiography system (axiom artis, siemens, germany). all angiographic procedures were performed using a transfemoral approach under local anesthesia using an electrocardiogram, arterial oxygen saturation, and blood pressure monitoring. four - vessel angiography was performed for estimation of collateral flow from the anterior and posterior communicating arteries, and to determine the presence of concomitant intracranial artery occlusion. the interventional treatment consists of two steps : first, emergent cas for athero - thrombotic occlusion of the cervical ica, and second, intracranial recanalization. a loading dose of 300 mg clopidogrel plus 300 mg prior to the therapeutic procedure, patients were administered a systemic heparinization and a bolus injection of heparin 3000 iu. an additional 1000 iu bolus of heparin was administered every hour in order to maintain an act of > 200 seconds throughout the procedure. coaxial catheter - flushing fluid was mixed with heparin at a concentration of 1000 iu of heparin per 1000 ml of saline. an 8 fr guiding catheter (envoy ; cordis, miami lakes, fl, usa) or 6 fr shuttle sheath was advanced to the common carotid artery, and it was connected to a continuous saline flush. a 0.014-in microwire (transend ; boston scientific, natick, ma, usa) was placed into the petrous ica passing the occluded segment. then, a self - expandable stent was advanced over the microwire and positioned across the occlusion. a stent deployment and balloon angioplasty were performed with or without distal protection device (spider device, ev3, plymouth, mn, usa) to prevent distal embolic event. the entire length of the ica and the intracranial area was then imaged for determination of whether there was any further stenosis or occlusion of the ica or mca. if there was no distal patency of the ica, we attempted restoration of distal flow using forced arterial suction thrombectomy using a 50-ml syringe. if an additional occlusion was present in the terminal segment of the ica or mca, a 6 fr guiding catheter was advanced as far as possible into the recanalized ica. the devices used for mechanical thrombectomy were the penumbra system (penumbra inc., alameda, ca, usa) or the solitaire ab / fr (ev3 europe sas, paris, france) with or without additional intra - arterial thrombolysis with urokinase. we initially attempted forced arterial suction thrombectomy using a penumbra reperfusion catheter, but switched to solitaire in difficult cases, which means failed thrombectomy despite several attempts at recanalization. immediately after the therapeutic procedure, we obtained the detailed neurologic history and performed a complete examination of all patients. the patients underwent non - enhanced brain ct imaging for evaluation of possible hemorrhagic complications. following endovascular stent placement, patients were transferred to a stroke intensive care unit for strict control of blood pressure for prevention of hyperperfusion syndrome. after the procedure, patients were administered aspirin 100 mg and clopidogrel 75 mg daily. low - molecular - weight nadroparin calcium (fraxiparine ; glaxosmithkline, marly - le - roi cedex, france) 2850 iu was also administered subcutaneously three times per day for at least three days. technical success of emergent cas was defined as restoration of ica flow with more than 50% recanalization of the cervical ica. intracranial arterial recanalization was assessed using the thrombolysis in cerebral infarction (tici) scores and was recorded prospectively at the end of each case by the treating interventionalist11). angiographic success of intracranial arterial recanalization was defined as restoration of ica flow with normal or near - normal perfusion in the ipsilateral ica territory (tici 2b flow). for clinical outcome evaluation, improvement of neurological function was defined as reduction of 4 points on the nihss at seven days after the procedure compared with baseline. in addition, clinical functional outcome was assessed upon discharge from hospital or at the last clinical visit using the modified rankin scale (mrs). medical records identified 17 patients who underwent emergent cas for treatment of acute stroke related to an athero - thrombotic cervical ica occlusion between 2009 and 2013. all patients underwent examination by computed tomographic (ct) perfusion imaging, followed by magnetic resonance (mr) imaging before conventional angiography. the indication for emergent cas was determined by mismatch between the diffusion - weighted mr imaging finding of localized high intensity areas and the ct perfusion finding of decreased cbf related to the occluded ica. patients who present within 4.5 hours of symptom onset and without contraindication to systemic thrombolysis are treated immediately with intravenous tpa (0.9 mg / kg) and, unless immediate neurological improvement is observed, are then transferred rapidly to the angiography suite. in contrast, patients who present between 4.5 and 6 hours after symptom onset do not undergo systemic thrombolysis, but are transferred immediately to the neurointerventional suite. patients matching the following criteria were included in our analysis : 1) onset of acute stroke symptom and arrival at the hospital within 6 hours, 2) national institutes of health stroke scale (nihss) score on admission 5, 3) athero - thrombotic occlusion of the cervical ica confirmed by catheter - based angiography : as the balloon configuration during inflation and features of residual stenosis after predilatation or the stent configuration before balloon angioplasty, and 4) negative findings on brain ct or limited hypodensities involving less than one - third of the middle cerebral artery (mca) territory. we excluded patients in whom a dissection of the ica was diagnosed due to the different nature of the disease. tia was defined as an acute transient focal neurological deficit caused by vascular disease, which showed a complete reversal within 24 hours, and caused no acute lesion, as confirmed by diffusion - weighted mr imaging. interventional procedures were performed in the neuroangiography room equipped with a digital subtraction angiography system (axiom artis, siemens, germany). all angiographic procedures were performed using a transfemoral approach under local anesthesia using an electrocardiogram, arterial oxygen saturation, and blood pressure monitoring. four - vessel angiography was performed for estimation of collateral flow from the anterior and posterior communicating arteries, and to determine the presence of concomitant intracranial artery occlusion. the interventional treatment consists of two steps : first, emergent cas for athero - thrombotic occlusion of the cervical ica, and second, intracranial recanalization. a loading dose of 300 mg clopidogrel plus 300 mg prior to the therapeutic procedure, patients were administered a systemic heparinization and a bolus injection of heparin 3000 iu. an additional 1000 iu bolus of heparin was administered every hour in order to maintain an act of > 200 seconds throughout the procedure. coaxial catheter - flushing fluid was mixed with heparin at a concentration of 1000 iu of heparin per 1000 ml of saline. an 8 fr guiding catheter (envoy ; cordis, miami lakes, fl, usa) or 6 fr shuttle sheath was advanced to the common carotid artery, and it was connected to a continuous saline flush. a 0.014-in microwire (transend ; boston scientific, natick, ma, usa) was placed into the petrous ica passing the occluded segment. then, a self - expandable stent was advanced over the microwire and positioned across the occlusion. a stent deployment and balloon angioplasty were performed with or without distal protection device (spider device, ev3, plymouth, mn, usa) to prevent distal embolic event. the entire length of the ica and the intracranial area was then imaged for determination of whether there was any further stenosis or occlusion of the ica or mca. if there was no distal patency of the ica, we attempted restoration of distal flow using forced arterial suction thrombectomy using a 50-ml syringe. if an additional occlusion was present in the terminal segment of the ica or mca, a 6 fr guiding catheter was advanced as far as possible into the recanalized ica. the devices used for mechanical thrombectomy were the penumbra system (penumbra inc., alameda, ca, usa) or the solitaire ab / fr (ev3 europe sas, paris, france) with or without additional intra - arterial thrombolysis with urokinase. we initially attempted forced arterial suction thrombectomy using a penumbra reperfusion catheter, but switched to solitaire in difficult cases, which means failed thrombectomy despite several attempts at recanalization. immediately after the therapeutic procedure, we obtained the detailed neurologic history and performed a complete examination of all patients. the patients underwent non - enhanced brain ct imaging for evaluation of possible hemorrhagic complications. following endovascular stent placement, patients were transferred to a stroke intensive care unit for strict control of blood pressure for prevention of hyperperfusion syndrome. after the procedure, patients were administered aspirin 100 mg and clopidogrel 75 mg daily. low - molecular - weight nadroparin calcium (fraxiparine ; glaxosmithkline, marly - le - roi cedex, france) 2850 iu was also administered subcutaneously three times per day for at least three days. technical success of emergent cas was defined as restoration of ica flow with more than 50% recanalization of the cervical ica. intracranial arterial recanalization was assessed using the thrombolysis in cerebral infarction (tici) scores and was recorded prospectively at the end of each case by the treating interventionalist11). angiographic success of intracranial arterial recanalization was defined as restoration of ica flow with normal or near - normal perfusion in the ipsilateral ica territory (tici 2b flow). for clinical outcome evaluation, improvement of neurological function was defined as reduction of 4 points on the nihss at seven days after the procedure compared with baseline. in addition, clinical functional outcome was assessed upon discharge from hospital or at the last clinical visit using the modified rankin scale (mrs). the 17 patients with acute stroke related to athero - thrombotic cervical ica occlusion were treated by emergent cas between 2009 and 2013. the mean age of the patients (15 men and 2 women) was 70.57.7 years (range, 54 - 82 years). the patients ' mean nihss score on admission was 15.15.4 (range, 6 - 23). six patients had contraindications to intravenous administration of tpa, however, the others received a standard 10% bolus of the maximum dose (0.9 mg / kg body weight). the mean time from stroke onset to recanalization was 277110 minutes (range, 120 - 460 minutes). some difficulties were encountered during passage of the guidewire through the occluded segment ; it was possible in all cases. in six patients, no further vessel occlusion was detected after successful recanalization of the cervical ica (fig. the stent used was protg (ev3, plymouth, mn, usa) in 15 patients and wallstent (boston scientific, natick, ma, usa) in two. of 17 patients, 11 (11/17, 64.7%) showed an additional intracranial occlusion after flow restoration in the cervical ica (fig. 2) : at the level of the terminal segment of the ica (n=3) and at the level of the mca in eight patients. intracranial arterial recanalization to a tici 2b flow was achieved in four of 11 patients (36.4%). the overall recanalization rate (cervical ica and intracranial artery) was 10 of 17 patients (58.8%). procedure - related complications developed in two patients (2/17, 11.8%). in patient 3, although a distal protection filter was used, distal embolism occurred after cas. immediate thrombolysis using a combination of mechanical thrombectomy and intraarterial urokinase led to recanalization to a tici 2b flow. however, afterward, relevant infarction was observed and his neurological status worsened. in one patient who suffered acute in - stent thrombosis, abciximab was administered intraarterially with a maintenance drip, yielding stent recanalization ; however, the patient subsequently developed massive intracranial hemorrhage (ich) and died (patient 12). thus, procedure - related mortality was 5.9% (1/17, patient 12) and morbidity was 5.9% ten of 17 patients (58.8%) showed improvement (decrease in nihss score of 4 points compared with admission score) during the hospital course at seven days after recanalization. follow - up ct scan obtained immediately after each intervention showed occasional enhancement around the area of ischemia, which had resolved by the time the scan was repeated later. the ich was symptomatic with neurologic deterioration, resulting in death (patients 9 and 12). the other patient suffered an asymptomatic hemorrhage in the basal ganglia (patient 2). one patient (patient 13) died secondary to pneumonia and sepsis 21 days after the procedure. thus, there were three in - hospital deaths (3/17, 17.6%), respectively. at the end of the observational period, nine patients showed a good clinical outcome, with an mrs score of 2 (9/17, 52.9%), while the other eight (8/17, 47.1%) were dependent or dead (mrs score, 3 - 6). all surviving patients were followed up for 6 - 21 months (mean, 12.5 months). no neurologic deterioration or ischemic event occurred during the follow - up period in all surviving patients. a summary of characteristics and clinical outcomes according to existence of intracranial tandem lesion is shown in table 3. near - complete occlusion of the cervical ica was identified in half of patients without an intracranial tandem lesion, while in only one patient with an intracranial tandem lesion. a favorable outcome (mrs 2) was obtained in four of the six patients with isolated cervical ica occlusion (66.7%) and five of 11 patients with intracranial tandem occlusion (45.5%) at the last follow up. no patient with isolated cervical ica occlusion suffered from a symptomtic ich or in - hospital mortality, however, there were two cases of symptomtic ich (18.2%) and three cases of in - hospital mortality (27.3%) in the patients with intracranial tandem occlusion. more favorable outcomes were achieved in patients without intracranial tandem lesion, compared to a patient population with intracranial tandem lesion. the 17 patients with acute stroke related to athero - thrombotic cervical ica occlusion were treated by emergent cas between 2009 and 2013. the mean age of the patients (15 men and 2 women) was 70.57.7 years (range, 54 - 82 years). the patients ' mean nihss score on admission was 15.15.4 (range, 6 - 23). six patients had contraindications to intravenous administration of tpa, however, the others received a standard 10% bolus of the maximum dose (0.9 mg / kg body weight). the mean time from stroke onset to recanalization was 277110 minutes (range, 120 - 460 minutes). some difficulties were encountered during passage of the guidewire through the occluded segment ; it was possible in all cases. in six patients, no further vessel occlusion was detected after successful recanalization of the cervical ica (fig. the stent used was protg (ev3, plymouth, mn, usa) in 15 patients and wallstent (boston scientific, natick, ma, usa) in two. of 17 patients, 11 (11/17, 64.7%) showed an additional intracranial occlusion after flow restoration in the cervical ica (fig. 2) : at the level of the terminal segment of the ica (n=3) and at the level of the mca in eight patients. intracranial arterial recanalization to a tici 2b flow was achieved in four of 11 patients (36.4%). the overall recanalization rate (cervical ica and intracranial artery) was 10 of 17 patients (58.8%). procedure - related complications developed in two patients (2/17, 11.8%). in patient 3, although a distal protection filter was used, distal embolism occurred after cas. immediate thrombolysis using a combination of mechanical thrombectomy and intraarterial urokinase led to recanalization to a tici 2b flow. however, afterward, relevant infarction was observed and his neurological status worsened. in one patient who suffered acute in - stent thrombosis, abciximab was administered intraarterially with a maintenance drip, yielding stent recanalization ; however, the patient subsequently developed massive intracranial hemorrhage (ich) and died (patient 12). thus, procedure - related mortality was 5.9% (1/17, patient 12) and morbidity was 5.9% (1/17, patient 3), respectively. ten of 17 patients (58.8%) showed improvement (decrease in nihss score of 4 points compared with admission score) during the hospital course at seven days after recanalization. follow - up ct scan obtained immediately after each intervention showed occasional enhancement around the area of ischemia, which had resolved by the time the scan was repeated later. the ich was symptomatic with neurologic deterioration, resulting in death (patients 9 and 12). the other patient suffered an asymptomatic hemorrhage in the basal ganglia (patient 2). one patient (patient 13) died secondary to pneumonia and sepsis 21 days after the procedure. thus, there were three in - hospital deaths (3/17, 17.6%), respectively. at the end of the observational period, nine patients showed a good clinical outcome, with an mrs score of 2 (9/17, 52.9%), while the other eight (8/17, 47.1%) were dependent or dead (mrs score, 3 - 6). all surviving patients were followed up for 6 - 21 months (mean, 12.5 months). no neurologic deterioration or ischemic event occurred during the follow - up period in all surviving patients. a summary of characteristics and clinical outcomes according to existence of intracranial tandem lesion is shown in table 3. near - complete occlusion of the cervical ica was identified in half of patients without an intracranial tandem lesion, while in only one patient with an intracranial tandem lesion. a favorable outcome (mrs 2) was obtained in four of the six patients with isolated cervical ica occlusion (66.7%) and five of 11 patients with intracranial tandem occlusion (45.5%) at the last follow up. no patient with isolated cervical ica occlusion suffered from a symptomtic ich or in - hospital mortality, however, there were two cases of symptomtic ich (18.2%) and three cases of in - hospital mortality (27.3%) in the patients with intracranial tandem occlusion. more favorable outcomes were achieved in patients without intracranial tandem lesion, compared to a patient population with intracranial tandem lesion. the natural history of acute cervical ica occlusion with serious neurologic symptoms is unfavorable1). for these types of lesions, treatment with standard intravenous thrombolysis alone leads to a good clinical outcome in only 17% of cases with a death rate of as high as 55%18). currently, there is no consensus treatment for patients with acute ischemic stroke presenting with severe clinical symptoms due to athero - thrombotic occlusion of the cervical ica. acute cervical ica occlusion resulting in ischemic stroke is different from other forms of acute occlusion of cerebral vessels. occluded segments in other cerebral vessels, such as the mca and the terminal segment of the ica, usually consist of an occlusive embolus in a normal vessel23). the ecass iii showed that intravenous tpa is effective in treatment of acute ischemic stroke if given to patients within 4.5 hours of stroke symptom onset. however, the recanalization rates achieved with intravenous tpa for large vessel arterial occlusion are low, ranging from 4% to 32%, depending on the vessel (4% for ica occlusions and in addition, cervical ica occlusion is associated with a high incidence of intracranial tandem occlusions. rubiera.26) reported on the presence of a tandem lesion (ica and mca) as an independent predictor of poor outcome after intravenous thrombolysis. according to linfante.,17) a significant difference was observed between the proportion of recanalization in mca - only occlusions (15 of 17 recanalized, 88%) at three days after tpa compared with that in ica - mca tandem occlusions (five of 16 recanalized, 31%). improvement in clinical outcome showed a close association with resumption of mca flow, and most ica occlusions did not recanalize after intravenous tpa therapy. the pathophysiologic processes involved in occlusion of the extracranial ica are similar to processes observed in acute occlusion of the coronary arteries. the occluded segment of the ica consists of a predominantly atherosclerotic plaque and a superimposed thrombus. therefore, large contributions of atherosclerotic plaque and platelet activation do not provide an ideal substrate for thrombolytics alone23,24). in acute myocardial infarction, the recanalization rate for carotid occlusion with intravenous tpa alone is very low, ranging from 15% to 35%. a good outcome has been documented for 20% to 44% of patients treated with intravenous tpa alone for acutely symptomatic ica occlusion. nevertheless, there is currently an interest in " early " cea after intravenous tpa for prevention of further stroke. however, cea is not suited for patients in an emergency situation such as acute stroke because control of the intracranial extension of the clot is extremely limited. surgical bypass may be a natural resolution for ica occlusion, however, a large international randomized ec - ic bypass trial failed to show any benefit22). recently, there has been a surge of interest in endovascular interventions for acute ica occlusion5,6,10,13,15,19,20,21). we have summarized the reported studies in which more than 10 patients with acute cervical ica occlusion were treated with cas in table 4. imai.13) treated 17 patients with either occlusion or a high - grade stenosis of the ica with emergent cas. median nihss scores before emergency stent placement and at seven days were 12 and 5, respectively (p<0.01). eleven patients (65%) showed favorable outcomes (mrs score, 0 - 2) at 90 days. dabitz.5) reported on their experience with 10 patients with an average nihss score of 21 using a combination of cas and intra - arterial thrombolysis for treatment of the cervical ica occlusion ; 70% had an mrs of 2 after 20 weeks. hauck.10) reported that 17 of 22 patients (77.3%) underwent successful revascularization with cas and 50% of patients showed good outcomes (mrs 2) after a median follow - up of three months. patients with complete cervical ica occlusion but partial distal preservation of the vessel were most likely to benefit from the intervention. papanagiotou.21) treated 22 patients with acute atherosclerotic extracranial ica occlusion with emergent cas and intraarterial thrombolysis using mechanical thrombectomy. fourteen of 22 patients (63%) underwent successful overall revascularization and nine patients (41%) had an mrs of 2 at 90 days. dalyai.6) reported on their experience with 17 patients using a combination of cas and intraarterial thrombolysis for treatment of proximal ica occlusion. the results of our study combined with the results of these series indicate that this approach is also feasible for athero - thrombotic occlusion of the cervical ica. after cas of an ica occlusion, an additional occlusion may be present in the terminal segment of the ica or the mca. in our study, acute cervical ica occlusion presented frequently (64.7%) as a tandem lesion with intracranial extension of the clot, usually into the mca branches or the carotid terminus. in the event of additional intracranial occlusion, further recanalization measures should be taken. however, the angiographic result was somewhat disappointing, with achievement of a recanalization rate of 36.4%. revascularization of intracranial artery occlusion requires a significant amount of time ; however, the therapeutic time window is narrow because of less collateral flow from the anterior communicating artery and/or posterior communicating artery, compared with that for isolated cervical ica occlusion. in some cases, we should have stopped further revascularization of intracranial artery occlusion because of exceeding the therapeutic time window. we found that patients without a tandem occlusion were more likely to show rapid neurologic improvement within the first seven days after cas and a mrs 2 at follow - up (table 3) ; this has also been previously reported in the literature10,19,21). however, due to the risk of reperfusion injury, such as hyperperfusion syndrome or hemorrhagic transformation, successful recanalization does not always result in a favorable outcome. although the incidence of hyperperfusion syndrome after cas is 5% to 7%, it is expected to be higher in emergent recanalization of the cervical ica. symptomatic ich rate of 12% (n=2) in the current study is similar to the previously reported rates for cas in acute stroke (6% to 18%)6,13,15,20,21). due to the aggressive anticoagulation required, the risk of ich is expected to be higher in acute cas compared with mechanical recanalization studies. in our series, there was one case of acute in - stent thrombosis ; prompt use of intraarterial abciximab yielded thrombolysis, however, subsequently developed massive ich and died. the diffusion / perfusion mismatch indicative of ischemic penumbra is useful for selection of patients who might benefit from recanalization. in our series, emergent endovascular recanalization of the cervical ica and distal branches was technically successful in 58.8% (10 patients) overall. at the last follow - up, good clinical outcome (mrs 2) with endovascular intervention was observed in nine of 17 patients (52.9%) overall and in seven patients (7/10, 70.0%) in whom revascularization was successful. our observation that endovascular revascularization of an acutely occluded ica at the cervical level has a fairly high chance of success exceeding the rate of recanalization with intravenous tpa alone is similar to the findings of several previous reports in the literature. the current study showed that patients with cervical ica occlusion with acute stroke presentation could benefit from endovascular revascularization, although these findings should be confirmed in a larger study with multivariate logistic regression analysis. emergent cas for acute stroke due to athero - thrombotic occusion of the cervical ica showed a good technical feasibility and favorable clinical outcome. in particular, emergent cas is more beneficial in patients with isolated cervical ica occlusion. treatment of intracranial tandem occlusions is an issue that must be resolved in order to achieve a favorable outcome. | objectivethe purpose of this study is to demonstrate the technical feasibility and clinical efficacy of emergent carotid angioplasty and stenting (cas) for acute stroke due to athero - thrombotic occlusion of the cervical internal carotid artery (ica).methodsreview of medical records identified 17 patients who underwent emergent cas for treatment of athero - thrombotic occlusion of the cervical ica with acute stroke between 2009 and 2013. eleven patients (64.7%) presented with concomitant intracranial artery occlusion, which was treated primarily by mechanical thrombectomy after cas.resultssuccessful revascularization of the cervical ica with emergent cas was achieved in all patients. after cas, intracranial recanalization with thrombolysis in cerebral infarction 2b flow was achieved in four of the 11 patients (36.4%). the overall recanalization rate (cervical ica and intracranial artery) was 10 of 17 patients (58.8%). symptomatic intracranial hemorrhage occurred in two patients (11.8%), resulting in death. ten patients (58.8%) showed improvement (decrease in nihss score of 4 points) at seven days after recanalization. nine patients (52.9%) showed a favorable outcome (mrs 2) at the last follow - up. a favorable outcome (mrs 2) was obtained in four of the six patients with isolated cervical ica occlusion (4/6, 66.7%) and five of 11 patients with intracranial tandem occlusion (5/11, 45.5%).conclusionemergent cas for acute stroke due to athero - thrombotic occusion of the cervical ica showed a good technical feasibility and favorable clinical outcome. |
choosing the parameters for the calculation of the fractal dimension of a discretized non - strictly self - symmetrical natural object is the first step in its surface roughness assessment. there is a strict connection between the density of points of the discretized set, the fractal scales used for the calculation of d, and the d value itself (figure 1). the choice of fractal scales may be summarized as follows : (i) choose exponential distances between the different scales ; (ii) select the smallest scale greater than the resolution of the discretization, otherwise d will be severely overestimated (figure 1b), and the largest scale to not exceed the size of the smallest object inspected, otherwise it will include a large amount of empty space which will result in underestimated d values. following these rules, we chose fractal scales [0.4, 0.8, 1.6, 3.2 ]. the largest one is about the size of a small ligand or amino acid side - chain diameter. the next step in parameterizing the calculation of d is the selection of a suitable density of the points on the ses generated by msms (figure 1a). while calculating d for different densities between one and 20 pp, we identified two intervals separated around 6 pp, in which d behaved differently. the first interval harbors densities that are too low for the selected fractal scales, so that the hyperspheres for the smallest can not reach any neighboring points, thus c(x,)=1/n, which leads to overestimating d. this interval has a large dispersion compared to the other one (figure 1a), but the orderings of the elements appear to be stable (figure 1d), meaning that a switch in density inside the interval would not drastically change the overall ordering of the set. the same assumption also holds for the second interval containing densities greater than 6 pp. it contains densities that are more favorable for a fractal dimension calculation, i.e. the production of stable regression lines, as well as the lower dispersion introduced by surface sampling effects. naturally, a decrease in point density reduces the qualities of the discretization and results in a more crude approximation of d, but it also speeds up the computations, so we found a compromise at a density of 7 pp. note that choosing different fractal scales shifts the limit between the two intervals, depending on the choice of the lowest. discrimination ability of the fractal dimension d depending on the fractal scale and point density (pp : points per) calculated for the 604 ligands of the main set of protein - ligand complexes. a) dispersion of d. a tendency of decreasing dispersion is apparent with increasing discretization density. a steep fall in d values as well as b, c) regression lines of the doubly logarithmic plot of the correlation sum for the ligand of the pdb i d : 1c5z complex for densities on both sides of the steep fall in d : b) 5 pp, (r=0.952) ; c) 7 pp, (r=0.999). d) gray scale coded matrix containing the spearman s rank correlation of the vectors of d values of the ligands (one 604-dimensional rank vector for each density). each filled square represents the pairwise rank correlation between two corresponding vectors, illustrating how the change in density shuffles the relative positioning of the d values. few changes in the ligands positions in the rank vector indicate stable discrimination ability (strong correlation, dark color). comprehensive definition of the model parameters : the analyzed spatial molecular data relied exclusively on prior crystallographic surveys on protein - ligand complexes, dna and rna. the pdb i d lists are provided as supplementary information. the protein - ligand complexes used in this survey were selected from two publicly available databases : (i) the pdbbind (www.pdbbind.org) was accessed to create the main set of complexes, the results of which were verified on (ii) a subset of the sc - pdb, termed the verification set for reference. a subset of 604 protein - ligand complexes based on the pdbbind database was selected according to the following criteria : (i) monomeric structures with predicted primary binding site volume not exceeding the volume of the corresponding ligand by more than 50 % ; (ii) structures for which the msms ses calculation failed due to software errors were excluded. the main set (supplementary listing 1) provided the following five sets of spatial data used in the survey at hand : (i) the proteins set contains spatial data gathered by stripping any atoms from the complexes that did not belong to a protein chain such as waters and ligands resulting in a set of 604 proteins ; (ii) the ligands set contains the ligands of the active sites of the protein - ligand - complexes resulting in a set of 604 small molecules ; (iii) the liganded pockets set contains the main pockets of the complexes resulting in a set of 604 active sites ; (iv) the protein surface clefts set consists of pockets selected by pocketpicker that are missing a ligand in the published crystallographic model ; (v) the background surface patches set comprises of protein points subsets extracted using msms at uniformly distributed random positions on the surfaces of the 604 proteins a subset of 8639 protein - ligand complexes based on the sc - pdb database was selected according to the following criteria : (i) the complexes are not present in the main set ; (ii) very large structures (more than 2.3510 surface points) and structures for which the msms ses calculation failed due to software errors were excluded. the surface patches for the pockets and clefts were excised using a hard proximity margin of 3 around the output of pocketpicker. for the sampling of the background surface patches 20 random atoms on the surface of each protein were selected. each single surface patch was then defined by using spheres with 6 radius around each randomly selected surface atom. the ppi set was extracted from the freely available dockground ppi database (dockground.bioinformatics.ku.edu/), benchmark 3.0. it contains 131 complexes with both bound and one available unbound structures for each entry, as well as 102 complexes, each with both bound and both unbound structures available and provided. thus, we were able to inspect 233 complexes with a grand total of 466 ppis. since we analyzed ppis of known bound complexes, we used the corresponding complex partners for the definition of the interface s position, and a hard proximity margin of 3.5. we chose this to be slightly higher than 3.0, because at 3.0 the sampled patches for the ppi exhibited cut - off artifacts such as holes in the surfaces influencing the roughness estimation. the protein, delivered in 20 mm acetate buffer, ph 5.0 with 200 mm nacl, 10 % (v / v) glycerol and 0.05 % 2-mercaptoethanol, was concentrated to a protein concentration of 3 mg / ml. 0.5 l of a 200 mm hiv protease inhibitor (compound 7) solution in dmso was added to 80 l of the concentrated hiv-1 protease solution. because it turned out that the inhibitor was poorly soluble in the resulting protein solution, an additional 2 l of a 50 mm inhibitor solution in peg400 was added to the last 20 l of the protein solution. 0.1 l of this latter protein solution was mixed with 0.1 l reservoir solution of 0.1 m nacitrate ph 3.5 and 3 m nacl and equilibrated at 20 c in a sitting drop setup. 20 % glycerol plus inhibitor was added to a crystal and the crystal was flash - frozen in liquid nitrogen. data were collected on beam - line id-29 at the european synchrotron radiation facility (esrf) in grenoble. data processing was done with xds and scaling with the ccp4 program scala, as implemented in the global phasing autoproc procedure. the crystal structure was solved by molecular replacement using the structure of hiv-1 protease (pdb i d : 5hpv) as a model. data collection and refinement statistics are listed in table 4. we found a peptide fragment fitted by perryman. (pdb i d : 4e43) in the active site of their 1.54 structure of, the peptide appeared to be in a large part cleaved by the protease and only the p1-p4 residues were fitted. weaker density indicated that partially also intact peptide was bound. similarly to the 4e43 structure, an acetate ion and glycerol molecule were found bound in the exosite. choosing the parameters for the calculation of the fractal dimension of a discretized non - strictly self - symmetrical natural object is the first step in its surface roughness assessment. there is a strict connection between the density of points of the discretized set, the fractal scales used for the calculation of d, and the d value itself (figure 1). the choice of fractal scales may be summarized as follows : (i) choose exponential distances between the different scales ; (ii) select the smallest scale greater than the resolution of the discretization, otherwise d will be severely overestimated (figure 1b), and the largest scale to not exceed the size of the smallest object inspected, otherwise it will include a large amount of empty space which will result in underestimated d values. following these rules, we chose fractal scales [0.4, 0.8, 1.6, 3.2 ]. the largest one is about the size of a small ligand or amino acid side - chain diameter. the next step in parameterizing the calculation of d is the selection of a suitable density of the points on the ses generated by msms (figure 1a). while calculating d for different densities between one and 20 pp, we identified two intervals separated around 6 pp, in which d behaved differently. the first interval harbors densities that are too low for the selected fractal scales, so that the hyperspheres for the smallest can not reach any neighboring points, thus c(x,)=1/n, which leads to overestimating d. this interval has a large dispersion compared to the other one (figure 1a), but the orderings of the elements appear to be stable (figure 1d), meaning that a switch in density inside the interval would not drastically change the overall ordering of the set. the same assumption also holds for the second interval containing densities greater than 6 pp. it contains densities that are more favorable for a fractal dimension calculation, i.e. the production of stable regression lines, as well as the lower dispersion introduced by surface sampling effects. naturally, a decrease in point density reduces the qualities of the discretization and results in a more crude approximation of d, but it also speeds up the computations, so we found a compromise at a density of 7 pp. note that choosing different fractal scales shifts the limit between the two intervals, depending on the choice of the lowest. discrimination ability of the fractal dimension d depending on the fractal scale and point density (pp : points per) calculated for the 604 ligands of the main set of protein - ligand complexes. a) dispersion of d. a tendency of decreasing dispersion is apparent with increasing discretization density. a steep fall in d values as well as b, c) regression lines of the doubly logarithmic plot of the correlation sum for the ligand of the pdb i d : 1c5z complex for densities on both sides of the steep fall in d : b) 5 pp, (r=0.952) ; c) 7 pp, (r=0.999). d) gray scale coded matrix containing the spearman s rank correlation of the vectors of d values of the ligands (one 604-dimensional rank vector for each density). each filled square represents the pairwise rank correlation between two corresponding vectors, illustrating how the change in density shuffles the relative positioning of the d values. few changes in the ligands positions in the rank vector indicate stable discrimination ability (strong correlation, dark color). the analyzed spatial molecular data relied exclusively on prior crystallographic surveys on protein - ligand complexes, dna and rna. the pdb i d lists are provided as supplementary information. the protein - ligand complexes used in this survey were selected from two publicly available databases : (i) the pdbbind (www.pdbbind.org) was accessed to create the main set of complexes, the results of which were verified on (ii) a subset of the sc - pdb, termed the verification set for reference. a subset of 604 protein - ligand complexes based on the pdbbind database was selected according to the following criteria : (i) monomeric structures with predicted primary binding site volume not exceeding the volume of the corresponding ligand by more than 50 % ; (ii) structures for which the msms ses calculation failed due to software errors were excluded. the main set (supplementary listing 1) provided the following five sets of spatial data used in the survey at hand : (i) the proteins set contains spatial data gathered by stripping any atoms from the complexes that did not belong to a protein chain such as waters and ligands resulting in a set of 604 proteins ; (ii) the ligands set contains the ligands of the active sites of the protein - ligand - complexes resulting in a set of 604 small molecules ; (iii) the liganded pockets set contains the main pockets of the complexes resulting in a set of 604 active sites ; (iv) the protein surface clefts set consists of pockets selected by pocketpicker that are missing a ligand in the published crystallographic model ; (v) the background surface patches set comprises of protein points subsets extracted using msms at uniformly distributed random positions on the surfaces of the 604 proteins. external test set. a subset of 8639 protein - ligand complexes based on the sc - pdb database was selected according to the following criteria : (i) the complexes are not present in the main set ; (ii) very large structures (more than 2.3510 surface points) and structures for which the msms ses calculation failed due to software errors were excluded. the surface patches for the pockets and clefts were excised using a hard proximity margin of 3 around the output of pocketpicker. for the sampling of the background surface patches each single surface patch was then defined by using spheres with 6 radius around each randomly selected surface atom. the ppi set was extracted from the freely available dockground ppi database (dockground.bioinformatics.ku.edu/), benchmark 3.0. it contains 131 complexes with both bound and one available unbound structures for each entry, as well as 102 complexes, each with both bound and both unbound structures available and provided. thus, we were able to inspect 233 complexes with a grand total of 466 ppis. since we analyzed ppis of known bound complexes, we used the corresponding complex partners for the definition of the interface s position, and a hard proximity margin of 3.5. we chose this to be slightly higher than 3.0, because at 3.0 the sampled patches for the ppi exhibited cut - off artifacts such as holes in the surfaces influencing the roughness estimation. the surface patches for the pockets and clefts were excised using a hard proximity margin of 3 around the output of pocketpicker. for the sampling of the background surface patches 20 random atoms on the surface of each protein were selected. each single surface patch was then defined by using spheres with 6 radius around each randomly selected surface atom. the ppi set was extracted from the freely available dockground ppi database (dockground.bioinformatics.ku.edu/), benchmark 3.0. it contains 131 complexes with both bound and one available unbound structures for each entry, as well as 102 complexes, each with both bound and both unbound structures available and provided. thus, we were able to inspect 233 complexes with a grand total of 466 ppis. since we analyzed ppis of known bound complexes, we used the corresponding complex partners for the definition of the interface s position, and a hard proximity margin of 3.5. we chose this to be slightly higher than 3.0, because at 3.0 the sampled patches for the ppi exhibited cut - off artifacts such as holes in the surfaces influencing the roughness estimation. the protein, delivered in 20 mm acetate buffer, ph 5.0 with 200 mm nacl, 10 % (v / v) glycerol and 0.05 % 2-mercaptoethanol, was concentrated to a protein concentration of 3 mg / ml. 0.5 l of a 200 mm hiv protease inhibitor (compound 7) solution in dmso was added to 80 l of the concentrated hiv-1 protease solution. because it turned out that the inhibitor was poorly soluble in the resulting protein solution, an additional 2 l of a 50 mm inhibitor solution in peg400 was added to the last 20 l of the protein solution. 0.1 l of this latter protein solution was mixed with 0.1 l reservoir solution of 0.1 m nacitrate ph 3.5 and 3 m nacl and equilibrated at 20 c in a sitting drop setup., 20 % glycerol plus inhibitor was added to a crystal and the crystal was flash - frozen in liquid nitrogen. data were collected on beam - line id-29 at the european synchrotron radiation facility (esrf) in grenoble. data processing was done with xds and scaling with the ccp4 program scala, as implemented in the global phasing autoproc procedure. the crystal structure was solved by molecular replacement using the structure of hiv-1 protease (pdb i d : 5hpv) as a model. data collection and refinement statistics are listed in table 4. we found a peptide fragment fitted by perryman. (pdb i d : 4e43) in the active site of their 1.54 structure of, the peptide appeared to be in a large part cleaved by the protease and only the p1-p4 residues were fitted. weaker density indicated that partially also intact peptide was bound. similarly to the 4e43 structure, an acetate ion and glycerol molecule were found bound in the exosite. g. s. is a co - founder of insili.com llc, zrich, and a consultant in the pharmaceutical and chemical industry. | quantifying the properties of macromolecules is a prerequisite for understanding their roles in biochemical processes. one of the less - explored geometric features of macromolecules is molecular surface irregularity, or roughness, which can be measured in terms of fractal dimension (d). in this study, we demonstrate that surface roughness correlates with ligand binding potential. we quantified the surface roughnesses of biological macromolecules in a large - scale survey that revealed d values between 2.0 and 2.4. the results of our study imply that surface patches involved in molecular interactions, such as ligand - binding pockets and protein - protein interfaces, exhibit greater local fluctuations in their fractal dimensions than inert surface areas. we expect approximately 22 % of a protein s surface outside of the crystallographically known ligand binding sites to be ligandable. these findings provide a fresh perspective on macromolecular structure and have considerable implications for drug design as well as chemical and systems biology. |
type 1 renal tubular acidosis (rta), or distal rta (drta), is a disorder of renal tubular acidification, characterized by hyperchloremic metabolic acidosis with a normal serum anion gap and a persistently high urinary ph. other than the hereditary and idiopathic varieties, most cases of drta are secondary to systemic disorders such as sjgren 's syndrome, hyperglobulinemia, chronic active hepatitis, or lupus. in nondiabetic adults, membranous nephropathy (mn) is the most common cause of nephrotic syndrome, accounting for up to one - third of biopsy diagnoses. about one - third of cases are secondary to infection, rheumatologic disorders, neoplasm, and exposure to drugs and toxins. here, we report a hitherto unusual combination of drta with mn presenting as hypokalemic paralysis and medullary nephrocalcinosis. a 20-year - old male presented with a 2-month history of on and off swelling of both legs and sudden onset weakness of all 4 limbs without any history of loss of consciousness, seizure, headache, sensory loss, or bowel and bladder involvement. additionally, there was no history of dryness of the mouth and eyes (suggestive of sjgren 's syndrome), no joint pain, oral ulcers, hair loss, or photosensitivity (suggestive of lupus). his deep tendon reflexes were diminished, with no plantar response ; however, sensory function remained intact. the patient 's laboratory profile was as follows : hemoglobin : 10.8 g / dl, total leukocyte count : 14,300/mm, platelet count : 2.8 10/mm, urinary protein : 3 +, urinary sugar : 0, urine microscopy : white blood cell count : 4 - 6/high - power field, red blood cell count : 2 - 4/high - power field, urinary ph : 6.5, 24-hour urinary protein : 3.3 g / day, serum albumin : 2.1 g / dl, serum cholesterol : 526 mg / dl, serum triglycerides : 346 mg / dl, c3 : 79.6 mg / dl (normal range : 80 - 160), c4 : 62.8 mg / dl (normal range : 20 - 40), hbsag : negative, anti - hcv : negative, hiv i and ii : negative, anti - ro and anti - la antibodies : negative, blood urea : 29 mg / dl, serum creatinine : 1.2 mg / dl, random blood sugar : 88 mg / dl, corrected serum calcium : 9.4 mg / dl, serum sodium : 143 meq / l, serum potassium : 2.0 meq / l, serum chloride : 120 meq / l, serum po4 : 4.3 meq / l, arterial blood gas : ph 7.2, pco2 : 31 mm hg, po2 : 104 mm hg, hco3 : 12.5 meq / l, anion gap : 10.5 meq / l (normal range : 10 - 12). the urine anion gap (uag) [na + k cl (70 + 25 65 = 30) ] was positive. urinary ph was 20 mmol / day suggests renal loss, as was the case in our patient. the uag represents an indirect index of urinary nh4 + excretion in patients with hyperchloremic metabolic acidosis. the uag is defined by the equation : uag = na + k cl. in our patient, though rta is a tubulointerstitial kidney disease, it may also be seen in primary glomerulopathies. primary glomerulopathies may cause damage to tubules and the interstitium with functional derangement. in glomerular diseases associated with proteinuria, activated tubular cells may synthesize proinammatory mediators, especially monocyte chemoattractant molecules (e.g. mcp-1, rantes, complement component, and fractalkine) and brosis - promoting molecules (e.g. endothelin, tgf-, and angiotensin ii). damage to the tubular basement membrane facilitates the passage of tubular - derived products into the interstitium and peritubular capillaries spaces. in the distal nephron glomerulus - derived cytokines activating tubular epithelial cells reduce peritubular blood flow, leading to tubulointerstitial ischemia and hyperfunction of remnant tubules. for example, drta occurring in immunological disorders such as sjgren 's syndrome and systemic lupus erythematosus is thought to result from an interstitial nephritis which various investigators have ascribed to mononuclear cell infiltration, deposition of antigen - antibody complexes along tubular basement membranes, and the formation of antibodies directed against the tubular basement membrane. a literature search revealed cases in which features of drta preceded other manifestations of systemic lupus erythematosus by 4 - 6 years. in our patient, lupus and sjgren 's syndrome were ruled out. a case of drta with mn has been reported. long - term correction of the hypokalemia in our patient was important not only to overcome the neuromuscular impairment, but also to prevent further deterioration of kidney function. it has been suggested that hypokalemia stimulates interstitial deposition of complement components (c3 and c5ba), which is conducive to the aggravation of renal interstitial damage. about 30 - 35% of patients with idiopathic mn eventually undergo spontaneous remission of nephrotic syndrome ; therefore, it is reasonable to delay specific immunosuppressive therapy for at least 6 months, instead utilizing supportive therapy including ras blockade unless the patient has unexplained rapid deterioration in kidney function or there are complications related to uncontrolled nephrotic syndrome. kdigo recommend that initial immunosuppressive therapy be started only in patients with nephrotic syndrome and when at least one of the following conditions is met : urinary protein excretion persistently exceeds 4 g / d and remains at over 50% of the baseline value, and/or it does not show progressive decline despite antihypertensive and antiproteinuric therapy during an observation period of at least 6 months (1b ; level 1 : recommend ; b : moderate quality of evidence). drta is a clinical syndrome consisting of hypokalemia, hyperchloremic metabolic acidosis, inability to lower urinary ph below 5.5 during acidemia nephrolithiasis, and nephrocalcinosis. hypokalemia is frequently seen in drta and may sometimes be severe enough to produce muscle weakness, as in our case. both hypercalciuria and especially hypocitraturia contribute to the development of nephrocalcinosis and nephrolithiasis. it is very evident that our patient had drta (type 1 rta) with medullary nephrocalcinosis. correction of acidosis with alkali therapy (usually potassium citrate) generally improves both calcium and potassium balance, and prevents stones and nephrocalcinosis. in contrast to the high alkali requirement in proximal rta, only daily acid load (1 - 2 meq / kg / day) has to be replenished with alkali therapy in drta. primary glomerulopathies may cause tubulointerstitial damage, which may subsequently lead to the development of rta. we have reported the case of an unusual association of drta with mn, with the rare presentation of hypokalemic paralysis, which was successfully treated. | type 1 renal tubular acidosis (rta), or distal rta (drta), is a disorder of renal tubular acidification, which is generally asymptomatic but may rarely present as hypokalemic paralysis. here, we report the case of a young male who presented with sudden onset weakness of all 4 limbs and a 2-month history of swelling of the legs. an investigation revealed hypokalemia, metabolic acidosis, and nephrotic syndrome. additional analyses revealed normal anion gap metabolic acidosis with a positive urine anion gap and drta. renal biopsy showed evidence of membranous nephropathy (mn). the patient 's weakness improved with potassium supplements. normalization of the serum potassium level and disappearance of proteinuria were established with an ace inhibitor and potassium supplementation. this case is an unusual combination of drta with mn coupled with the rare presenting symptoms of hypokalemic paralysis and medullary nephrocalcinosis. |
t regulatory (treg) cells are a t lymphocyte subpopulation that control the balance between immune activation and tolerance. treg cells can originate from two main sources : thymus - generated natural tregs (ttreg) and peripheral inducible tregs (ptreg), generated during immune priming. several factors are required for ttreg generation ; these cells are strongly dependent on tcr and cd28 signals and on several cytokines. cytokines contribute to treg maintaining via chain signaling of il-2 and il-15, and tgf increases foxp3 expression. however, certain cytokines like as tnf- have a controversial role in ttreg generation [2, 3 ]. on the other hand, ptreg generation requires stimulation in an anti - inflammatory milieu, a process where dendritic cells are critically involved [3, 4 ]. according to cytokine production, tregs have been further classified ; for instance, th3 cells are characterized by tgf production ; tr1 cells produce il-10, and tr35 cells produce il-35. while the expression of the transcription factor foxp3 can be transient in humans, this factor is associated with a suppressive function in mice. several action mechanisms by which treg cells control the immune response have been reported : (1) inhibition by immunoregulatory cytokines such as tgf, il-10, and il-35 ; (2) inhibition by cytolysis of effector cell by producing granzyme and perforin ; (3) metabolic interruption, including an inhibition of the proliferative response via il-2 receptor, camp - mediated metabolic inhibition, and immunomodulation mediated by the a2 adenosine receptor ; (4) interaction with dendritic cells that modulates their function and maturation. il-10 is an 18-kd protein produced by tr1 cells. while its production is not restricted to this cell line since monocytes, dendritic cells, neutrophils, other t lymphocytes, and b lymphocytes are able to release it, tr1 cells are the only regulatory cells to produce il-10 [5, 6 ]. il-10 has been described as the main immunomodulatory cytokine ; additionally, it can act as a paracrine or autocrine signal and can be induced by catecholamines [7, 8 ]. il-10 inhibits the production of inflammatory cytokines such as il-12, causing a decrease in the th1 response and in inf- production ; it also promotes the phagocytic activity, increasing the removal of cellular debris at the inflammation site. one of the best known molecular mechanisms of il-10 is the action on effector cells. the costimulatory molecule cd28 is involved in the interaction between effector cells and antigen - presenting cells. by binding its receptor, il-10 inhibits tyrosine phosphorylation in cd28, inhibiting pi3k / akt activation, which in turn inhibits the signaling cascade leading to nf-b translocation (figure 1) [10, 11 ]. as shown in figure 1, il-10 exerts its biologic function by activating jak1 and tyk2, both proteins associated to stat1, stat3, and in certain cases stat5 (figure 1) [10, 12 ]. in rats, the activation of the il-10 receptor in dendritic cells has been observed to promote the jak1-tyk2-stat3 pathway. when activated, this pathway inhibits dendritic cell maturation, decreasing the expression of mhc ii, cd11b / c, cd80, and cd86. in monocytes, il-10 has been demonstrated to induce the expression of the socs3 suppressor gene, which has influence on ifn - induced tyrosine phosphorylation in stat1. on the other side, in a lps - induced inflammation model, il-4 has been observed to promote c - maf expression in activated macrophages ; c - maf binds the promoter of the il-10 gene, favoring its production (figure 1) [14, 15 ]. by binding its receptor in tr1, il-10 induces jak1 and tyk2 phosphorylation, activating stat3, which is translocated to the nucleus, promoting socs3 ; in turn, socs3 inhibits the nf-b - induced factor myd88, resulting in the inhibition of the il-1, il-6, and tnf- cytokines (figure 1). moreover, it has been observed that il-10 promotes stat3 phosphorylation and its translocation to the nucleus in treg cells, mediating a further il-10 production (figure 1). il-10 exhibits a wide range of biological activities, including immunosuppression, anti - inflammation, and immunomodulation. il-10 is able to inhibit mhc i expression in b and t cells and also in dendritic cells, all of them involved in the inflammatory response. the transforming growth factor - beta (tgf) family includes several structurally and functionally related proteins acting as multifunctional factors in a wide range of biological processes. tgf is a 25 kda protein and a multifunctional cytokine, given its different effects on the cell [18, 19 ]. during secretion, tgf undergoes proteolysis by an endopeptidase, which cleaves the peptide bond linking the mature factor and the latency - associated peptide (lap). for tgf to be activated this process can be triggered by a number of factors, like temperature and ph changes. three tgf isoforms are known in humans, 1, 2, and 3, each coded in a different chromosome. functional tgf receptors are types i and ii (tgf ri and tgf rii), and nonfunctional receptors include types iii, iv, and v. tgf ri and rii bind tgf-1 and tgf-3 with greater affinity than tgf-2. tgf ri and tgf rii are responsible for the biological effects of tgf-1 in mammal cells. however, tgf riii is also capable of binding tgf-1. while tgf riii has no role in signal transduction, it has been suggested that it controls the availability of tgf-1 in the local extracellular microenvironment and regulates its active presentation to the functional receptors [21, 22 ]. in the extracellular space, tgf binds tgf riii, which then recruits tgf rii and phosphorylates itself ; alternatively, tgf can bind directly to membrane - anchored tgf rii and induce the attraction of tgf ri and its ensuing phosphorylation (figure 2(a)). both tgf ri and tgf rii have a cysteine - rich extracellular region, a transmembrane region, and a cytoplasmic region ; the latter includes a serine - threonine kinase domain. there is a functional dependence between tgf rii and tgf ri, since tgf ri requires tgf rii to bind the ligand, while tgf rii requires tgf ri for a functional signaling. when tgf binds tgf rii, the latter phosphorylates tgf ri in the serine- and glycine - rich domain near the transmembrane region. the activation of the ligand - receptor complex allows the direct interaction of smad proteins with the kinase domain of the type - i receptor, recruiting smad2 and smad3 and phosphorylating them, thus activating the canonical pathway and forming a complex with smad4. this protein acts as a convergence node for the signaling pathways induced by members of the tgf superfamily (figure 2(a)). the active complex formed by smad2, smad3, and smad4 is translocated to the nucleus, where it acts as transcriptional coactivator and regulates the transcription of several tgf-responder genes [21, 23 ]. tgf activates tak-1, a kinase of serine and threonine residues of the map kinase family, by the noncanonical pathway. ras, a member of the small g proteins, has also been involved in signaling. certain mapks, including those kinases regulated by extracellular signals such as erk-1 and erk-2 and stress - activated kinases such as jnk and pi3k, are also activated in some cell types (figure 2(a)). a number of mechanisms have been proposed : (i) it suppresses effector t cell differentiation ; (ii) it promotes the differentiation of nave t cells into regulatory t or th17 cells ; (iii) it inhibits t and b cell proliferation ; (iv) it inhibits the activity of macrophages, dendritic cells, and nk. the inhibitory activity of tgf on treg cells is due to the high lap / tgf expression in treg membrane. unlike t cells, monocytes and dendritic cells express tgf receptors, thus allowing for cell - to - cell interaction. tgf has been demonstrated as necessary to generate tolerogenic dendritic cells (dcs) by inducing ido, an enzyme that inhibits t cell proliferation. as another relevant suppressive mechanism, tgf inhibits the production of il-2 (figure 2(b)). the cis - actin protein, which suppresses il-2 production, is called tob ; this protein binds smad2, blocking the activation of runx 1/3. tgf modulates cell proliferation by controlling the expression of cell cycle - regulating factors, including cyclin - dependent kinases (cdks) like p15, p21, and p27 and cell cycle promoters like cmyc, cyclin d2, cdk2, and cyclin e. tgf also promotes t nave cell differentiation into treg cells by inducing foxp3 expression, or into th17 cells in the presence of il-6 (figure 2(c)). foxp3 then induces the transcription of inhibitory cytokines such as tgf (figure 2(c)). on the other hand, th17 differentiation from nave t cells signaling via il-6 activates stat3, which induces the expression of ror. ror then induces the transcription of il-17 (figure 2(c)). interleukin 35 (il-35) is a heterodimeric cytokine in the interleukin 12 (il-12) family. this cytokine family can be composed of one to five subunits (p19, p28, p35, p40, and another from epstein - barr virus gene 3, also called ebi3). il-35, an immunosuppressor cytokine formed by the p35 and ebi3 subunits, is produced by treg cells. il-35 has two known functions : to suppress the proliferation of t helper cells and to promote the conversion of nave t cells into highly suppressor treg cells (itr35). recently, this cytokine was demonstrated to be capable of inducing the conversion of b lymphocytes into b regulatory cells. the il-35 receptor is formed by the il-12r2 and gp130 subunits ; it can be heterodimeric or homodimeric. while gp130 is expressed in practically all cells, il-12r2 is expressed predominantly in activated t lymphocytes, nk cells, and to a lesser extent dcs and b cells. the il-35 signaling path has not been completely described yet ; however, it is known that the heterodimeric receptor activates stat1 and stat4, which induces ebi3 and p30 expression, causing nave t cells to convert into il-35-producing (itreg) cells, suppressing cell proliferation, blocking the shift to a th1 profile, and mediating il-10 production. treg cells are able to release the immunomodulatory cytokines il-10, tgf, and il-35. this action may target several immune cells expressing receptors for such cytokines. this way, several immune cells in different stages (activated, effector, or resting) could be affected by the receptor - ligand interaction. the effect of cytokines is probably not only local, since they could spread by the bloodstream throughout the body. thus, these cytokines play a role in the polarization of immune response during various pathologies. regulatory t (tregs) cells produce a serine protease called granzyme b, which allows them to induce apoptosis in effector t cells [2832 ]. during treg - effector cell interaction, directed exocytosis from treg granules to the extracellular space of both cells takes place ; these granules contain granzymes and perforins. once released from the treg, perforin molecules insert themselves into the lipid membrane of the target cell and polymerize in the presence of calcium ions to form a transmembranal cylinder ; each cylinder forms a pore through which granzymes enter the cell (figure 3(a)). granzyme can also enter the cell by an endocytosis process mediated by the manose-6-phosphate receptor. in this case, granzyme is sequestered in endosomes into the cytosol, and perforin acts to release it (figure 3(b)) [3337 ]. additionally, granzymes can bind to the cell surface in a way that granzyme recruitment is stimulated by perforin - mediated membrane damage (figure 3(c)). once within the target cell, granzyme b could induce apoptosis by caspase - dependent or independent mechanisms, as discussed elsewhere [3840 ]. cytolysis allows treg cells to act on several immune cell populations by cell - to - cell interaction. this mechanism is highly effective, since treg cells induce death by apoptosis on effector cells, thus decreasing the number of effector cells and controlling the immune response. interleukin 2 (il-2), chiefly secreted by t cells in response to antigenic stimuli, is the main cytokine for t cell proliferation. il-2 receptor is expressed by t lymphocytes, nk cells, b cells, macrophages, and monocytes ; however, only t lymphocytes are capable of producing this cytokine. il-2 receptor is a complex formed by three subunits : alpha chain (cd25), beta chain (cd122), and gamma chain (cd132) ; each subunit plays an important role in facilitating the transduction of il-2-dependent signals. alpha chain (il-2r) has a very short cytoplasmic domain and does not participate in signal transduction ; however, it is required to increase the affinity of il-2 to its receptor. on the other hand, beta (il-2r) and gamma (il-2r) chains play a crucial role in signal transduction. treg cells constitutively express high levels of il-2 alpha chain, having thus a higher affinity to il-2, and compete for this growth factor with proliferating cells. by depriving proliferating effector cells from il-2, treg cells do not only prevent them from continuing the proliferative process but also leave them without a vital cytokine, causing metabolic interruption and cell death (figure 4(a)) [3, 43 ]. cyclic adenosine monophosphate (camp) is a second messenger, capable of regulating the functional activity of effector cells and antigen - presenting cells. the high camp content in treg cells is due to the 50-fold higher expression of adenylyl cyclase 9 (ac9). additionally, the high cd25 expression in treg cells has been observed to favor adenylyl cyclase 7 activation and camp accumulation. the differential expression of cd25 and adenylyl cyclase in treg cells is controlled by foxp3. tregs show a low camp degradation rate due to a diminished expression of the phosphodiesterase 3b enzyme in this cell subpopulation. recent findings indicate that camp concentration is controlled by mir-142 - 3p, a microrna that regulates foxp3 transcription activity, and thus the expression of adenylyl cyclase. while mir-142 - 3p inhibits the production of adenylyl cyclase 9 in conventional t cells, this effect is not seen in treg cells since the foxp3 transcription factor negatively regulates mir-142 - 3p expression and keeps the ac9/camp pathway active. treg cells transfer camp to target cells by intercellular communications called gap junctions (figure 4(b)). in mammal cells, communicating junctions are used for the bidirectional traffic of ions, metabolites, and other molecules weighing less than 1 kd. the increase in intracellular camp activates protein kinase a (pka), since camp binds to the pka regulating subunit, activating it by releasing its catalytic subunit. on the cell membrane inner face, phosphorylation of tyrosine kinase c - src (csk) by pka increases its activity ; then, csk phosphorylates and inactivates the lymphocyte - specific protein tyrosine kinase (lck), an important protein in the proximal activation of t cell receptors. a number of signaling pathways can be regulated by pak. for instance, the camp response element - binding (creb) protein is phosphorylated by pak at serine 1343, which prevents it from forming a complex with the csk binding protein (cbp) and from binding to camp response elements (cre) ; these can be found in genes coding for t cell receptors or in other genes involved in t cell activation. additionally, pak regulates the activity of the nuclear factor in activated t cells (nf - at). when nf - at is phosphorylated by pak, binding sites for another protein called 14 - 3 - 3 are created. this new complex decreases nf - at transcription activity (figure 4(b)) [4749 ]. when cells are in resting state, nf-b is found in the cytoplasm, complexed with its inhibitor, ib, which prevents it from translocating to the nucleus. during cell activation, ib is phosphorylated by an ib kinase, which induces the nf-b / ib complex to split apart. conversely, when pka is phosphorylated, its catalytic subunit (pka - c) binds to the nf-b / ib complex, stabilizing it and keeping it inactive (figure 4(b)). nf-b regulates the transcription of a large number of genes, including those coding for the proinflammatory cytokines tnf- il-1b, il-6, il-8, vegf, and metalloproteases (mmp1, mmp2, mmp3, and mmp13). while the main target for camp is pak, camp has also been demonstrated to directly activate the exchange protein directly activated by camp (epac1 and epac2). this protein regulates the activation of a gtpase called rap-1, responsible for activating erk, thus inhibiting cell proliferation and differentiation (figure 4(b)). cd39 and cd73 are ectoenzymes, highly expressed on the surface of treg cells (figure 4(c)). cd39 is a nucleoside triphosphate diphosphohydrolase-1 (ntpdase 1) that degrades atp into amp. the expression of cd39 in tregs is regulated by the foxp3 transcription factor, and its catalytic activity is enhanced by tcr compromise. in turn, extracellular amp is rapidly degraded into adenosine by cd73, an ecto-5-nucleotidase bonded to the membrane of treg cells (figure 4(c)). the adenosine resulting from amp hydrolysis binds four different surface receptor subtypes coupled to gs proteins, called a1, a2a, a2b, and a3. the a2ar receptor is the main adenosine receptor associated to t and b lymphocytes, nk cells, macrophages, dendritic cells, and granulocytes (figure 4(c)). the outcome of stimulating these receptors is an intracellular amp accumulation ; through the camp - dependent protein kinase, these signals phosphorylate and activate creb. the latter binds the nuclear cofactor p300, producing a complex that regulates the expression of several genes in their promoter regions. creb is able to regulate indirectly the transcription of some inflammatory genes, competing with nf-b / p65 and then suppressing the expression of proinflammatory cytokines like tnf- (figure 4(c)) [51, 5355 ]. amp can also activate other substrates like epac1 and other kinases such as erk and jnk, altering the expression of proinflammatory genes through transcription factors responsible for synthesizing interleukins like il-12 and tnf- ; it promotes as well the production of anti - inflammatory cytokines like il-10 (figure 4(c)). the fact that tregs produce adenosine and respond to it at the same time means that this molecule acts as an autocrine factor to optimize the anti - inflammatory response. it also increases treg suppressor capacity, inhibits the expression of costimulatory molecules in dendritic cells, and inhibits the activation of effector cells [52, 55 ]. treg - mediated metabolic disruption occurs by competition for il-2, a growth factor for effector cells. under these conditions, treg cells control the immune response in a nonspecific but effective manner, since by consuming il-2 treg cells inhibit effector cell proliferation, actually impairing the immune response. on the other hand, immunosuppression mediated by camp and the 2a adenosine receptor induce several signaling pathways in effector cells that in turn impact transcription factors, controlling the effector response. this molecule has a high affinity by dendritic cell - expressed cd80 and cd86 ; thus, tregs compete with effector cells to bind these ligands. this interaction involves several events, including the production of inf-, a potent inducer of indoleamine 2,3-dioxygenase (ido). ido degrades tryptophan in a metabolic pathway starting with tryptophan oxidation to n - formylkynurenine, immediately followed by hydrolysis to kynurenine. this initial product follows various degradation pathways whose final products are kynurenic acid, 3-hydroxykynurenine, anthranilic acid, 3-hydroxyanthranilic acid, and quinolinate (figure 5(a)) [5961 ]. once tryptophan is degraded by ido, a signaling cascade starts. since there is a local tryptophan deficit, the level of transfer rna without amino acid load increases, and as a result the general control nonderepressible kinase 2 (gcn2) is activated, and it hyperphosphorylates the translation initiation factor (eif2), leading to a suppression in protein synthesis. the union of the ternary complex formed by eif2, gtp, and the initiation transfer rna for methionine (met - rnai) to the 40s ribosome subunit is inhibited, preventing the release of the binary complex eif2/gtp and inhibiting the eif2-promoted gtp / gdp exchange. the result is a cell cycle arrest in the g1 phase and thus anergy of effector t cells (figure 5(a)) [60, 62, 63 ]. although the mechanism is not well understood, 3-hydroxyanthranilic acid and quinolinic acid have been demonstrated to induce apoptosis in th1 cells by directly activating caspase-8 [64, 65 ] ; this truncates the bid protein, producing a small proapoptotic fragment called truncated bid (tbid). this active fragment can translocate to the mitochondria, causing the release of cyt c by interaction with bax, another member of bcl-2 family. in the cytosol, cyt c interacts with the proteins apaf1 and caspase-9 in the presence of atp, building the apoptosome, a catalytic complex that starts the caspase cascade activation, leading to the digestion of structural proteins, degradation of chromosomal dna, and phagocytosis of apoptotic bodies (figure 5(a)) [66, 67 ]. the metabolite 3-hydroxyanthranilic acid has also been reported to induce death in activated t cells by depleting glutathione, one of the main antioxidant molecules in animal cells. a decrease in glutathione promotes a misbalance between ros production and the antioxidant capacity. on the other side, t cell apoptosis due to oxygen free radicals produced by kynurenine and 3-hydroxykynurenine has been observed, leading as well to changes in the oxidant - antioxidant balance and promoting oxidative stress. oxidative stress causes a global collapse of mitochondrial function, reducing energy production and therefore contributing to cell death [64, 69 ]. another immunosuppression mechanism involves kynurenine binding to the aryl - hydrocarbon receptor (ahr), a transcription factor expressed in cell populations like t cells, resulting in a shift to a t regulatory cell phenotype (figure 5(a)) [70, 71 ]. lymphocyte - activation gene 3 (lag3, cd223) is a cell surface molecule expressed in tregs. structurally, lag3 is homologue to the cd4 receptor and binds to mhc ii with a significantly higher affinity than cd4 [7375 ]. the lag3-mhc ii union induces a signaling cascade starting with plc2 and p72syk phosphorylation and the activation of pi3k / akt, p42/44erk, and p38mapk (figure 5(b)). the latter kinase has been involved in the maturation process of dendritic cells ; these exhibit an increase in the expression of costimulatory molecules and a decrease in antigen capture [76, 77 ]. another study demonstrated that, after crosslinking mhc in the presence of lag3, itam - mediated inhibitory signals are induced, involving erk sequential activation and ship-1 recruitment similar to those proposed for fcr. the inhibitory signaling started by coengagement of mhc with lag3 along with cd4 and tcr could be mediated by several components, including ship-1, which may not be recruited by themselves by the cd4 or tcr signaling pathways. ship-1 has been labeled as a nf-b negative regulator and as a negative factor in cell activation. additionally, the conjoint union of ctla-4 and lag3 promotes a tolerogenic phenotype in dcs (figure 5(b)). dendritic cells being a key component of the immune response, the action implying cell - to - cell contact with dcs is one of the most important mechanisms for treg cells. depending on its phenotype, when interacting with a treg cell, a dc acquires a tolerogenic phenotype, which in turn promotes further treg cell generation, providing a suppressor microenvironment. the competition for dc ligands between effector and treg cells allows for an additional control mechanism of the immune response. the action mechanisms of tregs described above can act together or independently, according to the requirements of the immune system and homeostasis maintenance, or during the progression of various pathological processes. treg cells have been used as part of an escape mechanism by several pathogens, including viruses and helminthes, and they also modulate the immune response in noninfectious pathologies such as tumors. knowing the modulation molecular mechanisms and their effect according the pathological situation could help in identifying the therapeutic targets allowing an effective immune response. | t regulatory cells play a key role in the control of the immune response, both in health and during illness. while the mechanisms through which t regulatory cells exert their function have been extensively described, their molecular effects on effector cells have received little attention. thus, this revision is aimed at summarizing our current knowledge on those regulation mechanisms on the target cells from a molecular perspective. |
the association between physical activity and health is well - established. in line with this, many health institutions developed physical activity guidelines for health. for example, the world health organization (who ; world health organization, 2010) suggested that adults participate in at least 150 min of moderate - intensity physical activity, 75 min of vigorous - intensity physical activity, or an equivalent volume of moderate- to vigorous - intensity physical activity per week. nevertheless, the proportion of people who participate in the recommended level of physical activity is less than ideal in most developed countries. for example, moderate - intensity physical activity guideline adherence rate in 2013 korea national health and nutrition examination survey (knhanes ; ministry of health and welfare, 2014) was only 9.0% and 4.5% in males and females, respectively. similarly, accelerometer - measured physical activity adherence rate of us adults was 3.5% in 20032004 national health and nutrition examination survey (troiano., 2008). (2012) reported that physical inactivity, the fourth leading cause of death, caused 9% of premature mortality, 6% of coronary heart disease, and 7% of type-2 diabetes in 2008 worldwide. metabolic syndrome is a combination of at least three of the cardiovascular disease and type-2 diabetes risk factors : hypertension, impaired glucose tolerance, abdominal obesity, elevated triglyceride level, and decreased high - density lipoprotein cholesterol level. in korea, the number of people with metabolic syndrome increased from 24.9% to 31.3% during 1998 and 2007 (lim., 2011). epidemiologic evidences support the association between physical inactivity and the indicators of metabolic syndrome (barlow., 2006 ; durstine., 2001 ; thorogood., moreover, physical activity is a necessary component of intervention programs to prevent cardiovascular disease and type-2 diabetes for people who already have conditions of metabolic syndrome (dishman., 2013). meanwhile, wilkinson (2002) insisted that the prevalence of noncommunicable chronic diseases is associated with economic equity of the society. korean economic equity has decreased since late 1990 s national monetary crisis (kim and han, 2007). accordingly, inequity in metabolic syndrome prevalence among korean women had increased in 2005 compared to 1998 (kim and kim, 2007). moreover, research shows that socioeconomically disadvantaged groups in korea are more vulnerable to chronic diseases compared to better socioeconomic group. for example, kim and kim (2007) reported that the incident rate of metabolic syndrome was 38%85% higher in less educated women compared to their counterparts with higher education. among many behavioral risk factors, research on physical activity disparities showed that people in lower socioeconomic class are generally less likely to participate in health - enhancing physical activity. this means that different socioeconomic groups take advantage of different levels of physical activity resources such as knowledge, motivation, and social and physical environment. moreover, physical inactivity increases chances for not only onset of chronic diseases, but also recurrence and/or further development of such conditions (kang, 2015). therefore, it can be a double - difficulty for lower socioeconomic class with hypokinetic chronic diseases such as metabolic syndrome, because they may be less likely to participate in physical activity as a treatment purpose. therefore, this study was purposed to investigate physical activity adherence disparities by socioeconomic status that may exist among metabolic syndrome patients in korea. knhanes is a continuous annual surveillance system that consists of demographics, questionnaires, health examination, and laboratory test to assess the health and nutritional status of korean (ministry of health and welfare, 2014). it uses a complex, multistage, probability sampling design to enable estimations to be representative of korean population. this study analyzed adult subsamples (18 yr old) with sociodemographic information, physical activity questionnaire, and health examination data (n=19,831). socioeconomic status was measured using education level (elementary graduate or lower vs. middle school graduate vs. high school graduate vs. college graduate or higher) and household income quartiles (oakes and kaufman, 2006). in this study, metabolic syndrome was defined as having three or more of the following conditions (grundy., 2004) : elevated fasting glucose (110 or under treatment), central obesity (waist circumference90 cm for male, 85 cm for female), systemic hypertension (systolic blood pressure>130 mmhg or diastolic blood pressure>85 mmhg), elevated triglycerides (150 ml / dl), diminished high - density lipoprotein (40 ml / dl for male, 50 ml / dl for female) physical activity adherence was defined as meeting minimum criterion of the who s (2010) global physical activity recommendation : 150 min of moderate - intensity physical activity, 75 min of vigorous - intensity physical activity, or an equivalent volume of moderate- to vigorous - intensity physical activity (i.e., 600 mets - min / wk). physical activity was measured using international physical activity questionnaire (ipaq ; fogelholm., 2006). validity of korean version of the questionnaire was established by previous studies (chun, 2012 ; oh., 2007). sex- and age - group - specific physical activity adherence disparities controlling for age were analyzed using adjusted wald - test. knhanes is a continuous annual surveillance system that consists of demographics, questionnaires, health examination, and laboratory test to assess the health and nutritional status of korean (ministry of health and welfare, 2014). it uses a complex, multistage, probability sampling design to enable estimations to be representative of korean population. this study analyzed adult subsamples (18 yr old) with sociodemographic information, physical activity questionnaire, and health examination data (n=19,831). socioeconomic status was measured using education level (elementary graduate or lower vs. middle school graduate vs. high school graduate vs. college graduate or higher) and household income quartiles (oakes and kaufman, 2006). in this study, metabolic syndrome was defined as having three or more of the following conditions (grundy., 2004) : elevated fasting glucose (110 or under treatment), central obesity (waist circumference90 cm for male, 85 cm for female), systemic hypertension (systolic blood pressure>130 mmhg or diastolic blood pressure>85 mmhg), elevated triglycerides (150 ml / dl), diminished high - density lipoprotein (40 ml / dl for male, 50 ml / dl for female) physical activity adherence was defined as meeting minimum criterion of the who s (2010) global physical activity recommendation : 150 min of moderate - intensity physical activity, 75 min of vigorous - intensity physical activity, or an equivalent volume of moderate- to vigorous - intensity physical activity (i.e., 600 mets - min / wk). physical activity was measured using international physical activity questionnaire (ipaq ; fogelholm., validity of korean version of the questionnaire was established by previous studies (chun, 2012 ; oh., 2007). sex- and age - group - specific physical activity adherence disparities controlling for age were analyzed using adjusted wald - test. statistical significance test for adjusted wald - test was set at 0.05. using stata 12.1 (statacorp lp., college station, tx, usa) korean adults metabolic syndrome prevalence rate was 32.62% (95% ci, 31.6433.61) in 20102012. physical activity adherence rate of korean adult during this period was 36.87% (95% ci, 35.7538.01). among patients with and without metabolic syndrome, the proportion of people who met the who physical activity guideline were 36.28% (95% ci, 34.4538.16) and 37.96% (95% ci, 36.6439.29), respectively. sex- and age - group - specific metabolic syndrome prevalence rate and metabolic syndrome patients physical activity adherence rate estimation are presented on tables 1 and 2, respectively. metabolic syndrome patients physical activity adherence rate of the first (lowest), second, third, and fourth quartile household income group were 28.31% (95% ci, 26.1430.28), 34.68% (95% ci, 32.7136.70), 37.44% (95% ci, 35.6639.25), and 43.79% (95% ci, 41.8545.75), respectively. controlling for age and sex, adherence rate of the fourth income quartile group was statistically significantly higher than the first [f(1, 551)=9.45, p=0.002 ], second [f(1, 551)=8.64, p=0.003 ], and third quartile income groups [f(1, 551)=6.15, p=0.013 ]. this means that metabolic syndrome patients with higher income were more likely to adhere to the physical activity guidelines. physical activity adherence rate of metabolic syndrome patients with elementary or lower, middle - school, high - school, and college or higher education degree were 25.17% (95% ci, 22.9527.54), 38.20% (95% ci, 35.1341.00), 39.60% (95% ci, 38.2441.77), and 36.89% (95% ci, 35.7738.03), respectively. controlling for age and sex, adherence rate of the least educated group was statistically significantly lower than middle school [f(1, 551)=13.55, p<0.001 ], high school [f(1, 551)=11.31, p<0.001 ], and college or higher education degree groups [f(1, 551)=8.00, p=0.005 ]. this shows that disadvantaged social group in terms of education were less likely to meet the minimum levels of the physical activity guidelines. sex- and age - group - specific physical activity adherence rate by income and education level is presented on tables 3 and 4, respectively. this study investigated disparities in physical activity adherence rate among metabolic syndrome patients using 20102012 knhanes data. overall, it was found that physical activity inequity by income and education level does exist among metabolic syndrome patients. also, there were sex- and age - group - differences in the inequity patterns. as shown in table 1, metabolic syndrome prevalence rate was higher in older groups compared to younger counterparts, and such trend was stronger in females than males. even though physical activity is important to prevent progression of cardiovascular disease and type-2 diabetes in these population, adherence rate to the who physical activity recommendation is far less than ideal (31.90%44.33% among middle - age group ; 19.68%30.14% among older adults). the implication of this finding is twofold : (a) physical inactivity may contribute to increased metabolic syndrome prevalence ; and (b) patients with metabolic syndrome do not participate in the generally recommended amount physical activity for health. sex- and age - group - specific analyses of physical activity disparities by household income and education level (tables 3 and 4) suggest that lower income groups of middle age (4064 yr old) and older group s (64 yr old or older) adherence rate were significantly lower than higher income and/or education groups. among younger adults (1839 yr old), elementary and middle school education has been mandatory in korea since 1985, so very small fraction of population belong to elementary or less this may have contributed to the inflated standard error of estimation of the adherence rate in these subgroups. research on health inequity showed that socioeconomic inequity affects onset of chronic diseases (kim and kim, 2007 ; lim., 2011). this study added to the past research findings that physical activity as a treatment of such conditions to prevent further development may be unequal across different socioeconomic groups. future study should further explore psychosocial and biological mechanisms by which physical activity disparities affect incidence rates of cardiovascular diseases and type-2 diabetes among metabolic syndrome patients. meanwhile, research has shed lights on health disparities in developed countries, because it is an important index of social justice (kim and kim, 2007). health inequity directly affects health status of lower socioeconomic group of a society by lower quality of built and social environment, elevated behavioral risk factors, limited access to health care system, and psychological stress (berkman., 2014). declined social welfare causes social conflict, psychosocial stress, and consequently health status of the whole society members (wilkinson, 2002). therefore, not only lower but also higher socioeconomic groups may be at risk for chronic diseases when socioeconomic equity is impaired. according to kim and kim (2007), socioeconomic disparities in korean adults affected not only health outcomes but also health behaviors as mediators by which such social conditions affect health outcomes. according to yang. (2005), in addition, there were differences in korean adults physical activity level by their socioeconomic status in 2001. the current study findings suggest that physical activity disparities have not been alleviated during 20012012. therefore, reducing health inequity by promoting physical activity in lower socioeconomic group is still a public health priority. limitations of this study include cross - sectional survey design, subjective measure of physical activity, and relatively simplistic measure of socioeconomic status. due to cross - sectional design, this study results do not support cause - and - effect inferences. ipaq is a 7-day recall subjective measure of physical activity, which is vulnerable to recall bias and social justice bias. also, ipaq does not capture physical activity of daily life, but to what extent the participant was physically active during the past 7 days. measures of socioeconomic class in recent studies in health sociology include not only income and education but also community - based indicators such as quality of living place (berkman., 2014). | physical activity plays an important role in preventing further progression of metabolic syndrome conditions to cardiovascular disease and type-2 diabetes. this study investigated physical activity disparities by socioeconomic status among metabolic syndrome patients. the fifth korea national health and nutrition examination survey (20102012) data were analyzed (n=19,831). a revised definition of the us national cholesterol education program adult treatment panel iii was used for screening metabolic syndrome patients. using international physical activity questionnaire, physical activity adherence was defined as participating in 150 + minutes of moderate - intensity physical activity, 75 + minutes of vigorous - intensity physical activity, or an equivalent combination of moderate - to vigorous - intensity physical activity per week. socioeconomic status was measured by level of education and house - hold income. among metabolic syndrome patients, physical activity adherence rate of first (lowest), second, third, and fourth quartile house - hold income group were 28.31% (95% confidence interval [ci ], 26.1430.28%), 34.68% (95% ci, 32.7136.70), 37.44% (95% ci, 35.6639.25), and 43.79% (95% ci, 41.8545.75). physical activity adherence rate of groups with elementary or lower, middle - school, high - school, and college or higher education degree were 25.17% (95% ci, 22.9527.54), 38.2% (95% ci, 35.1341.00), 39.60% (95% ci, 38.2441.77), and 36.89% (95% ci, 35.7738.03), respectively. this study found that physical activity adherence rate was lower in socioeconomically disadvantaged metabolic syndrome patients, which may aggravate health inequity status of korean society. |
serum samples were collected in 9 of 17 provinces in laos from healthy ducks and chickens in live - bird markets, village backyard flocks, and layer duck farms. cloacal, tracheal, and environmental (fecal and water) swab specimens were also collected and placed immediately in transport medium (figure). swab specimens were screened in pools of 4 by using a real - time reverse transcription pcr for the matrix (m) gene segment (6). positive pools were reextracted individually, retested, tested for hemagglutinin 5 (h5) by real - time reverse transcription pcr (7), and injected into 1011-day - old embryonated chicken eggs. areas sampled and location of subtyped avian influenza viruses (h5n1), laos, 20092010. provinces that had previous outbreaks of highly pathogenic avian influenza and were part of the survey are indicated in gray, the province that had a previous outbreak but was not part of the survey is indicated in blue, and the province that had not had an outbreak but was part of the survey is indicated in green. colored dots indicate presence of viruses : light blue, anti - h5 (clade 2.3.4) ; gray, anti - h9 lineage g1 ; red, clade 2.3.4.1 ; orange, clade 2.3.4.2 ; green, anti - h5 (clade 2.3.2) ; black, anti - h9 lineage y280 ; white, anti - h4 ; yellow, anti - h6 ; purple, clade 2.3.2.1 or virus - specific antibodies. sequencing was conducted by using an illumina (san diego, ca, usa) platform (swabs and isolates) (8,9) and conventional sanger sequencing (isolates). illumina reads were first mapped to a database of publicly available human, avian, and swine influenza virus reference sequences from the western hemisphere americas and eurasia, and then remapped against references with the highest number of reads and best average coverage. final average coverage varied between samples and segments with ranges of 12324,163 (9 samples), 2118,671 (12 samples) and 9436 for sample a / duck / lao/670/10. mixed infections could not be excluded for direct sequencing in the absence of an isolate. phylogenetic analysis (clustalw [http://www.clustal.org/ ], neighbor - joining analysis, 1,000 bootstrap tests, maximum composite likelihood, pairwise deletions) was conducted by using mega version 5.02 (10). sequences are available in genbank (cy098294cy098334, cy098336cy098340, cy098342cy098368, and cy098370cy098464). serum samples were screened by using an elisa (flockchek multis screen ; idexx laboratories, westbrook, me, usa), and antibody - positive serum samples were tested by using a hemagglutinin inhibition assay for subtypes h3, h4, h5 (clades 2.3.2 and 2.3.4), h6, and h9 (lineages g1 and y280) as described (11). during march 2010, a total of 3,695 swab specimens were collected (1,928 duck and 279 chicken cloacal samples, 446 duck tracheal samples, 675 fecal samples, and 367 water samples). m gene prevalence was 4.0% (ducks), 1.8% (chickens), and 0.3% (environment samples). positive swab specimens were collected in 13 locations (8 backyards, 2 markets, and 3 farms) (table 1 ; figure) sample protection was suboptimal, and only 21 samples could be subtyped by real - time rt - pcr and sequencing (table 1). i d, identification ; pb, polymerase basic ; pa, polymerase acidic ; h, hemagglutinin ; np, nucleoprotein ; n, neuraminidase ; m, matrix ; ns, nonstructural ; na, not available ; env, environmental. pink shading, a / guizhou/1/2009-like clade 2.3.4.1 ; blue shading, a / whooper swan / mongolia/6/2009-like clade 2.3.2.1 ; purple shading, a / tree_sparrow / jiangsu/1/08-like clade 2.3.4 ; gray shading, a / environment / guizhou/4/2009-like clade 2.3.4.2. phylogenetic analysis identified 3 groups of viruses : clades 2.3.2.1, 2.3.4.1, and 2.3.4.2 (figure a1). two samples were closely related to a / chicken / lao / lh1/2010like virus (outbreak in vientiane in april may 2010) and to a / chicken / laos / c100209194-ptk/2009like virus (outbreak in phongsaly in february 2009) (clade 2.3.4.1) (figure a1 ; table 1). these viruses were closely related to a / guizhou/1/2009 and a / chicken / vietnam / ncvd-404/2010 (figure a1). sixteen surveillance viruses of clade 2.3.4.2 were highly homogeneous (figure a1 ; table 1) and closely related to a / environment / guizhou/4/2009 and a / chicken / vietnam / ncvd-394/2010like viruses, although they contained the polymerase basic 2 (pb2) gene of a clade 2.3.2.1 donor virus and were therefore interclade reassortants (figure a1 ; table 1). the genotype of the 4 isolates was verified by using sanger sequencing. these reassortants were detected in 3 locations in northern (backyard), central (backyard), and southern (farm) laos, suggesting multiple introductions of reassortants into laos (figure a1 ; table 1). three clade 2.3.2.1 viruses were detected in 2 ducks and 1 environmental sample (same trader). two (1 isolate and 1 direct sequence) contained 6 or 7 segments that were a / whooper swan / mongolia/6/2009 like (clade 2.3.2.1), and the nucleoprotein gene was a / tree_sparrow / jiangsu/1/08 like (clade 2.3.4). the environmental sample (direct sequence) contained a / whooper swan / mongolia/6/2009like hemagglutinin and m genes and a / guizhou/1/2009like pb2, nucleoprotein, and neuraminidase genes (clade 2.3.4.1) (table 1). this identity and the source of the 3 samples (1 trader) suggest that reassortment occurred recently, likely in laos. antibody titers to h5 and h9 and a subtype h3n8 virus isolate have been reported in laos (4,5). subtypes h4 and h6 also circulate in this region (1,2,12). for this study, 2,148 serum samples (1,899 from ducks, 200 from chickens, and 49 from unspecified species) were collected and 267 antibody - positive (seroprevalence 14%) duck and 15 (7.5%) chicken serum samples were detected. hemagglutination inhibition testing for specific antibodies against h3, h4, h5, h6, and h9 detected all antibodies but to h3 (table 2). antibodies against h9 were detected at highest titers and most frequently (1.1% of ducks for each h9 lineage), although some cross - reactivity between g1 and y280 likely occurred (table 2). these ducks were most widely distributed (18 [19% ] of 97 locations in all 9 provinces). antibodies against h5 clade 2.3.4 were found at the detection limit in few serum samples (0.4% of ducks in 3 northern and 2 southern provinces). antibodies against h5 clade 2.3.2 were detected in 0.4% of ducks in 4 northern provinces and the capital of vientiane. all titers for h3 (a / duck / laos - xaythany / a0573/2007) were 1 virus - positive or antibody - positive duck in each of the 9 provinces sampled. one village had 36% of sampled ducks exposed to this virus ; 18% shed 2.3.4 virus and 18% had antibody to 2.3.2 virus (table 2). exposure to 2.3.2 and 2.3.4 viruses was evident in ducks from locations in 3 other provinces (1 district each in champasak and xiengkhoung, and several districts in luang prabang). this study showed that 3 groups of avian influenza viruses (h5n1) were likely introduced into laos in 20092010, one of which resulted in 2 outbreaks (2009, 2010). in all 9 provinces where surveillance was conducted, several interclade reassortants were identified, demonstrating the high genetic mobility of these viruses in the region. since 2004, laos has had repeated outbreaks of highly pathogenic avian influenza viruses, which have also been detected in china and vietnam. the frequency of introduction, diversity, and extent of these viruses in laos suggests considerable movement of viruses into the country from surrounding territories (china and vietnam, but not cambodia) and within the country. | avian influenza viruses (h5n1) of clades 2.3.4.1, 2.3.4.2, and 2.3.2.1 were introduced into laos in 20092010. to investigate these viruses, we conducted active surveillance of poultry during march 2010. we detected viruses throughout laos, including several interclade reassortants and 2 subgroups of clade 2.3.4, one of which caused an outbreak in may 2010. |
it is characterized by progressive thinning of the scalp hair and a reduction in hair density and diameter. male aga presents with a typical pattern of bitemporal and frontal recession of the hair line or vertex thinning which gradually extends anteriorly. the prevalence increases with age, from 30% for men in their 30s to 50% for men in their 50s. nongenetic causes have received little scientific attention, and data on environmental factors that may aggravate male aga remain sparse. the present population - based cross - sectional study was carried out between may and september 2011. the study sample consisted of caucasian men aged 30 to 40 years who had uploaded a profile to one of two dating websites : jdate, which targets jewish subjects, and okcupid, a general dating service from which we selected non - jewish subjects using the search criteria. a total of 26,340 profiles were examined, each containing several photographs of the individual subject. the photographs were magnified and graded for the presence of alopecia according to the norwood classification. thereafter, randomly selected photographs were again evaluated by an independent dermatologist blinded to the results of the first observer. intra- and interobserver variability were analyzed with cramer 's reliability test and joint probability of agreement statistics. on the basis of the findings, subjects were divided into three groups : severe aga (norwood type vi or vii), non - severe aga (norwood type 90 cm, serum triglyceride level > 150 mg / dl, high - density lipoprotein cholesterol level 130/85 mmhg or treated hypertension. aga was found to be linked to cardiovascular diseases, coronary heart diseases, insulin resistance, hypertension, abnormal serum lipid profiles and obesity. su and chen noted that patients with severe aga (type v or above) had a 2.6-fold higher prevalence of mets than patients with moderate aga (types iii and iv), and pathomvanich. suggested that the increasing incidence of obesity in bangkok may be contributing to the higher prevalence of male aga compared to other asian countries. recently reported that the risk of acquiring norwood type iv aga or greater was not increased in subjects with mets relative to those without mets. we examined the bmi of 10,691men on jdate who had included data on height and weight in their profile. there was a positive association between the presence of severe aga and higher bmi, with an increased risk of 1.027 for every unit of bmi for men of the same age. if this finding is confirmed, severe aga may serve as a predictive factor for the early diagnosis of mets and aid physicians in the prevention of its complications. in conclusion, the present study describes a novel method for conducting epidemiological research of aga using photographs and data from the internet. to the best of our knowledge, this is the largest epidemiological study to investigate aga and the first to use this method. we focused on young caucasian men within a narrow age range of 30 - 40 years. our findings link aga with advancement in age and with higher bmi, and show significant differences in the prevalence of aga among countries pointing to some potential environmental risk factors. evaluation of photographs and data from the internet can serve as a novel method of studying the international epidemiology of androgenetic alopecia.high body mass index and exposure to high environmental levels of ultraviolet radiation may aggravate aga. evaluation of photographs and data from the internet can serve as a novel method of studying the international epidemiology of androgenetic alopecia. high body mass index and exposure to high environmental levels of ultraviolet radiation may aggravate aga. | background : the epidemiological evaluation of androgenetic alopecia (aga) is based mainly on direct observation and questionnaires. the international epidemiology and environmental risk factors of aga in young caucasian men remain unknown.aim:to use photographs and data from the internet to evaluate severe aga and generate greater understanding of the international epidemiology of the disorder in young caucasian men.materials and methods : a population - based cross - sectional study design was used. the sample included 26,340 caucasian men aged 30 to 40 years who had uploaded profiles to two dating websites. their photographs were evaluated for aga and graded as follows : severe aga (norwood type vi - vii), non - severe aga, and unknown. epidemiological data were collected from the sites. logistic regression was used to analyze the effect of risk factors on the prevalence of severe aga.resultsthe overall success rate for identifying severe aga by indirect evaluation of internet photographs was 94%. the prevalence of severe aga was 15.33% overall and varied significantly by geographical region. the risk of having severe aga was increased by 1.092 for every year of age between 30 and 40 years. severe aga was more prevalent in subjects with higher body mass index.conclusions:photographs from the internet can be used to evaluate severe aga in epidemiological studies. the prevalence of severe aga in young caucasian men increases with age and varies by geographical region. body mass index is an environmental risk factor for severe aga. |
various inflammatory diseases and benign or malignant tumors are known to cause distal common bile duct (cbd) strictures (1 - 3). it is important to determine whether a biliary stricture has a benign or malignant cause as each requires a different treatment approach. furthermore, as a normal distal cbd may show a narrow distal segment, it is difficult to assess the clinical significance of a distal cbd narrowing. generally, endoscopic retrograde cholangiopancreatography (ercp) has been considered the method of choice for diagnosing biliary strictures due to its accuracy in establishing the site and cholangiographic features of strictures (4). however, there are some conditions that ercp could not be successfully completed such as previous stomach surgery. in addition, other diagnostic approaches are required to evaluate the biliary stricture when tissue sampling is difficult using ercp. percutaneous transhepatic cholangioscopy (ptcs) might be useful for the diagnosis and treatment of biliary strictures (5, 6). however, there are no published studies examining the usefulness of ptcs for evaluating distal cbd strictures. the usefulness of ptcs for differential diagnosis of distal cbd strictures was evaluated in the present study. in addition, ptcs, computed tomography (ct), magnetic resonance cholangio pancreatography (mrcp) and direct cholangiography including ercp were compared in terms of their accuracy in diagnosing distal cbd strictures. between january 1995 and december 2004, 95 consecutive patients who underwent ptcs for the evaluation of distal cbd strictures at asan medical center, seoul, korea, were enrolled in this study. distal cbd stricture was diagnosed by endoscopic retrograde cholangiography or cholangiograms obtained by means of percutaneous transhepatic biliary drainage (ptbd) tubes, and radiologic finding including ct or mrcp. if the malignant distal cbd stricture was obvious, these patients promptly sent for operation. however, patients whose other masses were unable to be seen through radiologic findings and the cases of which the nature of distal cbd stricture was inconclusive as to benign or malignant underwent ptcs. cases in which malignancy could not be proven by tissue biopsy results were excluded from the malignant group, even if their strictures were clinically suspicious for malignancy. those patients with distal cbd stricture, which was diagnosed by postoperative cholangioscopy via t - tube tract, were also excluded. malignant distal cbd strictures were confirmed when malignant cells were observed in specimens obtained using ptcs - guided biopsy or surgery. the malignant distal cbd stricture group comprised of 35 patients (22 men and 13 women ; mean age, 66 yr) and the diagnosis of malignancy was confirmed by surgical resection with or without ptcs - guided biopsy in 29 and only ptcs - guided biopsy in 6. strictures were considered benign when malignant cells were not found in resected specimens from the patients who underwent surgery or when there was no clinical and radiology evidence of disease progression during more than 6 months of follow - up in patients with negative ptcs - biopsy findings who did not undergo surgery. the benign distal cbd stricture group comprised of 60 patients (45 men and 15 women ; mean age, 61 yr) and the median follow - up period for the benign group was 26 months (up to 60 months). in our study, 59 out of 95 patients could not undergo successful ercp due to a previous history of roux - en - y esophagojejunostomy or gastrojejunostomy (n=31), cannulation failure (n=19), cardiovascular disease (n=4), difficulty to assume a lateral position (n=2) and others (n=3). six patients underwent ptcs instead of ercp, because ercp had been performed at another hospital for evaluation of indeterminate distal cbd stricture suspicious for malignancy and they were referred to our tertiary referral center for cholangioscopy. thirty patients underwent ercp and in 8 out of 30 patients, we could not get the ercp - guided biopsy and brush cytology due to tight distal cbd structures (n=5), hemobilia (n=1) and others (n=2). in 8 patients, ercp - guided tissue sampling of distal cbd stricture the other 14 patients were unsuccessfully diagnosed because no additional information was obtained even though an ercp - guided biopsy and a brush cytology were done. two or three days after the ptbd, the percutaneous tract was dilated for the passage of the cholangioscope usually by one - stage dilation (eg, from 8.5 f to 16 or 18 f). for the sinus tract maturation, ptcs was performed using a 5.2 mm outer diameter cholangioscope (chf p20q : olympus optical ltd, tokyo, japan) or 4.9 mm outer diameter cholangioscope (fcn-15x ; pentax, tokyo, japan) with medication of meperidine (25 - 50 mg) and/or midazolam (3 - 5 mg). ptcs was performed by 3 endoscopists who had ptcs experience of at least 40 cases yearly for 2 - 7 yr. the distal cbd strictures were carefully examined for the presence of abnormal dilated vessel and mucosal abnormality. targeted biopsies were performed with a 1.8 mm diameter forceps (fb-19sx01, olympus) under direct vision. laboratory data such as ca 19 - 9 and carcinoembryonic antigen (cea) concentrations were compared, and ct, mrcp, direct cholangiography findings and ptcs findings were analyzed. direct cholangiography was defined as endoscopic retrograde cholangiography or cholangiograms obtained by means of ptbd tubes. mucosal findings such as tumor vessel, mucosal irregularity and mucosal color change were compared on cholangioscopy. a tumor vessel was defined as an abnormally proliferating and tortuous vascular structure in the bile duct stricture mucosa. mucosal irregularity was defined as granular mucosa, nodular mucosa, papillary mucosa, mucosal hyperplasia and irregular mucosa. ptcs - guided biopsies were performed at the sites of stricture, tumor vessel, mucosal irregularity and mucosal color change and three more biopsy specimens per patient were obtained. the sensitivity and specificity of ptcs - guided biopsy were calculated for the diagnosis of malignant versus benign distal cbd stricture. additionally the sensitivity and specificity of combination of ptcs - guided biopsy and tumor vessel on cholangioscopy were compared with those by ct, mrcp and direct cholangiography for diagnosis of malignant distal cbd stricture. the radiologist reviewed all radiologic images retrospectively by using a picture archiving and communication system (pacs) workstation monitor. the reviewer had no knowledge of clinical data or whether distal cbd strictures were diagnosed as malignant or benign following surgery or biopsy. the proportion of patients who underwent all three types of imaging study (ct, mrcp and direct cholangiography) was relatively small, thus the analysis included patients that underwent two or more imaging studies, which comprised 68 of the 95 patients (25 malignant, 43 benign). twenty - seven patients were excluded from this analysis either because there were no images before decompression, the images were older than the obligatory storage period expiry date (5 yr) or the quality of outside images was very poor. a stricture was assumed to be malignant if characterized by wall thickening, enhancement and abrupt narrowing according to the ct, and an irregular margin, asymmetric and abrupt narrowing according to mrcp and direct cholangiography. the sensitivity and specificity of diagnosis using ct, mrcp and direct cholangiography for malignant distal cbd strictures were determined. all procedures were explained to patients, and all patients gave informed written consent to the procedures. all statistical analyses were performed using the spss 12.0 statistical software program (spss, inc., potential predictive factors for ptcs findings that could be diagnosed as a malignant distal cbd stricture were analyzed with chi - square tests for univariate comparisons. factors that were deemed of potential importance on univariate analysis (p<0.05) were included in the multivariate analysis. student 's t - test was used to test for the differences between the benign and malignant groups in terms of laboratory test results, stricture length and upstream ductal diameters according to the ct. chi - square tests were used to compare the radiograpic findings according to mrcp and direct cholangiography. all statistical analyses were performed using the spss 12.0 statistical software program (spss, inc., potential predictive factors for ptcs findings that could be diagnosed as a malignant distal cbd stricture were analyzed with chi - square tests for univariate comparisons. factors that were deemed of potential importance on univariate analysis (p<0.05) were included in the multivariate analysis. student 's t - test was used to test for the differences between the benign and malignant groups in terms of laboratory test results, stricture length and upstream ductal diameters according to the ct. chi - square tests were used to compare the radiograpic findings according to mrcp and direct cholangiography. a p value of < 0.05 was considered to indicate a significant difference. baseline clinical findings are summarized in table 1. the mean serum values for alkaline phosphatase (alp), gamma glutamyl transpeptidase (ggt) and total bilirubin were higher in the malignant than those in the benign group (p < 0.05). the mean ca 19 - 9 levels of the benign and malignant groups were 227 /ml and 396 /ml, respectively, without a significant difference between the two groups (table 2). ptcs showed tumor vessels in 26, mucosal irregularity in 21 and mucosal color change in 12 of 35 malignant distal cbd stricture patients, while tumor vessels were not observed in benign groups and mucosal irregularity and mucosal color change were seen in 21 and 8 of 60 benign stricture patients. on univariate analysis, malignant distal cbd stricture was associated with tumor vessel, mucosal irregularity and mucosal color change (p<0.05). however, multivariate stepwise logistic regression analysis showed that the tumor vessel was only an independently differentiating variable (p<0.05) (table 3). in 35 patients of the malignant group, 33 underwent a ptcs - guided biopsy. for the remaining 2 patients, while biopsies were attempted up to four times, adequate biopsy specimens could not be obtained, and the malignancy was confirmed following surgical resection. of the 3 patients of malignant group, ptcs - guided biopsy diagnosed their strictures as chronic inflammation. sixty patients of benign group underwent ptcs - guided biopsy and they were diagnosed with benign distal cbd stricture. those patients with benign distal cbd strictures showed no significant interval change during the at least 6 months follow - up period. the diagnostic accuracies associated with ptcs - guided biopsy and tumor vessel are shown in table 4. the most valuable cholangioscopic finding for indicating the presence of a malignant distal cbd stricture was found to be the tumor vessel. furthemore, a combination of tumor vessel findings and ptcs - guided biopsy resulted in a superior preoperative diagnosis compared to tumor vessel or ptcs - guided biopsy alone, which provided 97.1% sensitivity, 100% specificity, 100% positive predictive value and 98.4% negative predictive value (table 4). the sensitivity and specificity of combined ptcs - guided biopsy plus tumor vessel were compared with those of ct, mrcp and direct cholangiography for the diagnosis of malignant distal cbd strictures (table 5). the sensitivity / specificity of ct, mrcp and direct cholangiography were 42.9%/65.8%, 53.3%/58.3% and 70.8%/47.6%, respectively, while it was 96%/100% for the combined assessment using tumor vessel and ptcs - guided biopsy findings. among 68 patients, 23 patients were suspected of malignancy according to at least one of ct, mrcp or direct cholangiographic images, and also shown to have malignant distal cbd strictures according to ptcs findings and biopsies. sixteen patients showed benign radiologic features in distal cbd strictures and also showed benign ptcs findings and biopsy results. for 27 patients, while radiologic images indicated malignant distal cbd strictures, ptcs and biopsy findings revealed benign strictures (fig. the mean values for alp, ggt and total bilirubin were 251 iu / l221.6, 205 iu / l198.5 and 3.2 mg / dl4.96, respectively, and these mean values were significantly lower than those of the malignant distal cbd stricture group (p<0.05). for one patient, radiology showed a benign stricture while ptcs and biopsy findings showed a malignant stricture (fig. three of 95 patients experienced some complications including deep sedation (n=1) and minor bleeding (n=2) during cholangioscopy. they recovered following conservative management and there was no other complication such as pancreatitis or cholangitis. the present retrospective study showed that combined use of ptcs - guided biopsy and tumor vessel observation resulted in a highly accurate method for diagnosing distal cbd strictures. these observations show that ptcs would be useful in diagnosing distal cbd strictures in cases that radiologic findings are ambiguous or ercp techniques are inadequate. after the mid 1970 's, ptcs was predominantly performed in asian countries such as taiwan, korea and japan, where there had been a high prevalence of intrahepatic stones and cholangiocarcinomas (7, 8). the disadvantages of this procedure are that it is invasive and requires sinus tract dilatation which takes a relatively long time (9). however, the advantages are direct visualization of the biliary mucosa and the ability to perform a ptcs - guided biopsy (10, 11). the sensitivity of ptcs - guided biopsy was reported as 80 - 89% (12, 13), and the present study found it to be 85.7%. the previous prospective studies reported that the sensitivity of ercp - guided biopsy and brush cytology was 61 - 75% for biliary tumors (14, 15). in some cases of distal cbd stricture, thus, cholangioscopic biopsy appears to be very useful since a clinician can precisely perform a targeted mucosal biopsy during continuous observation. in the current study, the number of patients who underwent ercp - guided biopsy and brush cytology was so small that we could not compare ercp with ptcs in terms of tissue diagnosis. sato. has also reported that ptcs - guided biopsy can detect invasive carcinomas only in the superficial layers of the bile duct and diagnosis of intramural extension of invasive carcinoma is difficult with ptcs even when the lesion is biopsied (16). therefore, reliable diagnostic criteria are required for accurate differential diagnosis in cases of difficult biopsies. ptcs is a useful method for distinguishing cholangiocarcinomas from other benign cbd strictures as it provides direct visualization of any tumor vessels. in the bile duct mucosa, the tumor vessel is an abnormal tortuous and serpentine vascular structure at the circumference of the cancer mucosa (17), and is important for tumor nutrition. of the 35 malignant group patients, biopsy results showed chronic inflammation in 3 patients, and adequate biopsy specimens could not be obtained in 2 patients. five patients could not initially be confidently placed in either the malignant or benign group and the malignant distal cbd strictures all were confirmed by surgical resection. four of those 5 patients had tumor vessels and only tumor vessel was associated with malignant distal cbd stricture on multivariate analysis. therefore, it appears that the presence of a tumor vessel is a valuable clue for identifying malignancy. it is meaningful that observation of a tumor vessel in distal cbd strictures by ptcs because observation of a tumor vessel by ercp is impossible. the present study documented ptcs - guided biopsy findings combined with tumor vessel observation provided the most precise preoperative diagnosis. this observation is consistent with that of a previous study involving 41 malignant biliary stricture patients that reported that the sensitivity of tumor vessel observation was 61%, the sensitivity of ptcs - guided biopsy findings was 80%, while the sensitivity of the two methods combined were 96% (12). in the present study, patients who failed ercp due to unsuccessful cannulation did not undergo peroral cholangioscopy which would have been much less invasive. in addition, endoscopic ultrasound - guided fine - needle aspiration (eus - fna) for the evaluation of distal cbd strictures was not performed. the diagnostic utility of peroral cholangioscopy has not yet been examined vigorously and the observation of far distal cbd on peroral cholangioscopy is techniqually difficult. the fragility of the equipment and technical difficulties hold back its popularity. eus - fna has been shown, in many series, to be highly accurate for the diagnosis of pancreatic masses ; sensitivity rates for malignancy range from 60% to 93% (18 - 23). in a prospective study, it has been demonstrated that the sensitivity of eus - based techniques for the diagnosis of malignant biliary stricture was only 25% and eus - guided biopsy was superior for pancreatic mass (14). in that study, if a mass lesion was noted around the bile duct, eus - fna was attempted and if a circumscribed mass was not demonstrated by eus, needle puncture was not attempted. there is few published data in respect to eus - guided tissue sampling of distal cbd stricture without definite cbd mass on radiologic imaging. eus - fna, therefore, may not be more valuable than ptcs with biopsy for the evaluation of distal cbd stricture without visible tumor in distal cbd. we suggest ptcs be undertaken in patients in whom it is difficult to determine whether the distal cbd strictures are benign or malignant by radiological findings and ercp and get ercp - guided tissue sampling are not feasible : billroth type ii surgery, cannulation failure, tight distal cbd strictures and etc. the present study was retrospective and therefore has the limitations inherent in such a study design. in addition, the number of patients who underwent all three imaging procedures (ct, mrcp and direct cholangiography) was relatively small, so analysis involved patients who had undergone at least two of the three procedures. furthermore, on the interpretation of ct, mrcp and direct cholangiography findings for distal cbd strictures, it is likely that observational disagreement for the evaluation of the results exist. moreover, sensitivity of ct may be underestimated because we did not adjust for the fact that, prior to march 2001, evaluation of biliary tract disease was performed by conventional abdomen ct, which was later replaced by dynamic biliary ct. thus, the outcomes of this study must be considered in the context of these limitations. in conclusion, the present study found that due to its ability to provide direct visualization of the mucosa and any tumor vessels, and to its ability to guide biopsies, ptcs was a useful method for identifying malignant distal cbd strictures compared to ct, mrcp and direct cholangiography. in particular, we recommend ptcs to obtain an accurate diagnoses in cases where laboratory findings, radiographic findings and tissue sampling results following ercp are inconclusive to exclude a malignant distal cbd stricture. | the diagnostic accuracy of percutaneous transhepatic cholangioscopy (ptcs) was compared to that of three radiologic modalities in distal common bile duct (cbd) strictures for the evaluation of clinical application. ninety - five patients who underwent ptcs for the evaluation of distal cbd strictures (35 malignant and 60 benign) whose masses were not obvious from radiologic imagings were included. confirmative diagnosis could not be reached by endoscopic retrograde cholangiopancreatography (ercp) or radiologic findings in all cases. specific findings on the computed tomography (ct), magnetic resonance cholangiopancreatography (mrcp) and direct cholangiography were analyzed among 68 (25 malignant and 43 benign) out of the 95 patients in order to determine the sensitivity and specificity of three radiologic studies for the diagnosis of malignant distal cbd strictures, and to compare those results with those by a combination of ptcs - guided biopsy and tumor vessel observation on cholangioscopy. the sensitivity / specificity of ct, mrcp and direct cholangiography including ercp in diagnosing malignant distal cbd strictures were 42.9%/65.8%, 53.3%/58.3%, and 70.8%/47.6% respectively, while it was 96%/100% for the combination of ptcs - guided biopsy and tumor vessel. ptcs is a useful method for differential diagnosis of distal cbd strictures, particularly when it is difficult to distinguish benign from malignant strictures by radiologic studies and when peroral approach is not feasible. |
since the production of restorative materials has rapidly developed, mechanical tests have played an important role in evaluating the bond strength of different materials to dental substrates. developing new techniques to increase bond strength in resin restoration is necessary to reduce marginal microleakage and discoloration due to failure in marginal integrity. so, measuring the enamel and dentin bond strength is important in the evaluation of mechanical fracture and therefore the prognosis of dental treatment. the application of alternative methods such as laser irradiation has been increased in restorative dentistry. among different lasers, the er : yag laser was approved by fda in 1997 for caries removal, cavity preparation and conditioning of the enamel or dentin. this laser with a wavelength of 2940 nm and its high coefficient of absorption in water and hydroxyapatite is more effective than the other lasers for preparing hard tissue. it produces minimal thermal effect on the tooth structures and surrounding tissues in comparison with other dental lasers especially when water spray is applied. this laser produces an irregular pattern on the surface of the enamel that can improve retention for bonding of restorative material. advantages such as biocompatibility, adhesion to tooth structure, fluoride release, reduced microleakage and lower polymerization shrinkage have led to wide use of glass - ionomer materials in restorative dentistry. different parameters and methodologies have guided to controversial results about the effect of er : yag laser on the shear bond strength of dental materials to enamel. souza - gabriel. in assessing the shear bond strength of resin - modified glass - ionomer to er : yag laser treated surfaces concluded that conventional bur - prepared samples provided better adhesion than samples prepared by er : yag laser. on the other hand, visuri. found higher values of bond strength in the irradiated surface by er : yag laser compared to those that were bur - treated.the aim of this study was to evaluate the shear bond strength of resin modified glass ionomer to enamel in er : yag laser - treated surfaces in comparison with bur - treated surfaces. all teeth were stored in distilled water with 0.4% thymol for 1 month to reduce the formation of microbial plaque. the teeth were embedded in clear acryl (repair material, dentsply international inc., milford, de) 2 mm under the enamel - cementum junction (cej). the buccal surfaces were flattened on the enamel level by carbon disc with 3 mm thickness and12.5 mm diameter. the first group was irradiated by er : yag laser (us2940d, deka, italy). this laser operates at a wavelength of 2940 nm accompanied by water and air spray. the laser irradiation was done with an average output power of 3.5 w, energy of 350 mj and frequency of 10 hz with a pulse duration of 230 s in a sweeping motion about 4 mm above the surface. the distance from the enamel surface was controlled by fixing an endodontic file to the laser handpiece. the spot size of laser was 1 mm. the second group was prepared by carbide bur (6 flutes and spiral angle of 30) under water spray in sweeping motion for 5 seconds. the dimension of the area, which was conditioned by two methods, was approximately 2 mm 2 mm. after surface treatment of the two groups, the conditioner (40% polyacrylic acid) was applied for 30s. then, self cure resin modified glass ionomer (fuji ix gc corporation, tokyo, japan) was placed on the surface in a 2.8 mm diameter and 3 mm height cylindrical mold. all the samples were placed in 37c distilled water for 24 h to provide the final setting of the restorative material. the samples were subjected to universal testing machine (zwick, germany) in order to measure the shear bond strength at a speed of 0.5mm / min and a 50 kgf load until fracture. the results of the two groups were analyzed by t - test at 0.05 confidence level. the mean and standard deviation of carbide group and er : yag laser group were 4.411.62 mpa and 6.751.99 mpa, respectively. there was significant difference between the shear bond strength of the lased and the bur group (p - value=0.01). according to the microretentive pattern obtained by er : yag laser irradiation, which is suitable for adhesion, in this study the shear bond strength of an rmgi to lased enamel surfaces in comparison with bur - treated surfaces was assessed. bond strength testing as a laboratory methodology has been proposed to evaluate the adhesion capacity of dental materials.the shear bond strength test is a simple procedure for experimental evaluation and also a screening mechanism for predicting clinical performance [9, 10 ]. laser technology has been presented as an alternative option to replace the conventional high speed turbine. this technology offers the patient comfort by reducing the pressure, heat, vibration and noise produced by a rotary instrument. using laser for etching enamel application of phosphoric acid for etching the enamel makes the surface more susceptible to caries because of demineralization of the superficial layer. the physiochemical changes by laser etching reduced the acid attack and the risk of caries. it may be related to changes in ca / p ratio, reduced carbonate and pyrophosphate formation accompanied by the reduction of water and organic component [1315 ]. er : yag laser acts on the dental substrate by thermo - mechanical ablation and vaporization of the water content which causes expansion followed by microexplosion that produces the ejection of both organic and inorganic tissue particles [1618 ]. therefore, it blocks the intra- and interprismatic spaces and restricts material interdiffusion into the enamel surface [19, 20].the micromorphology of laser treated surface shows less regular and homogeneous aspects with some fissures occurred in subsurface resulting from heat generated during irradiation. resin modified glass ionomer has many advantages and has been employed in dental clinic because of the physiochemical adhesion to the enamel and dentin. for a long period, it can release fluoride ions in adjacent enamel and may absorb fluoride from other sources like toothpastes. it also presents good adhesion, marginal seal and reasonable esthetics [22, 23 ]. most of the studies that evaluated the shear bond strength of laser treated surfaces in comparison with bur - prepared enamel showed higher bond strength in the bur - prepared group. measured the shear bond strength of resin composite to enamel treated by different energy intensities and frequencies of er : yag laser compared to phosphoric acid and concluded that acid conditioning of the enamel showed higher bond strength than laser. investigated the shear bond strength between light - curing nano - ionomer restorative and enamel or dentin after acid etching, after er : yag laser etching or after combined treatment. they concluded that etching with acid phosphoric increased the shear bond strength, but the laser group showed a lower bond strength that was in contrast with the results of the present study. when er : yag laser was used for surface treatment, compared to bur preparation it produced no smear layer leading to increased surface wettability and producing tag formation. on the other hand, turkmen. showed that er, cr : ysgg laser etches the enamel surface more effectively than phosphoric acid that is in agreement with our results. the higher values that were achieved from the laser group may be contributed to micro irregularities produced on the surface. there is need for more studies which evaluate the interaction of laser prepared surfaces with new generation of glass ionomers like light - curing nano - ionomer restoratives. based on the results of the present sudy, er : yag laser can be an alternative device for enamel preparation in restorative dentistry. | objective : the purpose of this study was to evaluate the effect of er : yag laser on the shear bond strength of resin modified glass ionomer (rmgi) to enamel.materials and methods : twenty extracted caries - free human premolars were selected. the teeth were embedded in acrylic resin. the buccal surfaces of each sample were ground to plane enamel with carbonated disc. the teeth were randomly divided in two groups. in the first group, the surfaces were treated by er : yag laser (350mj/10hz). the second group was prepared by carbide bur. fuji ix rmgi was adhered to surfaces of the samples in both groups in rod shape. the shear bond strength of samples was measured by a universal testing machine. the results of the two groups were analyzed by t- test.results:the means and standard deviations of shear bond strength of the laser - treated group and the bur - treated group were 6.75 1.99 and 4.41 1.62 mpa, respectively. there is significant difference in the shear bond strength of rmgi between the two groups (p - value=0.01).conclusion : the laser group showed better results. er : yag laser can be an alternative technology in restorative dentistry. |
epithelial ovarian cancer (eoc) represents one of the most common gynecologic malignancies worldwide. it has the highest mortality rate among malignant tumors in female reproductive system [1, 2 ]. because of the lack of specific early symptoms or effective tumor biomarkers, most patients with eoc are diagnosed at the advanced stages, and the prognosis of these patients is still poor, even though there has been great improvement on traditional treatments, such as surgery, supplemented with radiotherapy and chemotherapy. similar with other human malignancies, tumorigenesis and tumor progression of eoc are caused by numerous reproductive, environmental, and genetic risk factors. therefore, it is of great importance to discover and analyze the genetic changes and molecular events involving the initiation, progression, and metastasis of eoc. mammalian runt - related transcription factor (runx) family contains three members (runx1~3) which form the core - binding factor (cbf) complex and bind dna to either activate or repress gene transcription. as part of the cbf complex, the runx proteins are involved in the regulation of differentiation, survival, and growth in a variety of tissues. runx1 and runx2 are specifically essential for multiple haematopoietic lineages and osteogenesis, respectively, and runx3 is closely related with neurogenesis and gut development. accumulating studies have reported the oncogenic and tumor suppressive functions of the runx members. in the present study, we have chosen to focus on the role of runx2 in neoplastic disease. because runx2 plays a pivotal role in the process of bone formation or osteogenesis, it is no doubt that its deregulation is strongly associated with the development of osteosarcoma. in addition, the overexpression of runx2 has also been identified in several human malignancies, including prostate cancer, breast cancer, pancreatic cancer, colon carcinoma, and thyroid cancer. for example, tandon. indicated that runx2 is a potential therapeutic target to block tumor suppressor gene silencing in lung cancer cells. pratap. found that the expression of runx2 in breast cancer cells may be important for tumor cell invasion through matrigel chambers. the results of chua group revealed that runx2 nuclear staining was correlated with increased prostate - specific antigen (psa) levels, higher gleason scores and tumours with metastatic capability. identified runx2 as a potent prognostic factor in colon carcinoma patients through the promotion of cell proliferation and invasion properties. however, the expression patterns and involvement of runx2 in eoc are still unclear. therefore, the aim of this study was to investigate the clinical significance of runx2 expression in eoc. the study was approved by the research ethics committee of general hospital of pla, china. we collected formalin - fixed, paraffin - embedded tissues from the primary ovaries obtained during surgery from women with eoc (n = 116). as controls, we also obtained normal ovarian tissues (n = 5) from women who underwent hysterectomies for benign disease. all operations were performed in the department of obstetrics and gynecology at general hospital of pla from january 2005 to december 2006. all patients with only gynecology tumor were treated without preoperative radiotherapy, chemotherapy, or hormonal therapy. surgical staging was established according to the international federation of gynecology and obstetrics (figo) system. debulking status was defined according to the size of the nodules left in the peritoneal cavity after surgery. the paraffin - embedded tissues were cut at 3 m and then deparaffinized with xylene and rehydrated for further hematoxylin eosin (h&e) or 3,3-diaminobenzidine (dab) immunohistochemistry staining employing dako envision system (dako diagnostics, zug, switzerland). briefly, following treatment with 10% normal goat serum for 45 min to block nonspecific binding sites, the consecutive tissue sections were incubated for 180 min at 25c with mouse monoclonal antibody for human runx2 (dilution 1 : 1000, abnova corporation, taipei, taiwan). the specificity of the primary antibody has been validated by the previous studies [15, 16 ]. after washing, peroxidase - labeled polymer and substrate - chromogen were then employed in order to visualize the staining of the interested proteins. in tumor cells of eoc tissues, runx2 immunoreactivity was detected in the nucleus, and the immunoreactivity was evaluated as a labeling index (li) according to the methods of previous studies [15, 16 ]. briefly, runx2 immunoreactivity was evaluated in the nuclei of more than 1000 tumor cells for each case, and li was calculated as the percentage of runx2 positive cells per 1000 tumor cells counted at random in each section. this counting was performed under a 400 magnification. the software of spss version 12.0 for windows (spss inc., il, usa) and sas 9.1 (sas institute, cary, nc, usa) were used for statistical analysis. the pearson 's chi - square and fisher 's exact tests were used to assess the statistical significance of the association between runx2 expression and clinicopathologic parameters. kaplan - meier curves were plotted to assess the effects of runx2 expression on overall and progression - free survival. cox proportional hazard models were used to assess the prognostic significance of runx2 expression and several clinicopathologic parameters. the expression patterns and cellular localization of runx2 in 116 eoc and 5 normal ovarian tissues were assessed by immunohistochemical analysis. as shown in figure 1, runx2 immunoreactivity was predominantly localized in the nuclei of eoc cells (figure 1(a)), while almost negligible in normal ovarian tissues (figure 1(b)). the mean value of the runx2 li in 116 eoc tissues detected was 56.3% (range, 099%), which was significantly higher than that in normal ovarian tissues (11.7% ; range, 035.2% ; p 0.05). in order to investigate the prognostic implications of runx2 expression in overall survival and progression - free survival of eoc, the detailed clinical information of all 116 eoc patients in high runx2 expression and low runx2 expression groups was reviewed. median follow - up time was 66.8 months (range, 2.2118.9 months ; mean, 66.1 months). at last followup, 73 (62.9%) relapsed with a median time of 22.1 months (range, 2.885.2 months). as determined by the log - rank test, eoc patients with high runx2 li had significantly shorter overall (p < 0.001, figure 2(a)) and progression - free (p = 0.002, figure 2(b)) survival than those with low runx2 li did. moreover, the univariate analysis revealed that both the advanced stage (p < 0.001 and p = 0.008, resp.) and the high runx2 expression (p < 0.001 and p = 0.002, resp.) predicted poorer overall and progression - free survival of eoc patients (table 2). furthermore, the multivariate analyses identified the clinical stage (p = 0.01 and p = 0.03, resp.) and the runx2 li (both p = 0.01) in eoc cells as independent prognostic factors for overall and progression - free survival (table 3). interestingly, subgroup analyses according to clinical stage revealed that eoc patients with high clinical stages (iii~iv) in high runx2 expression group demonstrated a significantly worse clinical outcome than those in low runx2 expression group (figures 3(a) and 3(b)), but patients with low clinical stages (i~ii) had no significantly different prognosis between high and low runx2 expression groups (figures 3(c) and 3(d)). as the first leading cause of cancer death in female reproductive system malignant tumors, eoc has no characteristic early symptoms or tumor markers, leading to disappointing clinical outcome. the clinical course of remission and relapse is commonly seen in patients undergoing therapy for eoc. discovery and analysis of the genetic changes and molecular events have contributed largely to a better understanding of the molecular mechanisms of cancer ontogenesis. accordingly, it is of great significance to identify novel specific diagnostic or prognostic markers that contribute to progression and metastasis of eoc. in the present study, we detected the overexpression of runx2 in eoc tissues compared with normal ovarian tissues, and its upregulation was closely related with the clinical stage and poor prognosis of eoc patients, which led us to believe that runx2 might be used as a candidate biomarker for aggressive disease behavior. to the best of our knowledge, runx proteins have been demonstrated to play positive and negative roles in carcinogenesis according to different cancer types. as a member of runxs, runx2, also known as core - binding factor, runt domain, a - subunit 1, cbfa1, aml3, or osf2, is a lineage - specific transcription factor and the human homologue of mouse pebp2a. during embryonic development after birth, runx2 controls bone matrix deposition, especially collagen i, by differentiated osteoblasts. targeted disruption of runx2 in mice results in failure of osteoblast differentiation and bone formation, and runx2 haploinsufficiency in humans leads to the skeletal disorder cleidocranial dysplasia, with a similar phenotype observed in runx2 haploinsufficient mice.. reported that runx2 transcription factor may be involved in various aspects of luteal function by directly regulating the expression of diverse luteal genes in luteinizing granulosa cells of rat ovaries. in carcinogenesis, runx2 acts as a master regulator of tumor invasion and metastasis. nuclear runx2 is increased in malignant versus benign prostate tissue and is associated with tumor aggression in general and metastasis in particular. runx2 has the strongest basal expression among the runxs, and a significant increase in runx2 in carcinoma samples contributes to tumorigenesis in esophageal squamous cell carcinoma. the differential expression of runx2 has been found between invasive, and noninvasive breast cancer cells, and its upregulation is linked to the invasive phenotype of cancer cells. runx2 also plays an important role in angiogenesis by enhancing the endothelial cell proliferation, invasion and tube formation as well as indirectly by activating the promoter region of the vascular endothelial growth factor gene. conducted genome - wide expression studies in combination with gene - pathway analyses and cross - correlation to estrogen receptor- (er-) chromatin - binding sites, and demonstrated that runx2 expression was induced by er in human colorectal carcinoma cells. in the present study, our data found that runx2 significantly overexpressed in eoc tissues in contrast to normal ovarian tissues. we also showed that the eoc tissues with advanced clinical stage more frequently tend to express high runx2. these findings provide evidence that the up - regulation of runx2 expression might be important in the carcinogenesis of eoc. because of the poor prognosis in eoc patients, our study also focused on the prognostic implications of runx2 for this cancer. our survival analysis demonstrated that eoc patients whose tumors overexpressed runx2 had shorter overall and progression - free survival times than those with low runx2 expression tumors, especially those at advanced clinical stages. more importantly, further analysis using the cox regression model confirmed that the runx2 expression was an independent factor in predicting overall and progression - free survival times for eoc patients. these findings provide evidence that runx2 could be regarded as a biomarker for predicting the outcome of eoc patients. the function of runx2 is related with several molecules and multiple pathways, such as angiogenic factor, matrix metalloproteinases (mmps), transcription factors, the mapk pathway, the stat pathway and the pi3 k pathway, which are involved in ovarian carcinogenesis. for example, duan. indicated that the abnormal activation on pi3 k / pkb signaling pathway may be correlated with the occurrence and development of eoc ; chakrabarty and kondratick also suggested that activation on multiple cascades of the mapk pathway may promote the invasion and metastasis of eoc. in this study, although our results demonstrate the aberrant expression and important clinical significance of runx2 in eoc patients, the exact mechanism of runx2 upregulation in eoc is still not clearly understood. in this context, further studies are needed to determine the molecular mechanism of runx2 dysfunction in human ovarian carcinogenesis. in conclusion, our data suggest for the first time that runx2 overexpression is associated with advanced tumor progression and poor clinical outcome of eoc patients. | aim. to investigate clinical significance of runt - related transcription factor (runx)-2 in epithelial ovarian cancer (eoc). methods. runx2 protein expression and its subcellular localization were detected by immunohistochemistry in 116 patients with eoc. results. runx2 protein was predominantly expressed in cell nucleus of eoc tissues. the expression level of runx2 in eoc tissues was significantly higher than that in normal ovarian tissues (p < 0.001). in addition, the nuclear labeling index (li) of runx2 in tumor cells was significantly associated with the advanced clinical stage of eoc tissues (p = 0.001). moreover, eoc patients with high runx2 li had significantly shorter overall (p < 0.001) and progression - free (p = 0.002) survival than those with low runx2 li. especially, subgroup analysis revealed that eoc patients with high clinical stages (iii~iv) in high runx2 expression group demonstrated a significantly worse clinical outcome than those in low runx2 expression group, but patients with low clinical stages (i~ii) had no significantly different prognosis between high and low runx2 expression groups. conclusions. our data suggest for the first time that runx2 overexpression is associated with advanced tumor progression and poor clinical outcome of eoc patients. runx2 might be a novel prognostic marker of eoc. |
data were obtained from the international drug monitoring program of the world health organization (who). the who global individual case safety report (icsr) database, vigibase, is maintained by the uppsala monitoring centre and contains summaries of suspected spontaneous case reports originally summated by health care professionals and patients to national pharmacovigilance centers in 98 countries worldwide. as of may 2010, this database contained > 5 million case reports of suspected adrs regarding specific, but anonymous, patients. the reports contain administrative data, patient data, adr data, medication data, and additional information. the information in these reports is not homogenous, at least with regard to origin, completeness of documentation, or the likelihood that the suspected drug caused the adverse events (18). adrs are coded according to the adverse reaction terminology (who - art) and medical dictionary for regulatory activities (meddra ; www.who-umc.org). this study was designed as a nested case - control study. the base cohort consisted of all adrs associated with the use of any antidiabetic drug (anatomical therapeutic chemical [atc ] code a10), including oral antidiabetic drugs and insulins, in the period 1999 through 2009. infections were defined by means of meddra adverse reaction terms, including all relevant high - level group terms and lower - level terms. all infections from the system organ class (soc) infections and infestations and infections reported in other socs identified through a manual search were defined as cases. we grouped the infections on the first sublevel (high - level group terms) of meddra and looked at urti (e.g., sinusitis and nasopharyngitis), lower respiratory tract infections (lrti, e.g., bronchitis and pneumonia), and urinary tract infections (uti ; e.g., cystitis and pyelonephritis). antidiabetic drugs were subclassified based on the anatomical therapeutic chemical (atc) classification system of the who (www.whocc.no) : biguanides (atc code a10ba), sulfonylurea derivatives (a10bb), thiazolidinediones (a10bg), dpp-4 inhibitors (a10bh), insulins, and analogs (a10a). when multiple antidiabetic drugs were reported for a certain adr, this was classified as combination therapy, irrespective of whether a drug reported was classified as potential confounding factors retrieved from the case reports included age and gender of the patient, reporting year, reporting region (europe, north america, rest of the world), and reporter type, including physician, pharmacist, other caregiver, pharmaceutical company (indirectly obtained from a health care professional), and patient / consumer. concomitant use of medication affecting the immune system, defined as reporting one of these drugs as a concomitant drug for an adr, was taken into account when recorded, including antibiotics (atc code j01), corticosteroids for systemic use (h02), and immunosuppressants (l04). unconditional logistic regression analysis was used to estimate the strength of the association between use of antidiabetic drugs and reporting of infections and expressed as reporting odds ratios (rors) with corresponding 95% cis. owing to low numbers, combination therapy was analyzed on an aggregated level, not on an individual drug - drug combination level. we focused on infections in general and more specifically on the high - level group terms urti, lrti, and uti. more adrs were usually reported for one case report, and therefore it was possible that one case report contained more than one adr of an infection. to test the effect of multiple adrs reported in one case report, we analyzed the data on the level of case reports (one case report generally represented one patient). in the u.s., dpp-4 inhibitors are indicated for monotherapy for the treatment of diabetes mellitus, whereas in the eu, these medicines are only indicated for combination therapies. to check whether this affected the results of this study, we performed a sensitivity analysis by analyzing the data for the u.s. and the rest of the world separately. in addition, to study the effect of the type of reporter (health care professional or consumer) on the outcome, we performed a sensitivity analysis in which the reports were assessed according to the type of reporter. data were obtained from the international drug monitoring program of the world health organization (who). the who global individual case safety report (icsr) database, vigibase, is maintained by the uppsala monitoring centre and contains summaries of suspected spontaneous case reports originally summated by health care professionals and patients to national pharmacovigilance centers in 98 countries worldwide. as of may 2010, this database contained > 5 million case reports of suspected adrs regarding specific, but anonymous, patients. the reports contain administrative data, patient data, adr data, medication data, and additional information. the information in these reports is not homogenous, at least with regard to origin, completeness of documentation, or the likelihood that the suspected drug caused the adverse events (18). adrs are coded according to the adverse reaction terminology (who - art) and medical dictionary for regulatory activities (meddra ; www.who-umc.org). this study was designed as a nested case - control study. the base cohort consisted of all adrs associated with the use of any antidiabetic drug (anatomical therapeutic chemical [atc ] code a10), including oral antidiabetic drugs and insulins, in the period 1999 through 2009. infections were defined by means of meddra adverse reaction terms, including all relevant high - level group terms and lower - level terms. all infections from the system organ class (soc) infections and infestations and infections reported in other socs identified through a manual search were defined as cases. we grouped the infections on the first sublevel (high - level group terms) of meddra and looked at urti (e.g., sinusitis and nasopharyngitis), lower respiratory tract infections (lrti, e.g., bronchitis and pneumonia), and urinary tract infections (uti ; e.g., cystitis and pyelonephritis). antidiabetic drugs were subclassified based on the anatomical therapeutic chemical (atc) classification system of the who (www.whocc.no) : biguanides (atc code a10ba), sulfonylurea derivatives (a10bb), thiazolidinediones (a10bg), dpp-4 inhibitors (a10bh), insulins, and analogs (a10a). when multiple antidiabetic drugs were reported for a certain adr, this was classified as combination therapy, irrespective of whether a drug reported was classified as potential confounding factors retrieved from the case reports included age and gender of the patient, reporting year, reporting region (europe, north america, rest of the world), and reporter type, including physician, pharmacist, other caregiver, pharmaceutical company (indirectly obtained from a health care professional), and patient / consumer. concomitant use of medication affecting the immune system, defined as reporting one of these drugs as a concomitant drug for an adr, was taken into account when recorded, including antibiotics (atc code j01), corticosteroids for systemic use (h02), and immunosuppressants (l04). unconditional logistic regression analysis was used to estimate the strength of the association between use of antidiabetic drugs and reporting of infections and expressed as reporting odds ratios (rors) with corresponding 95% cis. owing to low numbers, combination therapy was analyzed on an aggregated level, not on an individual drug - drug combination level. we focused on infections in general and more specifically on the high - level group terms urti, lrti, and uti. more adrs were usually reported for one case report, and therefore it was possible that one case report contained more than one adr of an infection. to test the effect of multiple adrs reported in one case report, we analyzed the data on the level of case reports (one case report generally represented one patient). in the u.s., dpp-4 inhibitors are indicated for monotherapy for the treatment of diabetes mellitus, whereas in the eu, these medicines are only indicated for combination therapies. to check whether this affected the results of this study, we performed a sensitivity analysis by analyzing the data for the u.s. and the rest of the world separately. in addition, to study the effect of the type of reporter (health care professional or consumer) on the outcome, we performed a sensitivity analysis in which the reports were assessed according to the type of reporter. from the who vigibase we identified 305,415 suspected adrs related to the use of antidiabetic drugs in 106,469 case reports in the study period 1999 through 2009. patients were a mean age of 59.7 years (sd, 14.3) and 59.6% were women. a total of 288,434 reports (94.4%) reports involved one antidiabetic drug, 14,057 (4.6%) reported a combination of two antidiabetic drugs, and 2,924 (1.0%) reported three or more antidiabetic drugs (table 1). overall, the most commonly reported infections on the level of meddra lower - level terms were pneumonia (11.8%), nasopharyngitis (10.1%), utis (6.2%), infection not otherwise specified (5.5%), sinusitis (5.1%), and bronchitis (4.8%). all other types of infection were reported in < 4.5% of the reports related to infections. baseline characteristics of all spontaneous reports for antidiabetic drugs in the who vigibase (19992009) all data except patient age are shown as number (%). a total of 242 infections were reported as a meddra term in 212 case reports for dpp-4 inhibitors, of which 188 (88.7%) reported one infection. of the 24 case reports (11.3%) with multiple infections, 12 (50%) reported a nonspecific infection term, such as infection or urti, combined with a more specific infection term, such as nasopharyngitis or cystitis (see the supplementary data for a summary of the infections according to the meddra lower - level term). table 2 reports the rors of infections (overall) per antidiabetic drug compared with biguanides. the use of dpp-4 inhibitors as monotherapy (ror 2.3 [95% ci 1.92.7 ]), insulins as monotherapy (ror 1.6 [95% ci 1.41.8 ]), and the combination of any oral antidiabetic drug and insulin therapy (ror 1.8 [95% ci 1.32.4 ]) were all statistically significant associated with adr reports of infections compared with biguanides. a slightly increased reporting of infections for patients using thiazolidinediones was found (ror 1.2 [95% ci 1.11.4 ]), but no increased reporting of infections was found for sulfonylurea derivatives (ror 1.2 [95% ci 1.01.4 ]), combination therapy of two oral antidiabetic drugs (ror 1.0 [95%ci 0.81.2 ]), or for concomitant use of three or more antidiabetic drugs (ror 1.0 [95% ci 0.71.4 ]). adjustment for the potential confounding factors did not affect the results. crude and adjusted rors for any infection ads, antidiabetic drugs ; dpp-4 i, dipeptidyl peptidase inhibitors ; oad, oral antidiabetic drug ; su, sulfonylurea ; tzd, thiazolidinediones. adjusted for age, sex, reporting year, reporting region, reporter type, and comedication affecting the immune system : antibiotics (j01), corticosteroids for systemic use (h02), and immunosuppressants (l04). assessment of different types of infections (table 3) showed an increased reporting of urti (ror 12.3 [95% ci 8.617.5 ]) for the dpp-4 inhibitors, whereas the reporting for lrti, uti, and other infections was not increased. in addition, an increased reporting of urti was found for users of thiazolidinediones (ror 2.3 [95% ci 1.73.3 ]), and for concomitant use of three or more antidiabetic drugs (ror 2.5 [95% ci 1.34.7 ]). slightly increased rors were found for the use of insulin monotherapy and the reporting of urti (ror 1.5 [95% ci 1.12.2 ]) and uti (ror 1.7 [95% ci 1.12.5 ]). the reporting of lrti (ror 1.8 [95% ci 1.42.3 ]) and other infections (ror 1.7 [95% ci 1.42.0 ]) was also increased for insulin monotherapy. for the combination of an oral antidiabetic drug and insulin, increased reporting of uti (ror 3.5 [95% ci 1.77.2 ]) and other infections (ror 1.7 [95% ci 1.12.6 ]) was noted. crude rors for specific infections ads, antidiabetic drugs ; dpp-4 i, dipeptidyl peptidase inhibitors ; oad, oral antidiabetic drug ; su, sulfonylurea ; tzds, thiazolidinediones. the crude ror was lower for dpp-4 inhibitors but was still significantly increased (ror 1.6 [95% ci 1.31.9 ]) in the analysis of the data on the level of case reports. the sensitivity analyses showed that the country from which the case reports originates and the type of reporter did not have a major effect on the results (data not shown). the point estimates changed only slightly, but because of the decreased numbers the confidence intervals became wider this study showed that infections were approximately two times more frequently reported for dpp-4 inhibitors compared with biguanides in the who vigibase. in particular, urtis, including nasopharyngitis and sinusitis, were reported more frequently for dpp-4 inhibitors, although the reporting of urti was also increased for users of thiazolidinediones, insulin monotherapy, and concomitant use of three or more antidiabetic drugs, but to a much lesser extent than for the dpp-4 inhibitors. a hypothesis resulting from the current study is that the effect of dpp-4 inhibitors results in a slight imbalance of the immune system that causes an increased risk of common, less severe infections such as (viral) upper respiratory infections. this is supported by the results of the pivotal randomized clinical trials that also reported increased numbers of common infections rather than serious infections (13). as far as we are aware, no studies reporting serious infections in association with the use of dpp-4 inhibitors, the magnitude of the effects of dpp-4 inhibitors on the immune system may not be compared with the magnitude of the effects as seen, for example, with biologic agents, resulting in rather serious infections such as tuberculosis or histoplasmosis due to tumor necrosis factor- antagonists (19,20). with the current data, however, it was not possible to further differentiate between infections of different nature and viral, bacterial, or fungal causes. the strength of this study is that the who vigibase allows studying the association between use of antidiabetic drugs and infections outside the highly controlled environment of clinical trials. nevertheless, some limitations of this study need to be addressed : first, besides the known issue of underreporting in spontaneous reporting systems (21), the reporting pattern of adrs may differ between new and old drugs, with the most vigorous monitoring at the time of marketing and shortly thereafter, as described by weber (21). the dpp-4 inhibitors and thiazolidinediones were introduced in the study period (1999 through 2009), which might explain the relatively large number of reports for those drugs. however, it is unknown whether the type of adrs that are reported changes over time and how this affects the results of this study. nonetheless, second, the results of this study may be subject to reporting bias, because infections are listed in both the eu and u.s. this may have led to differential monitoring and reporting of infections for the dpp-4 inhibitors compared with other antidiabetic drugs. increased reporting (physicians report adrs that are likely to occur) or decreased reporting (physicians do not report adrs that are already mentioned in summaries of product characteristics) might have occurred. in absolute terms, the number of reported infections is low : only 3% of the cases for dpp-4 inhibitors reports involved an infection (242 reports of infection of 8,083 case reports). furthermore, differences in classification strategies or misclassification may have occurred by the translation from clinical terminology to the classification systems used by the who vigibase (who - art or meddra terms). we do not expect, however, that this misclassification is different for different antidiabetic medicines, and the effect of this nondifferential misclassification on the results of this study will therefore be limited. in several case reports, more than one infection term was recorded, mainly consisting of a specific infection term (e.g., nasopharyngitis) and an a - specific term (e.g., urti), which might influence the results. however, our analysis at the case - report level showed that the overall results did not change. unfortunately, we were not able to analyze the risk of infections for combination therapy on an individual drug - drug combination level owing to the low number of cases reporting each possible combination therapy. finally, we reasoned that the different indications for the dpp-4 inhibitors in the u.s. versus the rest of the world, and a possible effect of the type of reporter (health care professional, consumer, or industry) on the case reports, might have influenced the results of this study. however, excluding reports from the u.s. and excluding reporting from consumers and industry did not affect the results. another explanation for our results can be that diabetes itself, and its progression, are often associated with an increased risk of infections. although the literature is not yet conclusive on this, we could not exclude this possible association ; therefore, the current study was limited to case reports of antidiabetic medicines only, thereby eliminating the effect of the disease itself. some studies, however, suggest that more severe diabetes itself is also associated with higher risk of infections (14,22), although this is not supported by strong evidence. because disease severity is therefore possibly associated with both exposure and outcome, this can be a confounding factor. dpp-4 inhibitors are indicated as a second- or third - line therapy in combination with other oral antidiabetic drugs according to treatment guidelines in different parts of the world (23,24). therefore, patients who are treated with dpp-4 inhibitors may in general be more severely ill compared with patients being treated with, for example, biguanides or sulfonylurea derivatives. in the current study, however, most of the case reports indicated a dpp-4 inhibitor was the only antidiabetic drug, which indicates monotherapy with dpp-4 inhibitors. a question prompted by this study is whether the patients treated with dpp-4 inhibitors monotherapy were indeed the more severely ill patients, because one antidiabetic drug was sufficient for these patients. in addition, for users of a combination of an oral antidiabetic and insulin or combination therapy of two antidiabetics (i.e., usually the more severely ill patients), we did not found an increased risk of infections. this is in line with the studies that did not find an association between diabetes mellitus and infection. besides infections in general, the association between diabetes and uti has also been described (12,14,15,22), although we did not find significantly increased reporting of adrs for utis and use of any of the antidiabetic drugs. only for combination therapy of an oral antidiabetic and insulin did we find a slightly increased reporting for uti. channeling of this combination therapy toward the more severely ill diabetes patients, for which the use of insulin combined with an oral antidiabetic drug is a marker (25), may possibly explain this finding. this is in line with studies suggesting that the severity of the disease may play a role in the occurrence of uti (14,22). unfortunately, the subset of the vigibase used in this study did not contain information on the severity of the underlying disease, so the effect of this phenomenon in the current study remains unclear. postmarketing evaluation of safety concerns raised during the preregistration phase of medicines is import for the assessment of the benefit - risk balance of drugs. this study adds to the knowledge of this specific safety issue for the dpp-4 inhibitors. the results of the study, using data reported to national pharmacovigilance centres, are in line with the findings from the clinical trial program that included a much more selected patient population. however, more research is needed to further evaluate the clinical and regulatory consequences of this finding, such as severity of the infections. as a result of the observed increased risk in rcts, the risk management plans for dpp-4 inhibitors also address a possible increased risk of infections. the definition of the outcome (infection) in the postauthorization safety studies that are being conducted as part of the risk management plans is, therefore, of particular importance. because our study points out that several types of infections (urtis such as sinusitis and nasopharyngitis) are more frequently reported than others, the outcome should not be limited to infections in general but should also take specific types of infections into account. in addition, the nonserious infections seem to be neglected in the postauthorization safety studies because all studies aim to investigate the risk of serious infections (13). although from a regulatory viewport the focus on serious infections is understandable, the effect of (recurrent) nonserious infections on the quality of life can also be considerable. in conclusion, the results of this study show that there is an increased reporting of infections for users of dpp-4 inhibitors compared with users of other antidiabetic drugs. although the limitations of spontaneous reporting systems (e.g., underreporting, the weber -effect, reporting bias) should be taken into account, physicians and patients nevertheless should remain vigilant on the occurrence of infections and continue to report infections as possible adrs. infections may be related to diabetes, but a direct effect of the medication on the occurrence of infections should be considered. | objectivedipeptidyl peptidase-4 (dpp-4) inhibitors are a new class of antidiabetic drugs. they inactivate incretin hormones but also have many other effects throughout the body, among which are effects on the immune system. this might result in an increased infection risk. this study assessed the association between use of dpp-4 inhibitors and the reporting of infections.research design and methodsa nested case - control was conducted using vigibase, the world health organization - adverse drug reactions (who - adr) database. the base cohort consisted of adrs for antidiabetic drugs (anatomical therapeutic chemical code a10). cases were defined as adrs of infection according to the medical dictionary for regulatory activities (meddra) classification system. all other adrs were considered controls. reporting odds ratios (rors) were calculated to estimate the strength of the association between different classes of antidiabetic drugs and the reporting of infections.resultswe identified 305,415 suspected adrs involving antidiabetic drugs in 106,469 case reports, of which 8,083 involved dpp-4 inhibitors monotherapy. overall, the reporting of infections was higher for patients using dpp-4 inhibitors compared with users of biguanides (ror 2.3 [95% ci 1.92.7 ]). reporting of upper respiratory tract infections (ror 12.3 [95% ci 8.617.5 ]) was significantly associated with use of dpp-4 inhibitors.conclusionsthis study indicates an increased reporting of infections, in particular upper respiratory tract infections, for users of dpp-4 inhibitors compared with users of other antidiabetic drugs. however, the limitations of spontaneous reporting systems (e.g., underreporting, the weber - effect, reporting bias) should be taken into account. therefore, further research is needed to evaluate this suspicion and the underlying mechanism. |
obesity is one of the leading causes of overall morbidity and mortality in western societies and the prevalence of obesity continues to increase worldwide. it is quickly approaching pandemic proportions, currently afflicting nearly 100 million americans, with deleterious public health consequences. recent studies estimated that in 2005 the global population contained 937 million (922951 million) overweight and 396 million (388405 million) obese adults, respectively. by 2030, the respective number of overweight and obese adults is projected to be at least 1.35 billion and 573 million individuals. metabolic syndrome (mets) is a clustering of abnormalities that includes obesity, arterial hypertension, dyslipidemia, and insulin resistance. since the original publication of the national cholesterol education program (ncep) adult treatment panel iii (atp iii) diagnostic criteria for mets, two independent studies indicated that more than 1 in 5 adults in the us population meet these diagnostic criteria [6, 7 ]. prevalence of mets increases dramatically from 5%6% in normal - weight (body mass index (bmi) 25 kg / m) adults and 50%60% in obese (bmi 30 kg / m) individuals. mets is associated with an increased risk for developing cardiovascular disease and type 2 diabetes mellitus (t2 dm). a meta - analysis of 21 studies demonstrated that mets is associated with an increase in the risk of death from coronary heart disease (chd) (relative risk (rr) 1.4 ; 95% confidence interval (ci) 1.2 to 1.6) and overall mortality (rr 1.7 ; 95% ci 1.3 to 2.4). augmented oxidative stress appears to be an important component of both mets and atherosclerosis and may be an important pathway linking mets to the increased incidence of chd and t2 dm [11, 12 ]. oxidative stress markers are elevated in subjects with established chd and are associated with obesity and mets. it was reported that oxidative stress was augmented in otherwise healthy obese subjects with mets compared with bmi - matched obese individuals without mets. oxidative stress is generally considered to be a balance between the production of reactive oxidant species (ros) and antioxidant capacity. it has been previously reported that antioxidants such as vitamin e, beta - carotene, lycopene, and polyphenolic flavonoids can be associated with various lipoproteins, including low - density lipoprotein (ldl), in the circulation. the impact of these circulating antioxidants on oxidative stress is a topic of ongoing research. circulating oxidize ldl (ox - ldl) is generally believed to be proatherogenic, in part because of its lower affinity for the ldl receptor ; instead, it is recognized by a variety of scavenger receptors. uptake of ox - ldl by these scavenger receptors leads to accumulation of ox - ldl within the foam cells of atherosclerotic lesions, where ox - ldl induces endothelial activation and smooth muscle proliferation [1520 ]. plasma ox - ldl levels were found to be significantly higher in obese individuals with mets [13, 21 ]. in the atherosclerosis risk in communities (aric) study, circulating ox - ldl levels were adversely associated with individual mets components and increased risk of t2 dm. the atherosclerosis and insulin resistance (air) study reported that ox - ldl levels were associated with both subclinical atherosclerosis, as assessed by ultrasonography, and inflammatory markers, including c - reactive protein (crp) and tumor necrosis factor- tnf-. oxidative modification of ldl in the arterial wall is a complex process involving a number of biological pathways. oxidation can occur through many mechanisms including nadph oxidases, myoglobin, ros from the mitochondrial electron transport chain, hemoglobin, oxygenases, and peroxidases, among others. lipoprotein - associated phospholipase a2 (lp - pla2) catalyzes hydrolysis of the sn-2 ester bond of phospholipids in cellular membranes and lipoproteins (e.g., ldl) in settings of high oxidative stress [25, 26 ]. particularly in ox - ldl, in which the phospholipids have undergone extensive oxidative modification, the action of lp - pla2 results in the release of oxidized nonesterified fatty acids (fa) and lysophosphatidylcholine (lyso - ptdcho). lyso - ptdcho and oxidized nonesterified short - chain fa are highly proinflammatory lipid mediators which can stimulate macrophage proliferation, increase the expression of vascular adhesion molecules, and upregulate cytokines. lp - pla2 is produced by macrophages, and its production is regulated by inflammatory cytokines. in the circulation, 80% of the enzyme is bound to ldl particles, 1015% to high - density lipoprotein (hdl), and the remaining amount to very low density lipoprotein (vldl). small dense ldl particles (sdldl) are enriched in lp - pla2 compared to large buoyant ldl, and lp - pla2 activity is increased in sdldl [29, 30 ]. lp - pla2 was recently characterized as a novel inflammatory biomarker correlated with several components constituting mets and implicated in atherosclerosis and incident cardiovascular disease [31, 32 ]. myeloperoxidase (mpo) is an enzyme generally believed to be linked to oxidative stress. it is released by leukocytes in a state of inflammation and catalyzes the formation of ros which play an important role in host defense against microorganisms. on the other hand, various ros produced by mpo are unstable and promote oxidative modifications of ldl. in particular mpo - derived hocl modifies apolipoprotein b (apob), which is accompanied by oxidation of the lipid moiety of ldl and results in the formation of chloramines. the ldl - associated chloramines alter the charge characteristics of ldl particles, leading to the uncontrolled uptake of the modified ldl by macrophages. according to previous studies, antioxidant protection by vitamin e, carotenoids, and lycopene is susceptible to hocl - mediated ldl oxidation only at relatively high concentrations of hocl. interestingly, in vitro supplementation of vitamin e does not protect ldl against hocl - mediated modification ; it may even enhance the oxidation. vitamin e does not provide effective antioxidant protection against mpo - mediated oxidation of ldl. the only antioxidant able to defend against mpo - mediated ldl oxidation appears to be vitamin c, which scavenges hocl and regenerates amines from hocl - derived chloramines. vitamin c also protects ldl against lipid peroxidation initiated by mpo - derived tyrosyl radicals. apolipoprotein a - i present on hdl is a selective target for mpo - catalyzed oxidation, converting the lipoprotein into a dysfunctional form., in the european prospective investigation into cancer and nutrition (epic - norfolk) prospective population study, found a positive association of mpo levels with the risk of future chd in healthy individuals. we have previously shown that moderate acute weight loss of 57% with a very low calorie diet (vlcd) in obese subjects with mets was associated with dramatic improvement in all the clinical components of mets, even though the individuals remained markedly obese. as a part of this ongoing study, we focus on oxidative stress, as assessed by the measurement of circulating oxidative stress markers, and investigate the relationship between mets and oxidative stress in obese patients enrolled in a rapid weight loss program. obese individuals (56 women and 24 men, aged 47.1 0.9 years, bmi 38.3 0.7 kg / m) enrolled in a medically supervised rapid weight loss program and 20 lean controls (lc) were recruited for this study during a 12-month period (september 2001september 2002). the study protocol was approved by the baylor college of medicine institutional review board and written informed consent was obtained from each individual. the subjects were classified as mets positive (mets+) if they met 3 or more of the ncep atp iii criteria : waist circumference > 40 in (102 cm) for men or > 35 in (88 cm) (original criteria in inches, here listed with centimeter equivalents) for women, triglycerides (tg) 1.69 mmol / l (150 mg / dl), hdl - c < 1.03 mmol / l (40 mg / dl) for men or < 1.29 mmol / l (50 mg / dl) for women, blood pressure 130/85 mm hg or treated hypertension, and fasting glucose 6.1 mmol / l (110 mg / dl). however, for subsequent analysis in the current study, we adapted the diagnostic criteria for the mets revised in 2005, which redefines the fasting glucose criterion as 5.6 mmol / l (100 mg / dl) (see supplemental table 1 in supplementary material available online at http://dx.doi.org/10.1155/2013/729515). sequentially enrolled mets+ subjects (n = 40) were compared with 40 obese subjects matched for age and bmi without mets (mets). the study participants did not receive any medication known to affect glucose tolerance, insulin secretion, or insulin sensitivity during the active weight loss period. exclusion criteria were known eating disorder, cancer, use of lithium or corticosteroids, type 1 diabetes, active inflammatory bowel disease, active gout, liver disease, cardiovascular event within the past 3 months, endocrine causes of obesity, and pregnancy. the weight loss protocol description is as follows : the weight loss program was offered as a weekly follow - up commitment in an outpatient setting at a tertiary - care medical center site (the methodist hospital, houston, tx, usa) to individuals either self - referred or referred by health - care professionals. weight reduction was induced by a protein - sparing vlcd of approximately 800 kcal daily consisting of liquid beverages (nutrimed - plus, robard corp., mount laurel, nj, usa ; each serving contains 200 kcal, 6 g of fat, 26 g of protein, and 10 g of carbohydrate) alone or in combination with lean beef, fish, or poultry and daily fluid intake at a minimum of 2 l. study participants were encouraged not to use any over - the - counter dietary supplements while on the vlcd since the vlcd provided 140% of daily value (% dv) for all essential vitamins (including antioxidant vitamins), minerals, and trace elements. the mets+ individuals had scheduled follow - up visits at 46 weeks (visit 2) and 1220 weeks (visit 3) after the initial baseline evaluation. six subjects (3 men and 3 women) did not return for their final follow - up visit at visit 3. we used a multiple imputation method, a combination of mean and regression imputation, to fill in data for the missing variables on the final visit or these 6 individuals. anthropometric and biochemical measurements are as follows : all subjects were evaluated with a series of anthropometric measurements and tests for hematology, biochemistry, and hormonal functions after an overnight fast. biochemical measurements were performed at baseline for all 80 obese individuals and 20 lc and prospectively for the 40 mets+ subjects in visits 2 and 3 of active weight loss. all biochemical measurements were performed on frozen plasma samples obtained by centrifugation of freshly drawn blood (3000 g for 20 minutes at 4c) and subsequent storage at 70c. blood lipid profiles, including total cholesterol (tc, hdl cholesterol (hdl - c), calculated ldl cholesterol (ldl - c), tg), and nonesterified fa, as well as plasma -glutamyltransferase (ggt) concentrations, were determined by enzymatic assays using a hitachi 911 autoanalyzer. lp - pla2 activity was determined by colorimetric activity method using 1-myristoyl-2-(4-nitrophenylsuccinylphosphatidylcholine) as the enzyme substrate according to manufacturer 's protocol (diadexus, inc. plasma levels of ox - ldl were measured by a direct sandwich enzyme - linked immunoassay (mercodia ab, uppsala, sweden), which uses the monoclonal capture antibody mab-4e6 and a monoclonal detection antibody directed against a different epitope of the oxidized apob molecule. plasma concentrations of mpo were determined by direct sandwich enzyme - linked immunoassay according to manufacturer 's protocol (oxis research, portland, or, usa). plasma levels of insulin were determined on a luminex-100 multianalyte profiling system using commercially available immunoassay panels (linco research, inc., st. charles, mo, usa). measures of insulin resistance were obtained using the homeostasis model assessment of insulin resistance (homa - ir = fasting glucose (mg / dl) fasting insulin (u / ml)/22.5). statistical analyses were performed using stata version 11 (stata, college station, tx, usa). differences in baseline characteristics between groups (mets+, mets, lc) were tested using one - way analysis of variance (anova) and post hoc tests for differences between groups, fisher 's exact test, or the kruskal - wallis test when the data were not normally distributed. mpo, glucose, insulin, homa - ir, and tg were log - transformed to account for their right - skewed distributions. paired t - tests and wilcoxon matched - pairs signed - rank tests were applied to assess changes in ox - ldl, mpo, lp - pla2 activity, and other metabolic factors among mets+ subjects before and after weight loss. in order to maximize the data for analysis, any missing data points for certain metabolic factors were filled in using mean and regression imputation methods. multivariate linear regression and pearson 's correlation analysis were used to assess the independent effects of change in ox - ldl, mpo, lp - pla2 activity before and after the weight loss intervention. percent changes were applied for the above parameters. the efficacy of the ox - ldl, mpo, and lp - pla2 activity tests in distinguishing groups of mets+ versus mets was assessed by using logistic regression and receiver operating characteristic (roc) analysis. average age of our study participants was 46 years (range, 3071 years) at the time of recruitment. however, obese male participants were more likely to have mets compared to obese female participants. the lc - group had normal weight and bmi, normal systolic blood pressure (sbp), diastolic blood pressure (dbp), normal glucose and insulin levels, and normal lipid profiles. compared to the lc - group, mets and mets+ patients had significantly higher sbp and dbp, higher levels of glucose, insulin, and homa - ir values. the mets+ group had significantly higher levels of tg and lower levels of hdl when compared to the lc - group ; mets subjects did not differ significantly from the lc - group in regard to their lipid profile. the distribution of mets criteria among the participants based on the ncep atp iii criteria is shown in figure 1 and the percentage of participants in each group meeting the specific criteria for mets based on the more recent aha criteria is shown in figure 2. in our study cohort, obese study participants were less likely to have elevated fasting blood glucose than any of the other mets criteria. at baseline mets+ patients had significantly higher plasma ox - ldl levels when compared to the mets - group (64.26 2.2 versus 57.08 1.5 u / l, p = 0.010) and lc - group (64.26 2.2 versus 52.21 3.0 u / l, p = 0.002), whereas circulating levels of mpo were significantly higher in obese patients (with or without mets) when compared with the lc - group (330.79 45.0 and 375.28 65.1 versus 172.72 22.0 pm, p = 0.015). although lp - pla2 activity was the highest in the mets+ group, we did not find significant differences between groups at baseline (table 1). baseline correlations (pearson 's regression) between oxidative stress markers and mets components revealed that ox - ldl and lp - pla2 activity were adversely associated with cardiovascular lipid risk factors and showed a strong positive correlation with each other, while plasma mpo was associated with various measures of obesity and insulin resistance (table 2). we investigated the efficacy of the different oxidative stress markers to determine mets status in obese individuals (figure 3). using logistic regression, we calculated that the odds ratio (or) for mets as predicted by ox - ldl was 1.06 (95% ci : 1.0131.099, p = 0.010), indicating that for each 1 u / l increment in ox - ldl level the odds of having mets increased by 6%. roc analysis showed that the area under the roc curve (auc) for ox - ldl was 0.666. the or for mets as predicted by lp - pla2 activity was 1.02 (95% ci : 0.9991.036, p = 0.051) and the auc for lp - pla2 activity was 0.638. in contrast, mpo levels did not predict mets status (or = 1.00 ; 95% ci : 0.9981.001, p = 0.548). multivariate logistic regression analysis showed that a model including ox - ldl, lp - pla2 activity, and mpo improved prediction of mets status among obese individuals compared to each oxidative stress marker alone (auc = 0.701, likelihood ratio test, p = 0.026). although mpo did not predict mets status alone, it did add incrementally to the auc of a model including ox - ldl and lp - pla2 activity. the group of mets+ subjects on the vlcd reached an average weight loss of 16.9 kg and a 5.6 kg / m decrease in bmi, which coincided with a 10% reduction in waist circumference (table 3). furthermore, mets+ subjects showed a marked improvement in cardiometabolic risk factors, including blood pressure, lipid risk factors, fasting glucose, and insulin resistance following diet - induced weight loss. vlcd resulted in a reduction of ox - ldl levels from 64.3 to 54.7 u / l. this 12% reduction (p < 0.001) was associated with a decrease in tc, even after adjustment for age and sex (p = 0.019). dietary caloric restriction also led to a decrease in lp - pla2 activity by 4.7% from 136.6 to 127.7 nmol / ml / min (p = 0.024). the changes in ox - ldl and lp - pla2 activity associated with weight loss were positively correlated with changes in tc (figures 4(a) and 4(b)) and ldl - c (figures 5(a) and 5(b)). rapid weight loss resulted in a 15% decrease in plasma mpo levels, although this reduction did not reach significance. when we investigated the relationship between oxidative stress markers and glucose metabolism following rapid weight loss, we found a significant correlation of mpo with glucose (pearson 's r = 0.4317, p = 0.0054). in the current study, we confirmed findings from our previous report showing a significant improvement of metabolic factors following weight loss in subjects with mets. in addition, we found that baseline levels of individual oxidative stress markers did not consistently define mets status. however, the combination of the three oxidative stress markers (i.e., ox - ldl, lp - pla2 activity, and mpo) significantly improved prediction of mets status in our study cohort. although lp - pla2 activity was the highest in the mets+ group, we did not find significant differences between groups at baseline. baseline ox - ldl levels were significantly higher in mets+ obese individuals when compared to mets obese individuals and lc. this finding is in agreement with results from a previous study that reported elevated circulating ox - ldl levels in patients with mets, whereas ox - ldl levels did not differ between obese mets individuals and lc. we found that baseline mpo levels were significantly higher in obese individuals when compared with lc irrespective of presence of mets, which suggests that mpo may reflect an increased oxidative stress linked to obesity. taken together these findings indicate that an elevated oxidative stress status is present in obese individuals and particularly in those individuals with mets. there is a paucity of data from clinical studies investigating the role of oxidative stress in obesity and the effects of weight loss on oxidative stress status in metabolically healthy and metabolically abnormal individuals.. found a significant reduction of lp - pla2 activity after low - calorie diet associated weight loss in healthy obese women. on the other hand, results of hanusch - enserer., who examined the effect of weight loss after gastric banding surgery on cardiovascular risk factors, reported the lp - pla2 levels remained unchanged. since diet - induced weight loss also resulted in a significant reduction of lp - pla2 activity in our study, the combined data from these studies suggest that different modes of therapeutic intervention to achieve weight loss may lead to differing effects on oxidative stress status. investigated the influence of energy intake - restricted weight loss on endothelium - dependent artery dilation in overweight or obese nondiabetic men and women. shin. also reported a decrease in circulating ox - ldl levels following diet - induced weight loss in metabolically abnormal obese individuals, although they found no reduction of ox - ldl levels in a group of healthy obese subjects. the fact that weight loss resulted in a significant reduction of circulating ox - ldl levels in obese mets+ subjects in our study was in general agreement with the aforementioned studies. studying the effects of a 21-day diet and exercise intervention in men with mets, roberts and coworkers noted significant improvements in lipid risk factors and homa - ir as well as a reduction in circulating mpo levels. although differences in study population, dietary intervention, and time of follow - up exist between this study and ours, the general findings from both studies related to the effect of weight loss on metabolic parameters of lipid, glucose, and oxidative stress are similar in nature. in summary, weight loss induced by dietary caloric restriction reduces oxidative stress concurrent with an improvement in the clinical components of mets in these individuals. obese individuals with mets are in a state of hyperglycemia and chronic low - grade inflammation, conditions that promote increased production of ros resulting in increased oxidative stress. our data suggest that vlcd induced weight loss results in an improvement in glucose metabolism and may lead to decreased inflammation as a result of decreased adiposity and adipokine / cytokine secretion, thereby reducing the oxidative stress in these individuals. a limitation of our study is that we did not directly measure antioxidant capacity in study participants prior to or during the study. although it is unlikely that study participants would experience decreased antioxidant capacity during the vlcd intervention given more than adequate dietary intake of antioxidant vitamins (140% dv), we can not rule out that the 140% dv of antioxidant vitamins could have enhanced the antioxidant capacity in individuals who may have had an insufficient antioxidant capacity thereby contributing to decreased levels of oxidative stress markers. dietary caloric restriction resulted in a significant decrease in surrogate measures of body fat (i.e., bmi and waist circumference) in obese individuals. however, we did not investigate the effects of energy restriction on oxidative stress independent of changes in body fat mass in lean individuals. future studies are needed to address the utility of oxidative stress markers as not only predictors of mets in obesity but as markers of effective treatments that target cardiometabolic risk in the obese population. | objective. obesity is linked with a state of increased oxidative stress, which plays an important role in the etiology of atherosclerosis and type 2 diabetes mellitus. the aim of our study was to evaluate the effect of rapid weight loss on oxidative stress markers in obese individuals with metabolic syndrome (mets). design and methods. we measured oxidative stress markers in 40 obese subjects with metabolic syndrome (mets+), 40 obese subjects without metabolic syndrome (mets), and 20 lean controls (lc) at baseline and after three months of very low caloric diet. results. oxidized low density lipoprotein (ox - ldl) levels decreased by 12% in mets+ subjects, associated with a reduction in total cholesterol (tc), even after adjustment for age and sex. lipoprotein associated phospholipase a2 (lp - pla2) activity decreased by 4.7% in mets+ subjects, associated with a drop in ldl - cholesterol (ldl - c), tc, and insulin levels. multivariate logistic regression analysis showed that a model including ox - ldl, lppla2 activity, and myeloperoxidase (mpo) improved prediction of mets status among obese individuals compared to each oxidative stress marker alone. conclusions. oxidative stress markers were predictive of mets in obese subjects, suggesting a higher oxidative stress. rapid weight loss resulted in a decline in oxidative stress markers, especially in mets+ patients. |
the prevalence of spontaneous abortion is about 12 percent which increases in next pregnancy [35 ]. threatened - abortion occurs in 3040 percent of pregnancies and it is identified as vaginal spotting or bleeding under 20 weeks of pregnancy. the semiallogenic fetus antigens are in direct contact with the mother 's immune system during pregnancy ; therefore, immune suppression has a pivotal role in the maintenance of embryo. hla - g is a nonclassical hla - ib which has an important role in immune tolerance. this molecule has seven isoforms including hla - g1, hla - g2, hla - g3, hla - g4, hla - g5, hla - g6, and hla - g7, where hla - g1, hla - g2, hla - g3, and hla - g4 isoforms are surface molecules and hla - g5, hla - g6, and hla - g7 isoforms are soluble. in addition, soluble hla - g1 originated from surface hla - g1 by metalloproteinase protein activity [10, 11 ]. in physiologic conditions, expression of hla - g is restricted to fetal trophoblasts at the maternal - fetal interface, cornea, thymic epithelium, pancreatic islets, nail matrix, and the erythroid and endothelial precursors [1, 12 ]. this molecule is also expressed in malignant transformation, autoimmune and inflammatory diseases, transplantation, and infectious diseases [1, 11 ]. it has been proven that hla - g plays an important role in pregnancy. during pregnancy, these molecules can react with nk cells and t cell inhibitory receptors ; thus, it can protect the fetal trophoblast cells from maternal uterine natural killer cells invasion. totally, human natural killer (nk) cells include 1015 percent of circulating lymphocytes and are described phenotypically via their expression of cd56 and lack of cd3 expression. peripheral blood nk cells can be divided into two major subsets based on neural cell adhesion molecule cd56 (ncam) expression including cd56 and cd56 nk cells. cytolytic response is mostly restricted to the cd56 subset, while cytokine production is usually allocated to cd56 cells [13, 14 ]. based on peripheral blood nk cells level of cd56 expression and lack of cd3, nk cells can be divided into two major subsets. the two groups are defined as cd56 and cd56 nk cells. on the other hand, unk cells are defined with high expression of cd56 (cd56) and lake of cd16 (cd-16) and so they are phenotypically and functionally different from peripheral blood nk cells. nk cells could play an important physiological role in preventing fetal trophoblast invasion and also these cells under circumstances could react with trophoblast cells, leading to abortion. in this study, we evaluated hla - g1 and hla - g5 genes expression and unk cells percentage in vaginal discharge of threatened - abortion women in comparison with normal pregnant women. in a case - control study, 30 threatened - abortion women and 30 normal pregnant women with the age range of the 1942 years participated in the study (table 1). the criteria for threatened - abortion women had been light spotting or vaginal bleeding before 20 weeks of pregnancy. on the other hand, diabetes, hyper- and hypothyroidism, immunological disorders, anatomic anomalies, and microbial infections, such as toxoplasmosis, rubella, and chromosomal abnormalities, were exclusion criteria of this study. these items were evaluated using patients ' history, check - ups, and periodic examinations and collecting information through questionnaires approved by a gynecologist and an obstetrician. a sample of cervicovaginal discharge was taken from each woman with sterile gynecological collector and was directly transferred to 1 ml rpmi-1640 medium (sigma, usa). after 5 min centrifuge at 3000 rpm, the superior fluid was transferred to a microtube and kept at 70c for the next elisa analysis. the cellular suspension of vaginal discharge was added softly to ficoll - histopaque 1.077 (biosera, uk) solution in a falcon tube and centrifuged at 400 g (2000 rpm) for 20 minutes. 4 10 vaginal discharge mononuclear cells were labeled with 5 of fitc - anti - cd3 monoclonal antibody and pe - anti - cd56 monoclonal antibody (ebioscience inc., san diego, ca, usa). these cells were evaluated by flow cytometry using partec pas iii flow cytometer (partec gmbh, mnster, germany). the gate was set around the lymphocytes on forward scatter and side scatter dot plot to disclose the other cells of the following analysis. interference of fl1 and fl2 signals was adjusted by compensation using fitc and pe antibodies. total rna was isolated from vaginal discharge using cinnapure rna extraction kit (sinaclon bioscience co., tehran, iran), according to the manufacturer 's procedure. total rna was reversely transcribed using the accupower cyclescript rt premix (dn 6) kit (bioneer inc., qpcr was performed using specific primers for hla - g1, hla - g5 isoforms and elongation factor 1 (reference gene) genes (table 2) with sybr green qpcr master mix. quantitative pcr reactions were done in a 20 l volume including 1x sybr green pcr master mix (bioneer inc., seoul, south korea), 1 l of forward and reverse primers, and 2 l of cdna following the manufacturer 's instructions. after a primary 10 min at 95c as activation step, 40 cycles comprising of denaturation at 95c, 30 s, annealing at 59c, 30 s, and extension at 72c, 45 s were performed by biorad iq5 multicolor real - time pcr (bio - rad laboratories, hercules, ca, usa) detection system. il-10 level in superior fluid of vaginal discharge cellular suspension was measured by an elisa ready - set - go kit (ebioscience, usa) according to the manufacturer 's procedure. briefly, 100 l capture antibody in coating buffer (48 l capture antibody (250x) in 12 ml coating buffer per plate) per well of a 96-well plate (corning costar flat - bottom plates, ebioscience, usa) was incubated overnight at 4c. each well was then blocked with 200 l of 1x assay diluent (1 part 5x concentrated assay diluent with 4 parts di water) for 1 hour at room temperature, followed by overnight incubation with 100 l of sample at 4c. then, each well was incubated with detection antibody diluted in 1x assay diluent (48 l detection antibody in 12 ml 1x assay diluent) for 1 hour at room temperature. each incubation step was followed by 3 washes with wash buffer (pbs including 0.05% tween 20). 100 l of avidin - hrp diluted in 1x assay diluent was added to each well. following 7 washes with wash buffer, 100 l of substrate solution was added to each well and the plate was incubated for 15 min at room temperature. reactions were stopped by addition of 50 l stop solution (1 m h3 po4 : phosphoric acid). the p values were characterized in all cases and the p < 0.05 was considered to be statistically significant. totally, sixty pregnant women participated in this case - control study. out of the 30 threatened - abortion women, 26 pregnant women were followed up during their pregnancy. 53.84 percent (14/26) of the threatened - abortion women pregnancies resulted in abortion. the population of nk, nkt, and t cells in vaginal discharge mononuclear cells is presented in figure 1. the result showed that unk cells level significantly increased in vaginal discharge of threatened - abortion women in comparison with normal pregnant women (p = 0.01). moreover, the level of t cells in threatened - abortion women was significantly lower than control group (p = 0.003). there was no significant difference in nkt cells percentage between the two groups (p = 0.85) (figure 2). hla - g1 expression was significantly lower in the vaginal discharge of threatened - abortion women in comparison with normal pregnant women (p = 0.0001). however, threatened - abortion women had a decreased level of hla - g5 in comparison with control group, but this difference was not significant (p = 0.12, figure 3). the concentration of il-10 was determined in the vaginal discharge via elisa as an indicator of cytokine production. this cytokine production level in threatened - abortion women was lower compared with the normal pregnant control group (3.91 1.46 versus 4.76 2.5). at first, we carried out an evaluation on nk cell count and expression of hla - g isoforms expression (pan hla - g, hla - g1, hla - g5) in the vaginal discharge of the threatened - abortion women in comparison with control group, which was determined before 20 weeks of pregnancy. previously, it was observed that cell - bearing hla - g acquires tolerogenic potential to downregulate various immune reactions. our results showed that hla - g1 and hla - g5 isoforms decreased in the abortion - threatened women (figure 3). formerly, we showed that hla - g isoforms levels are different among individuals. in this research, we found that the hla - g1 expression in vaginal discharge was higher than hla - g5 in healthy pregnant women and hla - g1 levels in vaginal discharge of the threatened - abortion women are significantly higher in comparison with hla - g5 expression in healthy pregnant women. therefore, it is recommended that the evaluation of hla - g1 expression in vaginal discharge can be used as a normal pregnancy marker. probably, expression of hla - g1 isoform is controlled by key vital factors for preservation of fetus. in addition, hla - g1 may be a major isoform in the tolerance of mother 's immune system. furthermore, it is possible that the low expression of hla - g1 contributed to allogenic nk cell proliferative response in semiallogenic fetus. on the other hand, hla - g1 and hla - g5 have an important role in the upregulation of t regulatory cells in the pregnant women. also, our results showed that the high expression of hla - g resulted in an excess of il-10 in the vaginal discharge. thus, hla - g1 is an inhibitory constituent and may exert a key suppressive effect on t cell proliferation response to semiallogenic fetus with the cooperation of hla - g bearing cells inhibitory cytokine secretion. in this study, it was observed that the percentage of natural killer cells increased in the vaginal discharge of the threatened - abortion women in comparison with normal pregnant women which could be an indicator of fetus loss. in addition, low expression of hla - g1 immunotolerance molecule is a risk factor for spontaneous abortion in comparison with control group. furthermore, our data showed that hla - g1 expression was significantly lower in abortion - threatened women in comparison with the control group. however, hla - g5 expression level was not significantly different between the two groups, which propose that hla - g5 has inconsiderable characteristic in the mentioned population. several studies have shown a significant correlation between peripheral blood natural killer cell and hla - g in abortion - threatened women. in addition, it is reported that nk cell level can be a predictor of abortion in pregnant women [17, 18 ]. furthermore, there is a strong association between hla - g and nk cell level with spontaneous abortion. it is suggested that predictive value of hla - g or nk cell is superior to other established prognostic factors for fetal loss. reported that hla - g can be the most relevant marker for abortion screening due to t cell response. on the other hand, hla - g identified as a marker for tolerance and its plasma level was associated with better survival in pregnant women [23, 24 ]. our results show a significant correlation between vaginal discharge il-10 level with high hla - g and low nk cell count. these findings show a positive correlation between hla - g1 and hla - g5 expression with il-10 level and a negative correlation between nk cell counts with these cytokines. according to the results, hla - g molecules contribute to the regulation and control of the immune response. in conclusion, the results of our study showed that vaginal discharge, nk cell percent, and hla - g1 and hla - g5 expression were correlated with abortion in comparison with control. therefore, uterine nk and hla - g1 and hla - g5 have an important key role in fetal surveillance during pregnancy. | the immunotolerant human leukocyte antigen - g (hla - g) molecules have a major role in fetal - maternal tolerance during pregnancy. interaction between these molecules and uterine natural killer (unk) cells inhibitory receptors prevents nk cell invasion against fetus trophoblast cells. the aim of this study was to evaluate the percentages of unk cells and hla - g1 and hla - g5 isoforms expression in vaginal discharge of threatened - abortion women in comparison with control. in a case - control study, we investigated 30 threatened - abortion women with bleeding or spotting less than 20 weeks of pregnancy as compared to 30 normal pregnant women. unk cells percentage was assessed by flow cytometry. furthermore, we evaluated hla - g1 and hla - g5 isoforms expression by real - time pcr in these groups. the results of this study showed that threatened - abortion women had increased unk cells and decreased t cells percentage in vaginal discharge in comparison with normal pregnant women (p = 0.01, p = 0.003, resp.). in addition, hla - g1 isoform had lower expression in threatened - abortion women in comparison with control group (p = 0.0001). the increase of unk cells level with the decrease of hla - g expression in vaginal discharge of threatened - abortion pregnant women is an indicator of mother 's immune dysregulation. it is concluded that hla - g expression level with unk cells percentage can be determined as a diagnostic marker for threatened - abortion women. |
it was described for pathophysiological changes in neurological diseases such as alzheimer 's disease and brain trauma. more recent studies have suggested that this damage also occurs in the development of glaucoma [35 ]. loss of retinal ganglion cells in the retina and their axons that represent the optic nerve (3rd neuron) and a loss of astrocytes is the predominant finding in primary open - angle glaucoma. the axons of various retinal ganglion cell subtypes, differing in specific morphology and function, exit the eye ball and finally converge to anatomically distinguishable layers of the lateral geniculate nucleus (lgn) where a loss of neural cells has also been described in glaucoma. the lgn serves as a relay station that transmits the information via the 4th neuron to the primary visual cortex (v1). even in v1 neurons the loss of axons of the 3rd neuron and astrocytes, which are placed between the optic nerve fibers becomes visible by a functionally, glaucoma results in a loss of visual function, which is detectable by determination of the visual field (white - white perimetry) and of the spatial - temporal contrast sensitivity (frequency doubling test, fdt) [11, 12 ]. it is not clear if the loss of axons of the 3rd neuron causes directly an injury of the 4th neuron in humans. however, animal experiments recently have shown that glaucomatous loss of axons of the 3rd neuron is followed by a loss of the lgn volume which indicates a reduced number of 4th neurons. furthermore, with increasing loss of retinal ganglion cells an increasing loss in lgn neurons was described. in vivo detection of glaucomatous changes along the visual pathway and we report a new approach for in vivo visualization of pathological changes of the optic radiation (4th neuron) in glaucoma by use of diffusion tensor imaging (dti). dti is based on the random motion of water molecules, which is associated with their thermal energy at body temperature (brownian motion) and which is known as diffusion. in the presence of a strong magnetic gradient a loss of the magnetic resonance signal results as a consequence of the dephasing of spin coherence. in each voxel the eigenvectors can be characterized from the generated diffusion - weighted magnetic resonance images. in white matter consisting of axons in all directions of a three - dimensional space the diffusion of free water molecules is different (anisotropy) [15, 16 ]. predominantly the orientation of fiber tracts and their micro- and macrostructural features influences the diffusion anisotropy and provides the direction of the largest eigenvector. macroscopically the degree of anisotropy assigned to a definite voxel is affected by the variability in the orientation of all white matter tracts in this imaging voxel. even reconstructions of fiber crossing and branching within the optic chiasm and the lgn are possible by dti. thus, the entire visual pathway from the optic nerve to the visual cortex may be reconstructed. in our work we intended to introduce the dti as a suitable examination method to detect changes of the optic radiation in glaucoma patients. a case report in comparison to a healthy age - matched control subject is shown. subjects received a questionnaire requesting age, gender, known cardiovascular risk factors (i.e., arterial hypertension, diabetes, smoking history), and cardiovascular events (i.e., myocardial infarction, peripheral arterial disease, transient ischemic attack, and stroke). eyes were assessed by a full ophthalmological examination with dilated pupils and judgement of automated perimetry (octo 101 dg2, interzeag, schlieren, switzerland), of spatial - temporal contrast sensitivity (fdt, frequency doubling test, carl zeiss meditec ag, jena, germany), and nonmydriatic fundus images (kowa, nonmyd - alpha 45, japan). mri was performed on a 3 t high - field scanner (magnetom tim trio, siemens, erlangen, germany) with a gradient field strength up to 45 mt / m (72 mt / m effective). the anatomical data were obtained in a t1-weighted 3d - mprage sequence (tr = 900 ms, te = 3 ms, fov = 23 23 cm, acquisition matrix size = 512 256 reconstructed to 512 512, reconstructed axial plans with 1.2 mm slice thickness). for detection of microangiopathy, a heavily t2-weighted fluid - attenuated inversion recovery (flair) sequence covering the whole brain was acquired (tr = 10000 ms, te = 115 ms, matrix size = 512 512). dti was performed in the axial plane with 4 mm slice thickness and a 1 mm interslice separation using a single - shot, spin echo, echo planar imaging (epi) diffusion tensor sequence thus covering the whole visual pathway (tr = 3400 ms, te = 93 ms, fov = 23 23 cm, acquisition matrix size = 128 128 reconstructed to 256 256, number of signal averages = 7, partial fourier acquisition = 60%). diffusion weighting with a maximal b - factor of 1000 s / mm was carried out along 15 icosahedral directions complemented by one scan with b = 0. datasets were automatically corrected for imaging distortions and coregistered in reference to t1-weighted mprage images. these and further calculations such as determining the independent elements of the diffusion tensor, deriving the corresponding eigenvalues and eigenvectors, were performed with a dedicated software package (neuro 3d, siemens, erlangen, germany). the study was conducted in accordance with the declaration of helsinki on biomedical research involving human subjects. written informed consent the images of 14 patients were tested for the reliability of the corrected optic radiation segmentation. two observers postprocessed manually the automatically provided segmentation to exclude cerebral structures that are not a part of the optic radiation and the fractional anisotropy was calculated. cronbach- at the 95% confidence interval for fa was 0.990 (right optic radiation, f = 2.340, p = 0.150) and 0.886 (left optic radiation, f = 1.086, p = 0.316) with df1 = 1 and df2 = 13. thus, the intraclass correlation coefficients of reliability of the semiautomated segmentation were within a very good range of reliability. compared to the optic radiation of a healthy volunteer the dti of a patient with primary open - angle glaucoma appears rarefied (figures 1 and 2). the number of voxels in the optic radiation of the poag patient is on average a third smaller than in the control subject (table 1). calculations of the number of voxels in the manually segmented optic radiation have confirmed the rarefied optic radiation in glaucoma patients. the underlying pathophysiology of the glaucoma disease is based on a damage of the 3rd neuron with loss of function. most studies examined primate models of experimentally induced glaucoma. it was reported that in unilateral experimental glaucoma in addition to the damage of retinal ganglion cells also the post - retinal magno- and parvocellular layers of the lgn of the thalamus and the primary visual cortex may be impaired. transneuronal degeneration was also found in a postmortem human study of the optic nerve, lgn, and visual cortex. in a primate model of glaucoma the neuronal degeneration was shown to proceed from the lgn to the visual cortex [20, 21 ]. a loss of 5060% of the ganglion cells may be already present if visual field defects are detectable by perimetry. it was also shown in experimental glaucoma that some of the 4th neurons in the lgn have died and surviving neurons were atrophic [35 ]. because impairment of the visual field is associated with optic nerve degeneration a certain probability for neurodegeneration of the 4th neuron of the visual pathway may be adopted and even was confirmed. this case report demonstrates that the dti allows an in vivo visualization of the posterior part of the visual pathway. this new technique in ophthalmology may be applicable for earlier diagnosis of glaucomatous changes and for noninvasive therapy monitoring in the future. additionally, the impact for the different entities of optic nerve atrophy should be clearly validated prior to implementation into routine diagnostic. | glaucomatous optic nerve atrophy may continue to the linked optic radiation by transneuronal degeneration, as described in animal models of glaucoma. in vivo visualization of the visual pathway represents a new challenge in the field of ophthalmology. we present a new approach for illustration of the optic radiation by diffusion tensor imaging (dti) based on magnetic resonance imaging (mri). the dti was established by use of a 3 t high - field scanner. the case of a patient with primary open - angle glaucoma is opposed to this one of a healthy subject to demonstrate the visible rarefication of the optic radiation. the goal was to introduce the technique of the dti also in ophthalmology and to demonstrate that it may be useful to judge glaucoma - related differences. |
innate lymphoid cells (ilcs) are a heterogeneous population of immune cells with two defining characteristics : 1) they have lymphoid origin, differentiating from the common lymphoid progenitor (clp) and dependent on il-2rc signaling. 2) they lack antigen - specific receptors and therefore do not require the rag proteins for development. present throughout the body and enriched at mucosal surfaces in both human and mouse, ilcs promptly produce effector cytokines in response to stimulation with stat - activating cytokines and alarmins of the il-1 family [1, 2, and 3 ]. based upon similarities in effector cytokine secretion and developmental requirements, ilcs can be divided into three populations : group 1 ilcs, group 2 ilcs, and group 3 ilcs (figure 1). il-12, il-21 il-15 and il-18 activate group 1 ilcs to produce interferon (ifn-) ; this group includes subsets such as t - beteomes nk cells and t - beteomes ilc1. il-25, thymic stromal lymphopoietin (tslp), and il-33 trigger gata-3 ilc2 to produce type 2 cytokines such as il-5 and il-13. ilc3 subsets include lymphoid tissue - inducer like (lti - like) cells and others that express the nk cell marker nkp46. as potent innate cytokine producers that respond to changes in the cytokine microenvironment, ilcs have demonstrated roles in early infection control, adaptive immune regulation, lymphoid tissue development, and in tissue homeostasis and repair [1, 2, and 3 ]. group 1 ilc are composed of cell populations functionally defined by their capacity to produce ifn-. in mouse, cells within this group uniformly express the surface receptors nkp46 and nk1.1, markers characteristic of nk cells. however, nkp46nk1.1ifn- cells include a population, termed ilc1, which is developmentally and transcriptionally distinct from nk cells. ilc1, reported in the spleen, liver, intestine, and peritoneal cavity, can be discriminated from nk cells by the expression of the transcription factors (tf) t - bet and eomes : nk cells are t - beteomes while ilc1 are t - beteomes [4, 5, 6, 7, 8, and 9 ]. it was previously thought that t - beteomes cells were immature nk cells that acquire eomes upon maturation. however, it was recently shown that t - beteomes cells do not give rise to t - beteomes cells in steady state, suggesting that these cells are not immature but terminally differentiated. in further support of this conclusion, an ilc progenitor in the bone marrow was capable of reconstituting t - beteomes ilc1 cells but not t - beteomes nk cells. additionally, conditional eomes mice maintained ilc1 populations and lacked mature nk cells, while tbx21 mice lack ilc1 but maintained mature nk cells in the bm and gut [4 and 10 ]. ]. also, the transcription factor plzf is important for liver ilc1 but not liver nk cell development. finally, the transcription factor gata-3 has been shown to be critical for ilc1 development [12, 13, and 14 ], yet whether it is also important for nk cell development remains to be clarified. in addition to developmental differences, some ilc1 have been shown to be tissue resident, contrasting with nk cells, which recirculate in the blood [7 and 9 ]. surface markers can distinguish ilc1 and nk cells, although they may be variable depending upon the tissue examined. ilc1 have been found to be cd122cd49acd49b trailcd127cd69cxcr3cxcr6 while nk cells are cd122cd49acd49btrail cd127cd69cxcr3cxcr6 (table 1) [4, 5, 6, 7, 8, and 9 ]. importantly, although ilc1 express the il-7r (cd127) they do not require il-7 for their development and are present in il7 mice. instead, like nk cells, ilc1 critically require il-15 signaling and are absent in il15 mice [4 and 5 ]. although ifn- is the defining cytokine of both nk cells and ilc1, ilc1 are more proficient in the production of tnf-, and in the liver preferentially produce il-2 [5 and 7 ]. nk cells are well known for their capacity to kill target cells through the release of granules containing granzymes and perforin, but whether ilc1 also have cytotoxic capabilities is currently unclear. compared to liver nk cells, liver ilc1 show differential expression of granzymes and reduced expression of perforin, yet are equally capable of cytolytic degranulation and inducing target cell death in vitro [5, 7, and imn robinette., unpublished ]. given the phenotypic and functionally similarity between nk cells and ilc1, it will be important to see if these two cell subsets play complementary or distinct roles during antiviral and antitumor immunity. additionally, there are still other cell types that do not exactly fit into the nk cell / ilc1 dichotomy among various tissues. for example, the small intestine contains an intraepithelial ilc1 population which is developmentally t - bet dependent but only partially il-15 dependent. the small intestine lamina propria (silp) also contains a plastic subset of former rort ilc3 that converts to ifn--producing cells with surface markers difficult to distinguish from ilc1 (ex - rort ilc3). the salivary gland maintains a large population of cells which are both t - beteomes yet poor producers of ifn-. in the uterus, there are also nk cells that are still present in both t - bet and eomes deficient mice. more work is needed to delineate the developmental relations of these cell populations to each other and most importantly the functional role they may play during the immune response within the tissue where they reside. based on current evidence, group 1 ilcs are a spectrum of overlapping cell subsets that produce ifn- rather than bimodal, well defined cell lineages. ilc2 are the most homogenous ilc class, expressing largely conserved markers in all tissues, such as il-7ra, il2ra (cd25), sca-1, klrg1, and the il-33 receptor st2 (table 1). an additional innate il-25 responsive and type 2 cytokine producing population, called mpp, has also been reported. however, unlike ilc2, this il-7r and st2 negative population retains the ability to differentiate into myeloid cells [19 and 20 ] and is thus not strictly an ilc population. ilc2 are dependent on the tfs gata-3, ror, tcf-1 and notch for their development, which has recently been reviewed [2 and 21 ]. functionally, ilc2 are well known to produce il-5, il-13, and amphiregulin in a gata-3 dependent manner as well as gata-3 independent il-4 [12 and 22 ]. recent research has focused on the how ilc2 contribute to the normal immune response through crosstalk with stromal and other immune cells, both in the innate and adaptive responses. the first emerging mechanism that position ilc2 in the normal immune response is through positive feedback loops with other innate immune cells. early in the immune response, various cellular sources including stroma, macrophages, nkt cells, and mast cells produce il-33, a danger signal produced during necrosis and downstream of danger associated molecular patterns (damps) that has recently been reviewed. il-33 directly activates ilc2 and mast cells through its receptor st2, enhancing their recruitment. activated mast cells further enhance ilc2 activity through the secretion of the non - caspase proteases chymase and tryptase, which increase il-33 potency, and production of the strong ilc2 activator pdg2. this positive feedback cycle is fortified by ilc2 proliferative signals from basophils via il-4 production [29 and 30 ]. in turn, ilc2 produced il-5 feedback on eosinophils to promote innate effector responses, by regulating their recruitment and activity [31 and 32 ]. interestingly, the helminth h. polygyrus has developed mechanisms to circumvent this pathway, as its secretory products prevent il-33 release and subsequent ilc2 activation and eosinophil recruitment. thus, crosstalk between other innate immune cells and ilc2 promote an early feed forward type 2 response. a second theme investigated recently has been the role of ilc2 in the priming and development of an appropriate th2 response for example, in a papain model of allergic lung inflammation, ilc2-produced il-13 induces dc migration to the mediastinal lymph node, which was a key event responsible for th2 priming. similarly, ilc2 harvested from an atopic dermatitis model and adoptively transferred intradermally into wildtype mice led to enhanced th2 responses in the draining lymph node. ilc2 also directly lead to th2 responses via expression of mhc ii, which facilitates antigen presentation to t cells at a similar capacity to b cell [35 and 36 ]. functionally, this interaction is relevant, as mice depleted of ilc2 or lacking ilc2 expression of mhc ii had impaired ability to mount appropriate t cell responses during n. brasiliensis infection. ilc2 can also directly activate cd4 + t cells via il-4 production and ox40l costimulation. thus, ilc2 priming of the adaptive immune responses is an important component of their physiologic function and a mechanism of deregulation during pathology. this group of ilcs is highly complex and includes many reported subsets in adult mice, as well as additional fetal and neonatal lti subsets that have been long recognized as a key factor for lymphoid organogenesis. ilc3 are grouped together based on shared expression of the transcription factor rort, but vary in their expression of t - bet, cell surface markers, and cytokine production profiles. in the adult, these subsets include 4 populations found at greatest frequency in the silp (table 1) : cd4 and a cd4 subsets of ccr6 nkp46 rort lti - like cells ; nkp46 rort t - bet ilc3 progenitors ; nkp46 rort t - bet notch - dependent ilc3 [39, 40, 41, and 42 ]. one additional subset of il-17 and ifn- producing rort nkp46 ilc3 is present in the large intestine. similar to group 1 ilcs, group 3 ilcs are particularly complicated due to evidence of diverging progenitors for different subsets that share many of the same functions and cell surface markers. a current, simplified model of differentiation posits that there is progressive differentiation from nkp46 ilc3, to nkp46 ilc3, and finally to nkp46 nk1.1 ex - rort ilc3. in contrast, ccr6 nkp46 lti - like do not give rise to nkp46 ilc3 after transfer. recent gene expression profiling in our lab demonstrate that cd4 and cd4 nkp46 lti - like subsets have minimal transcriptional differences and are unlikely to be functionally distinct (imn robinette. it remains possible that the diversity of these subsets depends on the microenvironment or their origin from either fetal or adult progenitors. as all rort ilc3 can produce il-22, many recent studies have described new ways that this cytokine mediates barrier integrity during homeostatic conditions and mucosal remodeling during injury and infection. il-22 is a member of the il-10 family of cytokines, which acts through an il-22 dimeric receptor only expressed by non - immune stromal cells. il-22 is produced by ilc3 in response to il-23 stimulation by cx3cr1 inflammatory monocytes and cd11b dc [44 and 45 ]. this ilc3-dc priming has recently been shown to be dependent on cxcr6-cxcl16 and defects in cxcr6 lead to loss of protection from citrobacter rodentium, an attaching and effacing pathogen well known to be controlled by il-22. within the gi tract, ilc3 locally regulate commensals and pathogens by inducing intestinal epithelial fucosylation via il-22 in response to microbial signals [46 and 47 ]. systemic il-22 protects the gastrointestinal epithelium from potentially pathogenic symbionts, or pathobionts, after prior injury by inducing complement factor c3 production from the liver. additionally, il-22 in concert with il-18 are critical to the prevention and resolution of intestinal norovirus infection, demonstrating novel antiviral functions of this cytokine. although il-22 signaling appears to be mostly beneficial to the host, it can become pathogenic in certain circumstances, such as the anti - cd40 model of colitis in rag deficient animals, mediated in part by il-22-induced secretion of neutrophil chemoattractants by epithelial cells and dysregulated recruitment of neutrophils. another potential pathological effect of ilc3-mediated il-22 production is tumorigenesis, which occurs downstream of sustained stat3 activation in epithelial cells in mice. indeed, cells with ilc3 consistent phenotypes (cd3 il22) can be found in human adenocarcinomas. notably, this defect may be due in part to il-17, as loss of il-17 in rag deficient mice diminishes adenoma development. thus, ilc3 produced il-22 has both host protective and pathological roles within the gastrointestinal tract. beyond il-22, ilc3 also respond to the microbiome and host micronutrient status to modulate the immune response. in response to commensal flora driven il-1, ilc3 produce gm - csf, which expands colonic dcs and macrophages that support t - reg development. furthermore, recent functional analyses of ilc3 have demonstrated that their expression of mhc ii facilitates antigen presentation, which combined with the absence of costimulatory molecules, enables ilc3 to induce t cell tolerance. interestingly, recent evidence demonstrates that the immune response can also be modified through micronutrients availability. in this vein, vitamin a deficiency leads to a blunted ilc3 but enhanced ilc2 response, while vitamin d deficiency enhances the ilc3 response. as the availability of some micronutrients is directly regulated by the microbiome, this may represent a conserved pathway important for immunoregulation. within the bone marrow, the common lymphoid progenitor (clp) gives rise to all ilc lineages, as well as b and t cells. the earliest progenitor with restricted lineage potential all ilcs has recently been identified as the cxcr6 lp (47 expressing clp ; also known as the common innate lymphoid progenitor - cilp) (figure 1 and table 1). the lp shows multi - lineage differentiation, including nk, ilc1, ilc2, and all ilc3 both in vitro and upon transplantation in vivo. this progenitor is dependent on the basic leucine zipper tf nfil3, consistent with other reports describing a lack of all ilcs in nfil3 mice [58 and 59 ]. of note, the nfil3 requirement for group 1 ilc development can be bypassed following some infections and/or environmental factors [7 and 17 ]. moreover, group 1 ilc subsets in the salivary gland, uterus, and possibly liver appear to develop independently of nfil3. therefore the lp may not be completely dependent on nfil3 or some ilc may develop from a non-lp progenitor. after the lp, lineage divergence of ilc populations has recently been demonstrated by two reports [4 and 11 ]. one study used reporter mice for the tf id2 (a transcriptional repressor of e2-family tfs) to identify a id2linil-7ra47cd25plzf common helper - like innate lymphoid progenitor (chilp). upon transfer, the chilp gave rise to eomes ilc1, ilc2, nkp46 ilc3 and nkp46 ilc3 including the cd4 lti - like subset, but not eomes nk cells. although the chilp was identified by id2 expression, the importance of this tf may not be limited to this progenitor as id2 mice lack nk cells and some ilc1, ilc2, and ilc3 [61 and 62 ]. moreover, id2 identifies an early nk cell restricted progenitor, the pre - pro nk. in another study, lineage tracing with combined plzf rosa26-yfp reporter mice revealed that most ilcs were gfp (thus not expressing plzf). however, these cells were yfp (expressed plzf at some point) and found to derive from a plzflinil-7rackit 47 cxcr6 progenitor, called the innate lymphoid cell progenitor (ilcp). transfer of these ilcps with competitor clps demonstrated that ilcp cells gave rise to greater frequencies of ilcs, corresponding to a progenitor skewed toward ilc development. ilcps efficiently generated ilc1, ilc2, and some ilc3, but were poor at generating nk cells or lti - like subsets. one caveat is that this study identified ilc1s by the lack of expression of cd49b and not eomes expression, a more definitive maker. given the more restricted differentiation of the ilcp, the current model of ilc development would place it downstream of the chilp. plzf deficiency causes some defects in ilc1 and ilc2 development, suggesting that it is not essential for all ilc development. interestingly, gata-3 is first expressed in the ilcp and some models of gata-3 deficiency lead to selective reduction of ilc1, ilc2, and some ilc3, but not nk cells or lti - like cells, in agreement with the progenitor capacity of the ilcp [11, 12, 13,14, and 15 ]. with the increasing complexity of ilc subsets, it becomes important to clearly define the developmental origin and the functional impact of each of these subsets. while there may be redundancy among subsets, it is clear that each ilc group and some subsets within each group have specialized functions. current studies are shifting the focus on the interaction of ilc with the commensal flora, the relevance of this interaction in tolerance and, when interaction is deregulated, in the immune - mediated disease pathogenesis, including ibd, psoriasis, atopic dermatitis, and asthma. while ilcs were originally studied in gut, lung and lymphoid organs, more studies are investigating their roles in other organs and tissues, such as skin, liver and exocrine glands. it will be interesting to see how the developmental stages of tissues analyzed promotes the plasticity and activation of a given ilc population. better understanding of ilc cell biology will likely reveal new mechanisms of immunity and immunopathology and avenues of intervention in many human diseases. | here, we illustrate the complexity of ilc subsets, we discuss novel functions, focusing on emerging ilcs crosstalk with other immune cells and the microbiota. furthermore, we highlight recent insights into the development of ilcs, the common pathways they share as well as points of divergence between ilc groups and subsets. |
the myriad internal membrane structures that characterize eukaryotic cells were well known to nineteenth century cytologists, notably camillo golgi (golgi, 1898). however, with the perfection of techniques to preserve membranes for ultrastructural analysis by transmission electron microscopy, george palade focused our attention on the role of intracellular membranes in the maturation and secretion of proteins manufactured by cells of the exocrine pancreas. his pulse - chase autoradiographic tracing of newly synthesized zymogen proteins, marching progressively from the endoplasmic reticulum (er) through the golgi complex and storage granules to the cell surface, charted a pathway that only hinted at the complexity of intracellular membrane transactions (palade, 1975). nonetheless, in spite of the limitations of the technology, palade framed the basic questions that have engaged a generation of membrane biologists, biochemists, and geneticists. before any precise molecular probes became available, palade recognized that membrane assembly was achieved by expansion of a preexisting organelle, as opposed to the de novo creation of a membrane, and that traffic was mediated by transport carriers, vesicles that capture secretory cargo en route to the next station in the pathway. i learned of palade 's work in graduate school, but i was not trained as a cell biologist. my background was in the enzymology and physiology of bacterial dna replication. my mentor arthur kornberg greatly admired palade 's work, although the two had completely different styles and approaches. in considering where to apply my training in biochemistry, i was influenced by s. jonathan singer 's ideas about the dynamic structure of cellular membranes and lee hartwell 's genetic dissection of the yeast division cycle. after postdoctoral training in singer 's lab, i decided to focus my independent work on protein secretion in yeast. after my first year at uc berkeley, peter novick joined the lab as a beginning graduate student. he and i embarked on a search for conditional lethal, temperature - sensitive (ts) secretion mutants. after an aborted attempt to apply a clever genetic selection (schekman and novick, 2004), peter decided to inspect a random assortment of ts isolates, one of which, called sec1, proved to be defective in the last step of secretion as seen by the intracellular accumulation of mature secretory proteins and vesicles (novick and schekman, 1979). peter then found that sec1 mutant cells experience a dramatic increase in buoyant density that allowed him to use density gradient separation to enrich for more mutants that accumulate er, golgi structures, or vesicles at a restrictive temperature (novick., 1980). an entirely different genetic approach pioneered by ray deshaies, another one of my talented students, led to the discovery of the genes required for secretory and membrane protein insertion into the er membrane (deshaies and schekman, 1987). in the years since these discoveries, > novick and later postdoctoral fellow chris kaiser performed genetic and cytological analysis of single- and double - sec mutant cells to develop a temporal map of the secretory pathway (novick. the map proved to be similar to the path charted by palade for secretion of proteins in pancreatic acinar cells. of course, the clear advantage of the genetic approach is that it allowed the identification of the genes and proteins required to operate the secretory pathway. many of these genes were first identified in yeast, which allowed their mammalian homologues to be cloned and characterized. among the examples of striking evolutionary conservation of the sec genes are sec1, which encodes a regulatory protein required to control the activation of soluble n - ethylmaleimide - sensitive factor (nsf) attachment protein receptor (snare) proteins required for vesicle docking at multiple stages of vesicular traffic in all eukaryotic cells (novick and schekman, 1979) ; sec17 and sec18, which encode the yeast forms of the mammalian proteins nsf and -synaptosome - associated protein involved in membrane vesicle docking and fusion (kaiser and schekman, 1990) ; sec61, which encodes the major channel forming subunit of the secretory polypeptide translocase that is shared with the mammalian and bacterial equivalent function (deshaies and schekman, 1987) ; sec4, which encodes a ras - like guanosine triphosphate (gtp) binding protein, the first to be implicated in the targeting of vesicles to target membranes (salminen and novick, 1987) ; sec22, one of several ubiquitous snare proteins required to dock secretory vesicles (kaiser and schekman, 1990) ; and sec21 and sec23, two genes that encode subunits of distinct coat protein complexes that execute vesicle budding early in the secretory pathway (hicke., 1992 ; hosobuchi., 1992). yeast recombinant dna technology also permitted the application of reverse genetics to the functional evaluation of proteins whose physiological role had not been established. greg payne, a postdoc in the lab, was the first to clone the gene for clathrin heavy chain that had long been assumed to play a role in secretion as well as in receptor - mediated endocytosis. surprisingly, payne discovered that yeast cells survived deletion of the clathrin heavy chain gene (payne and schekman, 1985), although one genetic background yielded an inviable null phenotype (lemmon and jones, 1987). further characterization of viable null cells revealed a function for clathrin in the retention or retrieval of the mating pheromone processing endoprotease, kex2p, in the trans - golgi membrane (payne and schekman, 1989). in the late 1980s, hugh pelham suggested that the major atp - binding heat - shock proteins, hsp70, were involved in cytoplasmic protein refolding after denaturation (pelham, 1986). a genetic test of hsp70 function became possible when elizabeth craig 's lab characterized the hsp70 genes in yeast and found that one gene family, ssa14, encoded redundant essential functions. in collaboration with maggie werner - washburne in her lab, ray deshaies discovered that hsp70 is required to present incompletely folded secretory and mitochondrial precursor proteins that engage in posttranslational translocation into the er and mitochondrial membranes, respectively (deshaies., 1988). gunter blobel 's lab reported the same requirement using a reconstituted secretory protein translocation reaction (chirico., 1988). before our work, secretion mutants defective in mucocyst discharge in paramecium and tetrahymena were isolated and characterized morphologically (beisson., 1976). however, the difficulty of molecular and genetic analysis in protozoa limited the potential impact of these early, fascinating studies. a landmark study on muscle uncoordinated mutants of caenorhabditis elegans by sydney brenner yielded several mutants that were later found to affect neurosecretory processes (brenner, 1974). the locus of one such mutant, unc-18, was molecularly characterized and found to encode a protein of significant homology to the first yeast sec protein, sec1p (hosono., 1992 ; gengyo - ando., 1993). quite independently of our effort, a powerful biochemical approach was pioneered by james rothman, whose laboratory developed a cell - free reaction to reconstitute traffic of a viral glycoprotein among compartments of the mammalian golgi apparatus. rothman 's assays led to the isolation of a number of key proteins involved in vesicle biogenesis and fusion, including many that are encoded by the mammalian genes corresponding to the yeast sec genes (rothman, 2002). the genetic approach did not immediately yield molecular mechanistic insights. although it was clear that the yeast sec genes had mammalian homologues, among the first group cloned only one, sec4, showed a meaningful functional homology, in this case to the ras gtp - binding protein superfamily (salminen and novick, 1987). this discovery, which launched peter novick on his independent career, presaged the identification of dozens of rab proteins, each involved in targeting a vesicle to a unique intracellular destination. my hunch all along was that a functional biochemical approach of the sort that rothman 's lab had established, would be necessary to bridge the gap between genetics and molecular mechanism. by the mid - l980s, david baker in my lab and hannele ruohola (now ruohola - baker) in the lab of my former student susan ferro - novick had developed a cell - free reaction that reconstituted the translocation and vesicle - mediated traffic of a secretory protein (baker. the reaction faithfully reproduced the physiological event as evidenced by the reconstitution of sec mutant defects in vitro. given the likely biochemical complexity of the full transport reaction, we focused on an initial step in the formation of vesicles that bud from the er. michael rexach exploited the reaction devised by david baker to show that a gently prepared yeast lysate retains the er in a rapidly sedimentable form but that slowly sedimenting vesicles are produced that capture secretory cargo from the er lumen. rexach and another student linda hicke showed that the sec mutants that chris kaiser had demonstrated to retard transport vesicle formation from the er in vivo, do so in the cell - free reaction (rexach and schekman, 1991 ; hicke., 1992). hicke, christophe d'enfert, nina salama, and nancy prior then purified the cytoplasmic proteins required for vesicle budding, and charles barlowe put them all together to discover they comprise a novel coat complex that we called copii (barlowe., 1994). in late 1990, i received a phone call from lelio orci whose morphological work on insulin maturation and collaborative effort with jim rothman on vesicle traffic in golgi membranes reflected the highest standards of membrane ultrastructural analysis. he had some choice words about our published yeast morphology, but he offered his help, which i immediately accepted. he has been a valued colleague ever since that fateful call (figure 1). schekman and orci planning a review article in orci 's office at the university of geneva. further biochemical analysis by ken matsuoka, bruno antonny, and marcus lee revealed that the purified copii coat assembly subunits are sufficient to initiate and complete the membrane shape changes that accompany vesicle budding and scission (matsuoka. chris kaiser 's lab and yuval shimoni in my lab found that one copii subunit, sec24p, confers cargo sorting specificity to copii (roberg. liz miller then mapped multiple cargo binding sites on sec24p and presented the most direct evidence to date that membrane cargo sorting at the er is achieved by selective interaction of the coat and cognate sorting signals on the cargo proteins (miller., 2003). electron microscopic tomography and x - ray crystallographic analysis in the laboratories of william balch and jonathan goldberg has now provided an atomic resolution image of the coat and how it assembles and captures membrane cargo during vesicle budding (mossessova., 2003 ; after an initial setback with my first national institutes of health (nih) grant proposal, which was rejected because i lacked experience with yeast and had no preliminary data, my subsequent genetic and biochemical work on yeast was generously and continuously supported by national institute of general medical sciences. beginning in 1991, the howard hughes medical institute (hhmi) provided me an opportunity to extend this work in new directions, most recently into studies on membrane traffic in cultured mammalian cells (fromme., 2007 ; kim., 2007 ; merte., 2010). the added complexity and medical relevance of traffic in human cells will keep us busy for years to come. i am grateful to the funding agencies (national science foundation, nih, hhmi) ; to my academic home in the university of california ; to my patient and understanding family ; to my constant friends and collaborators bill wickner and lelio orci ; and particularly to the many dedicated students, postdoctoral fellows, and loyal staff who have made my lab an exciting place to come to every morning (figure 2) ! schekman 's 50th birthday party and lab reunion at the 1998 ascb meeting in san francisco. | george palade, a founding father of cell biology and of the american society for cell biology (ascb), established the ultrastructural framework for an analysis of how proteins are secreted and membranes are assembled in eukaryotic cells. his vision inspired a generation of investigators to probe the molecular mechanisms of protein transport. my laboratory has dissected these pathways with complementary genetic and biochemical approaches. peter novick, one of my first graduate students, isolated secretion mutants of saccharomyces cerevisiae, and through cytological analysis of single and double mutants and molecular cloning of the corresponding sec genes, we established that yeast cells use a secretory pathway fundamentally conserved in all eukaryotes. a biochemical reaction that recapitulates the first half of the secretory pathway was used to characterize sec proteins that comprise the polypeptide translocation channel in the endoplasmic reticulum (er) membrane (sec61) and the cytoplasmic coat protein complex (copii) that captures cargo proteins into transport vesicles that bud from the er. |
the cleaning and disinfection role of the irrigating solutions in the complex protocol of the endodontic therapy is essential for treatment success. as mccomb and smith10 (1975) demonstrated the presence of a dentin layer attached to the root canal walls, the irrigating solutions should act on both organic and inorganic compounds. among the irrigants used for removal of inorganic compounds are the chelating agents and inorganic acids represented by ethylenediaminetetraacetic acid (edta) and citric acid, respectively7,10,11,13. the biocompatibility of citric acid and edta in the pulp and periapical tissues induces an inflammatory response of variable intensity and depth depending on the methodology used. cotton and siegel5 (1978) and baumgartner,.3 observed no severe or irreversible damage on the human dental pulp. sterrett,.12 (1993) and malheiros,.8 (2005) stated that the effects observed on the human dentin may vary according to the concentration and application time. mcinnes - ledoux,.9 (1985) reported that, in monkeys, 1% citric acid showed severe effects on the pulpal tissues. leonardo,.6 (1984) observed a good tolerance of the periapical tissues to edta on dog 's teeth, while zina,.15 (1981) using trisodium edta in dog 's teeth did not observe any damage on the healing process of the pulp stump and periapical tissues. chan,.4 (1999) observed that in human pulp cell cultures, the toxicity of 0.5% and 1% citric acid is proportional to the concentration and exposure time, whereas other studies observed in rats that citric acid was less irritating than edta and ethyleneglycol - bis(2-aminoethoxy)-tetraacetic acid (egta)14. amaral,.1 (2007) observed that both edta and citric acid showed cytotoxic effects on macrophages, though citric acid produced less cytotoxic. malheiros,.8 (2005) demonstrated in cultured fibroblasts and in rats (rattus novergicus) in vivo, respectively, that, if applied at high concentrations, citric acid leads to a delay in cell growth and has an irritating effect in vivo. based on the endodontic biologic principles, the purpose of this study was to evaluate, histopathologically, the periapical tissue response of dog 's teeth submitted to root canal therapy with two demineralizing chemical substances : 1% citric acid and 17% trisodium edta salt. three different solutions were used in this study : 17% trisodium edta salt (, ph 8.0 ; chemical lab. ucpel, pelotas, rs, brazil), 1% citric acid (ph 2.0 ; curativa pharmacy, pelotas, rs, brazil) and saline (texon co., rs, brazil) as the control. six adult dogs from the central laboratory of ufpel were selected as the biological model of the study. the experimental sample comprised 56 roots of mandibular molars (first and second ; n= 13) and premolars (first, second and third ; n= 22). the animals were sedated with an intravenous injection of 2% virbaxyl (virbac co., sp, brazil) and then anesthetized with thiopental (cristlia chemical products, sp, brazil). after oral asepsis with 0.12% chlorhexidine access to the pulp chamber was gained with # 1012, 1015 and 1016 diamond burs (kg sorensen, so paulo, sp, brazil) and the convenience shape was given with an endo z bur (dentsply - maillefer, ballaigues, switzerland). pulpectomy was performed with a hedstren file and the working length was determined 1 mm short of the radiographic apex with k - files (dentsply - maillefer). root canals were instrumented with four k - files (dentsply - maillefer) of greater sizes than that of file used for root canal length determination. during biomechanical preparation, the number of irrigations and volume of the irrigating solution were standardized in 16 ml for premolars and 32 ml for molars with the aid of disposable syringes and 303, 304 and 305 hypodermic needles (becton - dickinson, so paulo, sp, brazil). in all phases of biomechanical preparation, either one of the test or the control substances were used as auxiliary irrigant. when instrumentation was completed, the root canal was filled with the test solution for 5 minutes to allow an intimate contact. after drying, the canal entrance was sealed with disinfected gutta - percha followed by composite prisma aph (dentsply ind. ltda, petrpolis, rj, brazil) below the occlusion line. after 24 and 48 hours of the treatment, the animals were euthanized and the teeth removed and fixated in 10% formalin solution. thereafter, the teeth were decalcified by immersion in 1:1 formic acid and sodium citrate solution followed by 5% nitrate acid. the specimens were embedded in fused paraffin histosec (merck kgaa, darmstadt, germany), cut with a microtome at the apical foramen in serial 6-m - thick sections, which were stained with hematoxylin and eosin and mounted on glass slides. the sections that best represented the area of pulp - periodontium communication were selected and examined with a light microscopy at 100 magnification. the histologic analysis considered the occurrence of an acute inflammatory response, so the presence of swelling, vasodilatation and inflammatory cells was evaluated and the degree of inflammation was determined for each case. for systematic examination of the specimens, a squared and numbered (with 100 indexed crosses (+) of equal dimension) eyepiece grid (ne21 mm ; graticules ltd, kent, uk), was introduced into one of the eyepieces of the microscope. of the total of 100 crosses that composed the net, approximately 50 would cover the periodontal space, being the remainder occupied by the root, dental canal and alveolar bone, which were excluded from the analysis. three fields of the periodontal ligament were evaluated : its most central area in relation to the apical foramen and two lateral margins. for each evaluated field, we considered the number of points coinciding with the occurrence of the three primary events of inflammatory reaction : swelling, vasodilatation and presence of inflammatory cells. the following criteria were used : mild inflammation - when each parameter was coincident with a maximum of 4 crosses ; moderate inflammation - when at least 2 parameters were coincident with at least 5 and at most 9 crosses ; severe inflammation - when at least 2 parameters were coincident with 10 or more crosses. chi - square test could have been used to assess differences in the efficacy of the three substances. however, as the expected frequencies for a few cells were less than 5, chi - square test was not deemed appropriate. alternatively, fisher 's exact test was used to overcome the problem due to small cell frequencies because it requires a non - zero frequency in each cell. hence, the category " severe " was eliminated because there was no observation for that level. the resulting contingency table was used for fisher 's exact test is reproduced as table 2 and 4. the data were analyzed with a confidence interval of 95% (p<0.05) using the spss software (spss 14.0, spss inc., the two tailed p value generated by fisher 's exact test for the above contingency table was 0.482 (for the 24-hour exposure time) and 0.377 (for the 48-hour exposure time). as both p values were greater than 0.05, the null hypothesis of independence was accepted, which means that the three substances did not have different efficacy at the either of the study intervals. the results of the histologic analysis of the inflammatory response observed in dogs ' teeth after contact with edta, citric acid and saline, after 24 and 48 hours, are presented in the tables 1 and 2. in order to provide the necessary adjustment for the statistical analysis, the scores 1, 2 and 3 were attributed to the mild (figure 2), moderate (figure 3) and severe (figure 4) inflammation degrees, respectively, which differ from the normal aspect (figure 1). there was no significant difference in the inflammation degree among the tested substances within the 24 hour (p=0.482) or 48 hour (p=0.377) periods. in the present study, the sample comprised the roots instead of molar and premolar teeth because, after pulpectomy, the surgical wounds behave independently even if submitted to the same treatment. the 24- and 48- hour periods were established because, though presenting a relative antimicrobial activity, the tested substances, are not indicated for this use and after longer observation periods there would be the risk of bacterial contamination, especially in the control group. the method proposed for inflammation degree evaluation consisted of using a net of eyepiece reticular scale adapted to the ocular of the microscope, which allowed the quantification of the events composing the inflammatory process, i.e., swelling, vasodilatation and presence of inflammatory cells in the apical region. therefore, the classification of the inflammatory response in mild, moderate and severe was determined by the counting. the results obtained with edta in relation to the control group demonstrated a good tolerance by the periapical tissues. similar findings have been reported by zina,.15 (1981) and leonardo,.6 (1984), the latter evaluating the tissue response in dogs ' teeth. the statistical analysis showed that even though there was a severe degree inflammation within 48 hours, there was no significant difference between both periods. citric acid showed a similar behavior as that of edta and saline at both periods and, though not statistically significant, it showed a higher degree of inflammation at 48 hours compared to the shorter period. this result differs from those of previous studies that used citric acid for cavity cleaning5,9 and evaluated the response to pulp tissues instead of to periapical tissues. the findings of the present study and of other authors1,8 disagree from those reported by chan,.4 (1999) who observed an increase in the inflammation degree with a longer exposure time in cell cultures. citric acid is an organic acid that takes part in the krebs cycle in the human cells, being therefore biologically acceptable. the results of the control group showed that an inflammatory response is a natural consequence of pulpectomy. comparison of the substances in both studied periods (24 and 48 hours) did not show statistically significant differences. this fact indicates that, although these two substances have a low superficial tension, even in multiple foramen conditions, other factors referring to their physicochemical properties and the site of application, control their diffusion. regarding edta, its activity is limited by the presence of organic material2, in addition to promoting the coagulation of proteins, which form of a mechanical barrier that avoids its diffusion towards the periapical tissues. citric acid has the property of not promoting collagen selling, which occurs with most acids. this justifies its use for histological preparations in the form of sodium citrate, in demineralization processes. this aspect could allow a higher diffusion of this substance, but its acidic characteristics provide cauterizing properties, due to its low ph. both edta and citric acid compared to the control group showed a potential for a mild inflammation, with no statistically significant difference to each other. the clinical use of either edta or citric acid should be done with caution to prevent their extrusion to the periapical tissues. citric acid showed similar conditions for clinical use as those of edta, as it has similar characteristics regarding the property as an inorganic solvent, removal of smear layer, improvements in naocl properties when associated to it and a satisfactory tissue tolerance. under the tested conditions, under the experimental conditions of this study, the obtained results showed that 17% edta and 1% citric acid caused inflammatory responses in dog 's periapical tissues with no significant differences from saline within the evaluated time periods (24 and 48 hours). 17% edta and 1% citric acid produced similar inflammatory reaction to each other at both the shorter and the longer time period. it may be concluded that the inflammatory response to the tested substances was of mild intensity. | the purpose of this study was to analyze the inflammatory response of dog 's periapical tissues to 17% trisodium edta salt (ph 8.0) and 1% citric acid (ph 2.0). saline was used as a control. six adult dogs were used as the biological model of the study. the experimental units comprised 56 roots of mandibular molars (first and second) and premolars (first, second and third). after coronal opening, pulpectomy and root canal instrumentation were performed using the above - mentioned irrigating solutions. after 24 and 48 hours, the animals were euthanized and the teeth and their supporting tissues were removed and histologically processed. the sections were stained with hematoxylin and eosin and analyzed histopathologically with a light microscope at x100 magnification. the histological analysis focused on the occurrence of acute inflammatory response. the presence of swelling, vasodilatation and inflammatory cells were evaluated and the degree of inflammation was determined for each case. data were analyzed by fisher 's exact test using the spss software with a confidence interval of 95% (p<0.05). 17% edta and 1% citric acid caused inflammatory responses in dog 's periapical tissues with no significant differences to each other or to saline (control) at either the 24-hour (p=0.482) or 48-hour (p=0.377) periods. it may be concluded that the inflammatory response was of mild intensity for the tested substances. |
post - transplant lymphoproliferative disorders (ptld) are a heterogeneous group of lymphoproliferative disorders associated with the immunosuppression in recipients of solid organ or allogeneic stem cell transplantation. the incidence of ptld varies from 1% to 20% according to the organ transplanted (1). the epstein barr virus (ebv) plays an important role in the pathogenesis of ptld. ptld is typically classified into three major categories : early lesions, polymorphic ptld, and monomorphic ptld (2). monomorphic ptld is defined by the fulfillment of the criteria for one of the recognized types of lymphoma. the majority of monomorphic ptld cases is non - hodgkin 's lymphoma of b - cell origin ; in contrast, t - cell lymphoma and hodgkin 's disease are rare. the recently updated world health organization classification separated classical hodgkin lymphoma type ptld from monomorphic ptld (3). although there are several reports on ptld, there are few data on monomorphic ptld in korea. the goal of our study was to investigate the incidence, clinical manifestation, treatment, and outcomes of monomorphic ptld, including hodgkin 's lymphoma, in korea. this study included all patients who were diagnosed with monomorphic ptld confirmed by tissue diagnosis among 5,817 recipients of solid organ or allogeneic stem cell transplantation from five institutions in korea between january 1987 and january 2009. the clinical factors evaluated included age, sex, transplanted organ, time from transplantation to diagnosis of ptld, pathology, ebv status in pathology, tumor stage, performance status, extent of extranodal disease, b symptoms, serum ldh level, graft organ involvement, treatment method, and clinical response to treatment. ebv detection methods included ebv - lmp (latent membrane protein), ebv polymerase chain reaction or epstein barr encoded rna (eber) in situ hybridization in accordance with the availability in the hospitals. staging was performed according to the criteria of the ann arbor system and the histological subgroups were classified according to who classification. this study was approved by the institutional review board of hanyang university hospital, asan medical center, soonchunhyang university hospital, busan paik hospital, and pusan national university hospital. overall survival was measured from the date of diagnosis of ptld to the date of death or last follow - up. the types of organ transplantation included renal (10 cases, 71.4%), liver (1 case, 7.2%), and allogeneic stem cell (3 cases, 21.4%). the overall incidence rate of monomorphic ptld was 0.24%. the incidence rates of monomorphic ptld according to the transplanted organ were 0.35% in kidney (10/2,880), 0.045% in liver (1/2,207), and 0.41% (3/730) in allogeneic stem cell transplantation. the study group included 11 men and three women, and the median age at the time of ptld diagnosis was 42.6 yr (range 24 - 60). the initial diagnosis of three patients who underwent allogeneic stem cell transplantation included 2 cases of aplastic anemia and one case of hemophagocytic syndrome. all cases of ptld after allogeneic hematopoietic stem cell transplantation occurred within 1 yr after transplantation (early onset ptld), whereas the majority of ptld after solid organ transplantation (10/11) occurred after 1 yr (late onset ptld). the most common disease type was diffuse large b cell lymphoma (50.0%), followed by peripheral t cell lymphoma (28.6%), hodgkin 's disease (14.3%), and one case of anaplastic large cell lymphoma. one patient had involvement of the graft organ and seven (50%) patients had extranodal involvement, most commonly in the liver (n=3). nine patients had elevated serum ldh, and ten patients demonstrated ebv positivity in the tumor. in ebv negative cases, two patients were diagnosed as diffuse large b cell lymphoma, one patient diagnosed with peripheral t cell lymphoma, and remaining diagnosed as anaplastic large cell lymphoma. according to the international prognostic index, six patients were low risk (42.9%), six patients were low - intermediate risk (42.9%), and two patients were high risk (14.2%). administration of immunosuppressive agents was reduced in all patients except for one patient who was not receiving immunosuppressive treatment at the time of diagnosis because the grafted organ had already been rejected. fourteen patients were treated with combination systemic chemotherapy as follows : chop (cyclophosphamide, adriamycin, vincristine, prednisone ; n=6), r - chop (rituximab, cyclophosphamide, adriamycin, vincristine, prednisone ; n=5), abvd (adriamycin, bleomycin, vinblastine, dacarbazine ; n=2), and chop followed by autologous stem cell transplantation (n=1). four patients received radiation therapy. because one patient died from infection during chemotherapy, 13 patients were assessed for response analysis. the overall response rate was 92.3% ; 10 patients achieved complete response (cr) and two achieved partial response (pr). among the 10 patients who achieved cr, one patient subsequently suffered relapsed diffuse large b cell lymphoma at 11.3 months. for the two patients who achieved pr, one patient diagnosed with hodgkin 's disease was treated with four cycles of abvd followed by radiation therapy (4,400 cgy) and remained in a state of pr at the time of the last follow up. the other patient diagnosed with diffuse large b cell lymphoma died due to disease progression. among 11 solid organ transplantations, there were five deaths during follow - up (35.7%) : three due to infection and two due to disease progression. nine patients were still alive (8 cr, 1 pr) at the time of the last follow - up. the types of organ transplantation included renal (10 cases, 71.4%), liver (1 case, 7.2%), and allogeneic stem cell (3 cases, 21.4%). the overall incidence rate of monomorphic ptld was 0.24%. the incidence rates of monomorphic ptld according to the transplanted organ were 0.35% in kidney (10/2,880), 0.045% in liver (1/2,207), and 0.41% (3/730) in allogeneic stem cell transplantation. the study group included 11 men and three women, and the median age at the time of ptld diagnosis was 42.6 yr (range 24 - 60). the initial diagnosis of three patients who underwent allogeneic stem cell transplantation included 2 cases of aplastic anemia and one case of hemophagocytic syndrome. all cases of ptld after allogeneic hematopoietic stem cell transplantation occurred within 1 yr after transplantation (early onset ptld), whereas the majority of ptld after solid organ transplantation (10/11) occurred after 1 yr (late onset ptld). the most common disease type was diffuse large b cell lymphoma (50.0%), followed by peripheral t cell lymphoma (28.6%), hodgkin 's disease (14.3%), and one case of anaplastic large cell lymphoma. one patient had involvement of the graft organ and seven (50%) patients had extranodal involvement, most commonly in the liver (n=3). nine patients had elevated serum ldh, and ten patients demonstrated ebv positivity in the tumor. in ebv negative cases, two patients were diagnosed as diffuse large b cell lymphoma, one patient diagnosed with peripheral t cell lymphoma, and remaining diagnosed as anaplastic large cell lymphoma. according to the international prognostic index, six patients were low risk (42.9%), six patients were low - intermediate risk (42.9%), and two patients were high risk (14.2%). administration of immunosuppressive agents was reduced in all patients except for one patient who was not receiving immunosuppressive treatment at the time of diagnosis because the grafted organ had already been rejected. fourteen patients were treated with combination systemic chemotherapy as follows : chop (cyclophosphamide, adriamycin, vincristine, prednisone ; n=6), r - chop (rituximab, cyclophosphamide, adriamycin, vincristine, prednisone ; n=5), abvd (adriamycin, bleomycin, vinblastine, dacarbazine ; n=2), and chop followed by autologous stem cell transplantation (n=1). four patients received radiation therapy. because one patient died from infection during chemotherapy, 13 patients were assessed for response analysis. the overall response rate was 92.3% ; 10 patients achieved complete response (cr) and two achieved partial response (pr). among the 10 patients who achieved cr, one patient subsequently suffered relapsed diffuse large b cell lymphoma at 11.3 months. for the two patients who achieved pr, one patient diagnosed with hodgkin 's disease was treated with four cycles of abvd followed by radiation therapy (4,400 cgy) and remained in a state of pr at the time of the last follow up. the other patient diagnosed with diffuse large b cell lymphoma died due to disease progression. among 11 solid organ transplantations, there were five deaths during follow - up (35.7%) : three due to infection and two due to disease progression. nine patients were still alive (8 cr, 1 pr) at the time of the last follow - up. in this retrospective analysis the incidence rate of monomorphic ptld was 0.24% and the median time between the transplant and diagnosis of ptld was 85.8 months. this might be because our study included only adult monomorphic ptld patients, and ptld is more common in children than in adults. in addition, the majority of patients in our study underwent kidney or liver transplantation (5,129/5,817), and the incidence of ptld is the lowest for recipients of renal or liver allografts and the highest for heart - lung, lung, or intestinal allografts (1, 3). previous studies on ptld typically included all ptld types, from early lesions to monomorphic ptld, and the incidence of monomorphic ptld is obviously lower than the overall incidence rate ; for example, in one report with an overall incidence rate of 1.2%, the incidence rate of monomorphic ptld was only 0.48% (4). finally, since this study was carried out in a retrospective manner, and since patients with only confirmed tissue diagnosis were included in this study, there may be possibilities that some potential candidates may have been left out. the majority of ptld occur less than 1 yr after transplantation ; however, the median time before the development of ptld in our study was longer than those in previous reports. ptld that occurs within 1 yr after transplantation is defined as early ptld, while that occurs after 1 yr is late ptld. early ptld is commonly ebv - positive, cd20-positive, and involves the graft organ (5). in our study, four patients had early ptld ; three of these were ebv - positive and cd20-positive, and one patient showed graft organ involvement. recently, a retrospective study was published which stated the clinical characteristics of ptld of kidney transplantation in korea (6). from 2,630 the mean time interval between the time of transplantation and diagnosis of ptld was 126.6 months. this report also represented low incidence rate and long time interval, which is similar to our results. our study revealed that all cases of ptld after allogeneic hematopoietic stem cell transplantation (hsct) were early ptld, whereas the majority of ptlds after solid organ transplantation (10/11) were late ptld. hou. reported that the majority of ptld after allogeneic hsct occurred within 6 months (7). in their study, the overall mortality rate was 92%, and the major causes of death were disease progression and infection. in addition, pulmonary ptld showed an aggressive clinical course and poor response to therapy (7). our study included three patients who received allogeneic hsct ; their time before the development of ptld was 5.0, 5.8, and 5.9 months, respectively. of these, one patient had ebv - associated ptld (diffuse large b cell lymphoma) with pulmonary involvement. although the patient was treated with r - chop, he died from infection 40 days after chemotherapy. suppression of t - cell activity interferes with immune surveillance and allows the proliferation of latently infected b lymphocytes, and the proliferation of a malignant b cell clone results in lymphoma (8, 9). however, the incidence of ebv - negative ptld appears to be increasing, and recent reports showed that 20 - 30% of ptld cases were ebv - negative. ebv - negative ptlds occur later than ebv - positive cases, have a higher proportion of monomorphic ptld, and show a more aggressive clinical course (10, 11). although the pathogenesis of ebv - negative ptld remains to be determined, changes in the immunosuppressive regimens and new, unidentified infectious agents might play a role (10). in our study, four cases were ebv - negative ; among these, three patients developed ptld more than 5 yr after transplantation. since monomorphic ptld fulfill the criteria for one of the recognized types of lymphoma, it is difficult to differentiate between monomorphic ptld and de novo lymphoma in patients who underwent transplantation. it could be a reasonable option to consider monomorphic ptld if it contains pleomorphism of the transformed cells, variability of cell size, and ebv positive cells (3). other treatment strategies for ptld included antiviral therapy, systemic chemotherapy, rituximab, interferon a, and radiation therapy (1) ; however, the treatment outcome and survival of ptld patients remains poor. in particular, monomorphic ptld requires more aggressive treatment strategies because of its poor prognosis. in our study, all patients received combination chemotherapy, and the overall response rate was 92.3%. although this study showed a better response rate than other reports, five of the 14 patients died due to infection or disease progression. our study has following limitations : the clinical data were analyzed retrospectively and a relatively small number of patients were included. nevertheless, our results provide further information on the clinical manifestation, treatment, and outcomes of monomorphic ptld. our results showed that a lower incidence rate and a longer median time before the development of ptld than those of previous reports. however, all cases of ptld after allogeneic hsct occurred within 1 yr after transplantation, therefore careful monitoring must be needed in this particular group of patients. compared with previous reports, our study showed better response rate and survival, and this might be due to the fact that all our patients received combination chemotherapy. in this aspect, we suggest that monomorphic ptld could lead to a better outcome with an aggressive combination treatment strategies. | post - transplant lymphoproliferative disorders (ptld) are a heterogeneous group of lymphoproliferative disorders associated with immunosuppression and epstein - barr virus infection. ptld is classified into three major categories : early lesions, polymorphic ptld, and monomorphic ptld. the majority of monomorphic ptld cases are non - hodgkin 's lymphoma of b - cell origin. this retrospective study was conducted to investigate the incidence, clinical manifestation, treatment, and outcomes of monomorphic ptld among 5,817 recipients of solid organ or allogeneic hematopoietic stem cell transplantation from five institutions. fourteen patients with monomorphic ptld were identified (male : female 11:3 ; median age 42.6 yr, range 24 - 60). the overall incidence rate was 0.24%. the most common disease type was diffuse large b cell lymphoma (n=7). the median time between the transplant and diagnosis of ptld was 85.8 months. however, all cases of ptld after allogeneic hematopoietic stem cell transplantation occurred within 1 yr after transplantation. ten of the 14 patients had ebv - positive tumor. fourteen patients received combination systemic chemotherapy and four patients were treated with radiation therapy. ten patients achieved a complete response (cr) and two patients a partial response (pr). the median follow - up period for surviving patients was 36.6 months. nine patients remain alive (eight cr, one pr). nine of 11 solid organ transplantations preserved graft function. the present study indicates a lower incidence rate and a longer median time before the development of ptld than those of previous reports. careful monitoring was needed after allogeneic hematopoietic stem cell transplantation for ptld. |
e - cadherin immunohistochemistry in foci of subpleural lipogranulomatous inflammation shows positive staining only in pneumocytes. | a 9-year - old, female, spayed, domestic longhaired cat presented to the ohio state university veterinary medical center oncology service with a 6-month history of changes in behavior and weight loss. the owner reported that the cat had become abnormally unfriendly and would growl or hiss when touched. the cat also had gait changes, manifested by left hind limb weakness, difficulty walking up stairs, and occasionally losing balance and falling down stairs. the cat was examined by its regular veterinarian and had abdominal radiographs, cbc, and biochemistry profile performed, which did not detect any abnormalities. on physical examination, the cat was underweight with an estimated body condition score (bcs) of 1/5. neurologic examination revealed a very dull mentation, severe generalized muscle atrophy, and a slow, reluctant, and crouched gait. cranial nerve examination revealed no abnormalities. there was absence of conscious proprioception in all limbs. a forebrain lesion was suspected. the cat was examined 8 days later because of progressive signs of neurologic disease. the cat was obtunded and had a right head turn with a mild right head tilt. the gait showed proprioceptive / right vestibular ataxia with knuckling of both thoracic limbs on ambulation, a crouched posture, and a splaying outward of the left thoracic limb. there was a decreased menace response bilaterally that was worse on the left and decreased nasal sensation bilaterally. spinal reflexes were normal in all limbs. neurolocalization was to the right - forebrain with possible brainstem involvement. an abdominal ultrasound was then performed at the owner s request to investigate the gastrointestinal tract as a possible explanation for the weight loss, but no abnormalities were identified. |
nasopharengeal carcinoma (npc) refers to a group of tumors originating from lymphoid tissues. the first symptom in half of the patients is cervical lymphadenopathy which represents a metastatic tumor. although rare in most parts of the world, npc is very common in southern china and southeast asia. npc is associated with epshteinbar virus infection, and other probable etiological factors are smoking and occupational exposure to wood, intake of some kinds of food specially the ones containing nitrosamines such as salted fish. treatment plan for npc patients varies from radiotherapy alone in primary lesions to adjuvant chemotherapy in advanced ones. exposure period and total treatment dose are both important factors in treating early stage npc. decreasing the total radiation time to less than 12 weeks and not exceeding a radiation dose of 75 gy has proved to show the best results. in such cases, results are comparable to those obtained with surgery because npc early lesions are very sensitive to radiation. although radiotherapy is preferred to surgery in early stages of treatment of npcs as it renders more chances of tissue preservation, it exerts complications in sensitive structures such as the oral mucosa, salivary glands, dentition and jaws. trismus is one of the common side effects after radiotherapy and chances for it are 8% to 38%. limited mouth opening can interfere with patient 's daily activities and compromise the quality of life. dental malpractice is also a sequel of this limitation because the practitioner would not have enough accessibility during dental treatments. xerostomia starts as soon as patients receive the first fractions of radiotherapy and can last up to 2 years. xerostomia can also cause discomfort especially in denture wearers. reduced salivary flow rate can develop an increased incident of radiation caries affecting incisal and cervical parts of teeth. dentists being part of the management team for npc patients can play a significant role. the dental treatments to these patients have to be done both before and after the radiation. dental practitioners must omit infection sources since the risk of new infection is high due to the compromised host defense after cancer therapy. there is also an increased risk of radio necrosis in the first few months after radiotherapy. the dentists should describe the condition and explain the post radiation complications to the patients to provide more compliance. during the time patients are undergoing radiotherapy, dentists must control acute infections and avoid invasive dental procedures as possible. at the time irradiation therapy for npc comes to end, patients need different sorts of dental treatments due to extensive caries caused by decreased salivary flow and poor oral hygiene as a sequence of compromised medical status. some dental materials including resin - modified glass - ionomer, compomers, and fluoride - releasing resin composites could be effective in preventing secondary caries.[1315 ] however, it should be emphasized that maintaining oral hygiene and patient 's diet surveying are important aspects of treatment. fluoride supplements are effective in reducing secondary caries and the patient should be asked to use it regularly. the patients who have received radiotherapy usually need full mouth rehabilitations since most of them have had their unrestorable teeth extracted before and after the radiotherapy to prevent various side effects. the limited mouth opening in these patients also causes challenges in choosing the type of the prosthetic treatment plan. this article describes a sequential approach to the interdisciplinary treatment of a partially edentulous patient with excessive tooth tissue loss in a 46-year - old man with a history of radiotherapy and chemotherapy. a 46-year - old man presented to the department of prosthodontics at tehran university, school of dentistry. his past medical history revealed that he has been under treatment for undifferentiated nasopharengeal carcinoma of neck (npc). he was irradiated with 60 gray (gy), at single doses of 2 gy. the patient had no muscle tenderness or facial asymmetry and denied any symptoms of temporomandibular joint disorder or myofacial pain dysfunction. functional manipulation did not show any sign of parafunction, but mandibular range of motion was not within normal limits and there was obvious limitation in mouth opening (30 mm). the saliva was thick and mucous and there was generalized caries and recurrent caries throughout dentition. all the teeth except maxillary incisors and first right premolar had received endodontic treatment, though all were faulty treatments and needed retreatment. the maxillary left and right canines, first and second premolars and mandibular left second premolar had complete coverage crowns [figure 1 ]. marginal opening of all complete crowns and breakdown and leakage of these restorations were noted. defective amalgam restorations with marginal breakdown and recurrent caries were present in teeth # 3, 14, and 15, and defective composite restorations with marginal leakage were present in teeth # 8, 9, and 10. the patient was referred to the ent department for evaluation. however, his medical consultation reported that he did not have any current signs or symptoms of npc. treatment plan options (fixed versus removable) were presented to the patient. however, considering the patient 's desires, complete mouth rehabilitation with tooth and implant supported metal ceramic restorations was suggested. however, the first step was controlling caries. therefore, diet evaluation was performed and he received daily fluoride treatment with a custom - made carrier and 0.4% stannous fluoride gel. besides diet survey and diet modification, oral health instruction including informing the patient about disease etiology and oral hygiene instruction, evaluation of salivary flow, and prescribing xylitol products were carried out for the patient. endodontic treatment and retreatment were done for all the remaining teeth except maxillary second left molar. this tooth was extracted because of limited mouth opening and therefore limited access to it [figure 2 ]. the treatment plan included initial preparation and caries removal and complete mouth clinical crown lengthening surgical procedures to expose 1.5 to 2 mm of tooth structure circumferentially for adequate ferrule for resistance and retention forms and to facilitate esthetic gingival levels. since there was no acceptable posterior support, the diagnostic phase involved a removable diagnostic overdenture. after the removal of failing restorations and caries removal, primary impressions were made and esthetic clinical crown lengthening was designed on the diagnostic casts. then a diagnostic wax up was done and a vacuum formed surgical template was fabricated as a reference for the prospective desired gingival levels during the crown lengthening surgical procedures. full - thickness flaps with scalloped incisions were elevated to preserve interdental papillae [figure 3 ]. osteotomy was performed according to surgical templates to develop 2.0 mm of biological width and 1.0 mm of sulcular depth. a centric relation record was obtained with record bases and occlusion rims using an interocclusal registration material (virtual ; ivoclarvivadent, schaan, liechtenstein). the casts were transferred into a semi - adjustable articulator (dentatus arh, stockholm, sweden) by the centric relation record. prosthetic teeth (major ; major prodotti dentari, torino, italy) were arranged for trial insertion. the overlay denture was processed and was adjusted on the remaining teeth [figure 4 ]. a radiographic stent for implants was fabricated (lucitone clear resin) by duplicating the overlay denture and coating its teeth surface with barium sulfate. then, tomographic radiographs were taken to check bone height and width and proper implant position [figure 5 ]. two 4.1 10 mm endosteal dental implants (institut straumann ag, waldenburg, switzerland) were placed in each posterior mandibular quadrant using the surgical guide. the following 5 days, 250 mg of cefuroxim (elobacts, bad oldeslohe, cascan, germany) was given twice orally and patients rinsed their mouth twice daily with 3% hydrogen peroxide ; no specific diet was followed. after 5 weeks, the stability was tested with osstell (integration diagnostics, gothenburg, sweden). the teeth were prepared and custom post patterns were fabricated according to a vacuum silicon guide made over overlay dentures. after finalizing cast post in the all remaining teeth, this impression in mandible not only record prepared teeth, but also was an implant level impression. for transferring the exact maxillo mandibular relationship established with overlay dentures to the final fixed restoration, the anterior section of the overlays were removed so that lucia jig could be made between incisors at the existing vertical dimension of occlusion (vdo). then, the centric relation was made between posterior maxillary prepared teeth and mandibular record base along with cross mounting with cast made of over impressions of overlay dentures. a custom incisal guide table was made from gc pattern resin (gc america) while overlay dentures were on the articulator. waxing was done according to the customized guide table on die casts and converted to heat - processed acrylic temporary restorations. the diagnostic interim restorations were modified until the patient was satisfied with phonetics, esthetics, and function. during the evaluation period, acrylic restorations on the implants first were made without occlusal contact, with occlusal contact on the abutments 4 weeks later, and full occlusal contact on the abutments and pontic after 8 weeks. the interim restorations were functioned for about 4 months to assess the patient 's adaptation to the proposed new vertical dimension of occlusion and the new clinical crown lengths [figure 6 ]. subsequently, the gingival tissue around the tooth preparations had matured and definitive impressions were made with vinyl polysiloxane impression material (affinis ; coltne / whaledent, inc, cuyahoga falls, ohio). centric occlusion, even protrusive contacts, and canine guidance were established in the definitive anterior restoration. after the fit of the cast crowns was verified intraorally, metal - ceramic restorations were completed (omega 900 ; vita zahnfabrik, bad sackingen, germany). a heat processed hard occlusal guard (lucitone clear, dentsply, york, pa, usa) was delivered to evaluate the patient 's tolerance of new vdo and providing the patient with a mutually protected occlusion [figure 7 ]. following the definitive cementation of all restorations, neutral ph sodium fluoride (previ dent 1.1% brush - on gel ; colgate oral pharmaceuticals, new york, ny) was prescribed to prevent secondary decay under the restorations. the patient has been followed for 8 months since the completion of his treatment and remains free of any complications. additional instruction was given on the use of floss threaders and superfloss under the fpd. (c, d) left and right lateral views panoramic radiograph after endodontic treatments crown lengthening procedure. (a, b) : maxillary and mandibular diagnostic casts ; (c) surgical templates ; (d) elevation of full thickness flap with scalloped incisions (a) the overlay denture ; (b, c, d) : adjustment of the overlay denture on the remaining teeth (a, b) : radiographic templates ; (c) evaluation of available bone in tomographic radiographs progressive bone loading. (a) right posterior temporary restorations out of occlusion ; (b) left posterior temporary restorations out of occlusion ; (c) three weeks later, temporary restorations in occlusion except pontic ; (d) three weeks later, full occlusal contact clinical view of post- treatment dentition. (a, b) occlusal views of maxilla and mandible ; (c) frontal view ; (d) smile view rehabilitation of npc patients involves several steps among which preventive measures such as oral hygiene instruction and diet survey are very important. as a sequel of xerostomia, not only there is an increased risk of caries, but also treatment plan is also affected. in these patients, the routine prosthetic treatments are not often successful because of reduced surface tension between mucosa and prosthesis. therefore, implant - supported prosthesis would be of choice and has demonstrated its efficacy and seems to be reliable.[1618 ] however, dosage and healing time after radiotherapy are significant factors to indicate implant in these patients. treatment of this case involved successful restorative treatment after a 3-month period of preventive evaluation phase. | this clinical report presents a 46-year - old man diagnosed with nasopharyngeal carcinoma with the chief complaint of masticatory and speech deficiency because of radiation therapy. after a period of controlling post radiation caries, the patient was rehabilitated with tooth and implant supported metal ceramic restorations following surgical and endodontic intervention. |
eukaryotes have been living in intimate contact with prokaryotes throughout the course of evolution. especially in the context of the gut, the host has developed highly conserved interrelated systems to protect itself from pathogenic attack whilst allowing beneficial microbes - symbionts - to thrive. past study of primordial immune mechanisms conserved in non - vertebrates has richly informed our understanding of the activation (and suppression) of innate immune networks. as adaptive immunity is restricted to vertebrates, our appreciation of how gut commensal communities interact with mucosal and systemic adaptive immunity is less understood. the gut / mucosal arm of the adaptive immune system provides humoral and cell - mediated immunity against ingested antigens and luminal organisms and is localized predominantly in the small bowel. effector lymphocytes are diffusely distributed in the lamina propria (lp) or in isolated lymphoid follicles or are organized into discrete structures termed peyer s patches, which are essentially mucosal lymph nodes overlaid with a specialized epithelial cell type, the m cell, which possesses the endocytic machinery for uptake of particulate antigens from the gut lumen. members of the normal flora, along with non - viable particulate antigens and all - too - viable pathogens, are continually being sampled by the m cells and perhaps other portals for processing by local dendritic cells or macrophages and subsequent education and activation of effector b and t cells. peyer s patches are grossly hypoplastic and iga responses are reduced in germ - free animals. it is also known that germ - free animals have reduced total cd4 t - cell populations and an inappropriate balance of th - cell subsets, which can be moderated within weeks upon colonization with a representative member of the normal flora (bacteroides fragilis) via dendritic cell recognition of a specific polysaccharide (polysaccharide a) component of b. fragilis. interestingly, the intestinal lp in healthy animals was shown to be a major location of a unique population of interleukin-17-producing cd4 t cells (th17 cells) distinct from th1- or th2-cell lineages. this work has spawned an explosion of interest in the development and functions of th17 cells in health and disease, especially in the gastrointestinal tract. while th17 cells are increased in inflamed intestinal tissue, it remains unclear whether these cells are pathogenic or protective during intestinal inflammation ; additionally, the mechanisms driving their development and differentiation are now only beginning to emerge. analyses in germ - free mice as well as antibiotic - treated conventional mice have shown that the intestinal microbiota can drive the development of th17 cells in the murine intestine. the mechanism by which the intestinal microbiota induces th17 cells is not via the classical toll - like receptor adaptor molecules myd88 or trif, but potentially via atp generation. to begin to probe which components of the diverse microbiota may preferentially induce th17 cells, ivanov. performed an immunophenotypic comparison of the same mouse strain (b6) obtained from two commercial vendors (taconic, tarrytown, ny, usa, and the jackson laboratory, bar harbor, me, usa) and found that the mice from the jackson laboratory showed a remarkable lack of th17 cells in the lp. interestingly, transfer of taconic flora (by direct gavage of fecal flora or by cohousing) to the mice from the jackson laboratory resulted in the induction of lp th17 cells, indicating a stark difference in the immunostimulatory potential of the distinct microbiota. this observation has been further developed in two recent papers by ivanov. and gaboriau - routhiau., both reaching the surprising conclusion that t - cell development in general and th17 differentiation in particular are largely induced by the action of a single (or at least limited) number of bacterial taxa. in the paper of ivanov and colleagues, the authors exploited the phylochip (affymetrix, santa clara, ca, usa) - a 16s ribosomal microarray platform that serves as a massive (300,000 probe) parallel survey of microbial taxa - and identified segmented filamentous bacteria (sfb), a non - culturable spore - forming gram - positive clostridia - related species present in b6 mice from taconic, but not the jackson laboratory. with the use of germ - free mice or jackson laboratory - sourced mice colonized (conventionalized) with sfb, this group studied th17 differentiation in lp lymphocyte populations and expression profiling to show that sfb could recapitulate the inductive effects of the intact taconic microbiota. an independent group headed by gaboriau - routhiau also used an expression profiling approach to characterize host responses of germ - free mice conventionalized with normal murine microbiota and found distinct patterns of innate (inflammatory) gene expression and adaptive immune stimulation (lp t - cell maturation). this signature of normal immune development was not induced by candidate mono - associated microbiota representatives or complex normal human microflora ; however, the host immune response pattern to normal murine microflora could be recapitulated by sfb as a mono - associated strain, further implicating this taxon as a primary driver of adaptive immune development (figure 1). recent reports implicate segmented filamentous bacteria (sfb) as the major component of the commensal microbiota which modulates mucosal t - cell development / responses. sfb, in particular, are potent inducers of th17 differentiation. the mechanisms by which these bacteria direct t - cell differentiation are poorly understood at present but may include direct interactions with intestinal epithelial cells, dendritic cells (dc), or macrophages (m) (or all three). sfb are not cultivable and must be maintained in monocultured mice and introduced to other mice by cohousing. thus, further characterization of the sfb is obviously necessary ; sequencing of their genome will be eagerly anticipated, as will be culture techniques that may allow them to be more readily introduced into test systems. it will be interesting to see what other taxa can mediate the immune stimulatory phenotype and whether there is a specific ligand (microbial - associated molecular pattern, or mamp) present on a subset (perhaps a very limited subset) of commensals. conversely, what are the specific receptors that the host uses to perceive this functional class of microbe and which cell types in specific regions of the intestine sense sfb in order to direct adaptive immunity ? in directing th - cell differentiation, do sfb and other commensal bacteria directly interact with lp dendritic cells and macrophages that augment th17 and treg (regulatory t) responses [14 - 17 ] or do they induce changes in intestinal epithelial cells that in turn imprint dendritic cells with this capacity ? it is also clearly important to identify the human sfb equivalent(s). as potential targets for modification, sfb may offer a clue to the identification of bacteria that could be added to (or suppressed from) the microflora to accrue benefits such as altered immune regulation relevant to human disorders such as inflammatory bowel disease and systemic autoimmunity. | commensal gut bacteria are necessary for the complete development of mucosal innate and adaptive immunity and thus may influence intestinal and systemic immune disorders. recent work has advanced our understanding of this association by identification of a single taxon of the murine microbiota which can stimulate t - cell development and differentiation. it is hoped that further characterization of the mechanisms involved will enable targeted manipulation of the microbiota - immune system relationship. |
serum samples for this retrospective case - control study were collected in the clinical department of neurology innsbruck between 2005 and 2014 and stored at 80c until use. serum samples from 366 individuals were included : 51 patients with adem, 41 patients with nmosd, 34 patients with clinically isolated syndrome (cis), and 89 patients with ms. nine patients with nmdar encephalitis, 94 patients with other neurologic diseases, and 48 healthy individuals were included as controls. most of the patients have previously been included in a study on nmdar antibody assay validation and in other studies on mog and/or aqp4 antibodies. nmosds were diagnosed as described by wingerchuk. in 2007, adem was diagnosed according to the criteria of the international pediatric ms study group, and ms and cis were diagnosed according to the 2005 revisions to the mcdonald criteria, as previously described. diagnosis of nmdar encephalitis was based on clinical assessment (e.g., new onset of neuropsychiatric symptoms) and demonstration of antibodies in serum or csf, as recommended recently. the relative frequency of symptoms suggestive of nmdar encephalitis was selectively increased in our cohort of patients with demyelinating diseases (figure). of these, 81 presented with seizures and/or behavioral symptoms whereas 134 did not. as expected, all 9 patients with nmda receptor (nmdar) encephalitis presented with seizures and/or behavioral symptoms. ab = antibody ; adem = acute disseminated encephalomyelitis ; aqp4 = aquaporin-4 ; cis = clinically isolated syndrome ; hc = healthy controls ; mog = myelin oligodendrocyte glycoprotein ; ms = multiple sclerosis ; nmdar - e = nmdar encephalitis ; nmosd = neuromyelitis optica spectrum disorder ; ond = other neurologic diseases. the present study was approved by the ethical committee of innsbruck medical university (study numbers am3041a and am4059). for the detection of autoantibodies against nmdar, aqp4, and mog we used live hek293a cells transfected with the respective complementary dna, as described previously. the following cutoff values were used : 1:20 for nmdar antibodies, 1:160 for mog antibodies, and 1:20 for aqp4 antibodies. to prove the specificity of antibody binding one csf sample was tested for nmdar antibodies as described above, except csf was applied undiluted but supplemented with mk-801 (sigma - aldrich, st. louis, mo) and further diluted (1:2, 1:4, etc.) in washing buffer containing mk-801. nmdar antibody testing with fixed cell - based assay (cba) and rat brain immunohistochemistry (ihc) was done as described recently. serum samples for this retrospective case - control study were collected in the clinical department of neurology innsbruck between 2005 and 2014 and stored at 80c until use. serum samples from 366 individuals were included : 51 patients with adem, 41 patients with nmosd, 34 patients with clinically isolated syndrome (cis), and 89 patients with ms. nine patients with nmdar encephalitis, 94 patients with other neurologic diseases, and 48 healthy individuals were included as controls. most of the patients have previously been included in a study on nmdar antibody assay validation and in other studies on mog and/or aqp4 antibodies. nmosds were diagnosed as described by wingerchuk. in 2007, adem was diagnosed according to the criteria of the international pediatric ms study group, and ms and cis were diagnosed according to the 2005 revisions to the mcdonald criteria, as previously described. diagnosis of nmdar encephalitis was based on clinical assessment (e.g., new onset of neuropsychiatric symptoms) and demonstration of antibodies in serum or csf, as recommended recently. the relative frequency of symptoms suggestive of nmdar encephalitis was selectively increased in our cohort of patients with demyelinating diseases (figure). of these, 81 presented with seizures and/or behavioral symptoms whereas 134 did not. as expected, all 9 patients with nmda receptor (nmdar) encephalitis presented with seizures and/or behavioral symptoms. ab = antibody ; adem = acute disseminated encephalomyelitis ; aqp4 = aquaporin-4 ; cis = clinically isolated syndrome ; hc = healthy controls ; mog = myelin oligodendrocyte glycoprotein ; ms = multiple sclerosis ; nmdar - e = nmdar encephalitis ; nmosd = neuromyelitis optica spectrum disorder ; ond = other neurologic diseases. the present study was approved by the ethical committee of innsbruck medical university (study numbers am3041a and am4059). for the detection of autoantibodies against nmdar, aqp4, and mog we used live hek293a cells transfected with the respective complementary dna, as described previously. the following cutoff values were used : 1:20 for nmdar antibodies, 1:160 for mog antibodies, and 1:20 for aqp4 antibodies. to prove the specificity of antibody binding one csf sample was tested for nmdar antibodies as described above, except csf was applied undiluted but supplemented with mk-801 (sigma - aldrich, st. louis, mo) and further diluted (1:2, 1:4, etc.) in washing buffer containing mk-801. nmdar antibody testing with fixed cell - based assay (cba) and rat brain immunohistochemistry (ihc) was done as described recently. demographic data and the nmdar, mog, and aqp4 antibody seroprevalence of all patients and controls are shown in the table. demographic data and prevalence of antibodies in patients with demyelinating diseases and controls serum nmdar antibodies were present in 9/9 (100%) patients with nmdar encephalitis, in only 1/89 (1%) patients with ms, and in none of the other cases. csf positivity of nmdar antibodies was confirmed in all patients with nmdar encephalitis by fixed cba and/or tissue ihc. csf from the patient with ms with low titer (1:20) serum nmdar antibodies at disease onset was negative for nmdar antibodies in our live cba but positive in the fixed cba and ihc. in contrast, serum was negative in the fixed cba despite being positive in live cba and weakly positive in ihc. at the age of 23, this woman presented with paresthesia of the left hand. mri showed multiple ms - typical t2 lesions, and investigation of csf showed the presence of oligoclonal bands and 15 cells/l. the patient was diagnosed with ms, and complete remission was observed after treatment with high - dose methylprednisolone. three months later, on coincided with a clear increase in t2 lesions on mri, and she was again treated with methylprednisolone followed by complete remission. however, on reoccurred 3 years after disease onset, and the patient had residual visual deficit despite methylprednisolone treatment. three months postpartum, at the age of 26, the patient presented with psychiatric (dysphoria, logorrhea) and cognitive (dyscalculia, frontal executive disorder, psychomotor slowing) symptoms. mri showed dramatic progression of t2 lesion load and serum was positive for low titer antinuclear antibodies. absorption of patient serum with nmdar - transfected cells abolished the nmdar - specific signal in the live cba, whereas incubation with nontransfected cells did not, proving the specificity of antibodies against nmdar. could be treated for an autoimmune cause of the psychiatric symptoms, she was transferred to an outside psychiatric hospital and no further clinical data were available. demographic, clinical, and antibody data of all patients with demyelinating cns disorders with seizures and/or behavioral symptoms, which are the major initial symptoms of nmdar encephalitis, are shown in the figure. overall, we found serum nmdar antibodies in 1 of 215 patients with cns demyelinating diseases, suggesting that the frequency of those autoantibodies is very low (< 1%). although a low false - positive rate of igg nmdar antibodies was reported recently, in our study serum igg nmdar antibodies were only present in patients with symptoms suggestive of nmdar encephalitis. the major limitation of our study was the small number of available csf samples, which could have possibly yielded a higher number of nmdar antibody positive patients with demyelinating diseases when using suitable testing methods. as demonstrated here, live cba might be more sensitive for detecting low titer serum nmdar antibodies but not for testing csf samples, as currently used. the disease course of the 1 patient with ms and nmdar antibodies was initially typical for relapsing - remitting ms with residual visual deficits after the second episode of on. because analysis of nmdar antibodies was done retrospectively, the occurrence of antibodies during the first episode of demyelination was not known at that time point. csf was positive for nmdar antibodies at disease onset, in line with studies suggesting csf to be more sensitive than serum. with the onset of psychiatric and cognitive symptoms, this case demonstrates that nmdar autoantibodies can be linked with demyelination, which has been shown in a recent study investigating a cohort of 691 patients with nmdar encephalitis, identifying a subgroup of patients with demyelinating episodes. most reported cases (18/23) of demyelinating syndromes overlapping with the presence of nmdar antibodies were positive for at least 1 antibody associated with demyelination, namely antibodies against mog or aqp4. in our case, those antibodies were not found. of note, nmdar encephalitis related symptoms occurred 3 months after the patient gave birth to a healthy child, paralleling a recently described case, although our case showed a less severe disease course. we found nmdar antibodies to be rare in demyelinating diseases, even when focusing on patients with symptoms suggestive of nmdar encephalitis. in the case described here, the presence of nmdar antibodies preceded the occurrence of symptoms associated with nmdar encephalitis. thus, serum nmdar antibodies could have diagnostic value for an overlapping nmdar encephalitis related disease course in patients with demyelinating diseases. conceived and designed the experiments : m. ramberger, g.b., m. reindl. performed the experiments : m. ramberger, k.s., this study was supported by research grants from the fonds zur frderung der wissenschaftlichen forschung, austria (fwf graduate program w1206 spin) and the austrian federal ministry of science and economy (grant big wig ms). m. ramberger, g. bsteh, k. schanda, and r. hftberger report no disclosures. k. rostsy is on the scientific advisory board for novartis and received speaker honoraria from merck - serono. lutterotti received travel funding from european charcot foundation and fundacio gaem and received research support from austrian ms society. f. deisenhammer is on the scientific advisory board for biogen - idec, novartis pharma, genzyme, merck, and teva ; received travel funding and/or speaker honoraria from bayer healthcare, biogen - idec, novartis, and genzyme ; was a section editor for multiple sclerosis and related disorders ; and received research support from biogen - idec and innovative medicine initiatives. m. reindl is an academic editor for plos one, was on the editorial board for current medicinal chemistry and autoimmune diseases, and received research support from fonds zur frderung der wissenschaftlichen forschung and austrian federal ministry of science, research and economy. reindl and medical university of innsbruck offer testing for mog antibodies for research purposes only (free of charge). the university hospital and medical university of innsbruck receive payments for antibody assays (nmdar, aqp4, and other autoantibodies) and for aqp4 antibody validation experiments organized by euroimmun. | objectives : to analyze the frequency of nmda receptor (nmdar) antibodies in patients with various inflammatory demyelinating diseases of the cns and to determine their clinical correlates.methods:retrospective case - control study from 2005 to 2014 with the detection of serum igg antibodies to nmdar, aquaporin-4, and myelin oligodendrocyte glycoprotein by recombinant live cell - based immunofluorescence assays. fifty - one patients with acute disseminated encephalomyelitis, 41 with neuromyelitis optica spectrum disorders, 34 with clinically isolated syndrome, and 89 with multiple sclerosis (ms) were included. due to a known association of nmdar antibodies with seizures and behavioral symptoms, patients with those clinical manifestations were preferentially included and are therefore overrepresented in our cohort. nine patients with nmdar encephalitis, 94 patients with other neurologic diseases, and 48 healthy individuals were used as controls.results:nmdar antibodies were found in all 9 patients with nmdar encephalitis but in only 1 of 215 (0.5%) patients with inflammatory demyelination and in none of the controls. this patient had relapsing - remitting ms with nmdar antibodies present at disease onset, with an increase in nmdar antibody titer with the onset of psychiatric symptoms and cognitive deficits.conclusion:in demyelinating disorders, nmdar antibodies are uncommon, even in those with symptoms seen in nmdar encephalitis. |
at the time of cataract surgery, astigmatism can be corrected combining incisional techniques, such as limbal relaxing incisions (lris) with conventional intraocular lenses (iols), or directly implanting a toric iol. both methods present strengths and weaknesses. lris are a relatively safe and inexpensive procedure inducing low amount of irregularity on corneal topographies, as well as minor glare and patient discomfort.14 however, we know that lris are surgeon - dependent and result in some degree of variability and unpredictability, which causes an increase in higher - order aberrations (hoas) and,5 occasionally, in dry eye and healing problems.6 on the other hand, toric iols are precise, predictable, and reliable, while requiring specialized surgical skills.79 indeed, toric iol effective correction of astigmatism relies on performing accurate keratometry and on the mastery of a perfect insertion technique, thus avoiding any postoperative (postop) rotation, even though rotations may be independently induced by postop capsular bag shrinkage.10 although several studies report that toric iols have advantages in comparison to lris in terms of astigmatism correction,1124 we wanted to assess the final changes in corneal wavefront aberration caused by an lri after cataract surgery through direct comparison before and after surgery on a single patient. the main objective of this study was to determine whether conventional lris using a diamond knife have an impact on corneal hoa and therefore on visual optical quality. this prospective, cumulative, interventional, nonrandomized, case study included astigmatic patients undergoing cataract surgery with iol implantation between october 2011 and february 2012. an informed consent form in accordance with the helsinki declaration was obtained from each patient. the institutional review board and the ethics committee of ospedale fatebenefratelli e oftalmico, milan, italy, approved the study. inclusion criteria were patients with cataract and lens opacity classification system iii severity of no1, c1, p1, or more;25 keratometric (k) astigmatism greater than 0.5 diopters (d) ; and no other ocular comorbidity that might influence visual outcome. exclusion criteria were patients with active ocular diseases, significant level of corneal hoa exceeding 0.350 m, using the zernike polynomials from the topography map (opd - scan ii ; nidek co., ltd., gamagori, japan), and a high irregular astigmatism index (> 0.54). irregular astigmatism index is the result of average sum of interring area - corrected dioptric variations along every semimeridian for the whole analyzed surface and normalized by average corneal power (acp) and the number of all measured points. it describes the short - range semimeridional fluctuation of power distribution and increases along with local irregular corneal astigmatism. length and number of lris were calculated on a web site (http://www.lricalculator.com, last accessed february 25, 2012) in order to obtain the minimum value of residual astigmatism. this calculator employs donnenfeld nomogram (dono) and automatically takes into account the vector from the phaco incisions. k readings, surgically induced astigmatism (sia), and tunnel location were required. k readings were simulated keratometry (sim ks) provided by corneal navigator, a function integrated into opd - scan ii. sim ks values include curvature radii along the steepest and the flattest meridians crossing each other at right angles and corneal refractive powers based in a 3 mm zone centrally on an axial map. a sia value of 0.20 d and a temporal tunnel location was also introduced (180 for right eyes and 0 for left eyes). in case of a sia cylinder of 00.5 d, the lri calculator software suggests a 30 phaco incision. lower than 0.5 d (including sia), the software automatically displays a message to avoid lris at all. all procedures were performed by the same surgeon (as), under topical anesthesia. preoperatively and before pupil dilatation, slit - lamp - marking technique was performed with patient sitting in upright position and staring at a distant target at head height using the contralateral eye. the finest slit allowed by the slit - lamp was centered on the corneal apex and rotated to the central axis of the planned incisions, using the slit rotation index. the tip of a 30 gauge needle was used to mark limbus at both ends. the length of lris was then marked by a fine - tipped marking pen at the beginning of surgery, using a mendez ring (martel inc., dover, de, usa). based on the procedure described by langerman,26 after proceeding to the operating room and under the operating microscope, a vertical limbal relaxing wound was created with a guarded micrometer diamond blade (meyco, biel - bienne, switzerland) by making a 0.5 mm groove inside limbus in clear cornea. the incision depth was standardized at 600 m. after making a single or paired incision as indicated by the nomogram, a 2.2 mm sutureless temporal clear corneal tunnel was created, avoiding overlapping with lris. as lris approximately have a 1:1 coupling ratio, no change in iol power was needed. the coupling ratio describes the amount of flattening induced in the incised meridian referring to the steepening occurring 90 away. in case of lris, negligible change in sphero - equivalent occurs, thus avoiding any adjusting of iol power.13 all patients underwent full ophthalmologic examination preoperatively and postoperatively (after 3 years), including refractive status, uncorrected distance visual acuity (udva) and best - corrected distance visual acuities (cdvas) for distance, slit - lamp examination, tonometry, and funduscopy. visual data were obtained using logmar units. additionally, corneal topography and aberrometry (opd - scan ii), endothelial cell count (non - con robo sp 6000, konan medical inc., irvine, ca, usa), corneal thickness (pentacam hr, oculus, inc., lynnwood, wa, usa), biometry (lenstar ls900, haag - streit ag, koeniz, switzerland), and macular optical coherence tomography (hra2, heidelberg engineering gmbh, heidelberg, germany) were performed (when preoperatively feasible). wavefront and topographic assessments were performed using opd - scan ii, which converts dynamic skiascopy data points (up to 1,440) into wavefront values, using zernike polynomials up to eighth order. the opd - scan ii measures the whole eye wavefront aberration and, through the placido - based corneal topography adoption, directly calculates corneal wavefront aberration. opd - scan ii has demonstrated similar reproducibility but lower interobserver variability when compared to other wavefront aberrometers.27,28 all tests were performed by an independent observer (gm). data analysis was performed by one of the authors (gm), upon completion of the data collection. on a 3-year follow - up visit, the following changes in corneal parameters were considered : 1) root mean square (rms) of wave aberration for total z(n, i)(1n8), astigmatism z(2,1), coma z(357,1), trefoil z(357,2), spherical z(468,0), and hoa z(3n8) with a 4.0 mm aperture diameter ; 2) sim ks ; 3) keratometric cylinder (cyl) ; 4) difference between pre - calculated (expected) and postop (obtained) cyl ; 5) acp (acp is an area - corrected average of corneal power ahead of the entrance pupil and generally equal to k spherical equivalent, except for decentered refractive surgical procedures) ; 6) surface regularity index (sri is correlated to potential visual acuity and measures local fluctuations in central corneal power) ; and 7) standard deviation of corneal power (sdp is calculated from distribution of all corneal powers in a videokeratograph ; it is often elevated in all situations involving a wide range of powers occurring in measured topography). postop udva and cdva were also reported. since preop and postop cyl presents different axes, comparison between preop versus postop, and expected versus obtained values was carried out using polar value analysis.29 statistical analysis was performed using spss windows software (version 15.0, spss inc., a sample size was determined from the data collected by the authors in previous studies. based on such information, a 95% confidence level, a 20% precision and a p 0.54). irregular astigmatism index is the result of average sum of interring area - corrected dioptric variations along every semimeridian for the whole analyzed surface and normalized by average corneal power (acp) and the number of all measured points. it describes the short - range semimeridional fluctuation of power distribution and increases along with local irregular corneal astigmatism. length and number of lris were calculated on a web site (http://www.lricalculator.com, last accessed february 25, 2012) in order to obtain the minimum value of residual astigmatism. this calculator employs donnenfeld nomogram (dono) and automatically takes into account the vector from the phaco incisions. k readings, surgically induced astigmatism (sia), and tunnel location were required. k readings were simulated keratometry (sim ks) provided by corneal navigator, a function integrated into opd - scan ii. sim ks values include curvature radii along the steepest and the flattest meridians crossing each other at right angles and corneal refractive powers based in a 3 mm zone centrally on an axial map. a sia value of 0.20 d and a temporal tunnel location was also introduced (180 for right eyes and 0 for left eyes). in case of a sia cylinder of 00.5 d, the lri calculator software suggests a 30 phaco incision. lower than 0.5 d (including sia), the software automatically displays a message to avoid lris at all. all procedures were performed by the same surgeon (as), under topical anesthesia. preoperatively and before pupil dilatation, slit - lamp - marking technique was performed with patient sitting in upright position and staring at a distant target at head height using the contralateral eye. the finest slit allowed by the slit - lamp was centered on the corneal apex and rotated to the central axis of the planned incisions, using the slit rotation index. the tip of a 30 gauge needle was used to mark limbus at both ends. the length of lris was then marked by a fine - tipped marking pen at the beginning of surgery, using a mendez ring (martel inc., dover, de, usa). based on the procedure described by langerman,26 after proceeding to the operating room and under the operating microscope, a vertical limbal relaxing wound was created with a guarded micrometer diamond blade (meyco, biel - bienne, switzerland) by making a 0.5 mm groove inside limbus in clear cornea. the incision depth was standardized at 600 m. after making a single or paired incision as indicated by the nomogram, a 2.2 mm sutureless temporal clear corneal tunnel was created, avoiding overlapping with lris. coaxial phacoemulsification was subsequently performed with acrysof iq sn60wf (alcon laboratories, inc., as lris approximately have a 1:1 coupling ratio, no change in iol power was needed. the coupling ratio describes the amount of flattening induced in the incised meridian referring to the steepening occurring 90 away. in case of lris, negligible change in sphero - equivalent occurs, thus avoiding any adjusting of iol power.13 all patients underwent full ophthalmologic examination preoperatively and postoperatively (after 3 years), including refractive status, uncorrected distance visual acuity (udva) and best - corrected distance visual acuities (cdvas) for distance, slit - lamp examination, tonometry, and funduscopy. visual data were obtained using logmar units. additionally, corneal topography and aberrometry (opd - scan ii), endothelial cell count (non - con robo sp 6000, konan medical inc., irvine, ca, usa), corneal thickness (pentacam hr, oculus, inc., lynnwood, wa, usa), biometry (lenstar ls900, haag - streit ag, koeniz, switzerland), and macular optical coherence tomography (hra2, heidelberg engineering gmbh, heidelberg, germany) were performed (when preoperatively feasible). wavefront and topographic assessments were performed using opd - scan ii, which converts dynamic skiascopy data points (up to 1,440) into wavefront values, using zernike polynomials up to eighth order. the opd - scan ii measures the whole eye wavefront aberration and, through the placido - based corneal topography adoption, directly calculates corneal wavefront aberration. opd - scan ii has demonstrated similar reproducibility but lower interobserver variability when compared to other wavefront aberrometers.27,28 all tests were performed by an independent observer (gm). data analysis was performed by one of the authors (gm), upon completion of the data collection. on a 3-year follow - up visit, the following changes in corneal parameters were considered : 1) root mean square (rms) of wave aberration for total z(n, i)(1n8), astigmatism z(2,1), coma z(357,1), trefoil z(357,2), spherical z(468,0), and hoa z(3n8) with a 4.0 mm aperture diameter ; 2) sim ks ; 3) keratometric cylinder (cyl) ; 4) difference between pre - calculated (expected) and postop (obtained) cyl ; 5) acp (acp is an area - corrected average of corneal power ahead of the entrance pupil and generally equal to k spherical equivalent, except for decentered refractive surgical procedures) ; 6) surface regularity index (sri is correlated to potential visual acuity and measures local fluctuations in central corneal power) ; and 7) standard deviation of corneal power (sdp is calculated from distribution of all corneal powers in a videokeratograph ; it is often elevated in all situations involving a wide range of powers occurring in measured topography). postop udva and cdva were also reported. since preop and postop cyl presents different axes, comparison between preop versus postop, and expected versus obtained values statistical analysis was performed using spss windows software (version 15.0, spss inc., chicago, il, usa). a sample size was determined from the data collected by the authors in previous studies. based on such information, a 95% confidence level, a 20% precision and a p<0.05 variability, the number of eyes necessary to detect the difference between preop and postop values was 64. normality of all data samples was assessed using kolmogorov smirnov test. for parametric analysis, a student s t - test for paired data was performed for comparisons between preop and postop examinations or expected versus obtained data. whenever parametric analysis was not feasible, wilcoxon rank - sum test was performed to assess the significance of difference between preop and postop conditions, or expected versus obtained data. out of 55 patients recruited, seven were excluded on account of : central retinal vein occlusion (one patient), concomitant cardiovascular diseases (three patients), no - show on follow - up visits (three patients). sixty - four eyes of 48 patients (27 female and 21 male) were included in this study., no statistical difference was found in terms of rms of total aberrations z(n, i)(1n8) (p=0.61) which decreased mainly due to reduction in astigmatism z(2,1) (p=0.10). no significant changes were found in the rms value of hoa z(3n8) (p=0.13) except for the trefoil z(3,2), which showed a significant increase (p=0.004). in table 2, the visual and refractive data are reported. analysis of kp90 and kp135 polar values did not show a statistical decrease (p=0.22 and p=0.24, wilcoxon rank - sum test). the target cyl was not reached, as confirmed by the analysis of the difference between expected and obtained cyl (p<0.01, wilcoxon rank - sum test). lris did not induce alteration and fluctuations in central corneal power (acp, sdp, and sri). at the time of cataract surgery, astigmatism can be corrected using incisional techniques, such as lris. yet, the aim of this study was to assess long - term changes induced by lris on hoa and corneal optical quality. three years after procedure, as shown in table 1, no significant differences were found in terms of the rms value of hoa between preop and postcorneal aberrations (figure 1). such a result contrasts sharply with the conclusions of a previous report by monts - mic where the authors found that astigmatic keratotomy (ak) increased hoa, both coma - like and spherical - like, in a population of patients treated for high myopic astigmatism.5 this could confirm that lris are more conservative than astigmatic keratotomy in terms of aberrations, however, probably less effective. indeed, in our study, only one value showed a significant increase : the trefoil. this figure could relate to the findings of denoyer showing how corneal trefoil increases along with size of incision during cataract surgery.30 finally, it can be assumed that if an incisional procedure, such as lris, is added during phacoemulsification surgery, third - order aberrations in cornea are only partially affected. if the unaltered indices of corneal irregularities such as sri and sdp are taken into account, it may be assumed that lris can be considered safe, thus not causing significant irregularities in corneal morphology. total wavefront aberrations decreased mainly due to reduced astigmatism (figure 2). the result has been confirmed by refractive data, as shown in table 2. cyl value decreased to a mean value of 40.1%, comparable to the one reported in literature by several authors. budak reported an absolute decrease in mean astigmatism by 44%,31 bayramlar 52%,32 kaufmann 25%,33 carvalho 50%,34 and kim 32%.35 however, analysis of kp90 and kp135 polar values did not show, in our study, a significant reduction. target postop cyl was not reached, as confirmed by the analysis of the difference between expected and obtained cyl. only sharma reported positive results using dono.36 we think that the nomograms proposed in the literature, including the one we used, are not able to thoroughly account for and correct all the tissue factors influencing the results. personalized nomograms probably are not a viable solution because they are not exact and are far too dependent on the surgeon s specific medical expertise, thus proving counterproductive. these limitations should be improved by femtosecond laser, even if recent reports disagree, probably because the technique involves the same biologic aspects.37,38 average central corneal power was not significantly modified by lris, thus confirming the value of the coupling ratio of approximately 1:1 previously published. to our knowledge previously, lindstrom found a 1:1 coupling ratio when a straight 3 mm keratotomy or a 4590 arcuate keratotomy incision is performed, thus showing that coupling ratio depends on the length, location, and depth of incision.13 thornton stated that a true 1:1 coupling ratio can only occur when corneal incisions act as an added tissue but at the same time, corneal circumference is not changed ; the latter result is achieved only in case of short, concentric, and arcuate incisions.39 the authors are aware that the present study is limited by data resulting from a 3-year assessment. this means that they can not rule out the possibility that part of the lri effect could be due to surgical regression. yet, the aim of this study was to assess the final outcome, not the evolution in time. our study demonstrated that lris during cataract surgery do not induce significant changes in corneal hoa. for this reason, though the variability of refractive correction is still a major drawback, lris can still be considered a safe and viable means to correct astigmatism in those cases where toric iols are contraindicated or not available. | purposeto compare the final changes in corneal wavefront aberration by limbal relaxing incisions (lris) after cataract surgery.methodsthis prospective cumulative interventional nonrandomized case study included cataract and astigmatic patients undergoing lris and phaco with intraocular lens implantation. lris were planned using donnenfeld nomogram. the root mean square of corneal wave aberration for total z(n, i)(1n8), astigmatism z(2,1), coma z(357,1), trefoil z(357,2), spherical z(468,0), and higher - order aberration (hoa) z(3n8) was examined before and 3 years after surgery (optical path difference - scan ii [opd - scan ii) ]. uncorrected distance visual acuity and best - corrected distance visual acuity (cdva) for distance, keratometric cylinder, and variations in average corneal power were also analyzed.resultssixty-four eyes of 48 patients were included in the study. age ranged from 42 to 92 years (70.68.4 years). after lris, uncorrected distance visual acuity and best - corrected distance visual acuity improved statistically (p<0.01). the keratometric cylinder value decreased by 40.1%, but analysis of kp90 and kp135 polar values did not show any decrease that could be statistically confirmed (p=0.22 and p=0.24). no significant changes were detected in root mean square of total (p=0.61) and hoas (p=0.13) aberrations. lris did not induce alteration in central corneal power confirming a 1:1 coupling ratio.conclusionlris determined a nonsignificant alteration of corneal hoa. therefore, lris can be still considered a qualitatively viable mean in those cases where toric intraocular lenses are contraindicated or not available. yet, the authors raise the question of nonpersonalized nomograms, as in the present study, lris did not reach the preset target cylinder. |
globally, oral squamous cell carcinoma (oscc) is one of the ten most frequently diagnosed cancers. all the modern treatment modalities have been unable to modify the survival rate of oscc. the factors that are presently considered to influence prognosis of oral cancers are tumor size, histologic type, and lymph node metastasis. malignant tumor cells have a tendency to invade the surrounding matrix and penetrate the basement membrane. proteases are thought to play an important role in tumor invasion and metastasis because they degrade extracellular matrices and basement membranes. aberrant signaling cascades leading to selective over - expression of a number of proteinases cause the highly invasive and metastatic phenotypes of malignant cells. such proteinases include aspartic proteases (cathepsin d [cd ]), cysteine proteases (cathepsin b), matrix metalloproteinases (mmp-1, mmp-2, mmp-3, mmp-7, and mmp-9), serine proteinases, and urokinase - type plasminogen activator. cathepsins are lysosomal endopeptidases, which, in normal cells, participate in the proteolysis of endocytosed proteins and proteolytic activation of secretary proteins. they are powerful inducible proteinases with broad substrate specificity ; some of them may be induced by oncoproteins (ras, fos / jun), hormones, and cytokines (interleukin-1, granulocyte - macrophage colony stimulating factor). secretion and alteration in the subcellular localization of cathepsins in malignant cells is presumed to function in the digestion of the extracellular matrix components. cd, a lysosomal acidic protease, was first identified as a 52 kda estrogen dependent glycoprotein in mcf-7 cells (michigan cancer foundation-7 cells)). three forms of cd include the 52 kda procathepsin d (pcd) (enzymatically inactive), an active intermediate form (48 kda) and mature 34 kda and 14 kda dimer form. the mature forms of cd proteolytically degrade extracellular matrices and proteoglycans. in the last decade ; however, an increasing number of studies demonstrated that enzymatic function of cd is not restricted solely to its proteolytic action, but also involves regulation of apoptosis along with a significant role in mitogenesis ; numerous studies found that pcd / cd level represents an independent prognostic factor in a variety of cancers and is therefore, considered as to be a potential target of anti - cancer therapy. the purpose of this study was to find out any association between the cd with lymph node metastasis and to study the correlation of cd with various clinicopathological parameters to aid in assessment of its role as a prognostic indicator. twenty primary oscc specimens were retrieved from department of oral and maxillofacial pathology of a tertiary care teaching dental hospital of north india. the specimens included previously untreated, surgically resected tumors for which complete clinico - pathological data was available. clinicopathological information on each case including age, sex, and tumor size, nodal status, tumor grading, and staging was collected. out of 20 patients, 13 (65%) patients were males and 7 (35%) patients were females. grossly, identified cervical lymph nodes were examined microscopically to classify patients as node - negative or node - positive. patient and tumor characteristics paraffin - embedded sections (5 m thickness) were deparaffinised and rehydrated, and endogenous paraffin activity was blocked with 3% h2o2 in methanol. for cd antibody immunostaining (biogenex), antigen retrieval was achieved by boiling tissue specimens for 5 min at 95c and 5 min at 98c in antigen retrieval machine (ez v.2.1 antigen retriever system) in citrate buffer (ph 6). staining was performed using standard avidin - biotin method with appropriate secondary antibody.. a reproducible semi - quantitative assessment of ihc staining was used to evaluate the expression level of cd in tumor samples. cd expression was measured by examining 100 tumor cells in four randomly selected high power fields. the number of cd positive tumor cells was calculated along with the intensity of staining ranging from 0 to 4. score (0 - 4) for cd staining in each tissue section was then calculated by multiplying and proportion of positive cells at each staining intensity by the numerical value of that intensity. the cd expression was graded from 0 to 4 and expression levels of each cathepsin in tumor samples were graded based on the total score as follows : negative expression = score 0 - 0.5 ; low expression = score 0.6 - 1.0, high expression = score > 1.0. h and e sections were graded as well differentiated, moderately differentiated and poorly differentiated oscc on the basis of anneroth classification, which included degree of keratinization, nuclear polymorphism, number of mitosis, pattern of invasion, stage of invasion and lymphoplasmocytic infiltration for histologic grading of malignancy of tumor - host relationship. in each case, the lympho - plasmocytic infiltration was graded as marked, moderate, slight or none. first group without lymph node metastasis included 4 (20%) cases of well - differentiated osccs, 3 (15%) cases of moderately differentiated and 3 (15%) cases of poorly differentiated osccs, and second group with lymph node metastasis included 10 (50%) cases of well - differentiated osccs. both non - parametric and parametric statistical procedures were used in analyzing data, depending upon variable of interest (i.e., pearson chi - square analyses, spearsman correlation coefficient, mann - whitney test, kruskal wallis test and student t test). twenty primary oscc specimens were retrieved from department of oral and maxillofacial pathology of a tertiary care teaching dental hospital of north india. the specimens included previously untreated, surgically resected tumors for which complete clinico - pathological data was available. clinicopathological information on each case including age, sex, and tumor size, nodal status, tumor grading, and staging was collected. out of 20 patients, 13 (65%) patients were males and 7 (35%) patients were females. grossly, identified cervical lymph nodes were examined microscopically to classify patients as node - negative or node - positive. patient and tumor characteristics paraffin - embedded sections (5 m thickness) were deparaffinised and rehydrated, and endogenous paraffin activity was blocked with 3% h2o2 in methanol. for cd antibody immunostaining (biogenex), antigen retrieval was achieved by boiling tissue specimens for 5 min at 95c and 5 min at 98c in antigen retrieval machine (ez v.2.1 antigen retriever system) in citrate buffer (ph 6). one section from each sample was stained with hematoxylin and eosin for concurrent histopathologic evaluation. a reproducible semi - quantitative assessment of ihc staining was used to evaluate the expression level of cd in tumor samples. cd expression was measured by examining 100 tumor cells in four randomly selected high power fields. the number of cd positive tumor cells was calculated along with the intensity of staining ranging from 0 to 4. score (0 - 4) for cd staining in each tissue section was then calculated by multiplying and proportion of positive cells at each staining intensity by the numerical value of that intensity. the cd expression was graded from 0 to 4 and expression levels of each cathepsin in tumor samples were graded based on the total score as follows : negative expression = score 0 - 0.5 ; low expression = score 0.6 - 1.0, high expression = score > 1.0. h and e sections were graded as well differentiated, moderately differentiated and poorly differentiated oscc on the basis of anneroth classification, which included degree of keratinization, nuclear polymorphism, number of mitosis, pattern of invasion, stage of invasion and lymphoplasmocytic infiltration for histologic grading of malignancy of tumor - host relationship. in each case, the lympho - plasmocytic infiltration was graded as marked, moderate, slight or none. first group without lymph node metastasis included 4 (20%) cases of well - differentiated osccs, 3 (15%) cases of moderately differentiated and 3 (15%) cases of poorly differentiated osccs, and second group with lymph node metastasis included 10 (50%) cases of well - differentiated osccs. both non - parametric and parametric statistical procedures were used in analyzing data, depending upon variable of interest (i.e., pearson chi - square analyses, spearsman correlation coefficient, mann - whitney test, kruskal wallis test and student t test). cd staining was found to be positive in all the cases, although there was a diverse expression with intensity and number of positive cells varying from very low to very high levels. chief pattern of the staining was paranuclear punctuate type among the differentiated cells at the center of the tumors with the undifferentiated cells at the infiltrating margins, showing diffuse cytoplasmic and cell surface staining. 6 cases (30%) of oscc showed a low level of cd expression [figures 1 and 2 ], whereas 14 (70%) cases exhibited high levels of cd expression [figures 3 and 4 ]. photomicrograph showing low cathepsin d expression in an tumour island of oscc without metastasis (cathepsin d immunostaining, original magnification 40) photomicrograph showing extremely focal cathepsin d expression in another case of oscc without metastasis (cathepsin d immunostaining, original magnification 40) photomicrograph showing a high level of cathepsin d expression in oscc with metastasis (cathepsin d immunostaining, original magnification 40) photomicrograph showing a high level and intensity of cathepsin d expression in another case of oscc with metastasis (cathepsin d immunostaining, original magnification 40) sex of the patient and lymph node metastasis were not significantly associated. the cases with lymph node metastasis showed a significantly increased intensity of cd expression (p < 0.05). oscc with lymph node metastasis showed only slight lymphoplasmocytic infiltration in a highly significant manner (p < 0.001). a significantly increased cd expression was observed in patients with lymph node metastasis (100% cases) compared to patients without lymph node metastasis (p < 0.01) [table 2 ]. although, the correlation of tumor duration and cd expression with the sex of the patient was not significant, number of cd positive cells was significantly increased in male patients without lymph node metastasis (p < 0.05) [table 3 ]. qualitative analysis of clinical parameters in scc correlation of sex with duration cathepsin d expression and number of cathepsin d + cells no significant association was observed between sex of the patient and other variables such as tumor duration, cd expression, and the number of cd positive cells in any case [table 3 ]. the intensity of cd staining was not significantly related to duration of the tumor and number of cd positive cells in all the cases [table 4 ]. no significant relation could be observed between size of the tumors and other parameters including cd expression and number of cd positive cells in cases without lymph node metastasis. however, increasing cd expression was significantly associated with tumor size in cases with lymph node metastasis [table 5 ]. furthermore, tumor size did not correlate to the cd intensity, and the number of cd positive cells in scc [tables 5 and 6 ]. the intensity of cd staining did not correlate with the increasing tumor grading in any case [table 7 ]. the number of cd positive cells and cd expression did not significantly rise with increasing tumor staging [table 8 ]. although, the correlation between cd expression and intensity of expression to tumor grading was found to be insignificant, number of cd positive cells correlated significantly with increasing tumor grade in squamous cell carcinoma (scc) without lymph node metastasis (p < 0.05) [table 9 ]. lymphoplasmocytic infiltration did not correlate with cd positive cells, cd expression, and staining intensity in cases without lymph node metastasis [table 10 ]. no correlation was found between lymphoplasmocytic infiltration and cd staining intensity [table 11 ]. correlation of staining intensity with duration, number of cathepsin d + cells and age correlation of tumor size with cathepsin d expression and number of cathepsin d + cells correlation of tumor size with intensity in scc correlation of staining intensity with staging of scc correlation of staging with the number of cathepsin d + cells and cathepsin d expression correlation of histopathological diagnosis with the number of cathepsin d + cells and cathepsin d expression correlation of lymphoplasmocytic infiltration with number of cathepsin d + cells and cathepsin d expression correlation of lymphoplasmocytic intensity with staining intensity cd staining was found to be positive in all the cases, although there was a diverse expression with intensity and number of positive cells varying from very low to very high levels. chief pattern of the staining was paranuclear punctuate type among the differentiated cells at the center of the tumors with the undifferentiated cells at the infiltrating margins, showing diffuse cytoplasmic and cell surface staining. 6 cases (30%) of oscc showed a low level of cd expression [figures 1 and 2 ], whereas 14 (70%) cases exhibited high levels of cd expression [figures 3 and 4 ]. photomicrograph showing low cathepsin d expression in an tumour island of oscc without metastasis (cathepsin d immunostaining, original magnification 40) photomicrograph showing extremely focal cathepsin d expression in another case of oscc without metastasis (cathepsin d immunostaining, original magnification 40) photomicrograph showing a high level of cathepsin d expression in oscc with metastasis (cathepsin d immunostaining, original magnification 40) photomicrograph showing a high level and intensity of cathepsin d expression in another case of oscc with metastasis (cathepsin d immunostaining, original magnification 40) the cases with lymph node metastasis showed a significantly increased intensity of cd expression (p < 0.05). oscc with lymph node metastasis showed only slight lymphoplasmocytic infiltration in a highly significant manner (p < 0.001). a significantly increased cd expression was observed in patients with lymph node metastasis (100% cases) compared to patients without lymph node metastasis (p < 0.01) [table 2 ]. although, the correlation of tumor duration and cd expression with the sex of the patient was not significant, number of cd positive cells was significantly increased in male patients without lymph node metastasis (p < 0.05) [table 3 ]. qualitative analysis of clinical parameters in scc correlation of sex with duration cathepsin d expression and number of cathepsin d + cells no significant association was observed between sex of the patient and other variables such as tumor duration, cd expression, and the number of cd positive cells in any case [table 3 ]. the intensity of cd staining was not significantly related to duration of the tumor and number of cd positive cells in all the cases [table 4 ]. no significant relation could be observed between size of the tumors and other parameters including cd expression and number of cd positive cells in cases without lymph node metastasis. however, increasing cd expression was significantly associated with tumor size in cases with lymph node metastasis [table 5 ]. furthermore, tumor size did not correlate to the cd intensity, and the number of cd positive cells in scc [tables 5 and 6 ]. the intensity of cd staining did not correlate with the increasing tumor grading in any case [table 7 ]. the number of cd positive cells and cd expression did not significantly rise with increasing tumor staging [table 8 ]. although, the correlation between cd expression and intensity of expression to tumor grading was found to be insignificant, number of cd positive cells correlated significantly with increasing tumor grade in squamous cell carcinoma (scc) without lymph node metastasis (p < 0.05) [table 9 ]. lymphoplasmocytic infiltration did not correlate with cd positive cells, cd expression, and staining intensity in cases without lymph node metastasis [table 10 ]. no correlation was found between lymphoplasmocytic infiltration and cd staining intensity [table 11 ]. correlation of staining intensity with duration, number of cathepsin d + cells and age correlation of tumor size with cathepsin d expression and number of cathepsin d + cells correlation of tumor size with intensity in scc correlation of staining intensity with staging of scc correlation of staging with the number of cathepsin d + cells and cathepsin d expression correlation of histopathological diagnosis with the number of cathepsin d + cells and cathepsin d expression correlation of lymphoplasmocytic infiltration with number of cathepsin d + cells and cathepsin d expression correlation of lymphoplasmocytic intensity with staining intensity the goal of this study was to determine whether high levels of cd expression in surgical specimens of oral carcinomas are associated with increased propensity for local invasive growth and lymph node metastasis. oral carcinomas with high levels of cd expression showed intense cytoplasmic and cell surface staining of tumor cells, most often concentrated at the invasive front of tumors. in our study, high levels of cd expression was observed in oral carcinomas with regional lymph node metastasis (n1/n2) compared with node - negative tumors (no) (p < 0.05). moreover, we observed that cd expression also correlated with tumor size in oscc with lymph node metastasis [table 5 ]. though, cd expression did not correlate with the stage of the tumor, but the cd positive cells correlated with the tumor grade [table 9 ]. spyratos. stressed on the role of cd as a prognostic marker for the disease - free interval in patients with breast cancer and emphasized that cd was a more effective prognostic indicator than lymph node metastasis, in the case of lymph node negative patients. the relationship between cd expression and poor survival has been known to differ among lymph node positive and negative patients. in a study by henry., high levels of cd have been associated with good outcome in breast cancer, whereas high levels of cd have been found both to correlate with aggressive disease and to show no relationship with patient outcome in other studies in node negative patients. some of these contradictory findings could be attributed to different methodology used for pcd / cd quantification, subjectivity, different types of tissue, that is fresh versus formalin fixed and paraffin embedded, patients and diagnosis selection, and the length of follow - up period. a study by vigneswaran. in oral cancer patients correlated increased expression of cd with the presence of metastasis, poor histologic malignancy grade and high proliferation rate. high levels of cd expresssion have been observed in oral carcinomas with the regional lymph node metastasis compared to node negative tumors. kawasaki. also found that higher cd immunostaining was related to invasion, cell proliferation and to expression of growth factor receptors in oscc. they also observed that cd had a significant correlation with stage of invasion and lymphoplasmocytic infiltration.. found a higher risk of metastatic disease and poor outcome in laryngeal cancer patients with higher expression of cd. earlier clinical studies have found pcd / cd related to metastasis - free survival and disease - free survival in breast cancer patient. since then, numerous clinical studies have been conducted to find an association between pcd / cd level and tumor size, tumor grade, tumor aggressiveness, incidence of metastasis, prognosis, and a degree of chemoresistance in a variety of solid tumors including head and neck tumors, but some of them have given acutely contradictory results. moreover, studies dealing with pcd / cd diagnostic and prognostic value in cancer are complicated by the fact that there are several forms of cd in a cell at the same time pcd, intermediate enzymatically active cd and mature heavy and light chain cd. presently some of the researches are also looking for the expression of cd in dysplastic cases, and that could serve as an early attempt for cancer prevention. patients with lymph node metastasis showed an increase in cd expression, which was statistically significant. increasing tumor size seemed to correlate with increased cd expression. there was a statistically significant association between increasing number of cd positive cells and increasing grades of oscc in cases without lymph node metastasis. furthermore, there was the presence of only slight lymphoplasmocytic infiltration in cases with lymph node metastasis. thus, based on the active potential of cd in regulating the prognosis of oscc the design and synthesis of specific cd inhibitors can have significant research and therapeutical consequences. however, standardization of measurement techniques for cd and its other forms is recommended for further clinical usefulness in the field of oncology. | background : the lysosomal protease cathepsin d (cd) has been associated with tumor progression in malignant tumors including oral squamous cell carcinoma (oscc). the purpose of this study was to find out any association between the cd and lymph node metastasis and to study the correlation of cd with various clinicopathological parameters to aid in assessment of its role as a prognostic indicator.materials and methods : immunohistochemical staining was performed on 20 oscc samples with polyclonal antibody against cd. positive results indicative of the presence of cd were further analyzed to determine any correlation between the cd and other clinicopathological parameters. pearson chi - square analyses, spearsman correlation coefficient, mann - whitney test, kruskal wallis test and student t test were used for statistical analysis (p < 0.05).results : patients with lymph node metastasis showed statistically significant increase in cd expression (p < 0.01). increasing tumor size seemed to correlate with increased cd expression (p < 0.05).conclusion : based on its association with other clinicopathological variables, cd expression can be used for the assessment of patient survival in cases of oscc. |
cancer is the leading cause of death in economically developed countries and the second leading cause of death in developing countries. advancements have been made in traditional treatment modalities that have been used for decades, namely, surgery, radiotherapy, and chemotherapy. in addition, with the value of early diagnosis in cancer therapy recognized, the technology of early diagnosis is also advancing. although these treatment modalities play an important role, the results are not entirely adequate, especially for advanced cancers. cancer is still a major public health problem worldwide, and new treatment modalities and strategies are still needed to optimize patient outcomes. cancer immunotherapy, which can be generally classified as passive or active, has always been an attractive and potentially efficient treatment for cancer patients. passive immunotherapy, consisting of infusion of donor t lymphocytes and transfer of anticancer monoclonal antibodies, has been proven to be an effective treatment for a variety of cancers [3, 4 ], and continued advances in t - cell engineering and antibody should further enhance their clinical impact. however, vaccines, which represent active immunotherapy, are based on the manipulation of the host immune system to fight cancer and provide a path to obtain long - lasting responses in cancer patients. as one of the major players in active immunity, cytotoxic t lymphocytes (ctls) play a critical role in immunity against cancers. a variety of vaccine strategies have been designed to meet this goal, and recent phase ii / iii clinical trials using these vaccines have achieved promising results. cancer vaccines enhance the antitumor immune response by providing the early signals of activity ; dendritic cells (dcs) play an important role in this immune response activation, which involves a number of complex processes. first, dcs must capture tumor antigens, process the captured antigen for presentation on major histocompatibility complex (mhc) molecules (either class i or class ii), and then migrate to draining lymph nodes. if capture and processing is accompanied by a suitable activation signal, dcs will enhance the activation of the immune response. if not, dcs will instead induce tolerance. second, in lymphoid organs, tumor - antigen - loaded dcs are capable of triggering protective t - cell responses, especially ctl responses. in this process, dcs require a maturation signal (i.e., a stimulatory adjuvant) in order to elicit the desired ctls [9, 10 ]. without a maturation signal, dcs present antigens in a stable state, which promotes tolerance by inducing regulatory t cell (treg) production and thereby thwarting an antitumor response [1113 ]. in addition, the ability of dcs to promote a ctl response also depends on the interaction of a positive t - cell costimulatory molecule (i.e., cd28, ox40) with dc surface receptors (i.e., cd80/cd86, ox40l) ; the interaction of the negative t - cell costimulatory molecule (i.e., ctla-4, lag-3) with the dc surface receptors can limit the activity of ctls by promoting treg formation. for these reasons, the appropriate utilization and regulation of dcs correlate with the success or failure of vaccine design. all in all, increasing immune activation by dcs is a critical step for improving ctl antitumor immune responses (figure 1). targeted therapy is a typical representative of selective, mechanism - based therapy and has become a new treatment option. based on the molecular mechanisms of ctl activation, targeted drugs can not only promote the capture of tumor antigens by dcs can also enhance the expression of costimulatory molecules such as cd40, cd80, and cd86 on the dc surface. moreover, targeted drugs block important inhibitory signals for activated t - cells, thereby maintaining t cell activation and potentiating tumor destruction. in general, through regulation of dcs and activated t cells, targeted drugs effectively boost ctl activation [1517 ]. furthermore, activated ctls must enter the tumor microenvironment to perform their functions, at which point a variety of negative regulatory signals suppress the immune response ; ctls must overcome the tolerance induced by tumor cells in order to mediate their cytotoxic effects. therefore, the effects of drugs that target the processes that generate ctl antitumor immune responses can be complementary and synergistic with cancer vaccines. combinatorial therapy provides a new treatment modality that enhances the vaccine - induced ctl antitumor immune response. however, greater focus on the appropriate dosage, sequencing, and timing of the targeted drugs is needed, as it will probably be crucial to the success of combinatorial strategies. another key issue for combinatorial therapies is the toxicity of these regimens. in general, the ultimate goal of enhancing vaccine - induced ctl antitumor immune responses encouraged by positive preclinical and clinical data [2125 ], the research into the appropriate utilization and regulation of dcs in vaccine design continues, and the study of the combination of vaccines and targeted drugs aims to solve the above - mentioned problems. dcs, as the most efficient antigen - presenting cells (apcs), have been increasingly used in various strategies of vaccine design to enhance their unique ability to activate antitumor ctls. these strategies facilitate the capture of tumor antigens by dcs, which helps to stimulate the activation of ctls in the next step. in the past few decades, vaccine design strategy has included several antigen - loading techniques to activate dcs, such as (i) pulsing dcs with peptides or proteins, (ii) transfecting dcs with dna or rna, (iii) loading dcs with tumor - cell lysates or tumor cells, and (iv) infecting dcs with bacterial, viral, or yeast vectors. (figure 1(a)) in contrast, vaccines that do not utilize dcs tend to depend on the immunogenicity of the vaccines itself, which are not enough to elicit a stronger ctl response [27, 28 ]. dcs transfected with the human telomerase reverse transcriptase (htert)-hil18 gene were capable of eliciting a stronger htert - specific ctl response in vitro than those stimulated with the htert construct only. this strategy of vaccine design utilizes dc activation in vitro in order to avoid the complex regulation of dc exposure to antigen in vivo. on antigen encounter in vivo, dcs require a suitable activation and maturation signal to promote immunity (enhanced capture, processing and presentation of tumor antigen - derived peptides) rather than tolerance [7, 30 ]. these signals include endogenous tumor cell lysates (e.g., high mobility group proteins or atp) and exogenous molecules (e.g., toll - like receptor (tlr) ligands or antibodies). like tlr ligands (which will be discussed later in this paper), monoclonal antibodies can been used to facilitate the capture of tumor antigens by dcs. trastuzumab and cetuximab are clinically efficacious monoclonal antibodies that are directed toward the tumor - associated receptor tyrosine kinases her2 and egfr, respectively. these antibodies not only downregulate oncogenic signaling, but also enhance the induction of tumor - specific ctls [31, 32 ]. these antibodies coat tumor - associated receptors on tumor cells and bind fc receptors expressed on dcs simultaneously to form immune complexes that contribute to the capture of tumor antigens by dcs. further, fc receptor - mediated opsonization enhances the expression of costimulatory molecules (i.e., cd40, cd80, and cd86) on the dc surface, boosting t - cell activation. therefore, direct inoculation of patients with vaccines that activate dcs in vivo requires combining this targeted therapy in order to promote a desired immunogenic phenotype. confirmed that combining a cancer cell vaccine and her2 antibodies resulted in significantly longer survival rates than either therapy alone in a transgenic mouse model of breast cancer. this may be due to the fact that the combination therapy increased the capture of the cancer vaccine by dcs, enhanced the expression of costimulatory molecules (i.e., cd40, cd80, and cd86) on the dcs, and elicited greater tumor - specific t - cell responses. recently, a phase i / ii clinical trial of trastuzumab combined with a her2 peptide vaccine showed that 69% of patients developed ctl immune responses to tumor cells that expressed her2. similarly, the egfr antibody cetuximab also enhances dc - mediated phagocytosis, increases the expression of the mhc class ii molecules, costimulatory molecules on dcs and induces a highly efficient antigen - specific ctl response in vitro (figure 1(b)). tumor - antigen - loaded dcs are capable of triggering protective t - cell responses, especially by activating the ctl response in lymphoid organs. however, without a maturation signal (i.e., a stimulatory adjuvant), dcs can not elicit the desired ctl response. when dcs present antigens in a stable state without an immunogenic maturation signal, regulatory t cells (treg) are induced, which both promote tolerance of antitumor responses and directly counters the antitumor response. what 's more, at the same time, stimulatory adjuvant can induce costimulatory molecules (i.e., cd40, cd80, and cd86) on the surface of dcs and the release of cytokines (i.e., ifn-, ifn-, and il-12) by dcs, which are required for t - cell activation and differentiation (figure 1(c)). many initial attempts at cancer vaccines lacked a consideration of this immune process, specifically the requirement of dcs for a stimulatory adjuvant in order to induce the desired ctls. peptide - based vaccines that were used to treat thousands of patients often lacked an effective dc - activating adjuvant. on the one hand, free peptides may be rapidly cleared before being loaded onto dcs because of their poor pharmacokinetic properties, so their half - lives may also be short. on the other hand, without an adjuvant, dcs might remain in the steady state and gradually promote tolerance rather than immunity. as a result, the ctl response to the selected tumor antigens is poor and therapeutic benefit is minimal. because the function and importance of dcs in stimulating ctl responses is well known now, effective dc - activating adjuvants are increasingly being used in vitro and in vivo. for example, il-2 is an important stimulatory adjuvant for maintaining dc growth and has received fda approval for use in melanoma and renal cell cancer. granulocyte - macrophage colony - stimulating factor (gm - csf) is necessary for the growth and differentiation of dcs, and tumor - necrosis factor- (tnf-) can promote dc maturation and antigen - presenting capabilities [38, 39 ]. tlr ligands are the ideal adjuvants, allowing a variety of functions required for ctl activation. secondly, tlr ligands induce the expression of costimulatory molecules (cd40, cd80, and cd86) on dcs that are required for t - cell activation. thirdly, tlr - induced cytokines, principally il-12, ifn-, and ifn-, guide t - cell differentiation toward either cd8 + cytotoxic lymphocytes or cd4 + t helper type 1 (th1) cells. several tlr ligands have shown significant promise for the treatment of cancer for example, the tlr7 agonist imiquimod that is currently approved for treatment of superficial basal cell carcinoma. although monotherapy with tlr agonists may provide a benefit in certain types of cancer, our interest is centered on the role of tlr agonists as stimulatory adjuvants. encouraging results have been reported in human melanoma with an antitumor peptide vaccine using cpg - odn (a tlr agonist) as an adjuvant, which promotes the generation of rapid and strong ctl antitumor responses. researchers also confirm that tlr agonists facilitate vaccine - induced ctl responses in many different mouse tumor models. therefore, its use as a vaccine adjuvant is perhaps the most extensively explored application for tlr agonists. after capture, processing and presentation of tumor antigens by dcs and activation of ctls, activated ctls must enter the tumor microenvironment to perform their functions ; at this point, a variety of negative regulatory signals interact and form a network to resist the immune response. these negative regulatory signals mainly originate from tumor cells and stromal cells such as immune cells, inflammatory cells, endothelial cells, and fibroblasts. tumor - induced mechanisms of immunosuppression are as follows : (i) tumor cells expansion or enabling the local accumulation of treg cells, which release suppressive cytokines and serve to silence immune responses [47, 48 ], (ii) tumor cells also encourage the formation of myeloid - derived suppressor cells (mdscs), which use indoleamine 2,3-dioxygenase (ido) released by tumor cells to silence the responses of cytotoxic cd8 + t cells and helper cd4 + t cells while simultaneously promoting the formation of treg cells, and (iii) tumor cells secrete a variety of products including the above - mentioned ido, vascular endothelial growth factor (vegf) and surface molecules such as pd - l1. vegf promotes the expansion of mdscs, blocks the maturation of dcs, and inhibits the expression of costimulatory molecules [50, 51 ]. pd - l1 engages receptors on the surfaces of activated t cells and causes t - cell anergy or exhaustion [52, 53 ] (figure 2(a)). in addition, t cells express other receptors that negatively regulate the immune response in the tumor microenvironment. for example, ctla-4 is a well - studied key negative regulator. when dcs present peptide epitopes to the t - cell receptor (tcr) and simultaneously present b7 costimulatory molecules (cd80, cd86) to cd28 on specific antitumor t cells, these t cells become activated. ctla-4 is subsequently upregulated and competitively engages b7 to attenuate t - cell responses (figure 2(b)). lag-3 is another negative receptor on the t cell surface that not only limits the activity of cd8 + and cd4 + t cells but also augments the activity of treg cells [55, 56 ]. moreover, macrophages polarize to type 2 macrophages (m2) because of hypoxia in the tumor microenvironment. m2-like macrophages secrete and release cytokines such as ccl17, ccl22, or ccl24, which recruit treg cells to the local environment. however, there are still a large number of immunosuppressive mechanisms in the tumor microenvironment which are not mentioned in this paper. thus, even if pathologically induced auto - ctls could avoid negative regulatory signals and overcome diverse immunosuppressive networks within the tumor microenvironment, they would still be too weak to promote tumor destruction. although vaccines (e.g., sipuleucel - t) can be used to increase the number of ctls, immunosuppression still occurs in the tumor microenvironment and can attenuate vaccine - induced ctls. for this reason, a larger number of targeted drugs have been designed and used in clinical trials to counteract negative regulatory mechanisms. imatinib can decrease ido expression and lead to an increased ratio of ctls to treg cells in a mouse model of gastrointestinal stromal tumors. reported that imatinib has a direct inhibitory effect on treg cells, including decreased numbers of treg cells and weak immunosuppressive capacity. the combination of imatinib plus a dc vaccine resulted in decreased numbers of treg cells, increased t - cell - derived ifn production and fewer metastases compared with either therapy alone in a bcrabl lymphoma model. the targeted drug sunitinib, a multityrosine kinase inhibitor that blocks vegfr function, decreased the function and number of mdscs in the tumor microenvironment of a mouse model of colon cancer. consequently, sunitinib inhibited the number and function of treg cells and augmented the activation of cytotoxic cd8 + t cells and helper cd4 + t cells. furthermore, sunitinib diminished the expression of pd-1, pd - l1, and ctla-4. the combination of a dc vaccine plus sunitinib prolonged survival compared with either agent alone in a b16 melanoma model. the anti - ctla-4 antibody ipilimumab has recently been approved by the fda for use as a first - line or second - line therapy in patients with advanced melanoma. previous studies have confirmed the fundamental importance of ctla-4 in controlling t - cell function, and ctla-4 ligation is also important for the immune suppressive function of treg cells [62, 63 ]. reported clinical trial data that led to the fda approval of anti - ctla-4 for the treatment of metastatic melanoma. their data indicates that ipilimumab, most notably, achieves durable benefits for more than 2.5 years. a subsequent clinical trial confirmed that ipilimumab plus standard care improved overall survival compared with standard care alone (11.2 months versus 9.1 months) and significantly increased the proportion of surviving patients for at least 3 years of followup (20.8% versus 12.2%). as the most important function of vaccine - induced ctls, ctl - mediated cytotoxic effects play a critical role in immunity against cancers. ctls have the ability to kill tumor cells following the interaction of their tcr with a specific mhc - i complex on tumor cells. this process is mainly mediated by the ability of effector ctls to release perforin, granzyme, and tnf-, or to express fas ligand [66, 67 ]. cytotoxicity proceeds through a multistep mechanism : (i) the tcr combines with a specific mhc - i complex on tumor cells, and then the remodeling of the actin cytoskeleton, reorganization of the cell surface, and repositioning of cytoplasmic proteins result in the formation of a so - called immune synapse, (ii) polarization of the microtubule organizing center (mtoc) toward the target occurs, and lytic granules move along the microtubules toward the mtoc, and (iii) lysis occurs through the polarized release of the content of cytotoxic granules (a process also referred to as degranulation) or expression of fas ligand on the ctl surface. the lytic pathway uses mainly two types of mechanisms to kill target cells : one is perforin mediated and another is fas ligand (fasl) based. the perforin - mediated mechanism is believed to involve secreted perforin, which forms pores in the target membrane. then co - secreted granzymes pass through the pores, enter cells and activate a cascade of caspases including caspase-3 and caspase-8 [7072 ]. the fasl - based mechanism involves crosslinking of the cell surface death receptor fas expressed on target cells induced by cell surface fasl expressed on ctls. cross - linked fas rapidly induces the assembly of an intracellular death - inducing signaling complex (disc), which recruits and activates caspase-8 and ultimately activates caspase-3, which serves as a common substrate for these two killing mechanisms [7275 ]. both of these mechanisms eventually induce apoptosis of target cells. although ctls have the capacity to efficiently kill tumor cells, tumor cells may develop escape mechanisms to evade ctl - mediated killing. for instance, tumor cells acquire resistance to perforin and granzyme or alter the expression of death receptors. in addition, the genetic instability of tumor cells can lead to the alteration of proapoptotic regulators, one of the most important being p53. it has been demonstrated that the p53 status of tumor cells has a key role in determining the fate of the antitumor ctl response, because it regulates fas receptor expression, cellular flice / caspase-8 inhibitory protein (cflip) short protein degradation, and cd95-mediated apoptosis. in addition, tumor cells can lower the expression of mhc class i molecules to avoid detection or increase the expression of antiapoptotic proteins in order to resist cytotoxic mechanisms. therefore, the combination of vaccines and some targeted drugs may counteract these escape mechanisms and improve ctl - mediated tumor destruction. (figure 3) in contrast to the escape mechanism described above, some targeted drugs enhanced sensitization of tumor cells to ctl - mediated destruction. for example, the proteasome inhibitor bortezomib sensitizes tumor cells toward adoptive ctl attack. a preclinical trial of bortezomib plus vaccination with dna encoding a tumor - specific protein is planned. the combination of an hpv e7-based vaccine and bortezomib prolonged the steady state of the disease, in contrast to either therapy alone, which had minimal effects. the increased survival correlated with enhanced sensitivity of the bortezomib - treated tumor cells to killing by e7-specific ctls. the possible mechanisms are as follows : (i) bortezomib may enhance the baseline activity of granzyme and caspase-8 in tumor cells, which could provide sensitization towards apoptosis, (ii) bortezomib may sensitize tumor cells to ctl - mediated lysis through enhanced expression of noxa, a bh3 protein that sequesters potent antiapoptotic proteins such as myeloid cell leukemia sequence 1 (mcl1), or (iii) bortezomib may increase tumor cell surface expression of the tnf - related apoptosis - inducing ligand receptor dr5 and fas, which further augments nk cell cytotoxicity. in addition, some evidence suggests that bortezomib can also sensitize tumor cells to nk cell - mediated lysis by downregulating peptide loading and mhc class i molecule expression. this is due to bortezomib inhibiting the proteasome that generates peptide epitopes for mhc class i molecules, rendering tumor cells more susceptible to killing by nk cells. pi3 k - akt inhibitors are another class of targeted drugs that sensitize tumor cells to ctl - mediated destruction through pi3 k - akt pathway inhibition ; this eliminates some prosurvival signals such as expression of antiapoptotic proteins, thereby increasing tumor cell lysis by perforin and granzymes released from ctls [83, 84 ]. confirmed that tumor cells in which akt can be inducibly expressed showed enhanced resistance to ctl killing upon akt activation compared with noninduced cells. the enhanced resistance involved an increase in mcl1 expression, which is an antiapoptotic protein. demonstrated that a tumor cell line selected for resistance to vaccination therapy upregulated akt compared with wild - type cells that are sensitive to vaccination therapy. this result was associated with increased levels of bcl-2, ciap1, and ciap2, which are also antiapoptotic proteins. moreover, noh. demonstrated that inhibiting the upregulation of the akt pathway increased the ctl - mediated killing of akt - upregulated tumor cells in vitro and akt inhibition in combination with vaccine - augmented ctl responses. a variety of strategies have been used to design vaccines that can induce a durable and long - lasting host immune response to fight cancer. however, a series of processes are necessary in order for a vaccine - induced ctl immune response to play a positive role in destroying cancer. in any step in these processes even when ctls are activated, negative signals causing t - cell anergy or exhaustion may occur in the tumor microenvironment. ctl - mediated cytotoxic effects also are affected by the escape mechanisms of tumor cells. for these reasons, when designing vaccines, we attempt to utilize and regulate dcs to elicit a much more potent ctl immune response. this strategy includes antigen - loading techniques in vitro, supplying targeted drugs that facilitate the capture of tumor antigens by dcs in vivo, and using stimulatory adjuvants that promote the whole development and function of dcs. in addition, targeted drugs can be used which block important immunosuppressive signals in the tumor microenvironment, and overcome the tolerance of tumor cells. the strategy of combining vaccines and targeted drugs increases ctl - mediated tumor cell lysis. in the context of advances in the understanding of how immunity, immunosuppression and tolerance regulate antitumor immune responses, immunotherapy and targeted drugs might have complementary and synergistic roles in cancer treatment ; therefore, the combination of vaccines and targeted drugs may become a more accepted practice in cancer treatment. | vaccine - induced cytotoxic t lymphocytes (ctls) play a critical role in adaptive immunity against cancers. an important goal of current vaccine research is to induce durable and long - lasting functional ctls that can mediate cytotoxic effects on tumor cells. to attain this goal, there are four distinct steps that must be achieved. to initiate a vaccine - induced ctl antitumor immune response, dendritic cells (dcs) must capture antigens derived from exogenous tumor vaccines in vivo or autologous dcs directly loaded in vitro with tumor antigens must be injected. next, tumor - antigen - loaded dcs must activate ctls in lymphoid organs. subsequently, activated ctls must enter the tumor microenvironment to perform their functions, at which point a variety of negative regulatory signals suppress the immune response. finally, ctl - mediated cytotoxic effects must overcome the tolerance induced by tumor cells. each step is a complex process that may be impeded in many ways. however, if these steps happen under appropriate regulation, the vaccine - induced ctl antitumor immune response will be more successful. for this reason, we should gain a better understanding of the basic mechanisms that govern the immune response. this paper, based on the steps necessary to induce an immune response, discusses current strategies for enhancing vaccine - induced ctl antitumor immune responses. |
hybridization is a controversial topic in coral reef ecology 1, 2. while small numbers of hybrid embryos can be produced in a few species in vitro, the evidence for hybrids in the field is often equivocal because the genetic techniques used for corals can not distinguish between hybridization and incomplete lineage sorting. in fact, only one of the over 1300 species in the order is generally accepted to be unequivocally of hybrid origin : acropora prolifera 1, 5. nonetheless, hybridization is often invoked as a source of evolutionary novelty in the order scleractinia 6, 7. here, we report an incidental observation on the potential for hybridization between two closely related scleractinian corals species in the family fungiidae, ctenactis echinata and ctenactis crassa. these species are sympatric, often dominating large multi - specific assemblages of fungiid corals throughout the central indo - pacific. colonies of these species are superficially very similar (figure 1a and b) but can readily be distinguished by the shape of the costal dentitions. both species are gonochoric, that is each colony is either male or female, and reproduce by broadcast spawning, releasing gametes into the water column for fertilization (figure 1c and d). at our study site on sesoko island (2638'13.00"n ; 12751'56.24"e), okinawa, japan, spawning occurs following the full moons from july to august. furthermore, both species release gametes at the same time and consequently there is the potential for hybridization. in the days before the predicted date of spawning in july 2013, we collected four colonies of c. echinata and six colonies of c. crassa, to produce larvae for other experiments. coral species in the family fungiidae, such as these colonies of fungia repanda, are gonochoric broadcast spawners : each individual releases either eggs (c) or sperm (d) into the water column where fertilization takes place (arrows indicate gametes). while the species are relatively easy to identify, determining the sex of each individual prior to spawning is impossible without destructive sampling to expose the gametes. consequently, we placed each individual in a separate 20 l bucket containing sea water in the open air at approximately 20:00 h in order to sex each individual once gametes had been released. on the night of 27 july between 22:30 and 23:30 h three c. echinata and five c. crassa spawned revealing that the three spawning c. echinata were female, while four c. crassa were females and one was a male. the size of the eggs of each species at the time of release was distinct with a range in maximum diameter of 244266 m in c. echinata and 133155 m in c. crassa. in contrast to earlier work on c. echinata, we saw no symbiotic algae in the eggs of either species. we collected approximately 5000 eggs from the three c. echinata females and mixed them with sperm from the c. crassa male. as a positive control we mixed eggs from the four c. crassa females with the c. crassa sperm. approximately 100 eggs were observed under a stereo - dissecting microscope for cleavage, indicating fertilization, every 2 to 6 h over the next 24 h. at no point did we observe cleavage in the cross between species indicating that no hybrid embryos were produced. in contrast, over 90% of c. crassa eggs in the positive control were fertilized within 2 h. we conclude that despite synchrony in the time of gamete release between these two closely related sympatric species there appears to be strong pre - zygotic mechanism to avoid hybridization. while our observations are preliminary and in only one direction (i.e. we did not cross c. echinata males with c. crassa females) we predict that hybridization between these species is unlikely. this observation adds to a growing body of evidence indicating strong pre - zygotic barriers to hybridization in many scleractinian corals 11 13. | hybridization is often cited as a potential source of evolutionary novelty in the order scleractinia. while hybrid embryos can be produced in vitro, it has been difficult to identify adult hybrids in the wild. here, we tested the potential for hybridization between two closely related species in the family fungiidae. we mixed approximately 5000 eggs of ctenactis echinata with sperm from ctenactis crass. no hybrid embryos were produced. this observation adds to a growing body of evidence for pre - zygotic barriers to hybridization in corals and challenges the claim that hybridization is a major source of evolutionary novelty in the order. |
a 71-year - old man presented at our institution 's emergency department with worsening quadriparesis. prior to this presentation, he had a 2-week history of upper respiratory tract infection as well as neck pain, which were both treated symptomatically. one week before admission, he began experiencing progressive weakness in all four limbs. on presentation upon examination the left side had grade 0 power, and the right side was slightly stronger with grade 2 power in both upper and lower limbs. blood investigations showed a total white count of 16,500/mm, c - reactive protein of 73, and erythrocyte sedimentation rate was 100 mm / h. cerebrospinal fluid analysis showed low glucose and high protein levels but failed to grow any organism. imaging included plain radiographs, cervical spine computerized tomography (ct), brain and cervical spine magnetic resonance imaging (mri). ct scans were suggestive of erosive lesions in the left - sided c1c2 and c2c3 facetal joints. mri showed evidence of an epidural abscess on the left side abutting the cervicomedullary junction as well as osteomyelitis and myositis of the structures around the atlantoaxial joint (figs. 1 2 3 4 5). there were also radiological features suggestive of meningitis and pyogenic ventriculitis with no hydrocephalus or brain abscesses. sagittal sections of magnetic resonance imaging (t2-weighted) showing extension of the abscess from c0c3. axial section of magnetic resonance imaging (t2-weighted) at c1c2 level showing a left - sided hyperintense cervical cord collection. sagittal sections of the magnetic resonance imaging (t1-weighted) showing extension of the abscess from c0c3. sagittal sections of the magnetic resonance imaging (contrast enhanced) showing extension of the abscess from c0c3. contrast - enhanced axial images showing ring enhancement of the collection. in view of the radiological findings and clinical deterioration, the posterior arch of c1 was drilled and removed while protecting the extradural portion of the vertebral artery. the epidural space did not have an abscess, but there were congested epidural veins. at this stage, an intradural abscess was suspected, and the dura was opened on the left side with stay sutures ; the left c1 nerve root was cut to get a window to decompress the abscess cavity (fig. care was taken to identify and retract the intramural portion of vertebral artery and the spinal part of the accessory nerve. dissection was limited on the anterior aspect due to proximity of the anterior spinal artery. durotomy was closed with ethilon 50 (johnson & johnson, somerville, nj) and sealed with tissue glue. cultures grew methicillin - susceptible staphylococcus aureus, which was treated with an extended course of intravenous cloxacillin. the patient subsequently recovered urinary bladder and bowel function, and on the latest follow - up was noted to have grade 5 power on the left side of the body. a 71-year - old man presented at our institution 's emergency department with worsening quadriparesis. prior to this presentation, he had a 2-week history of upper respiratory tract infection as well as neck pain, which were both treated symptomatically. one week before admission, he began experiencing progressive weakness in all four limbs. on presentation upon examination the left side had grade 0 power, and the right side was slightly stronger with grade 2 power in both upper and lower limbs. blood investigations showed a total white count of 16,500/mm, c - reactive protein of 73, and erythrocyte sedimentation rate was 100 mm / h. cerebrospinal fluid analysis showed low glucose and high protein levels but failed to grow any organism. imaging included plain radiographs, cervical spine computerized tomography (ct), brain and cervical spine magnetic resonance imaging (mri). ct scans were suggestive of erosive lesions in the left - sided c1c2 and c2c3 facetal joints. mri showed evidence of an epidural abscess on the left side abutting the cervicomedullary junction as well as osteomyelitis and myositis of the structures around the atlantoaxial joint (figs. 1 2 3 4 5). there were also radiological features suggestive of meningitis and pyogenic ventriculitis with no hydrocephalus or brain abscesses. sagittal sections of magnetic resonance imaging (t2-weighted) showing extension of the abscess from c0c3. axial section of magnetic resonance imaging (t2-weighted) at c1c2 level showing a left - sided hyperintense cervical cord collection. sagittal sections of the magnetic resonance imaging (t1-weighted) showing extension of the abscess from c0c3. sagittal sections of the magnetic resonance imaging (contrast enhanced) showing extension of the abscess from c0c3. in view of the radiological findings and clinical deterioration, the decision was made for surgical intervention. the posterior arch of c1 was drilled and removed while protecting the extradural portion of the vertebral artery. the epidural space did not have an abscess, but there were congested epidural veins. at this stage, an intradural abscess was suspected, and the dura was opened on the left side with stay sutures ; the left c1 nerve root was cut to get a window to decompress the abscess cavity (fig. care was taken to identify and retract the intramural portion of vertebral artery and the spinal part of the accessory nerve. dissection was limited on the anterior aspect due to proximity of the anterior spinal artery. durotomy was closed with ethilon 50 (johnson & johnson, somerville, nj) and sealed with tissue glue. cultures grew methicillin - susceptible staphylococcus aureus, which was treated with an extended course of intravenous cloxacillin. the patient subsequently recovered urinary bladder and bowel function, and on the latest follow - up was noted to have grade 5 power on the left side of the body. spinal cord dural anatomy and blood flow patterns unlike those of the brain are not conducive to intradural infections. absence of dural sinuses, width of the epidural space, and centripetal direction of blood flow in the spinal vasculature all contribute toward the low incidence of these abscesses within the spinal dura.5 to date, only 65 cases of ssa have been reported since sittig 's first described case in 1927.2 6 only nine of these cases were primary cervical spine collections with no thoracic or lumbar involvement (table 1).1 2 3 4 7 8 9 10 11 abbreviations : c, cervical ; e. coli, escherichia coli ; ivda, intravenous drug abuse ; lp, lumbar puncture ; s. aureus, staphylococcus aureus ; ?, data not available ; +, mild recovery ; + +, moderate recovery ; + + +, full recovery. hematogenous spread from the respiratory tract is the most common etiology followed by contiguous spread from adjacent infections.1 congenital dermal sinus or other congenital conditions with open dural defects may also result in intradural collections. recent reports have pointed toward iatrogenic causes, such as lumbar puncture, injection of a local anesthetic agent, and discography.12 intravenous drug abuse also appears to be a major risk factor, especially in the cervical spine where 3 of 10 patients were drug abusers (table 1). the proposed etiology in our patient was a possible community - acquired respiratory tract infection complicated by meningitis causing bacteria seeding beyond the dura resulting in an intradural collection. culture growth of s. aureus from the surgery specimen was not unexpected as s. aureus is the most commonly involved pathogen.1 2 3 4 clinically and radiologically it is difficult to distinguish epidural and intradural abscesses.3 4 13 like their more common epidural counterpart, suspicion of ssa should be aroused when a febrile patient with back or neck pain reports a recent history of infection. levy described a characteristic triad of fever, neck or back pain, and signs of cord compression, which was present in 18 of their 47 reviewed cases of intradural spine collections.3 this triad was also manifested by our patient upon presentation. typical mri findings of an intradural spinal abscess have been well described.4 13 in t1-weighted images, the intradural lesion is usually isointense with the cord. on t2-weighted images, the contents appear hyperintense and well demarcated from the other intradural contents. on gadolinium enhancement, a typical ring - enhancing lesion is seen in the setting of an abscess.4 13 however, an epidural abscess may also have some of these characteristic features and therefore diagnosis purely on imaging can be misleading. in our case, it was only after an intraoperative durotomy that the true location of the abscess was revealed. surgical drainage, together with systemic antibiotics, is the treatment of choice.1 2 3 without intervention, patients can develop significant neurological deficit below the lesion, and surgery performed after this stage may not reverse the deficits. surgical decompression and washout were performed among all the cervical intradural abscess cases reported to date, with a majority showing good recovery (table 1). postsurgery he improved considerably, and the only deficit that remained was a mild right - sided hemiporasis. he is able to walk independently with the help of a walking stick within 6 months after surgery. cervical intradural abscess is an extremely rare but a well - described entity that requires prompt surgical intervention. it can be clinically and radiologically indistinguishable from the more commonly occurring epidural collection. thus if an exploration of a suspected epidural abscess with progressive neurological deficits is performed, one must always be aware of the possibility of an intradural or a combination abscess and therefore keep a low threshold for performing a durotomy to look for a subdural collection that maybe mimicking the epidural abscess. | intradural spinal cord abscesses especially in the cervical spine are a rare occurrence. we report a rare presentation of an intradural extramedullary abscess at the atlantoaxial level, initially misdiagnosed as an epidural collection. the patient presented with worsening quadriparesis preceded by a 2-week history of upper respiratory tract infection and neck pain. magnetic resonance imaging showed evidence of an epidural abscess on the left side abutting the cervicomedullary junction. we performed occipitocervical fixation and surgical decompression. absence of a suspected epidural abscess led us to consider a durotomy, and an intradural abscess was recognized and drained. presence of an intradural abscess, though extremely rare, must always be considered in suspected spinal epidural collections as radiological and clinical findings are indistinguishable between the two conditions. |
panic attacks are defined as sudden and shortlived anxiety spells with various somatic and cognitive symptoms. according to dsm - iv, these discrete periods of intense fear or discomfort furthermore, at least four of the following thirteen symptoms evolve : palpitations or accelerated heart rate ; sweating, trembling, or shaking ; sensations of shortness of breath or smothering ; feeling of choking, chest pain, or discomfort ; nausea or abdominal distress ; feeling dizzy, unsteady, lightheaded, or faint ; derealization or depersonalization ; fear of losing control or going crazy ; fear of dying ; paresthesias ; chills or hot flashes. panic attacks can occur sporadically in healthy man, but also in the context of anxiety disorders (if the panic attacks are not due to the direct physiological effect of a substance or a general medical condition). diagnostically, recurrent panic attacks are the hallmark of panic disorder, which is a disabling anxiety disorder that has a lifetime prevalence of about 5%. the interest in the neurobiology of panic attacks has considerably been stimulated by the discovery that these spontaneous anxiety paroxysms can be provoked experimentally in susceptible subjects in the laboratory under controlled conditions. the seminal report about neurochemical provocation of panic attacks in man was published by pitts and mcclure in 1967. based on the observation that patients with anxiety neurosis were exercise - intolerant and developed high blood levels of lactic acid during standardized workload, these researchers developed the idea that the lactate molecule might be the elicitor of anxiety attacks in vulnerable individuals. in a double - blind study with intravenous infusion of 10 ml / kg body weight of 0.5 molar sodium lactate over a maximum of 20 minutes, 13 out of 14 patients with anxiety neurosis (all of them with a history of spontaneous anxiety spells), but only 2 out of 10 healthy controls, developed typical anxiety attacks. this observation laid the foundation for extensive research efforts on the experimental psychopathology of panic, which offers a unique opportunity in the field of psychiatry. the scientific and clinical promises of such symptom provocation studies for the pathophysiology and psychopharmacology of psychiatric disorders have been drafted as follows : in this paradigm investigators administer a psychopharmacologic agent or psychological challenge procedure to patients under controlled conditions to probe psychiatric symptoms and other neurobiological responses. the principal scientific rationale behind this approach is to learn more about the underlying pathophysiological mechanisms responsible for the symptomatic expression of psychiatric illnesses. in addition, the knowledge gained from this type of study might lead to better predictors of treatment response or identification of novel therapeutic interventions. a quintessential ethical framework of challenge studies includes preserved decision - making capacity, informed consent, potential scientific and future clinical benefits, consent of an ethical committee, a favorable or acceptable risk : benefit ratio, absence of severe or long - lasting effects of the challenge agent, and follow - up studies on the effects of participation in symptom - provoking studies. in addition to lactate infusion, several further methods to provoke experimental panic attacks in patients with panic disorder by pharmacological means have been developed during the past decades (overview in ref 4). they include other agents that influence respiration, such as carbon dioxide inhalation or doxapram infusion. further established panicogens act specifically on neurotransmission, such as the noradrenergic substances yohimbine and isoprenaline, the serotonergic agents metachlorophenylpiperazine (mcpp) and fenfluramine, benzodiazepine - receptor agents, such as the inverse agonist fg 7142 and the antagonist flumazenil, agonists at the cck-2 (formerly type b) receptor, such as cholecystokinin tetrapeptide (ckk-4) and pentagastrin, and the adenosine receptor antagonist caffeine. the following criteria for an ideal panicogen for human use have been proposed (compiled according to gutmacher and gorman) : it should mimic naturally occurring panic attacks it should foster both central and peripheral manifestations of panic it should be replicable the phenomena should be either short - lived or readily reversible it should differentiate between healthy subjects and those with pathology it should reflect the potential for a state response ; those who have been successfully treated clinically should not respond or respond far less than those who have had no treatment the effects should not be blocked by drugs, which do not work against spontaneous panic. concerning the claimed potential for a state response in patients after successful anti - panic treatment, the further discourse of this paper will be restricted to drug treatment (and will not address findings after psychotherapeutic treatment or spontaneous remission). the use of experimental panic provocation by panicogens after psychopharmacological anti - panic treatment may be a more advantageous means to assess drug effects than just waiting for days and weeks for spontaneous panic attacks to occur, and having the patients keep panic diaries to characterize panic frequency and severity. in some regard, this procedure resembles the role of the well - known exercise stress test in diagnosis and treatment of angina pectoris in internal medicine. several studies, mostly with lactate, carbon dioxide, or cck-4, have demonstrated that the established antipanic drug treatments with nonselective serotonin reuptake inhibitors (the tricylic antidepressants imipramine and clomipramine), various selective serotonin reuptake inhibitors (ssris) and benzodiazepines (particularly alprazolam) indeed diminish experimentally induced panic in patients with panic disorder. in detail, in studies with sodium lactate infusions prior medication with the benzodiazepines diazepam and alprazolam as well as with the tricyclic antidepressant imipramine significantly decreased induction of panic anxiety in panic patients and thus appeared to increase the threshold for lactate - induced panic attacks. carbon dioxide (35%)-induced panic was attenuated in panic disorder patients after treatment with the benzodiazepines clonazepam and alprazolam, with imipramine or clomipramine, paroxetine, sertraline, or fluvoxamine. cck-4-elicited panic was decreased in panic patients after imipramine treatment and after citalopram or fluvoxamine. the response to the further panicogens after treatment with standard anti - panic drugs are less intensively studied in patients alprazolam blocked the panic symptoms provoked by mcpp (piperazine) and yohimbine, long - term imipramine did not alter yohimbine - induced increases in ratings of anxiety - nervousness, but fluvoxamine reduced yohimbine - induced anxiety. for some investigational drugs experimental panic provocation has been used in patients with panic disorder (without prior testing in panic models in healthy man, as described in the following paragraphs). the emerging data might give valuable information for decision making as to whether further study on their action on spontaneous attacks is worthwhile. however, a definite evaluation of the informative value of this approach at this stage is not yet possible due to the paucity of studies. a single oral dose of 50 mg of l-365,260, a central cck receptor antagonist, had shown a differential action on cck-4- and lactate - induced panic attacks in patients with panic disorder. in a double - blind, placebo - controlled, crossover study in 29 patients, cck-4 90 minutes after l-365,260 significantly reduced the number and sum intensity of provoked panic symptoms. in contrast, a double - blind, placebo - controlled parallel - group study in 24 patients with sodium lactate infusion given after the same dosage of the compound and in the identical time frame failed to reveal statistical differences on these panic attack parameters. in a multicenter, placebo - controlled, double - blind trial with l-365,260 30 mg qid for 6 weeks no clinically significant treatment effects in panic attack frequency or intensity were found and testing higher doses was suggested, but has not been performed so far. in a double - blind, placebo - controlled, crossover design, nine panic patients were given an intravenous infusion of 150 g of atrial natriuretic peptide (anp) followed by experimental panic induction using cck-4. the rationale was derived from observations that anp is released during panic attacks in humans and has anxiolytic - like effects in preclinical studies. the cck-4 response as per acute panic inventory (api) ratings was significantly reduced after anp versus placebo pre treatment. these findings of anti - panic activity of anp were replicated in another study in ten panic patients under comparable experimental conditions with a lower dose of cck-4. unfortunately, until now no study about the action of anp (or another agonist at the type a natriuretic peptide receptor) on spontaneous panic attacks in patients with panic disorder has been reported. an early study in outpatients suffering from panic disorder using the panic response to cck-4 challenge as the primary outcome parameter was conducted with the novel neurokinin-3 (nk-3) receptor antagonist sr142801 (osanetant). fifty - two patients who had developed a panic attack with cck-4 were randomized to 4 weeks of treatment (200 mg / d orally) in a double - blind, placebo - controlled design and then a second cck-4 challenge was performed. however, with regard to the primary efficacy end point, ie, the increase of api total score, osanetant was not significantly different from placebo. on the panic and agoraphobia scale no significant treatment effects of this compound were detected during these 4 weeks. for many panic patients it is quite aversive and frightening to undergo an experimental panic challenge and to be treated with an investigational product due to catastrophizing disorder - immanent cognitions, fears of side effects, and the possibility of being randomized to placebo treatment. to bridge the gap between preclinical panic models and studies in patients, experimental panic provocation in healthy human subjects might serve as a valuable tool for assessment of novel anti - panic compounds during the early phase of drug development in proof - of - concept studies. healthy volunteer translational studies might deliver valuable information on whether it is worthwhile to consider further, more thorough studies in patients. however, standard sodium lactate panic is not an apt panic model in healthy subjects, because, as already mentioned, in contrast to patients with panic disorder, only a small percentage of healthy humans develop panic symptoms to it. interestingly, sinha investigated, in a single - blind pilot study, whether additional pretreatment with naloxone, an opioid receptor antagonist, could render healthy controls who are nonresponsive to panic induction by lactate infusion sensitive to the latter panicogen. indeed, substantial increases in the api scores were displayed by 8 out of 12 subjects during such treatment ; naloxone alone did not result in panic symptoms. in a following more sophisticated investigation in 25 volunteers (using a crossover, randomized design) further evidence was shown that impairment of the endogenous opioid system by naloxone accentuates symptomatic response to lactate, but no significant differences in api ratings were detected. notwithstanding, the authors suggest testing the specificity of the naloxone - lactate model in healthy man comparing specific anti - panic medications with ineffective anti - panic agents, and furthermore screening for putative anti - panic agents with this method. further studies will demonstrate whether this complex model is applicable for translational panic research in healthy humans. the further discourse will be restricted to these two panicogens, because we are not aware of any published studies testing anti - panic drugs in normal volunteer challenge studies using other substances. although patients with panic disorder show an enhanced sensitivity to intravenous bolus injection of cck-4, increasing its dose brings about a substantial panic - like reaction also in normal controls. while the panic rate after injection of 25 g was 91% for patients and only 17% for controls, 50 g of cck-4 induced a fullblown panic attack in 100% of patients and in a sizable 47% of controls. among healthy volunteers significant dose - related differences were also found for the number of panic symptoms and their sum intensity, which makes cck-4 a useful research model for dimensional aspects of panic also in the nonclinical subjects who do not develop a full - blown panic attack. also, with a single breath of 35% carbon dioxide inhalation panic patients show significantly stronger symptoms of panic anxiety than normal controls. the induced cluster of symptoms in healthy volunteers is, however, similar to those elicited in panic attacks naturally occurring in patients affected by panic disorder ; but the panic signal obtained with one breath of 35% carbon dioxide seems to be weaker than with injection of 50 g of cck-4 in healthy subjects : direct comparison of only 25 g of cck-4 and 35% carbon dioxide revealed significantly more intense symptoms with cck-4, but not a significantly greater number of symptoms ; the incidence of panic attacks was similar : 21% for co2 and 17% for 25 mg of cck-4. a methodological problem is that psychometric assessment of induced panic does not follow consistent rules different panic rating scales, such as the api and the dsm - derived panic symptom scale (pss) are used and different criteria to divide panickers from non - panickers. to provide a basis for the use of the cck-4 model in proof of concept studies in healthy volunteers, the psychometric, cardiovascular, and neuroendocrine responses to 50 g of cck-4 were studied in 85 healthy men. the api - derived panic rate was 78.8% and thus 10.6% higher than that derived from the pss ratings (68.2%). this should be taken into account when comparing studies and when choosing a categorical instead of a dimensional outcome parameter of panic provocation. another result of this study was that cardiovascular and hormonal alterations to cck-4 challenge are not valuable as an objective readout of panic. we must bear in mind to depend on relatively weak data from self - report when assessing panic anxiety. because the vast majority of studies on pharmacological modulation of experimental panic in healthy volunteers was performed using cck-4, the focus here will be on this panicogen (for synopsis of results, please see table i). in the 35% carbon dioxide model of panic in healthy volunteers an acute dose of 1 mg alprazolam 2 hours before inhalation resulted in significant anti - panic effects in a double - blind, placebo - controlled, three - way crossover study in 12 healthy subjects. with an ssri, only one study in healthy man using the 35% co, challenge has been published. in this 2-week double - blind, placebo - controlled trial in 24 subjects, who were at high risk for panic disorder because of a personal history of panic attacks or a family history of treated panic disorder, and who had reacted with a panic attack to prior carbon dioxide testing however, the caveat must be applied that time of treatment with an ssri of only 14 days might not be long enough to manifest anti - panic action, because clinical benefits for ssri in panic disorder typically take longer. further studies must clarify, whether the 35% carbon dioxide panic model is sensitive to modulation with serotonergic antidepressants and other anti - panic drugs in healthy man. the acute inhibitory effect of benzodiazepines on cck4 panic in normal man has been demonstrated in two studies. in an early, small exploratory open - label study de montigny showed that pretreatment with lorazepam (1 mg at 4 pm and 2 mg at bedtime on the preceeding day, 1 mg at 8 am 1 hour before cck-4 challenge) prevented the psychic effects of cck-4 (doses between 75 and 150 g) in four subjects who had experienced a panic - like attack with the same dose of this peptide before. in a double - blind, placebo - controlled, parallel - group study in 30 healthy volunteers treated with 1 mg alprazolam 1 hour prior to a 50-g cck-4 challenge, a significant reduction of api and pss scores and of the number of reported symptoms compared to placebo pretreatment were found. a recent study (following an unbalanced, three - arm, two - period, crossover, double - blind, placebo - controlled design) in 21 male volunteers who received 1 mg of lorazepam 2 hours before cck-4 did not show an attenuated panic signal in any pss parameter. however, this dose of lorazepam was considerably lower than in the two previous studies reported above. concerning anti - panic non - selective serotonin reuptake inhibitors (imipramine or clomipramine) no study on cck-4 panic in healthy volunteers has been published. with an ssri, kellner could not demonstrate an inhibitory effect of escitalopram (6 weeks of 10 mg / d) on a 50-g cck-4 challenge in a double - blind, placebo - controlled, randomized, within - subject crossover design in 30 healthy young men. induced panic under escitalopram was even significantly more pronounced in the subgroup of subjects with the short / short genotype for the serotonin transporter linked polymorphic region in this study. another investigation with an identical dose and duration of escitalopram pretreatment also failed to show a significant inhibitory effect on cck-4 panic in healthy man (i. tru, personal communication). to test the effectiveness of a single oral 100 mg dose of the cholecystokinin b antagonist ci-988 in attenuating panic symptoms induced by cck-4 a randomized, placebo - controlled, double - blind, three - way crossover design was used in 30 healthy men. a small (14%), but significant decrease of sum intensity scores of panic symptoms was observed under ci-988. in contrast, a subsequent study in 14 patients with panic disorder who were given 50 or 100 mg of ci-988 in a double - blind, two - period incomplete block design 2 hours before injection of cck-4 failed to show a statistically significant treatment effect on the total intensity score on the pss (the primary efficacy parameter), as well as on the number of panic symptoms, time to and occurrence of the first panic symptoms, duration of symptoms, intensity of apprehension, and the percentage of patients who did not have a panic attack. in a randomized, double - blind, placebo - controlled trial with 100 mg tid of ci-988 in panic patients no superiority to placebo in reducing panic attacks could be shown. after the metabotropic glutamate 2/3 receptor agonist ly544344/ly354740 had shown acute anxiolytic - like action in preclinical studies, a pilot study on panic anxiety induced by cck-4 was performed in healthy humans. twelve male volunteers were treated with 80 mg bid ly544344 orally for 1 week in a randomized placebo - controlled double - blind crossover design. while no significant treatment effect for the number of cck-4-induced panic symptoms and subjective anxiety ratings emerged in the entire sample, the ten subjects who showed an endocrine response to the test substance displayed a significant reduction on these two measures. unfortunately, due to emerging problems in long - term preclinical safety, no subsequent clinical studies were performed with this compound. the finding of a significant anti - panic effect of atrial natriuretic peptide (anp) in patients with panic disorder has already been mentioned above. in this double - blind, placebo - controlled, crossover study also nine healthy control subjects matched for sex and age were included and they were given an intravenous infusion of 150 g of anp followed by cck-4 panic induction. however, no significant treatment effect of anp on api ratings was observed in healthy man. the -blocker propranolol (0.2 mg / kg given intravenously over 20 minutes) has been observed to significantly decrease the cck-4 response (sum intensity and number of panic symptoms) in a study in 30 healthy male volunteers who were randomly assigned to propranolol or placebo. in panic patients no study with a -blocker has been reported using the cck-4 model and in a 5-week double - blind placebo - controlled study, no efficacy of propranolol on spontaneous attacks was detected. interestingly, using the cck - b receptor agonist pentagastrin for panic provocation in a double - blind, randomized, placebo - controlled study with identical dose and application of propranolol as above in a predominantly female group of 16 healthy adult subjects, no significant effect on total symptom intensity as per the api was observed. regarding gaba (-aminobutyric)-ergic drugs other than benzodiazepines both the gaba reuptake inhibitor tiagabine and the gaba transaminase inhibitor vigabatrin have been studied in this experimental panic paradigm. fifteen healthy volunteers received 15 mg tiagabine daily for 1 week in an open study. both api - and pss - scores showed a significant reduction to a cck4 stimulus that was performed before and after treatment. in a following double - blind placebo - controlled pilot study with 4 weeks of tiagabine in 19 patients with panic disorder a subset of seven patients (three treated with tiagabine, four treated with placebo) was challenged with 25 g of cck-4 at baseline and after 14 and 28 days. patients of the tiagabine vs the placebo group showed considerably decreased sensitivity to cck-4 (as per api ratings). however, clinical benefits of tiagabine on the panic and agoraphobia scale were not detected. also a 10-week open study suggested that tiagabine maybe of little benefit on this measure. vigabatrine (2 mg / d) was given for 7 days to ten healthy volunteers in an open - label study after placebo - controlled administration of cck-4 and a second cck-4 challenge followed after the treatment period. a marked and significant attenuation of cck-4 induced panic symptoms (as per api and pss scores) and of anxiety was observed with vigabatrine. however, no placebo - controlled and double - blind study has followed so far and the effect of vigabatrine has not been investigated in the cck-4 paradigm in panic patients. recently, the translocator protein (18 kd) ligand xbd173, which enhances gabaergic neurotransmission via induction of neurosteroidogenesis, was tested in 71 healthy male volunteers who had shown a clear panic response to an initial cck-4 challenge. in this double - blind study the subjects were randomized to 7 days of treatment with placebo, 10, 30, or 90 mg / day of xbd173 or 2 mg / d alprazolam as active control condition. a significant difference from placebo in the difference of the api ratings between the first and the second challenge (on day 7) with cck-4 the acute inhibitory effect of benzodiazepines on cck4 panic in normal man has been demonstrated in two studies. in an early, small exploratory open - label study de montigny showed that pretreatment with lorazepam (1 mg at 4 pm and 2 mg at bedtime on the preceeding day, 1 mg at 8 am 1 hour before cck-4 challenge) prevented the psychic effects of cck-4 (doses between 75 and 150 g) in four subjects who had experienced a panic - like attack with the same dose of this peptide before. in a double - blind, placebo - controlled, parallel - group study in 30 healthy volunteers treated with 1 mg alprazolam 1 hour prior to a 50-g cck-4 challenge, a significant reduction of api and pss scores and of the number of reported symptoms compared to placebo pretreatment were found. a recent study (following an unbalanced, three - arm, two - period, crossover, double - blind, placebo - controlled design) in 21 male volunteers who received 1 mg of lorazepam 2 hours before cck-4 did not show an attenuated panic signal in any pss parameter. however, this dose of lorazepam was considerably lower than in the two previous studies reported above. concerning anti - panic non - selective serotonin reuptake inhibitors (imipramine or clomipramine) no study on cck-4 panic in healthy volunteers has been published. with an ssri, kellner could not demonstrate an inhibitory effect of escitalopram (6 weeks of 10 mg / d) on a 50-g cck-4 challenge in a double - blind, placebo - controlled, randomized, within - subject crossover design in 30 healthy young men. induced panic under escitalopram was even significantly more pronounced in the subgroup of subjects with the short / short genotype for the serotonin transporter linked polymorphic region in this study. another investigation with an identical dose and duration of escitalopram pretreatment also failed to show a significant inhibitory effect on cck-4 panic in healthy man (i. tru, personal communication). to test the effectiveness of a single oral 100 mg dose of the cholecystokinin b antagonist ci-988 in attenuating panic symptoms induced by cck-4 a randomized, placebo - controlled, double - blind, three - way crossover design was used in 30 healthy men. a small (14%), but significant decrease of sum intensity scores of panic symptoms in contrast, a subsequent study in 14 patients with panic disorder who were given 50 or 100 mg of ci-988 in a double - blind, two - period incomplete block design 2 hours before injection of cck-4 failed to show a statistically significant treatment effect on the total intensity score on the pss (the primary efficacy parameter), as well as on the number of panic symptoms, time to and occurrence of the first panic symptoms, duration of symptoms, intensity of apprehension, and the percentage of patients who did not have a panic attack. in a randomized, double - blind, placebo - controlled trial with 100 mg tid of ci-988 in panic patients no superiority to placebo in reducing panic attacks could be shown. after the metabotropic glutamate 2/3 receptor agonist ly544344/ly354740 had shown acute anxiolytic - like action in preclinical studies, a pilot study on panic anxiety induced by cck-4 was performed in healthy humans. twelve male volunteers were treated with 80 mg bid ly544344 orally for 1 week in a randomized placebo - controlled double - blind crossover design. while no significant treatment effect for the number of cck-4-induced panic symptoms and subjective anxiety ratings emerged in the entire sample, the ten subjects who showed an endocrine response to the test substance displayed a significant reduction on these two measures. unfortunately, due to emerging problems in long - term preclinical safety, no subsequent clinical studies were performed with this compound. the finding of a significant anti - panic effect of atrial natriuretic peptide (anp) in patients with panic disorder has already been mentioned above. in this double - blind, placebo - controlled, crossover study also nine healthy control subjects matched for sex and age were included and they were given an intravenous infusion of 150 g of anp followed by cck-4 panic induction. however, no significant treatment effect of anp on api ratings was observed in healthy man. the -blocker propranolol (0.2 mg / kg given intravenously over 20 minutes) has been observed to significantly decrease the cck-4 response (sum intensity and number of panic symptoms) in a study in 30 healthy male volunteers who were randomly assigned to propranolol or placebo. in panic patients no study with a -blocker has been reported using the cck-4 model and in a 5-week double - blind placebo - controlled study, no efficacy of propranolol on spontaneous attacks was detected. interestingly, using the cck - b receptor agonist pentagastrin for panic provocation in a double - blind, randomized, placebo - controlled study with identical dose and application of propranolol as above in a predominantly female group of 16 healthy adult subjects, no significant effect on total symptom intensity as per the api was observed. regarding gaba (-aminobutyric)-ergic drugs other than benzodiazepines both the gaba reuptake inhibitor tiagabine and the gaba transaminase inhibitor vigabatrin have been studied in this experimental panic paradigm. fifteen healthy volunteers received 15 mg tiagabine daily for 1 week in an open study. both api - and pss - scores showed a significant reduction to a cck4 stimulus that was performed before and after treatment. in a following double - blind placebo - controlled pilot study with 4 weeks of tiagabine in 19 patients with panic disorder a subset of seven patients (three treated with tiagabine, four treated with placebo) was challenged with 25 g of cck-4 at baseline and after 14 and 28 days. patients of the tiagabine vs the placebo group showed considerably decreased sensitivity to cck-4 (as per api ratings). however, clinical benefits of tiagabine on the panic and agoraphobia scale were not detected. also a 10-week open study suggested that tiagabine maybe of little benefit on this measure. vigabatrine (2 mg / d) was given for 7 days to ten healthy volunteers in an open - label study after placebo - controlled administration of cck-4 and a second cck-4 challenge followed after the treatment period. a marked and significant attenuation of cck-4 induced panic symptoms (as per api and pss scores) and of anxiety was observed with vigabatrine. however, no placebo - controlled and double - blind study has followed so far and the effect of vigabatrine has not been investigated in the cck-4 paradigm in panic patients. recently, the translocator protein (18 kd) ligand xbd173, which enhances gabaergic neurotransmission via induction of neurosteroidogenesis, was tested in 71 healthy male volunteers who had shown a clear panic response to an initial cck-4 challenge. in this double - blind study the subjects were randomized to 7 days of treatment with placebo, 10, 30, or 90 mg / day of xbd173 or 2 mg / d alprazolam as active control condition. a significant difference from placebo in the difference of the api ratings between the first and the second challenge (on day 7) with cck-4 despite ample exciting research efforts, we are still far from having reliable information on model validity of experimental panic provocation paradigms in healthy man as tools to test novel anti - panic drugs. a few false - negative or false - positive findings question the usefulness of this approach. existing preliminary data need replication using exclusively double - blind, placebo - controlled designs. especially for multicenter trials, standardization of the test environment and subjects ' instruction need careful attention. many findings were obtained with relatively small samples and few studies had included women. rarely have dose - response aspects been investigated. challenge studies with genetically precharacterized and homogenized samples are worth considering and may achieve clearer results. another problem is that our growing understanding of the complex pathophysiology of panic suggests that there may be no unitary model but possibly different phenocopies, leading to a similar pathophysiological phenomenon. hopefully, further research will eventually lead us to more definite knowledge on which panicogens in healthy man are capable of predicting the usefulness of various anti - panic drugs for treatment in panic disorder. | experimental neurochemical provocation of panic attacks in susceptible human subjects has considerably expanded our knowledge of the pathophysiology and psychopharmacology of panic disorder. some panicogens also elicit short - lived panic - like states in healthy man. this offers the opportunity to assess the anti - panic action of drugs in proof - of - concept studies. however, from current data it is still unclear whether experimental panic in healthy man is a valid translational model. most such studies in healthy volunteers have been performed using a cholecystokinin tetrapeptide (cck-4) challenge. while cck-4 panic was blocked by alprazolam pretreatment, escitalopram showed negative results in healthy man. preliminary findings on novel investigational drugs and a few problematic results will be reviewed. small sample sizes in many panic provocation studies, lack of dose - response aspects, and still - insufficient knowledge about the biological underpinning of experimental and spontaneous panic limit the interpretation of existing findings and should inspire further research. |
endosonographic procedures, including endobronchial ultrasound (ebus)-guided transbronchial needle aspiration (tbna) and endoscopic ultrasound (eus)-guided fine - needle aspiration (fna) are routinely used for accessing mediastinal and hilar lymph nodes and masses. in patients with hypoxemia, excessive cough, raised intracranial tension, or with central airway obstruction due to lesions compressing the trachea ; eus - fna may be safer than ebus - tbna as it employs the esophageal route. however, patients with central airway obstruction requiring mechanical ventilation are often managed in respiratory care units, where eus - fna may not be available. in this situation, the pulmonologist can use the ebus scope for performing a technique known as eus (with an echobronchoscope)-guided fna (eus - b - fna). this procedure has been reported to have a good diagnostic performance in the evaluation of mediastinal lesions. herein, we describe a patient with respiratory failure due to airway obstruction by a mediastinal tumor, who was being mechanically ventilated. a 50-year - old man presented to the emergency department with stridor of 1-week duration. there was a significant reduction in appetite and he had lost 5 kg of his body weight. on admission to our facility, physical examination revealed an afebrile patient with a heart rate of 120 beats / min, respiratory rate of 38 breaths / min, and blood pressure of 80/50 mmhg. contrast - enhanced computed tomography (ct) of his chest revealed the presence of a mass in the subcarinal region encasing the lower trachea and main bronchi and consolidation in the right upper lobe [figure 1 ]. mass in the subcarinal region encasing both the main bronchi and causing compression of the pulmonary vessels the patient was intubated with an endotracheal tube (7.0 mm internal diameter) and mechanically ventilated. he was initiated on intravenous co - amoxiclav (1.2 g thrice a day). however, the patient could not be weaned off the ventilator over the next 5 days. on the 6 day, eus - b - fna of the subcarinal mass was performed. cytological examination showed loosely cohesive clusters and dispersed population of small - sized tumor cells with hyperchromatic nuclei showing focal nuclear molding suggestive of small cell carcinoma of the lung [figure 2 ]. photomicrograph of the aspirate from the mediastinal mass obtained by endoscopic ultrasound (with an echobronchoscope)-guided fine - needle aspiration showing loosely cohesive clusters and dispersed population of small - sized tumor cells with hyperchromatic nuclei showing focal nuclear molding suggestive of small cell carcinoma. background shows nuclear debris (h and e, 40) the final clinicoradiological stage was iiib (t4 n3 m0), but the patient had a poor performance status (eastern cooperative oncology group score of 4). placement of a metallic tracheobronchial y - stent was considered but was not performed as the patient 's legal representative refused consent. chemotherapy with cisplatin 100 mg (85% of 60 mg / m of body surface area [bsa ]) and irinotecan 160 mg (90% of 100 mg / m of bsa) were administered. after a week, the patient was successfully weaned off the ventilator and was discharged to home care. however, the disease progressed and the patient succumbed to his illness 3 months after the diagnosis. the index case illustrates the utility of eus - b - fna in accessing mediastinal lesions in mechanically ventilated patients, where ebus - tbna may be technically difficult or not feasible. a diagnosis of advanced lung cancer was made and the administration of palliative chemotherapy enabled liberation from mechanical ventilation. mediastinal or hilar lesions in critically ill patients can be approached endoscopically by the pulmonologist through the airway using tbna that can be performed with or without ebus guidance. both conventional and ebus - guided tbna have been described in patients with respiratory failure, who are mechanically ventilated through an endotracheal tube ; however, they are not performed frequently. this is because a small - sized endotracheal tube does not allow the entry of the echobronchoscope (insertion tube diameter, 6.9 mm). further, as the procedure requires a few minutes, significant hypoxia may occur in patients with a poor respiratory reserve. the index patient was being ventilated with a small - sized endotracheal tube (internal diameter of 7.0 mm) and he had significant hypoxemia. eus may be useful in such patients as the access is through the esophagus, thus not compromising ventilation. eus offers access to lymph node stations 5 (para - aortic), 8 (paraesophageal), and 9 (pulmonary ligament) in addition to stations 4 l (left lower paratracheal) and 7 (subcarinal). however, this procedure is usually performed by gastroenterologists and many respiratory intensive care units may not have easy access to this technology, especially at the bedside. eus - b - fna, which is performed by the pulmonologist with the same echobronchoscope used to perform ebus - tbna overcomes this limitation. moreover, the introduction of the ebus scope (6.9 mm in diameter) may be easier than the introduction of the eus scope (1214 mm in diameter) in patients who also have esophageal narrowing. due to these reasons, ct - guided fna was not employed as it would have entailed a high risk of pneumothorax due to the central location of the mass. a systematic review of the pubmed database using the search string : (ebus or endobronchial ultrasound or transbronchial needle aspiration) and (icu or intensive care unit or critically ill or mechanical ventilation or mechanically ventilated or intubated or endotracheal tube) yielded 10 reports (62 patients) of endoscopic interventions (conventional tbna, ebus - tbna, eus - fna, or eus - b - fna) for mediastinal / hilar lesions in critically ill patients [table 1 ]. forty - six patients could be diagnosed successfully resulting in an overall yield of 74.2% ; there were no major complications. moreover, the diagnostic information obtained from the procedure led to a change in the treatment decision in most of these patients (49/62 ; 79%). in our case, eus - b - fna allowed institution of appropriate chemotherapy and facilitated liberation of our patient with advanced lung cancer from mechanical ventilation, an event not commonly encountered in clinical practice. published reports of endoscopic diagnostic interventions for accessing mediastinal / hilar lesions in critically ill patients the index case demonstrates that eus - b - fna is a valuable addition to the armamentarium of the pulmonologist for diagnosing mediastinal lesions in critically ill patients, including situations such as respiratory failure, which preclude a diagnostic bronchoscopy and ebus - tbna. | endobronchial ultrasound (ebus)-guided transbronchial needle aspiration (tbna) is routinely used for accessing mediastinal lymph nodes and masses. however, in patients with respiratory failure, who are being mechanically ventilated through an endotracheal tube, ebus - tbna may not be feasible due to several reasons. in such patients, the esophageal route offers a useful alternative for accessing mediastinal lesions. herein, we describe a 50-year - old man with a mediastinal mass, who was being invasively ventilated for respiratory failure. endoscopic ultrasound (with an echobronchoscope)-guided fine - needle aspiration was performed, which revealed a diagnosis of small cell carcinoma. appropriate cancer chemotherapy resulted in successful liberation of the patient from mechanical ventilation. we have also performed a systematic review of literature for reports of endoscopic diagnostic procedures for mediastinal / hilar lesions in critically ill patients. |
helicobacter pylori (h. pylori) causes a long - term infection of the human gastric and duodenal mucosa (1). mucosal colonisation predisposes for peptic ulcer disease, atrophic gastritis and distal (antral) stomach cancer (2), with various effects on gastric acid secretion. genetic variability of h. pylori is high (3). several genes have been identified that may play a role in the pathogenicity (4, 5). most important is the cytotoxin - associated gene a (caga), which is associated with peptic ulcer disease (6), and intestinal type adenocarcinoma of the stomach (7). patients with duodenal ulcer often have high basal gastrin levels, high peak acid output and high 24-hour intragastric acidity (8 - 10). in contrast, patients with h. pylori - associated gastric ulcer often have hypochlorhydria (11). several reports suggest that the prevalence of h. pylori and especially the most pathogenic form caga might be lower in patients with gastroesophageal reflux disease (gerd), including barrett s oesophagus (be) than in the rest of the population (12, 13). one explanation for the negative association between mucosal colonisation with h. pylori and gerd is the effect of h pylori on acid production, since extensive gastritis involving the corpus may lower acid secretion by impairing parietal function. the aim of this study was to determine the prevalence of h. pylori infection and its possible protective role in the appearance of gerd and its progression to be. the time of investigation was june 2009december 2011. in this prospective study, from 120 patients, 70 patients were with gerd, and 50 patients with be. all the patients were interviewed for their age, sex, reflux symptoms, chronicity, medications used, the presence of h. pylori infection, weight, family history, smoking. endoscopic reflux changes was performed, according to los angeles (la) classification (14, 15, 16). diagnosis of infection with h. pylori was making by biopsy for hut test (astra zeneca gmbh). histological processing was performed in the institute of pathology in prishtina and skopje. during the recognition of intestinal metaplasia by biopsy, especially goblet cells can facilitate the use of alcian blue stain of ph 2.5 (17, 18, 19). dysplasia is categorized as : low and high based on cytological and histological architectural abnormalities (20, 21, 22, 23, 24, 25, 26, 27). the study included patients with positive anamnestic data, endoscopic findings positive for the presence of erosive gastroesophageal disease, which last month did not receive any ppi treatment, or nonsteroidal anti - inflammatory drugs. the study excluded patients who did not have typical anamnestic data for gastroesophageal reflux disease, those who have gastroesophageal erosive changes during endoscopy, and patients with pre - existing histopathological proven esophageal adenocarcinoma the results were processed by modern statistical methods. this study included 50 patients with be, 70 patients with gerd and 50 healthy persons or persons with ulcer of the duodenum, as a control group. (sd 10.8 yr.) in the gerd group average age was 40.8 years. (sd 13.5 yr.), whereas in control group average age was 42.1 years. the age difference between groups was significant (one way anova f = 13.91, p < 0.001). among patients with be, the most represented age group was 50 - 59 years, in the gerd - group the most representd age group was from 40 - 49 years. in all groups included in this study, men were more represented than women. in the group with be 78.0% were men, in the group with gerd 64.3%, and in the control group 60.0%. however, the difference was not statistically significant (2-test = 4.08, p = 0.130). average body height of respondents in group be was 174,8 cm (sd 8,2 cm), in the gerd group it was 168,5 cm (sd 8,9 cm) and among patients of the control group it was 170, 4 cm (sd 9,5 cm). the difference was stetistically significant (one way anova f = 7.45, p < 0.001). patients in group be were higher than those of group gerd (be vs gerd p < 0,001), and also higher than the control group (be vs control group : p < 0,05), and between the average heights of gerd group and the control group, the difference was not statistically significant (gerd vs. g control.. patients in group be smoked larger number of cigarettes (60%) than patients in the group with gerd (37,1%), as well as comparing to patients of the control group (50.0%). the difference was statistically significant (2 = 6.26, ss = 2 p = 0.044). regarding alcohol consumption, the difference was not significant (2 = 0.316, ss = 2, p = 0.854). as for the level of education, the difference was significant (2 = 6.48, ss = 2, p = 0.039) and the family history showed a significant difference (2 = 9.44, ss = 2, p = 0.009). patients in be group (4.0%) received less medications than patients in the gerd group (5.7%), as well as comparing to the control group (14.0%). patients in be group and gerd group had higher bmi, in comparison with patients of the control group ; one way anova obtained a significant difference (one way anova f = 23,27, p < 0.001). also in patients from be group, average value of bmi was higher than in patients of gerd group (be vs. gerd., p < 0.001), and in both groups bmi was higher than in patients of the control group (be vs control g. p < 0.001, gerd vs. g control. the difference between the duration of symptoms was significant (one way anova f = 161.5, p < 0.001 ; be vs. gerd p < 0.001, be vs. g control. it was concluded that hiatal hernia was more common in the group with be, with a statistically significant difference (2-test = 15.1, p < 0.001). in patients included in the study h. pylori infection was present in 16.0% of patients. in gerd group, h. pylori infection was present in 42.9%, and in patients of the control group, in 52.0% of cases. so, in be group, the prevalence of h. pylori infection showed less significant difference, compared to the control group (be vs. control group, -test = 12.87, p = 0.003) and in gerd group (be vs. gerd ; 2-test = 8.52, p = 0.0035). between gerd group and the control group -test = 0.64, p = 0.421, (table 1, figure 1). prevalence of infection with h. pylori in patients with be and gerd the prevalence of infection with h. pylori in patients with be vs. patients with gerd presence of infection with h. pylori by endoscopic type be regarding endoscopic type of be and the presence of infection with h. pylori, there was no significant difference (fisher exact test, p = 0.665). infection with h. pylori was present in 17.9% of patients with ssbe and 9.1% of patients with lsbe the management of gerd and be remains a challenging problem and this is partly due to a limited knowledge of its natural history. the relationship between gerd, be and h. pylori is very complex (1). there might also be connection between prolonged proton pump inhibition and the rate of progression to atrophic gastritis, leading to hypochlorhydria (12, 13, 14). h. pylori, in contrary to overweight and hiatal hernia, may interact with the risk of be rather in physiological aspect, than anatomically. h. pylori can reduce the risk for be by possible reduction of acidity in the stomach by the action of urease. the fact that h. pylori may protect against be is contrary to the established status of risk factors for peptic ulcer and gastritis. h. pylori infection was present in 16.0% of patients in be group, comparing to 42.9% of patients in the group with gerd, and to 52.0% of cases in the control group. results from one study (28) showed low prevalence of h. pylori infection in patients with be (12%). data from the literature also showed low prevalence of h. pylori infection in these patients. in the same study, of 251 patients who underwent endoscopy, caga + h. pylori was present in 44% of examinations, 36% of 36 patients with gerd. 20% of 10 patients with ssbe, and in 0% of 18 patients with lsbe. a limitation in our study was lacking of the laboratory method for determination of caga + types of h. pylori. h. pylori infection may be associated with increased acid secretion, but in contrast with achlorhydria resulting in atrophic gastritis, depending on the bacterial species and the inflammatory response that causes it. studies showing that h. pylori negative patients have more severe esophagitis compared with h. pylori positive, suggesting that this bacterium may have a protective role in patients with gerd. in fact, infection with h. pylori can induce atrophy and thus reduction of acidic secretion, which ultimately results in reduced risk of developing gerd. in contrast, the eradication of h. pylori infection may result in normal acid production and exacerbation of gerd. however, recent clinical studies can not provide strong enough evidence for a possible role of h. pylori infection in the development of gerd and erosive esophagitis. in clinical practice, since h. pylori infection is associated with an increased risk of peptic ulcer and gastric cancer, existing guidelines recommend its eradication, regardless of the potential effect on gerd (28, 29). h pylori, in particular the caga phenotype, through gastritis and associated hypochlorhydria might be a protective factor against gerd and its complications (30). in recent years it has become clear that a significant number of patients will develop reflux oesophagitis after apparently successful eradication (30). the findings are consistent with the hypothesis that the declining infection rates of h. pylori in the general population have led to a rise in the occurrence of gerd and associated oesophageal adenocarcinoma. the prevalence of caga phenotype was also lower in patients with complicated gerd (such as be), than in the rest of population (31). the prevalence of h. pylori infection in patients with be was lower in comparison with patients with gerd and with control group (p < 0.01). the prevalence of h. pylori infection in patients with be, especially those with lsbe was very low, which indicates the possible protective role of this microorganism. h. pylori infection was present in 17.9% of patients with ssbe and in 9.1% of patients with lsbe, p = 0665). | abstractintroduction and aim : the role of helicobacter pylori in esophageal disease has not been clearly defined. to clarify this issue, we analyzed 120 patients with histologically confirmed esophageal disease.material and methods : in this prospective study, 120 patients who underwent upper endoscopy examination were included ; among them 70 patients with clinically, endoscopically and histologically confirmed gerd, and 50 patients with be. this investigation was performed in the clinic of gastrohepatology in prishtina, during the period : june 2009december 2011. each patient was investigated for h. pylori infection, by performing biopsy for hut test.results:in be group, h. pylori infection was present in 16.0% of patients. in gerd group, h. pylori infection was present in 42.9%, and in patients of the control group, in 52.0% of cases. so, in be group, the prevalence of h. pylori infection showed less significant difference, compared to the control group (p = 0.003) and in gerd group (p = 0.0035). between gerd group and the control group there was no significant difference (gerd vs. g control. p = 0.421).conclusion : the prevalence of h. pylori infection in patients with be (16%) was lower in comparison with patients with gerd (42.9%) and with control group (p < 0.01). the prevalence of h. pylori infection in patients with be, especially those with lsbe (9.1%) was very low, which indicates a possible protective role of this microorganism. |
trachoma, caused by ocular infection with chlamydia trachomatis, is the leading contagious cause of blindness globally. estimations by mariotti. in 2008 suggested there were 40.6 million people worldwide suffering from active trachoma, leading to trichiasis in 8.2 million. trachoma is responsible for visual impairment in 2.2 million and clinical blindness in 1.2 million individuals. trachoma, a neglected tropical disease (ntd), spreads efficiently within households and in areas with poor sanitation and hygiene. the musca sorbens fly, which breeds on human faeces, may also act as a vector for transmission. the disease is highly correlated with poverty, lack of clean water for washing, and limited access to healthcare. trachoma infections occur mainly in children aged 1 to 5, who act as reservoir for the bacterium. in older age groups, the development of immunity limits infection, although reduced exposure as a result of behavioural change also contributes to the declining prevalence of infection with age. repeated ocular infections by chlamydia trachomatis inflame the eyelids, leading to scarring of the conjunctival lining of the upper eyelid. when the lid margin is distorted, eyelashes turn inwards touching the eye surface, termed entropion trichiasis. unresolved repeated abrasions of the cornea contribute to disabling pain, corneal opacification, and ultimately the loss of vision [1, 5 ]. in 1997, who established the alliance for global elimination of trachoma by the year 2020 (get 2020), a partnership with the aim to eliminate blindness caused by trachoma. the get 2020 alliance recommends interventions for trachoma control, known by the acronym safe, which stands for surgery for trichiasis, antibiotics, facial cleanliness, and environmental improvement, including clean water and latrines. the aim of antibiotic use is not only to treat the affected patients, but also to limit disease transmission to others. it was estimated that the ultimate intervention goals would require antibiotic treatment for some 340 million people and trichiasis surgeries for 8.2 million [1, 5 ]. there are concerns about the possibility of antibiotic resistance, especially among bacterial pathogens such as streptococcus pneumoniae, which could potentially undermine the safe strategy. invasive pneumococcal diseases associated with streptococcus pneumoniae are a major cause of morbidity and mortality worldwide, resulting in around half a million deaths in children under 5 in 2008, mostly in developing countries. azithromycin is commonly used for the treatment of community - acquired pneumonia in adults and clinical cure is compromised in patients infected with resistant strains. resistance to azithromycin (azm) has not been documented in isolates of chlamydia trachomatis following mass treatment in trachoma control programmes. however, correlation between macrolide use and resistance in streptococcus pneumoniae has been well documented [10, 11 ]. for example, a randomized controlled trial by malhotra - kumar. showed a causal effect between azm use in individuals and resistance in streptococci, which remained significant until 6 months after treatment. several studies have been conducted to examine the possibility of antibiotic resistance in streptococcus pneumoniae after mass trachoma treatment with oral azm. here, we attempt to summarize their findings and quantify the impact of azm. this systematic review considers studies that involved participants of any age in trachoma - affected regions, who received oral azithromycin as part of trachoma eradication program, where outcome measures included streptococcus pneumoniae presence and antibiotic resistance detected from nasopharyngeal swabs of participants. searches were performed by derek k - h. ho and christian sawicki on electronic databases medline and web of science up to 23rd of january 2014. cochrane and dare (database of abstracts of reviews of effectiveness) databases were also consulted [12, 13 ]. studies of community - wide trachoma treatment with azithromycin that measured the prevalence of streptococcus pneumoniae carriage and azm sensitivity based on nasopharyngeal swabs were included in the analysis. participants who received other drugs such as tetracycline ointment or took azithromycin in forms other than oral were not included in the analysis. primary outcome measure is the prevalence of antibiotic resistance to azithromycin in streptococcus pneumoniae isolated from nasopharyngeal swabs of study participants before and after community - wide administrations. secondary outcome measure is the prevalence of streptococcus pneumoniae carriage in nasopharyngeal samples from participants. the following studies were excluded : reports of antibiotic resistance in species other than s. pneumoniae ; use of azithromycin other than for treatment and prevention of trachoma ; mathematical modeling ; surveillance reports ; review articles ; case reports or series with a study size of less than 50 ; studies without a consistent laboratory protocol for resistance testing ; studies without sufficient information on the number of samples tested. ho extracted the data from the studies that met the eligibility criteria, and then recorded the primary and secondary outcome measures as well as the participant numbers and demography, geographic areas, azithromycin regimes, and its background usage in the regions. ho also assessed the included studies for risk of bias using the cochrane risk of bias tool. odds ratios (or) were calculated and pooled across studies using the mantel - haenszel random effects model implemented in review manager 5.2. results were summarized in forest plots and heterogeneity across studies assessed using the i statistic. exact 95% confidence intervals on proportions were calculated using the clopper - pearson method. for the purpose of analysis, we defined resistance to azithromycin as reported in the studies. searches on medline and web of science provided 28 and 43 results, respectively, giving a total of 71 citations. after adjusting for duplicates, 45 remained. of these, 18 studies were discarded as they did not fit the inclusion criteria, having reviewed their titles and abstracts. the full text of the remaining 27 citations was retrieved and examined in detail, and 19 of them were rejected by the exclusion criteria. a total of 8 remaining studies were identified for inclusion in the systematic review [1623 ]. 6 were community - based studies and 2 were individual - based studies (table 1). one study recruited children under 5, three studies [17, 19, 20 ] recruited 1- to 10-year - olds, two studies [18, 21 ] recruited all nonpregnant residents over 1 year old, and two studies [16, 19 ] recruited children with trachoma and their household contacts. in four studies [18, 2022 ], participants who were ineligible, including pregnant women, infants less than 1 year old, and individuals allergic to azm, were given tetracycline ointments instead. all studies took place in less - developed countries or disadvantaged communities. two studies took place in ethiopia [21, 22 ], three studies took place in nepal [17, 19, 20 ], and two studies took place in tanzania [18, 23 ]. there was considerable variation in the administration regimes used ; azithromycin was administered once only at the beginning of five of the studies [1619, 23 ], 3 monthly for 4 times in one study, annually for 3 times in one study, and biannually for 6 times in one study. all specified the dosage as 20 mg / kg, while two studies also administered 1 gram for adults [19, 21 ]. there were largely two categories of sampling criteria for the eight included studies : random selection from a predefined age range [17, 19, 2123 ] or all children within a specified age range [16, 18, 20 ]. only one study specifically sampled children with trachoma ; other studies did not specify disease status. the resistance status of streptococcus pneumoniae to azm was tested using different commercial tests : e - test strips (ab biodisk, sweden and usa) [16, 18, 19, 22, 23 ] and broth dilution sensititre mic plates (trek diagnostics inc. one study mentioned the use of broth dilution mic testing without specifying whether it was a commercial product. resistance status was determined by the mic values as per test kit instructions ; this was explicitly stated in six studies [16, 1820, 22, 23 ]. the two remaining studies [17, 21 ] made reference to national committee for clinical laboratory standards, usa, and clinical and laboratory standards institute, usa, respectively.. however, only four of the studies described the process of masking samples to laboratory workers [2023 ]. five studies [1618, 22, 23 ] measured the baseline prevalence of resistance before azm administration. the eight studies performed measurements at various time points after the antibiotic therapy, varying from once only at month 3 in skalet 's study to month 6, year 1, and year 2 in haug 's study. as previously mentioned, haug 2010 and skalet 2010, both based in ethiopia, administered the antibiotic at higher frequencies, biannually and quarterly, respectively, than the rest of the included studies. all but two studies (haug 2010 and cole 2013) recorded low baseline prevalence of s. pneumoniae resistance to azm (0% to 5.3% of isolates), which rose within three weeks following drug intake (0% to 54.6% of isolates) (figure 2(a)). these studies showed the resistance figures dropping below 20% by 6 months and below 5% by 12 months. haug, skalet, and coles ' studies showed much higher resistance rates at over 75% by 6 months [2123 ] (figure 2(b)). for haug 2010 and skalet 2010 studies, this was likely due to the more frequent azm administration as discussed earlier, whilst, for coles 2013 study, similarly high resistance values at baseline have also been demonstrated by the control group. these three studies appeared to display similar trends in the prevalence of resistance, with a prolonged peak of resistance at around 80% even at 6 months. five of the studies took baseline measurements [1618, 22, 23 ] and four of the studies had a control arm [2023 ]. however, due to the fundamental differences in the nature of these studies, both in terms of frequencies of azm provision and varied baseline resistance, we did not combine the data for meta - analysis. it is of particular note that coles 2013 demonstrated a high baseline resistance of 36% even without the prior dosing as in haug 2010 study. examining the five studies where baseline resistance was recorded, the prevalence of resistance at months 2 to 3 appeared highly correlated with that of baseline, with a correlation coefficient of 0.759 (figure 3). these included streptococcus selective media [19, 21, 22 ], morphology [16, 18 ], observation for -haemolysis [18, 19 ], optochin susceptibility [16, 18, 19, 2123 ], and bile solubility test [18, 19, 21, 22 ]. two studies only described the use of media and sample freezing, but not the type of test used [17, 20 ]. prevalence of pneumococcal carriage in six of the eight studies ranged from 68% to 85% initially (figure 4) and fell within days after azm administration, returning to original values from 2 months onwards. batt 's and coles ' studies remained below 15% and 52% prevalence throughout the 6 months of their study periods, respectively. two of the studies [16, 19 ], instead of providing blanket azm coverage to population, administered the antibiotic only to individuals suffering from active disease and their household contacts. the who simplified trachoma grading scale was used in one of these studies, but the number of assessors and the degree of their agreement were unknown. leach did not report the criteria used at screening. for studies with control groups, this was absent in gaynor 's and coles ' studies ; however. in coles ' study, the treated villages were chosen for their high trachoma prevalence (> 10%), whilst the ineligible villages served as control group. gaynor appeared to have included two randomly selected untreated villages only at the end of the study. there was no masking of the study personnel, as the use of placebo was not mentioned in any of the included studies. assessments of nasopharyngeal streptococci carriage and antibiotic resistance are relatively easy to mask, as it is straightforward to anonymize laboratory samples. three out of eight studies [2022 ] reported masking of the outcome assessors (the laboratory workers) to the nasopharyngeal samples. five out of eight studies reported sampling at baseline (i.e., before azm was administered). there was no suggestion from the published reports that the outcomes at any other time points were undisclosed due to selective reporting, although the lack of sampling at baseline made results interpretation more difficult due to the heterogeneity in the prevalence of resistance in different regions. we judged that there was no selective reporting of outcomes because the data was presented in full in all the included studies. we included all studies irrespective of the language of publication ; however, we can not exclude the possibility that relevant studies published in languages other than english were not picked up in the initial search or that studies with negative findings were published in less accessible journals. background use of azm or other erythromycins in the study regions could also render results less reliable, as the selection pressure would have been present prior to the studies. for the two studies in western nepal [17, 20 ], around 3000 children were administered azithromycin as part of a clinical trial in 1998. for coles ' study, the reported use of (unspecified) drugs to treat suspected infections in the 30 days prior to study was over 65%. however, comparison with baseline or with a control arm in these studies mitigates against bias introduced by the use of antibiotics outside the study intervention. azithromycin coverage also varies in different trials, as denoted in table 1. for example, batt administered azm to all nonpregnant residents over 1 year old, while coles administered the antibiotic only to children under 5 years old. streptococcus pneumoniae is the most common causative pathogen for community - acquired pneumonia (cap). published guidelines from the infectious diseases society of america (idsa), european society of clinical microbiology and infectious diseases (escmid), and british thoracic society (bts) all reaffirm the role of macrolide as part of the initial empirical treatment for cap in both outpatient and hospital settings [2830 ]. this is particularly so in the us due to the higher prevalence of atypical organisms, whilst more emphasis is placed on penicillins in europe and uk. an european study on outpatient antibiotics use demonstrated a significant positive correlation between the volume of penicillin consumption in 19 countries and the prevalence of antibiotic resistance in s. pneumoniae. this is of significance, because there may come a time for routine macrolide use when pneumococcal infections become commonly penicillin resistant. a study that compared between annual and twice - yearly azithromycin regimes demonstrated that while a twice - yearly treatment can hasten the mean elimination time of ocular chlamydial infection by 7.5 months, the two groups showed no difference in disease prevalence from 18 months onwards. our systematic review faced some challenges as a result of limitations in the included studies. four studies did not include a control arm and only 2 studies followed up their participants beyond 6 months, with gaynor 2003 measuring 0% resistance to azm among isolates at 1 year and haug measuring 30.6%, which was similar to the baseline value of 28.2%. in terms of the strength of the included studies, all were cohort studies except skalet 2010 and haug 2010, which were randomized controlled trials. a recent mathematical modeling study, based on the study data from haug 2010, estimated that within 5 years of the last antibiotic dose there would be a 95% chance of macrolide resistance being eliminated by intraspecies competition. however, it has also been suggested that sustained antibiotic use below a critical threshold may encourage the persistence of antimicrobial drug resistance. despite these limitations, it appears that in communities where baseline resistance to azithromycin in pneumococcus is low, mass azm administration increased resistance only transiently, with the proportion of resistant cases gradually reducing as measurements were taken at further time points. a lack of long - lasting pneumococcal resistance may be somewhat reassuring for azithromycin - based trachoma eradication programs. we however also noted two studies [21, 23 ] that demonstrated high baseline and subsequent antibiotic resistance in streptococcus ; one is the only study that implemented a high - intensity regimen for a prolonged period (twice a year for three years), whilst the population examined in the other study appeared to have high background antibiotic use (65%73% in control and treatment groups). these results from the field may give us an insight as to the potential adverse outcome when a critical threshold of antibiotic use in the region is exceeded. our analysis, which showed a certain level of correlation of resistance prevalence between baseline and subsequent time point with a coefficient of 0.759, appears to be in agreement with this hypothesis. woolhouse and farrar recently reiterated the importance of global efforts in combating antimicrobial resistance, with one of the possible solutions being the investigation of dosing regimens that can stall resistance development. health authorities in trachoma - affected regions should therefore be mindful of the selective pressure asserted by mass antibiotic use when implementing the safe strategy. | trachoma is caused by chlamydia trachomatis and is a leading cause of blindness worldwide. mass distribution of azithromycin (azm) is part of the strategy for the global elimination of blinding trachoma by 2020. although resistance to azm in c. trachomatis has not been reported, there have been concerns about resistance in other organisms when azm is administered in community settings. we identified studies that measured pneumococcal prevalence and resistance to azm following mass azm provision reported up to 2013 in medline and web of science databases. potential sources of bias were assessed using the cochrane risk of bias tool. a total of 45 records were screened, of which 8 met the inclusion criteria. we identified two distinct trends of resistance prevalence, which are dependent on frequency of azm provision and baseline prevalence of resistance. we also demonstrated strong correlation between the prevalence of resistance at baseline and at 2 - 3 months (r = 0.759). although resistance to azm in c. trachomatis has not been reported, resistance to this commonly used macrolide antibiotic in other diseases could compromise treatment. this should be considered when planning long - term trachoma control strategies. |
neuroendocrine tumors (nets) are considered a class of rare neoplasms accounting 20%) grade, whereas in the usa, tumors are graded as well equates to low - intermediate grade and poorly equates to high - grade tumors [3, 4 ]. up to 80% of gep nets express somatostatin receptors (sstr2 and sstr5 primarily). therefore, somatostatin analogues have been used for both diagnosis and treatment of nets. in - labeled sst - analogues spect and ga sst - analogues pet / ct represent an accurate methods for nets diagnosis peptide radioreceptor therapy (prrt) indication and patients management [58 ]. when beta - emitters isotopes as y (t1/2 of 2.67 days, maximum range of tissue irradiation of 12 mm) or lu (t1/2 of 6.73 days, maximum range of irradiation of 1.5 mm) are used to label sst - analogues linked to a chelator, prrt may be performed. after the i.v. injection, the radiopharmaceutical will distribute in the body, selectively bind to sstrs, and actively be taken up by the cells through a process called receptor - ligand internalization [9, 10 ]. the internalization will ultimately lead to a selective accumulation of radioactivity in the tumor, thus determining cell death. the majority of clinical trials data available is from non - randomized retrospective case series. due to variation in patients selection, dosing, scheduling, and total number of treatments it can be challenging to draw firm conclusions from the literature. however, it seems to be a benefit for selected patients with response rates in the range of 40% [1114 ]. here we present the results of a phase ii study designed to treat disseminated or nonoperable nets patients with prrt. patients demonstrated enhanced sstr expression at pet / ct with ga - peptide (dotatoc / dotatate). this was a prospective nonrandomized single - arm clinical trial performed at the department of nuclear medicine, santa maria nuova hospital, reggio emilia (italy). all patients with advanced, progressive net fulfilling the study inclusion criteria were first evaluated with ga - peptide pet / ct followed by in - peptide dosimetric evaluation to determine both the presence of sstr expression as a target for the following treatment and eligibility to prrt, that is in presence of provisional adsorbed doses : (a) > 10 gy to tumor, (b) 18 years;histological confirmation of net ; inoperable or metastatic disease;presence of at least one measurable lesion ; positive 68ga - peptide pet / ct defined as radiopharmaceutical uptake in tumor and/or metastasis higher than liver, evaluated within 3 months before prrt (qualitative analysis);adequate hematological parameter : hemoglobin level (hb) 10 g / dl ; leucocytes (wbc) 2.5 10/ml ; platelets (plt) 100 10/ml;adequate liver and renal function : bilirubin levels 25% ; other concomitant tumors, except in situ basal cell carcinoma and tumors of the uterine cervix treated with radical surgery. other treatment (such as chemotherapy or radiotherapy) or participation in any investigational drug trial within 1 month of prrt and for the following 2 months ; pregnancy or lactation ; bone marrow involvement > 25% ; other concomitant tumors, except in situ basal cell carcinoma and tumors of the uterine cervix treated with radical surgery. additionally, before each prrt cycle the following parameters should be maintained : hb 10 g / dl, wbc 2.5 10/ml ; plt 100 10/ml, creatinine levels 99.8%. the radiolabeling of ga - peptide was performed by means of a modular lab synthesizer (eckert & ziegler, berlin, germany) as already described. briefly, the fraction of about 2 ml of the ge / ga - generator eluate containing about 80% of the ga activity in 0.1 m hydrochloric acid was selected and directed to a reactor vial containing a 20 l of peptide solution (1 mg / ml) and 200 l of a 1.5 m sodium formate solution or 140 l of a 1.5 m sodium acetate solution in order to obtain a ph ranging between 3.2 and 3.5. the mixture was heated at 100c for 5 minutes and, then, passed through a light c-18 cartridge. ga - peptide was eluted with 0.51 ml of a 50% ethanol solution and diluted with 8 ml of 0.9% sodium chloride solution. the synthesis was carried out in 14 minutes with a mean yield of 63 3% (not corrected for decay). quality controls were performed by chromatographic methods as already described, obtaining a radiochemical purity always > 95%. for this study, pet / ct scans were acquired on a ge discovery at 60 min after injection of about 120 mbq of ga - peptide. seven or eight bed positions with 5 slices overlap were acquired for 4 min emission time in 3d. the ct - exposure factors for all examinations were 120 kvp and 80 ma in 0.8 seconds. mean and maximum suv (activity concentration corrected for patient weight and total injected dose) was determined in all lesions and recorded. ga - peptide pet / ct was considered positive in patients who showed uptake in the tumor lesions at least two - times higher than the liver ; thus they were considered eligible for prrt and, therefore, admitted to dosimetric evaluation. injection of 185 mbq of in - peptide with a dual - head gamma camera (genesys, philips, the netherlands) using parallel - hole, medium - energy, general - purpose collimators. the windows were centered over both in- photon peaks (247 and 172 kev with a window width of 20%), whereas scatter fraction was evaluated at 140 kev (width 20%). in all the patients, whole - body scan and, in selected cases, spot images of the abdomen were obtained after 1, 4, 20, 48, and 72 hours for control of biodistribution. to determine blood clearance, we drew blood samples at 30 and 60 minutes and at 4, 20, and 48 hours after injection. radioactivity in blood was measured with a hpge spectrometer (dspec jr 2.0ortec). for dosimetric calculations, regions of interest were drawn manually on the whole - body scans from anterior and posterior projections and ulmdos software (university of ulm, germany) was used. scans were corrected for background, self - absorption, patient thickness attenuation, and organ overlapping. whole - body activity acquired immediately after injection was defined as 100% of the injected activity. the resulting time - activity points were fitted to a monoexponential or multiexponential curve for whole - body, kidneys, liver, spleen, and red marrow to calculate residence time. the estimated doses delivered to critical organs and to the tumor were obtained by the software olinda / exm. the activity in blood was fitted to a biexponential curve to determine the residence time in blood. the dose to the red marrow was calculated from the residence time in blood, assuming no specific uptake, a uniform distribution of activity, and clearance from red marrow equal to that from blood. in case of lu - prrt the dosimetric evaluation was performed acquiring images during the first cycle of therapy, thanks to the low gamma emission of this isotope. a fractionated treatment protocol was followed with the intravenous administration of an average activity of 2.6 gbq and 6.0 gbq per cycle for y - prrt and for lu - prrt, respectively, with an interval of about 2 months. for each cycle thirty minutes before administration of the radiopeptide 2 l of amino acid solution of hartmann - hepa 8 (ringer 's lactate hartmann, proteinsteril hepa 8%, mg 5-sulfat) were infused, which were continued up to 3 hours after injection to inhibit tubular reabsorption of the radioactive tracer. repeated treatments were performed in case of response and significant improvement in symptoms and quality of life, except in cases of renal toxicity and rejection by the patient for further treatment within 3 months. additional cycles were suspended in case of pd. in order to evaluate the biodistribution of therapeutic activity, after each treatment, planar imaging was performed with a dual head spect gamma camera (genesys, philips, the netherlands) or with a dual - head spect / ct gamma camera (symbia - t, siemens, germany) using parallel - hole, medium - energy, general - purpose collimators. the windows were centered over lu - prrt photon peaks (208 kev and 110 kev width 20% in both cases ; scatter window at 160 kev) in case of treatment with lu - prrt ; while at 170 kev (20%) and 80 kev (55%) in case of treatment with y - prrt, as bremsstrahlung planar scan. whole - body scans (acquisition time : 25 minutes) and spot images (acquisition time : 10 minutes) were obtained. clinical benefit was assessed comparing baseline clinical conditions with end - treatment parameters. in the clinical benefit evaluation the worsening of clinical conditions (i.e., appearance of new sign(s)/symptom(s)) were considered as pd. indeed any significant variations in baseline clinical conditions was defined as stable disease (sd). clinical benefit was defined as non - pd / sd. for the follow - up blood tests were evaluated, as described in the clinical protocol, repeated before and after each treatment cycle and every two weeks. baseline and end - treatment serum cga values were compared and the trend was defined as increased, stable (variation over time 10%) or decreased. acute and long - term adverse events were graded according to the common terminology criteria for adverse events, version 3.0 of the national cancer institute. to assess response to treatment pet / ct studies performed at baseline and at the end of treatment were considered. treatment responses assessed by pet / ct scan were defined as follows : complete response (cr) : disappearance of radiopharmaceutical uptake in all detectable lesions;partial response (pr) : reduction of radiopharmaceutical uptake (> 50%) in all detectable lesions in absence of appearance of new lesion(s);stable disease (sd) : no variation or reduction of radiopharmaceutical uptake (25% of radiopharmaceutical uptake in one or more lesions or appearance of new lesions and/or > 10% increasing of tumor marker. complete response (cr) : disappearance of radiopharmaceutical uptake in all detectable lesions ; partial response (pr) : reduction of radiopharmaceutical uptake (> 50%) in all detectable lesions in absence of appearance of new lesion(s) ; stable disease (sd) : no variation or reduction of radiopharmaceutical uptake (25% of radiopharmaceutical uptake in one or more lesions or appearance of new lesions and/or > 10% increasing of tumor marker. in this series of patients, we did not assess treatment response based on the size of lesions using the ct component of pet / ct images or ct scan, but as described above we evaluate only the functional response. all values are expressed as median and range, as customary for nonparametric data.correlation analysis was performed using the mann - whitney test. ga - dotatoc / dotatate pet / ct was performed in all the patients to evaluate the eligibility. baseline ga - dotatoc / dotatate pet / ct demonstrated at least one site of radiopharmaceutical uptake. ga - dotatoc positive lesions were preferentially localized at liver, lymph nodes, lung, and skeleton. an end - treatment ga - peptide pet / ct was performed in all treated patients about 36 months after the last prrt administration except for 6 patients in which pd was determined on the basis of worsening of clinical conditions. figures 2 and 3 represent examples of pre- and posttherapeutic ga - dotatoc pet / ct. prrt cycles were administered at 70 24.6 days apart (range 35140) with a median cumulative activity of 5.5 gbq (range 3.67.4 gbq). thirty - five patients received 4 or 5 prrt cycles, 10/59 more than 5 cycles while 14/59 patients had 2 cm, lu for lesion(s) < 2 cm, and y / lu in presence of both conditions) and on dosimetric estimates. administration of y - prrt (average activity of about 2.6 gbq / cycle) and lu - prrt (average activity of about 6.0 gbq / cycle) induced disease control in 83% of patients (1 cr, 24 pr, and 24 sd) with a duration of response of at least 6 months. in the majority of cases, objective response was associated to symptomatic response with an improvement of quality of life. these responses rates are comparable with data from literature [11, 24, 25 ] demonstrating radiological response of 34.1% and clinical response in 29.7% for y - prrt with longer median survival in responders compared to nonresponders (44.7 versus 18.3 months) and response rates of up to 30% with median time to progression of 40 months for lu - prrt. interestingly, in these studies the degree of uptake on the pretreatment in - peptide was found to be predictive of response to treatment and overall survival. despite the small number of patients, combined treatments with labeled peptide using both y and lu seem to perform better (no evidence of pd) when compared to single radionuclide prrt (8 and 2 cases of pd administering only y - peptide and lu - peptide, resp.). in our patients population, the suvmax value in tha main lesion at baseline ga - peptide pet / ct examination compared to end - treatment scan was in line with the functional response evaluation. additionally, in our series of patients, cumulated administered activity was significantly different in responders and no - responders. in agreement with previous reports in literature our data supported the hypothesis that progression at baseline could be a prognostic factor of objective response to prrt. indeed previous reports showed also that both sd and objective response (cr+pr) in previously progressive patients showed the same favorable trend. finally, prrt showed beneficial effect on symptoms in the majority of patients (36%) and all asymptomatic patients (46%) remained stable over the time. in 39% of our patients discordant results between serum cga trend and ga - peptide findings were observed. particularly, cga values increased (with variation up to + 263%) in 68% of patients in which prrt determined pr or sd. our results on cga trend and antitumor activity are in contrast with previous reported ones in the literature. however, the majority of our patients assumed proton - pump inhibitor as prophylactic therapy and as well known these drugs may cause substantial increase of blood cga levels underling the need of a more reliable biomarker to monitoring nets. the prevalence of partial responses and stable disease obtained is mainly related to the advanced stage of the disease when patients are referred to prrt. in fact, the majority of patients have entered the protocol after the failure of multiple types of treatment situation when more affords are needed to avoid toxicity (i.e., accurate dosimetric evaluation). in this setting of patients an individualized dosimetry has been demonstrated as the ideal method to plan prrt in order to combine the highest possible dose of radiation to the tumor with the maximum tolerated dose by the dose - limiting organ. in fact, since the absorbed dose to the kidneys and bone marrow might vary considerably between patients, using fixed dose regime side effects may be reduced but at the cost of undertreatment for certain patients, thus, reducing the potential effectiveness of the prrt. the fractionated schedule that we apply in the protocol was selected based on literature data and personal experience. treatment with this schedule was generally well tolerated with the most common side effects of nausea and vomiting being caused by the administration of amino acid solutions. reversible bone marrow suppression was seen in 20% of patients. however, in 3 patients we also observed delayed nephrotoxicity requiring in one case dialysis. according to literature data we observed nephrotoxicity in case of y - prrt ; however, the presence of high - risk comorbidities in 2/3 cases (only one kidney and blood hypertension plus diabetes each) is not negligible especially if we consider that a low bed threshold (< 28 gy) have been maintained in these specific patients. in all these patients prrt no myelodysplastic syndrome or acute leukemia occurred. despite these interesting results, our study presents some limitations. first the choice of radionuclide used for prrt was based in some cases on radiopharmaceutical availability at our center. the reason for this choice lies in the fact that the majority of patients (42/59) had liver lesions and their sizes were difficult to accurately be measured using the ct component of pet / ct images without contrast medium. finally, as previously described, histopathological features of nets including grading were not comparable avoiding the possibility to further speculate on results. our study demonstrated that prrt using a fractionated treatment protocol with the intravenous administration of an average activity of 2.6 gbq / cycle for y - prrt and 6.0 gbq / cycle for lu - prrt, respectively, and with an interval of about 2 months is a feasible therapeutic option for patients with neuroendocrine tumors able to induce disease control in up to 83% of patients, associated with significant clinical response. the use of ga - peptide pet / ct as first - selection procedure to determine the presence of high sstr expression followed by standard dosimetric estimates may be used for patients selection. however, there is the clinical need of randomized clinical trials to determine what is optimal treatment schedule based on the specific biological and molecular features of the tumor. future therapeutic trials should also aim to include patients at earlier stage of disease and to investigate the best setting where to introduce radioreceptor therapy in combination, rather than an alternative, to other treatment options. | the aim of this study was to assess the efficacy of prrt in patients with advanced neuroendocrine tumors (nets). patients and methods. from january 2007 to august 2011, we enrolled 65 patients (m / f 38/27 ; mean age 65 years, range 3383) with advanced nets having enhanced sstr expression, treated with prrt. the enhanced expression of sstr was assessed using 68ga - dotatoc / dotatate pet / ct. among all the enrolled patients, 6 of them were excluded from the present analysis since they voluntarily interrupted treatment. mean activity / cycle of 2.6 gbq (90y - dotatoc / dotatate) or 6.0 gbq (177lu - dotatoc / dotatate) was administrated intravenously (max 9 cycles). results. complete response (cr) was found in 1/59 (2%) patients, partial remission (pr) in 24/59 (40.5%) patients, stable disease (sd) in 24/59 (40.5%), and progression (pd) in 10/59 (17%) patients. the overall tumor response rate (cr + pr) was 42.5%. in 40.5% of patients, the disease could be stabilized. overall, 49 out of 59 patients had no tumor progression (83%). twelve patients out of 59 (20%) had grade 2 - 3 hematological side effects including anemia, thrombocytopenia, and leukopenia. long - term nephrotoxicity was observed in 3 patients (2 moderate, 1 severe). conclusions. prrt is a promising perspective for patients with advanced nets. |
approximately 10 million americans have osteoporosis and 80% of those are women whereas 20% are men. peak bone mass (pbm) is a major determinant of bone mass later in life and is attained during the first two decades of life [3, 4 ]. therefore, maximizing the amount of bone gained during childhood and adolescence is essential in preventing osteoporosis and decreasing the risk of fractures later in life [59 ]. physical activity is one of the most important factors in increasing pbm and preventing osteoporosis. when examining the effects of physical activity on bone mineral density (bmd), type of exercise is important. most tennis players have significantly greater bone mass and bone area in their dominant arm compared to their nondominant arm [4, 1115 ]. however, most of these earlier studies examined bmd of adult tennis players. the purpose of this study was to determine differences in bmd among adolescent female tennis players (tps) and nontennis players (ntps). nineteen female tps and 19 ntps between 14 and 18 years of age participated in this study. this study was performed with the approval of the university of mississippi 's institutional review board. tps were included in the study if they had been playing tennis for a minimum of two years and were playing at least three hours per week. the control group included female adolescents who did not play tennis ; however, they may have been involved in other sports competitively or recreationally. all of the subjects were healthy with no known diseases and were not receiving medications known to effect bone metabolism, such as, corticosteroids. body mass index (bmi) plays an important role in bmd [16, 17 ] ; therefore, bmi of tps and ntps was matched. tps were recruited from local high school tennis teams and a local tennis academy, while the ntps were recruited from local high schools. a letter was sent home with the potential participant giving information on the methods of the study and that participants would need parent 's permission to enroll in the study. once complete, participants were scheduled to come into the bone density laboratory where weight and height were measured using a standard doctor 's scale. total hip, ap lumbar spine, and both forearms bmd and body composition were assessed using a hologic delphi - w (bedford, ma) dxa machine. the forearm scans included the area of the radius and ulna and wrist bones (carpals). tanner stages were assessed using a questionnaire that is related to the adolescent 's pubertal status. menstrual status was assessed by asking participants if they had regular menstrual cycles and if they had ever experienced amenorrhea. participants who indicated that they did not have regular menstrual cycles or experienced amenorrhea were eliminated from the study. physical activity (pa) participants were asked to recall physical activities they engaged in during the previous seven days as being either light (activities of daily living), moderate (active but nonexhausting), or vigorous (made their heart beat rapidly). they recorded only those activities and durations that fell within the moderate or vigorous categories. a weekly physical activity score was computed for each participant as metabolic hours per week (met hours / week) calculated by multiplying the met score for each moderate or vigorous activity by the listed duration for each activity and then summed across all seven days. moderate activities were assigned a met value of 4 and vigorous activities a met value of 7. one - way anova was used to assess differences between the groups for all bmd, body composition, and physical activity measurements. multiple regression analysis was used to determine the best predictor of bmd amongst weight, total body lean mass, and total body fat mass. both z scores and bmd raw scores were analyzed to determine differences in spine and hip measurements between the groups whereas only bmd raw scores were used to obtain differences in forearms bmd since the dxa software used for this study did not have the database of z scores for forearms. significance level was set at = 0.05 and data were analyzed using spss version 18.0. there were no significant differences between the tp and ntp for height, body mass, age, and tanner stages. tp had significantly greater femoral neck bmd than the ntp (p = 0.02 for z scores and p = 0.02 for raw scores). although tp had greater bmd for lumbar spine and total hip measurements, these differences were not statistically significant (p = 0.37 and 0.12, resp.). when total forearm bmd of the dominant arm and nondominant arm was compared within each group, only tps had statistically significant differences indicating that dominant arm bmd of the tps was significantly greater than non - dominant arm bmd (p < 0.001). in addition, a side - to - side comparison between tps and ntps was not significant (f = 0.39, p = 0.53). however, a comparison of mean differences of distal 1/3 radius and ulna was compared between tps and ntps, a significant difference was found (t = 2.03, p = 0.05). we did not find a significant difference between the ulna distal region (p = 0.97). there were no significant differences for total body percent fat (p = 0.72), total body lean mass (p = 0.07), and total body fat mass (p = 0.59) between the groups. regression analysis revealed that the combination of the three independent variables (weight, total fat mass, and total lean mass) significantly predicted bmd at the femoral neck and total hip (p < 0.001, r = 0.38 ; p = 0.03, r = 0.23, resp.) with lean mass being the best predictor for both measurements. results of pa data and tennis playing history are summarized in table 4. the difference in physical activity between tp and ntp expressed as met - hours / week did not reach statistical significance (p = 0.24). this study assessed the differences in bmd between adolescent female tps and ntps who may have been involved in sports other than tennis. to the best of our knowledge, this is the first study to assess femoral bmd in adolescent female tps, which includes a physically active nontennis playing control group. the ntp control groups in previous studies have been sedentary. in the present study, the main finding of this study was that tps had significantly greater femoral neck bmd than ntps, even though the ntps participated in significant amounts of nontennis physical activity. this result supports previous research findings suggesting that tennis players have significantly greater femoral neck bmd than controls [12, 21 ]. however, tennis players in the previous research were adult male and female tennis players. previous data have shown that high - impact exercise, such as jumping, increases bmd in the femoral neck. tennis requires mostly anaerobic, rapid accelerations, and decelerations with twisting components and can produce ground reaction forces 5 to 10 times a person 's body weight. observed that badminton players have significantly greater femoral neck bmd than ice hockey players and controls and stated that this difference might be due to the movements executed in the sport. badminton players are generally subjected to short high - impact bursts when jumping and high strains in unusual directions ; during leg lunges forward and fast direction changes from side to side. furthermore, the ground reaction forces created by a jump may be absorbed first in the feet and joints of the lower extremities in which the created high strains may be a powerful stimuli to increase bmd. (2005) found that female athletes who were subjected to different loading modalities had stronger femoral neck measurements than those not performing these modalities. again, in 2010, nikander found that odd - impact exercises were significantly associated with a 20% thicker cortex around the femoral neck. therefore, it can be suggested that the movements of tennis playing that produce great ground reaction forces might increase bmd in the femoral neck. although these cross - sectional data do not allow us to quantify the minimum amount of physical activity necessary to bring about increases in bmd, it is worth noting that the tennis averaged 4.8 tennis sessions / week and 83.6 minutes / session. thus, of the 53.3 met hours per week of physical activity they engaged in, 88% (46.8 met hours / week) was accounted for by their tennis playing. whether lesser amounts of weekly tennis playing cause favorable changes in bmd will need to be examined in prospective investigations. according to the national osteoporosis foundation 297,000 osteoporosis related hip fractures occurred in the united states in 2005. furthermore, approximately 293,000 americans aged 45 years and older were admitted to hospitals with a fracture of the femoral neck in 2005. developing high femoral neck and total hip bmd by playing tennis during teenage years might play an important role in osteoporosis prevention and decreasing the risk of fractures at the hip later in life. in this study juzwiak. also observed no significant differences in lumbar spine bmd between adolescent male tps and controls. however, haapasalo. reported that lumbar spine bmd was significantly greater only in tennis players who were at tanner stages 4 and 5 compared to control group participants who did not participate in sports. we did not find a significantly greater spine bmd for tps which may be because participants of the ntp group were not sedentary and were involved in activities that might affect their spine bmd. in the current study, all of the tps and ntps were at tanner stages 4 and 5. when the mean bmd values for the lumbar spine in both studies are compared, tps in the current study had greater values than the tennis players of haapasalo. (0.980 g / cm versus 0.971 g / cm for tanner stage 4 and 1.045 g / cm versus 0.963 g / cm for tanner stage 5, resp.). the ntps in this study were as active as tps and also had higher mean bmd of the lumbar spine when compared to the tennis players in the research of haapasalo.. in the present study, there were no significant differences between tps and ntps in forearms bmd. the difference between the dominant arm bmd and the nondominant arm bmd was significant only in tps. this result supports previous studies which report that side - to - side differences were significantly greater in tennis players [3, 14, 25, 26 ]. found that adolescent male tennis players have significantly higher lean body mass content and lower fat mass than controls. in contrast, calbet. observed no significant differences in body mass, total lean body mass, total body fat, and the percentage of body fat between tennis players and sedentary controls. in the present study, there were no significant differences in fat mass and lean body mass variables between the tps and ntps. data have shown that lean body mass is greater in boys than girls, whereas fat mass is greater in girls than boys in healthy children and adolescents. furthermore, in the present study, there were no significant differences between the groups for the total minutes of weekly pa, and ntps had performed significantly greater amounts of moderate pa than tps. this may explain why there were not significant differences between the groups in body composition measurements. on the other hand, researchers have suggested that in children, lean mass in relation to body height (lean mass / height) should be assessed since sole usage of age and sex specific charts for bmd limits the clinical interpretation of dxa output, and the relation of lean mass to height provides an understanding of the origin of low bmd / age. when lean mass was calculated related to body height (lean mass / height) for this study, tps had significantly greater total body lean mass than ntps (p = 0.02). the results of the regression analysis are in agreement with the results of the previous data. in the present study, all of the independent variables were significant predictors of bmd with lean mass being the best predictor among the three independent variables. previous studies have also shown that lean mass is a significant predictor of bmd in young females [16, 2830 ]. the significant associations between lean mass and bmd have been shown to be performed during force generation by the muscle mass. on the other hand, seeman. suggested that the significant positive correlations between lean mass and bmd could be explained by genetic determinants rather than nongenetic factors. it has been noted previously that adult tennis players have greater femoral neck bmd than their inactive peers. our study extends this premise and shows that adolescent female tennis players also have significantly greater femoral neck hip bmd than adolescent female nontennis players who are as physically active as tennis players. osteoporosis prevention measures are aimed at women who already show the signs of depleted bone mass [32, 33 ]. | the purpose of this study was to determine differences in bone mineral density (bmd) among adolescent female tennis players (tps) and nontennis players (ntps) and to assess body composition as a predictor variable of bmd. nineteen female tps and 19 female ntps, ages 14 to 18 years, participated in this study. lumbar spine, total hip, femoral neck, forearms bmd, and body composition were assessed using dual - energy x - ray absorptiometry (dxa). lumbar spine and total hip bmd measurements for tp were greater than ntp. however, these differences were not statistically significant (p = 0.37 and 0.12, resp.). tp had significantly greater femoral neck bmd than ntps (p = 0.02). this difference might play an important role in preventing osteoporosis and decreasing the risk of fractures at the hip later in life. |
well - differentiated thyroid carcinomas, including papillary (ptc) and follicular (ftc) carcinomas, are characterized by a favorable prognosis, while undifferentiated / anaplastic carcinoma (atc) is an uncommon and highly aggressive form, which usually results in the death of the patient [24 ]. the 5-year survival ranges from 0 to 14%, with a median survival of 26 months [59 ]. atc arises more commonly in female patients, with a mean age of 70 years, usually affected by nodular goiters or with a history of well - differentiated thyroid carcinoma or with nodal or distant metastases. the patients usually complain of hoarseness due to a large - sized and rapidly expanding neck mass, which, at the time of presentation, is often surgically unresectable due to the invasion of surrounding thyroid structures, such as the laryngeal nerve, esophagus and trachea, and/or documentation of distant metastases. the most important prognostic factor is the degree of the extent of disease at diagnosis. small - sized atcs or foci of atc arising in the context of well - differentiated thyroid carcinomas have a better prognosis [911 ]. obviously the prognosis also depends on the ability to eradicate the disease by surgery [7, 12 ]. in fact, if the eradication surgery is associated with radiotherapy and adjuvant or neoadjuvant chemotherapy with doxorubicin, survival may slightly increase [7, 9, 1315 ]. unfortunately wide surgical resection usually fails to provide benefits due to the local spread of tumor, while tracheostomy is often performed to ensure the patent of upper airway, invaded and/or obstructed by massive tumor. grossly, thyroid parenchyma is widely or completely replaced by a fleshy mass, whitish in color, with multiple areas of necrosis and hemorrhage, which diffusely infiltrates adjacent tissues [3, 5, 6 ]. histologically, the tumor is composed of a variable mixture of spindled, epithelioid, and large pleomorphic / bizarre giant cells exhibiting different growth patterns such as solid, trabecular, and fascicular patterns [2, 3, 5, 6, 10 ]. the overall appearance of atc is usually closely reminiscent of a high - grade pleomorphic sarcoma. hemorrhage and necrosis, sometimes with palisading configuration, are often seen. there may be an inflammatory infiltrate, predominantly of granulocytes, which occasionally can invade the cytoplasm of tumor cells. although the above mentioned features represent the common basic morphological aspects of atc, several morphological variants have been described over time, some of which appear to be rather uncommon : (i) squamous cell carcinoma variant (tumor consisting of dominant / pure squamous differentiation) ; (ii) adenosquamous carcinoma variant (in addition to squamous differentiation, tumor contains foci of glandular differentiation with mucin production) ; (iii) lymphoepithelioma - like carcinoma variant (tumor sharing morphological features with the nasopharyngeal undifferentiated carcinoma) ; (iv) rhabdoid variant (tumor exhibits cells with clear - cut rhabdoid morphology) ; (v) osteoclastic variant (tumor contains reactive cd68 + osteoclast - like multinucleated giant cells intermixed to cancer cells) ; (vi) carcinosarcoma variant (tumor with a mixture of carcinomatous and heterologous mesenchymal components, such as cartilage, bone, or skeletal differentiation) ; (vii) paucicellular variant (hypocellular tumor with diffuse sclerosis, mimicking riedel thyroiditis) ; (viii) angiomatoid variant (tumor mimicking angiosarcoma). despite the poor morphological differentiation, the epithelial nature of atc is demonstrable in 4580% of cases by staining for cytokeratins, especially using cytokeratin ae1/ae3. as atc is refractory to conventional chemotherapy, radiotherapy, and radioiodine (i) therapy, new therapeutic approaches are urgently needed in the future. in this regard, some original or review articles about genetic mutations, chromosomal instability, and identification of potential biomarkers exploitable against atc are emerging in the literature [1724 ]. however, while for ptc several potential gene and protein therapeutic targets have been identified [2529 ], only a few options seem to be available for atc in the literature. waiting for the advent of new genomewide approaches, such as next - generation sequencing (ngs), the analysis of the molecular mechanisms involved in the pathogenesis of atc still remains the only available tool for planning any target therapy. there is increasing evidence that follicular cell - derived thyroid carcinomas represent a biological continuum of the same disease that progresses from the curable well - differentiated thyroid carcinomas (ptc and ftc) to fatal atc. in fact, although atc may derive de novo, many cases seem to arise from preexisting ptc or ftc [3133 ]. this is supported by morphological evidence showing the gradual loss of papillary and follicular growth patterns associated with a concurrent increase in the presence of solid growth pattern, mitoses, necrosis, and nuclear pleomorphism that is typically observed in atc. moreover, most of atcs exhibit residual foci of differentiated thyroid carcinoma, including both ptc and ftc. notably, atc may also develop as a recurrence months or years after the removal of a well - differentiated neoplasm [5, 34 ]. apart from this morphological evidence, it has been previously demonstrated that the development of chromosomal instability underlies the progression to more aggressive phenotypes of thyroid cancer. recurrent gains at 3p13 - 14 and 11q13 and loss of 5q1131 were identified exclusively in atc, suggesting they may be markers for anaplastic transformation. for atc with minor ptc or ftc components, it is likely that the mutations typically occurring in the latter tumors (e.g., ras and braf mutations) may represent only early events in the tumorigenesis of atc, while others, including tp53, catenin beta 1, and pik3ca, may contribute later to the acquisition of a phenotype responsible for the extremely aggressive behavior of atc [32, 3638 ]. generally, the genes coding proteins differently involved in the transduction pathway, such as ret, ras, braf, pi3k, pten, and akt, are mutated or aberrantly expressed in atc, providing conditions for uncontrolled cellular proliferation and carcinogenesis via the map kinase pathway. ras point mutations involving specific regions (codons 12, 13, and 61) of the three ras oncogenes, h - ras, k - ras, and n - ras, by activating both the map kinase pathway and the pi3k / akt pathway, are associated with aggressive thyroid tumor phenotypes including atc [3942 ]. braf, which belongs to the raf family of serine / threonine kinases, by regulating the map kinase / erks signaling pathway, affects cell division, differentiation, and secretion. the most frequent braf mutation involves nucleotide 1799 and results in substitution of valine for glutamate at residue 600 (v600e). this point mutation leads to constitutive activation of braf kinase and chronic stimulation of the mapk pathway, playing tumorigenic activity for thyroid cells [30, 32, 43 ]. inhibition of braf v600e by using vemurafenib has shown promising clinical responses in metastatic ptc. although braf mutation (v600e) is reported in approximately only 25% of atc, suggesting its involvement in tumor progression together with other genetic markers, it could be exploitable as potential therapeutic target. in this regard a dramatic response to vemurafenib has been obtained in a 51-year - old man with braf - mutated anaplastic thyroid cancer. this single case report provides evidence for testing atcs for braf mutation (v600e), treating the positive cases by using vemurafenib. this approach could be suggested, at least, as empirical treatment in rapidly progressive cases. anyway, the results need to be confirmed in larger series of acts. different alterations of pten / pi3k / akt pathway that regulates several cellular processes, including cell cycle progression, adhesion, and motility, are also commonly observed in atc, and then they could be exploitable as potential therapeutic targets. in this regard the missense mutations of pik3ca, which encodes the p110 catalytic subunit of phosphatidylinositol 30-kinase (pi3k), have been frequently detected [32, 38, 46 ]. aberrant activation of pi3k / akt pathway has been suggested to promote progression of a thyroid adenoma to ftc and/or atc, while activation of akt has been observed in most of the atcs with pik3ca mutation [32, 38, 46 ]. molecular mechanisms involved in tumor cell dedifferentiation are thought to be mediated by loss / inactivation or mutation of tumor suppressor gene, p53 [30, 32, 36, 38, 4851 ]. it has been suggested that, unlike ras and braf gene alterations, p53 mutations are crucial in accelerating genomic instability, triggering tumor dedifferentiation toward atc [36, 51 ]. redifferentiation of tissues from atc upon the reintroduction of wild type p53 and the restoration of cellular responsiveness to physiologic stimuli, such as thyroid stimulating hormone and reexpression of thyroid peroxidase [38, 52 ], strongly support this hypothesis. the biological process of dedifferentiation from well - differentiated thyroid carcinomas toward atc is also underlined by -catenin expression. -catenin acts as cell - cell adhesion molecule that complexes with e - cadherin proteins in normal epithelium. derangement of the e - cadherin / catenin complex, as well as low membrane -catenin expression or its nuclear localization, is associated with transformation of differentiated carcinomas into atc [38, 5357 ]. new diagnostic and therapeutic opportunities are emerging by the analysis of micrornas (mirnas). mirnas are a heterogeneous class of small noncoding but functional rnas involved in posttranscriptional regulation of target genes, playing a control role in development, proliferation, apoptosis, and stress response [58, 59 ]. as mirna expression is frequently altered in several tumors, they are recently emerging as promising prognostic biomarkers and therapeutic agents for many tumors [60, 61 ]. mirna - chip - microarray analysis demonstrated aberrant mirna expression profile, especially decrease of some of them (mirnas-30d, -125b, -26a, and 30a-5p), which clearly differentiates atc from normal thyroid tissues and ptc. subsequently, mitomo. confirmed downregulation of some mirnas, such as -26a and -138, but they also noticed upregulation of others, including mirnas-21, -146b, -221, and -222. the association of specific mirnas deregulation with atc transformation is the rational approach for further challenging investigations. in fact, mirna-125b has a different expression in the human tumors, being upregulated in pancreas and stomach carcinomas, whereas it is downregulated in breast cancer and atc, suggesting that it can act in different ways depending on the cellular context. moreover, mirna-125b and others, which significantly decreased in atc, have, among the predicted regulated target genes, also hmga1 and hmga2, which are proteins expressed at very high levels in several malignant tumors, including thyroid carcinomas [68, 69 ]. again, mirna-21, described to be upregulated, targets e2f (involved in cell cycle and apoptosis) and inhibits pten ; mirna-138, found to be downregulated [63, 64 ], targets the human telomerase reverse transcriptase (htert) gene which is also found to be totally downregulated in both atc and ptc cell lines in comparison with normal thyroid tissues. nevertheless, mirnas are emerging as promising new strategy with therapeutic potential for many aggressive cancers, such as atc. preclinical studies, through in vitro and in vivo analyses, are providing helpful information in the therapeutic approach of atc, especially the analysis of the mouse model closely recapitulating the clinic - pathological features of human atc. while most genetically engineered mouse models gave significant advancements about differentiated thyroid carcinomas, such as ptc [7072 ] and ftc [7376 ], only recently atc mouse models have been developed. firstly obtained a mouse model of atc by combining, in the mouse thyroid follicular cells, two molecular hallmarks of human atc, namely, activation of pi3k (via pten deletion) and inactivation of p53. by the age of 9 months, over 75% of the compound mutant mice developed aggressive, undifferentiated thyroid tumors, displaying all the features of their human counterpart, including pleomorphism, epithelial - mesenchymal transition, aneuploidy, local invasion, and distant metastases. it was shown that the tumors developing in this animal model undergo the glycolytic shift known as warburg effect and are highly sensitive to the therapeutic use of glycolytic inhibitors, which synergize with standard chemotherapy. later, nehs. elegantly demonstrated the remarkable efficacy of plx4720 compound, which is an atp analog that selectively inhibits b - raf by stabilizing it in an inactive conformation, to induce significant regression in an orthotopic mouse model of atc even when administered at a very late therapeutic intervention stage. it is well known, in fact, that atc tends to be resistant to traditional approaches such as standard chemotherapy, radiation, and radioiodine (i) due to loss of the sodium iodide symporter through malignant dedifferentiation. if downregulation of braf with anti - braf therapy also causes sodium iodide symporter upregulation, as suggested by in vitro data, it could be expected that patients treated with anti - b - raf therapy may undergo both reduction of tumor size / invasiveness and possible redifferentiation, thus making radioactive iodine administration possible to control an additional metastatic burden [78, 8183 ]. a detailed description of an approach establishing an orthotopic mouse model of atc has been reported by sewell., which mainly emphasized thyroid tumor metastasis and disease related cachexia and respiratory distress. just recently, mcfadden. have genetically engineered a mouse model of braf - mutant atc and demonstrated that combination treatment with mek and braf inhibitors results in enhanced antitumor activity as compared to treatment with a braf inhibitor alone, suggesting that this combination could be useful as a component of treatment regimens also in human. given these results, it must be stressed that the animal model is a tool of unquestionable benefit for the development of appropriate therapeutic approach against complex diseases. orthotopic mouse models seem to be ideal and commonly used for preclinical and translational studies of compounds and therapies, not only because of the fact that they may mimic key aspects of human diseases (e.g., metastasis), but also because of their reproducibility and the possibility to evaluate systemic effects of treatments. thus, even if aggressive thyroid tumors, such as atc or poorly differentiated thyroid carcinoma, carry several complex genetic alterations, likely explaining disease progression and resistance to single - compound approaches, orthotopic models of human thyroid cancer also hold the potential to be good models for testing novel combinatorial therapies. among the biomarkers which have been identified in atc, by using different approaches, and that can be exploitable as potential therapeutic targets, we focus on type i receptor for transferrin (tfr1/cd71). tfr1, also known as cd71, is a type ii cell membrane - associated glycoprotein involved in iron homeostasis and cell growth [8789 ]. although ubiquitously expressed on the cell surface, tfr1/cd71 is commonly upregulated in cells with high proliferative index, including cancer cells that need iron as cofactor of many enzymatic reactions, such as dna synthesis [8790 ]. tfr1/cd71 overexpression has been reported in several human malignant tumors, including lymphomas, carcinomas, neuroendocrine, and brain tumors. among carcinomas, tfr1/cd71 overexpression has been documented in colon, stomach, pancreas, breast, lung, liver, bladder, oral cavity, and uterus. notably a close correlation of tfr1/cd71 expression level and tumor proliferation index, histological grading, stage, and prognosis has also been largely demonstrated [8791 ]. based on our previous studies which showed an increased transferrin expression by ptc cells in comparison with thyroid cells from benign tissues, we performed pcr, western blotting, and immunohistochemical studies on fresh, paraffin - embedded thyroid tissues, as well as in thyroid cell lines, to assess whether tfr1/cd71, the receptor for transferrin, is upregulated in malignant thyroid tumors. conventional rt - pcr revealed the presence of tfr1/cd71 mrna in all thyroid samples examined, suggesting that this receptor is transcribed in both benign and malignant tissues but differently expressed in malignant versus benign tissues. in fact, western blot analyses showed that although tfr1/cd71 protein was detected in all examined samples, its relative abundance appeared substantially higher in malignant tissues, especially ptc and atc, when compared to their benign counterparts. immunohistochemical results paralleled the findings of western blot, revealing an overall overexpression of the receptor in malignant tissues as compared to benign tissues which, by contrast, did not or only weakly and focally showed low levels of expression. all the above mentioned findings suggest that synthesis and membrane incorporation of tfr1/cd71 occur at low levels in normal thyroid tissues, whereas it becomes part of an aberrant gene / protein expression pattern upon neoplastic transformation and malignant progression. in particular, tfr1/cd71 overexpression was observed in all cases of atcs tested (10 out 10 cases), and similarly to most cases of ptc, a combined strong and diffuse cytoplasmic, as well as, cell membrane immunostaining was observed (figures 1 and 2). similar results were also obtained in atc cell lines (figure 3). our unpublished immunohistochemical data, showing that most neoplastic cells of atc exhibit strong and diffuse cytoplasmic staining for transferrin (figure 4), suggest that the overexpression of this protein concurs with the increased expression of its cognate receptor. thus, it could be speculated that, as already seen in ptc, an autocrine and/or paracrine regulatory loop of transferrin - tfr1/cd71 does exist also in atc. however we admit that this hypothesis needs to be confirmed by mrna analyses to exclude the possibility that immunohistochemically detected transferrin can derive from internalized protein present in blood or outside the cell through interaction with its receptor. interestingly, more than 30 years ago, it was largely known that gallium-67 citrate scintigraphy was helpful in identifying malignant thyroid tumors, especially primary and metastatic atcs [92, 93 ]. at that time, it was hypothesized that there was a close correlation between gallium-67 uptake and degree of malignancy of thyroid tumor cells, even if the mechanism of tumor localization of gallium-67 was still unclear [92, 93 ]. nowadays it is commonly accepted that gallium-67 citrate is preferentially uptaken by high - grade malignant tumors, through its ability to bind, in place of transferrin, tfr1/cd71 [9496 ]. this is supported by the evidence that only malignant tumors that overexpressed tfr1/cd71 at a tissue level were also marked in vivo by gallium [9799 ]. based on our findings, it is likely that the high uptake of gallium-67 citrate by atc cells might be explained by the high expression of tfr1/cd71 in this aggressive neoplasm. because of its upregulation in malignant tissues, extracellular accessibility, and constitutive ability to internalize into cells, tfr1/cd71 is currently attracting wide interest as potential direct or indirect therapeutic target [8789, 91 ]. firstly, tfr1/cd71 can be targeted by direct interaction with conjugates of its ligand transferrin (tf), the iron transporting protein. in this regard, gallium nitrate binds avidly to transferrin to form transferrin - gallium complexes, which in turn bind tfr1/cd71 on the surface of neoplastic cells [94, 100 ] (figure 5). gallium nitrate or alternatively gallium compounds, a group iiia metal salt, have been described to inhibit the proliferation of tumor cells in vitro and in vivo. antitumoural activity by novel organogallium (iii) through induction of apoptosis has also been described in 8505c anaplastic thyroid cancer cell line. gallium nitrate cytotoxicity may be due to its capability to interfere with the release of iron from endocytic vesicles, depending on not only tfr1/cd71 receptor density on cellular surface but also tfr1/cd71 cycling. in addition, cytotoxicity of gallium may be due, at least in part, to the inhibition of iron uptake. gallium nitrate binds transferrin and thus, iron can not bind and is not taken up by the cell [103, 104 ]. thus, the transferrin - gallium - tfr1/cd71 molecular complex may represent a promising therapeutic approach against atc. tfr1/cd71 can be also specifically targeted by monoclonal or recombinant antibodies (figure 5). in this regard, there are two different types of antibodies : (i) directly cytotoxic antibodies and (ii) therapeutic agents delivery antibodies. the former, binding tfr1, inhibit the function of the receptor by inducing its sequestration and subsequently degradation in sensitive cells. it has been shown that tfr1 level reduction on the cell surface results in decreased transferrin uptake and induction of lethal iron deprivation (lid) in hematopoietic malignancies [105, 106 ]. other monoclonal or recombinant antibodies have been developed to target tfr1/cd71 for delivering chemotherapeutic drugs, protein toxins, radionuclides, liposomes, modified viral particles, and nanoparticles to kill malignant cells [8789, 107, 108 ] (figure 5). the combinations of such antibodies against tfr on human tumor cells have been demonstrated to have antiproliferative effects both in vitro and in vivo [106, 109114 ]. lastly, tfr1/cd71 may be exploitable as specific target for small interfering rna (sirna) approach (figure 5). this technology represents a powerful genetic tool for sequence - specific inhibition of target proteins capable of modulating cell growth, apoptosis, chemoresistance, and chemosensitivity. in this regard the use of transferrin should ensure specific targeting of sirna - containing complexes to atc cells in situ and the consequent uptake by tfr1/cd7-mediated endocytosis of the delivered particles. recently a sirna clinical trial has successfully targeted nanoparticles containing transferrin, which engage tfr on the surface of cutaneous melanoma cells. however, the targeting specificity reported in this study has been questioned by other authors who failed to demonstrate the overexpression of tfr1/cd71 in a large series of cutaneous melanomas. accordingly it was contemplated the possibility that neoplastic cells might internalize nanoparticles conjugated with transferrin through a mechanism independent of the activity of the cognate receptor. in conclusion, although these potential tfr1/cd71-based therapeutic strategies appear to be intriguing, the question of whether this receptor will remain accessible in vivo in atc is still to be elucidated. for these reasons we advise that future in vitro and preclinical studies will be performed to confirm the idea of using tfr1/cd71 as a meaningful molecule for target therapy against atc, which continues to be one the most aggressive human tumors. | anaplastic thyroid carcinoma (atc) is one of the most aggressive human cancers. actually, atc is refractory to conventional therapies, including surgery, chemotherapy, radiotherapy, and radioiodine (131i) therapy. accordingly, genetic and molecular characterizations of atc have been frequently and periodically reviewed in order to identify potential biological markers exploitable for target therapy. this review briefly focuses on main molecular events that characterize atc and provides an update about preclinical studies. in addition, the overexpression of transferrin receptor 1 (tfr1/cd71) by neoplastic cells of atc is emphasized in that it could represent a potential therapeutic target. in this regard, new therapeutic approaches based on the use of monoclonal or recombinant antibodies, or transferrin - gallium - tfr1/cd71 molecular complexes, or lastly small interfering rnas (sirnas) are proposed. |
it is characterized by a thickening at the bifurcation of the plantar digital nerve, typically developing in the third intermetatarsal space and, less frequently, in the second intermetatarsal space. histology usually reveals endoneural and perineural fibrosis at the level of the heads of the metatarsals, and it is therefore considered to be an entrapment neuropathy in which the intermetatarsal ligament could play a key role. morton neuroma has been associated with various overload mechanisms and particularly with the use of inadequate footwear. morton neuroma is eight to ten times more common in women than in men ; this may be related to the use of shoes with high heels or with a constricting toe box [5, 6 ]. morton neuroma is named after thomas g. morton who described it in 1876, though earlier descriptions exist. the symptoms are characterized by plantar pain of neuropathic characteristics in the forefoot, typically between the third and fourth toes. the pain is more intense during standing and walking and is exacerbated by the use of footwear with a high heel or narrow toe. it is described as a stabbing pain that radiates to the medial and lateral borders of the affected toes in the territory supplied by the plantar digital nerves. on physical examination there is usually a tender point on the dorsal aspect of the corresponding intermetatarsal space. there may be altered sensation, with the hyperaesthesia characteristic of neuropathic pain, which can be provoked by testing light touch sensation over the medial and lateral surfaces of the affected toes. clinical diagnosis can be confirmed by sensory nerve conduction studies of the interdigital nerve, ultrasound or magnetic resonance imaging (mri) (fig. 1). an mri increases the range of detectable differential diagnoses, which include intermetatarsal bursitis, arthritis, synovitis, osteomyelitis, foreign body granuloma, stress fracture, freiberg disease and metatarsophalangeal subluxation.fig. 1magnetic resonance image of a morton neuroma (arrows) magnetic resonance image of a morton neuroma (arrows) treatment of morton neuroma can be conservative or surgical. in addition, corticosteroid injection and nerve blocks with local anaesthetic or alcohol are recommended. the principal surgical option indicated for cases refractory to conservative treatment systematic reviews suggest that insufficient studies have been performed to demonstrate the efficacy of the different treatments employed or whether any one treatment is superior to the others. botulinum toxin a is a peptide formed by a light and a heavy chain linked by a disulphide bridge. it acts as a protease at the presynaptic nerve terminal, blocking acetylcholine release at the motor endplate. because of this it has been used for decades in the treatment of disorders characterized by muscle hyperactivity, such as spasticity or dystonia. its analgesic potential was also observed when, in addition to decreasing the hyperactivity, it was found to improve the pain in patients with dystonia. this initial finding was later confirmed when its analgesic therapeutic effect was observed in other conditions such as epicondylitis, low back pain, piriformis syndrome, migraine and plantar fasciitis. in recent years, this analgesic effect has also been investigated in the field of neuropathic pain [1827 ]. the analgesic effect of the toxin may be related to inhibition of neuropeptide release in the nociceptive terminals. the objective of the present study was to investigate the effect of treatment with onabotulinumtoxina on the neuropathic pain of morton neuroma. we present an open pilot study performed on patients with morton neuroma who attended the rehabilitation and orthopaedics outpatients clinic of alicante university general hospital, alicante, spain, and in whom the symptoms had not improved after treatment involving footwear modification and orthoses. the following inclusion criteria were applied : diagnosis of morton neuroma, pain of more than 3 months duration and with a pain intensity on walking 5 on a visual analogue scale (vas). all patients received detailed information about the nature of the study, its objective and the therapeutic procedures involved. they all agreed voluntarily to participate in the study and signed an informed consent form. the following clinical variables were studied : body mass index, type of footwear (narrow - toed or broad - toed), occupational activity requiring standing, number of hours walking per week (more or less than 3 h per week), previous treatment with corticosteroid injections, type of footprint, foot morphology, web space affected and nocturnal pain. the primary outcome measure was pain intensity on walking, evaluated using a vas with a score range from 0 to 10 points. secondary variables were studied using the foot health status questionnaire, for which there is a validated version in spanish. this questionnaire offers four domains related to foot health : foot pain, foot function, general foot health and footwear. optimum foot health is considered to be a score of 100 points and lower values indicate the percentage deterioration in the dimension being evaluated. all patients received a single injection of onabotulinumtoxina (botox, allergan, irvine, ca, usa) in the area of the neuroma. the site of injection was selected by anatomical skin marking based on the findings of the mri scan and concordance with the site of pain. all patients received a dose of 50 units of onabotulinumtoxina dissolved in 0.5 ml of normal saline. the statistical study was performed using the spss software package version 15.0 (spss inc., the values of the dependent variables before treatment and at 1 and 3 months after treatment were compared using repeated measures anova. the possible influence of the clinical variables was studied by calculating the percentage of patients in whom the pain had improved at 3 months after treatment in each subgroup defined by the clinical variables ; the percentages were then compared using contingency tables and the chi - square test to determine the influence of each clinical variable on outcome. seventeen patients [ten women (59 %) and seven men (41 %) ] with a mean (standard deviation) age of 59.29 (2.65) years were included in the study. analysis of the results of the overall group (table 2) showed a significant improvement in pain on walking measured using the vas (fig. 2) and in the foot pain and foot function (fig. 3) dimensions of the foot health status questionnaire ; the improvements in the other dimensions evaluated by the questionnaire (general foot health and footwear) did not reach statistical significance.table 1characteristics of the sample (n = 17 patients)characteristicn (%) mean (sd)age (y)58.19 (2.56)sex female7 (41.2) male10 (58.8)body mass index (kg / m) 255 (29.4) > 2512 (70.6)site of the neuroma second intermetatarsal space6 (35.3) third intermetatarsal space11 (64.7)standing activity prolonged9 (52.95) normal8 (47.1)footwear narrow toes4 (23.5) broad toes13 (76.5)walking activity (h / week) 312 (70.6) 254 (80)1 (20)site of the neuroma second intermetatarsal space4 (66.7)2 (33.3)0.7 third intermetatarsal space8 (72.7)3 (27.3)standing activity prolonged6 (66.7)3 (33.3)0.5 not prolonged6 (75)2 (25)footwear narrow toes2 (50)2 (50)0.3 broad toes10 (76.9)3 (23.1)walking activity (h / week) 39 (75)3 (25)0.4 <33 (60)2 (40)previous corticosteroid injection yes3 (100)0 (0)0.3 no9 (64.3)5 (35.7)footprint normal5 (83.3)1 (16.7)0.6 pes cavus6 (66.7)3 (33.3) pes planus1 (50)1 (50)foot type greek3 (50)3 (50)0.2 egyptian9 (81.8)2 (18.2)nocturnal pain yes5 (71.4)2 (28.3)0.6 no7 (70)3 (30)overall sample12 (70.6)5 (29.4) distribution of the patients who had improved at 3 months, according to different subgroups the size of the neuroma also had no effect on the percentage of patients who presented an improvement in the pain score on the vas 3 months after treatment. the largest and smallest diameters of the neuromas showed correlations of 0.16 and 0.19, respectively, with the outcome measure. nor was treatment outcome related to patient age (correlation, r = 0.15). none of the patients reported any local, distant or generalized adverse effects after the injection. this is the first study of the effect of onabotulinumtoxina on the symptoms of morton neuroma. the data obtained show an improvement in pain at rest and on walking and an improvement in foot function ; these improvements were detectable at 1 month after treatment and persisted at significant levels until the end of the study (3 months) in the sample analysed. local corticosteroid injection is usually indicated when the first - line treatment of morton neuroma by footwear modification and the use of orthoses does not achieve adequate control of symptoms. favourable results have been reported in 76 % of cases 1 week after corticosteroid injection. these good immediate results have not always been confirmed after a longer follow - up of months or years the percentage of patients with continued improvement varied between 30 and 66 %, depending on the series [2, 31, 32 ]. one classic series reported improvement in only 40 % of patients despite repeating the injection on up to four occasions. the injection of an alcohol solution into the morton neuroma has also been proposed ; published series have reported improvements in 6984 % of patients [11, 3335 ]. in some studies, it should be noted that this procedure can lead to an increase in symptoms in the initial weeks in up to 16 % of patients. a number of studies have been published in which onabotulinumtoxina has been shown to have analgesic activity in conditions associated with neuropathic pain, such as postherpetic neuralgia, carpal tunnel syndrome, occipital neuralgia, diabetic polyneuropathy, trigeminal neuralgia and phantom limb syndrome. morton neuroma, a well - known cause of neuropathic pain, is a localized lesion that can be observed on imaging studies, and it therefore represents a good model to study the possible beneficial effect of the local injection of different drugs. in this open pilot study, onabotulinumtoxina improved the foot pain and foot function scores both at 1 month and at 3 months ; this would suggest a potential beneficial therapeutic effect on the symptoms of morton neuroma. the mechanism of action of onabotulinumtoxina in neuropathic pain must be considered in the context of its activity as a protease of the cytoplasmic transport proteins. this activity could lead to a reduction in the concentration of some of the nociceptive neurotransmitters such as substance p, glutamate and calcitonin gene - related peptide. it is also possible that it could inhibit the transport proteins of the membrane receptors. this blockade of neurotransmitter release could alter sensitization phenomena, interrupting the cascade of local events that precipitates the situation that perpetuates the pain. this modulation of sensitization may also be implicated when botulinum toxin is used in the treatment of myofascial pain syndrome [3944 ]. the results of this study show that 29.4 % of the sample showed no benefit whatsoever from the treatment. this result contrasts with the marked and sustained improvement in 70.6 % of the patients. an analysis of the clinical variables studied did not identify a specific profile of the patients who did not respond, as it was not possible to find any variable that was predictive of the success or failure of treatment. nor was there a correlation between the size of the neuroma and the outcome. as the injections were directed by anatomical skin marking, it is possible that the therapeutic target may not have been adequately reached in all patients. for this reason, further studies of this treatment should be performed with ultrasound guidance in order to reduce the risk of treatment failure due to an incorrect site of injection of the drug. another aspect that must be considered is the suitability of a dose of 50 u. this dose is the same as has been used at each site of blockade in other series on the treatment of neuralgia. the percentage of therapeutic success would suggest that this dose could be appropriate, although it is not currently known whether any variation in the dose might improve the results. another issue concerns the onset, peak and disappearance of the analgesic effect of onabotulinum toxin. in movement disorders, the clinical onset of action of botulinum toxin typically occurs between 12 and 72 h after injection ; the effect peaks between 1 and 3 weeks and this is followed by a plateau phase that for lasts for 12 months. patients therefore often require re - injection approximately every 3 months. however, our patients presented better results at 3 months than at 1 month. this long - term improvement is not uncommon in pain treatment and improvements have even been reported at up to 6 months. this has been observed in patients with neurogenic bladder, as those who received treatment with abobotulinumtoxin injection presented less bladder wall fibrosis than those who received other treatments. in addition to pain reduction, botulinum toxin treatment may also produce a decrease in the size of neuroma. we plan to study changes in morton neuroma size after botulinum toxin injection in the future. the treatment was well tolerated and an exacerbation of symptoms was not observed in any case. a further relevant consideration is that these results were achieved after a single injection, whereas other injectable treatments have required repetition of the procedure on up to four occasions. the fundamental limitation of this study is the absence of a control group and the data presented can not therefore be extrapolated to other populations. this was an initial clinical investigation in the form of an open pilot study, the main aim of which was to open a new line of investigation for the treatment of neuropathic pain in morton neuroma. the small sample size and the lack of objective outcome measures can be rectified in further studies. the injection of onabotulinumtoxina in morton neuroma has been shown to be useful to relieve pain and improve function in a 3-month open pilot study. this finding opens the door to further clinical research to determine the optimal dose, possible improvements in the outcome through the use of ultrasound - guided injection, and comparison of the results of this new procedure with other procedures commonly used in morton neuroma. it would also be interesting to determine the possible effects of treatment on the size and characteristics of the neuroma. to establish the role of onabotulinumtoxina in morton neuroma, it will be necessary to perform clinical trials that compare this new therapeutic option with the established management options in order to determine its superiority or inferiority, its profile of adverse effects and its relative cost. such studies would enable us to determine whether onabotulinumtoxina offers any advantage over the other known injectable treatments. allergan was not informed of the undertaking of the study until after its completion and after the analysis of results. the company did not exert any control over the preparation of the manuscript or the presentation of the results. | background and objectivemorton neuroma is a common cause of metatarsalgia of neuropathic origin. systematic reviews suggest that insufficient studies have been performed on the efficacy of the different treatments available. onabotulinumtoxina has shown a degree of usefulness in other conditions associated with neuropathic pain. the aim of this study was to investigate the therapeutic potential of onabotulinumtoxina in morton neuroma.patients and methodswe present an open - label, pilot study with 17 consecutive patients with morton neuroma and pain of more than 3 months duration that had not responded to conservative treatment with physical measures or corticosteroid injection. patients received one onabotulinumtoxina injection in the area of the neuroma. the main outcome measure was the variation in the pain on walking evaluated using a visual analogue scale (vas) before treatment and at 1 and 3 months after treatment. the secondary outcome was the change in foot function, which was assessed using the foot health status questionnaire.resultsin the overall group, the mean initial vas score on walking was 7. this mean score had fallen to 4.8 at 1 month after treatment and to 3.7 at 3 months. twelve patients (70.6 %) reported an improvement in their pain and five patients (29.4 %) reported no change ; exacerbation of the pain did not occur in any patient. improvements were also observed in two of the dimensions of the foot health status questionnaire : foot pain, which improved from a mean of 38.88 before treatment to 57 at 3 months, and foot function, which improved from a mean of 42.27 before treatment to 59.9 at 3 months. clinical variables including age, sex, site and size of the lesion, standing activity, weekly duration of walking, footwear, foot type and footprint had no influence on the outcome. no adverse effects were reported.conclusionsin this pilot study, injection with onabotulinumtoxina was shown to be of possible usefulness to relieve the pain and improve function in morton neuroma. this finding opens the door to further clinical research. |
herpes simplex virus encephalitis (hse) is an acute infection accompanied by significant morbidity and mortality with the diagnosis often made by cerebrospinal fluid (csf) polymerase chain reaction (pcr) testing. we report a case of a healthy 35-year - old woman presenting with altered mental status. due to suspicion of herpes encephalitis, a csf pcr for herpes virus was sent for examination and acyclovir was started. the patient had an immediate response to acyclovir ; however, when the pcr returned negative she was discharged without therapy. the altered mental status returned and she was started on acyclovir therapy and a second csf pcr sample was sent and was again negative. mri performed at initial hospitalization was negative, but a repeat mri demonstrated bilateral temporal lobe involvement suggestive of herpes encephalitis. csf pcr for herpes virus is highly sensitive and specific and remains the standard for diagnosing herpes encephalitis. clinicians should be aware of the pitfalls of csf pcr testing, specifically false - negative results. herpes simplex virus encephalitis (hse) is an acute infection accompanied by significant morbidity and mortality. clinically, hse has a prodrome of fever, headache, seizure, focal neurologic signs, and altered consciousness ; however, mild or atypical presentations have been reported [3, 4, 5, 6, 7, 8, 9 ]. the diagnosis of hse is generally clinical with confirmatory tests including lumbar puncture and cerebrospinal fluid (csf) polymerase chain reaction (pcr) testing. auxiliary tests to aid diagnosis include electroencephalogram (eeg), computed tomography (ct), and especially magnetic resonance imaging (mri). we present a patient initially suspected to have hse although with a negative mri and csf examination including pcr for herpes simplex virus (hsv). the patient was started on acyclovir with an initial response but was discharged after the pcr was negative. the patient returned with worsening symptoms, this time with mri and eeg both suggestive of hse ; however, repeat csf pcr for hsv was again negative. the high sensitivity and specificity of pcr for hsv leads physicians to rely on this test for making or excluding the diagnosis of hse, although false - negative results may occur. cases with a high pre - test probability for hse and suggestive mri and/or eeg examinations, although with two false - negative pcr tests, are seldom encountered or reported in the literature. a 35-year - old hispanic female without a significant past medical history arrived at the emergency department complaining of headache, dizziness, nausea, and vomiting for 2 days prior to admission. the headache was localized to the frontal region and accompanied by photophobia and phonophobia and six episodes of vomitus. immediately prior to admission, the patient had had a witnessed generalized seizure of 30 - 45 s duration without bowel or bladder incontinence and developed postictal confusion without postictal lateralized neurological deficits. at the time of admission, she was disoriented to person, place, and time with a gcs of 9. lumbar puncture revealed clear fluid with glucose 69 mg / dl, protein 31 mg / dl, 2 wbc, 3 rbc, no monocytes or lymphocytes, and without xanthochromia. urine analysis, toxicology, culture of blood, csf, and urine were all negative. acyclovir (10 mg / kg i.v., 3 times per day) was started as empiric treatment in addition to levetiracetam for seizure prevention. eeg showed generalized slowing suggestive of encephalopathy although it was extremely limited due to patient movement and inability to cooperate. twenty - four hours after initiation of acyclovir the patient was lethargic but not confused. forty - eight hours following admission, the patient 's mental status was reported to be at baseline, gcs 15, and she was eager to return home. acyclovir was switched to oral preparation and after one dose, the csf hsv - pcr returned negative, and the patient was discharged without further need to continue antiviral medication. within 24 h, she returned to the emergency department complaining of severe headaches, vomiting, and an episode of unresponsiveness, slurred speech, and blank starring as reported by family members. at the time of admission, she had intermittent episodes of confusion ; however, the remainder of the physical examination was normal. acyclovir (10 mg / kg i.v., 3 times per day) was restarted. repeat lumbar puncture was performed with glucose 79 mg / dl, protein 24 mg / dl, 50 wbc, 17 rbc, and negative for xanthochromia, cryptococcal antigen, vdrl, rpr, bacterial antigens, lyme, west nile antigen, and cmv antigen. after careful consideration of the differential diagnosis of our patient, attempts were made to exclude limbic encephalitis with nmda receptor antibody test, which was ultimately negative. overnight, the patient became increasingly confused, delirious, and extremely agitated followed by a witnessed tonic - clonic seizure. respiratory distress ensued, and due to the inability to protect her airway, intubation and mechanical ventilation was initiated and a loading dose of valproic acid was started for seizure control. she became febrile to a maximum of 101.3f (38.5c) and developed thick endobronchial secretions identified as gram - positive cocci and gram - negative rods. she remained febrile with a chest x - ray finding suggestive of developing infiltrate at the right lung base. repeat eeg demonstrated absence of normal background activity with generalized slowing and presence of periodic lateralized epileptiform discharges. repeat mri had increased signal intensity in the medial aspect of the temporal lobes, more marked on the left than on the right, and was felt to be compatible with an acute phase of herpes encephalitis (fig. 1, fig. the patient remained encephalopathic for 1 week with minimal improvement during which time her pneumonia resolved. three weeks after the second admission, the patient had returned to baseline and was able to leave the medical center. written informed consent was obtained from the patient for use of medical information and images for publication of this case report. a copy of the written informed consent is available for review by the editor - in - chief of this journal. with the discovery of pcr, diagnosis of central nervous system infections has become more sensitive and specific, particularly with infections caused by the herpes virus. pcr is considered the standard for the diagnosis of hse, although specific csf serologic testing can be employed in cases where suspicion remains high in the setting of a negative pcr test. retrospective diagnosis can be made with the presence of anti - hsv igg in the csf. pcr allows for the detection of minute quantities of viral dna or rna present in fluids and tissues by nucleic acid amplification over a short time period and has nearly replaced brain biopsy for diagnosis of hse in regions where the test can be performed. negative pcr tests are associated with low csf protein and leukocyte counts, and in such cases, clinical suspicion should dictate the treatment course. the sensitivity and specificity of csf pcr for hsv exceeds 95%, allowing exclusion of hse in patients with high pre - test probability should the result return negative. in the study by lakeman and whitley, pcr sensitivity and specificity were reported as 98 and 94%, respectively, with positive and negative predictive values of 95 and 98%, respectively, when compared with brain biopsy. however, in patients with high clinical probability of hse, a negative pcr result, while decreasing the likelihood of hse, does not entirely exclude the possibility of infection. few reports exist identifying hse in patients with negative pcr [13, 14, 15 ]. reported a series of 3 cases of hse confirmed by csf antibody testing in the setting of negative pcr tests. possible explanations for negative pcr results have been suggested and include : pcr sent very early in the disease development (days 1 - 4 with neurological symptoms) and viral load too low for detection, although this is unlikely as the pcr technique by virtue of the amplification process allows for the detection of minute amounts of viral copies, as low as 10 copies of viral inhibitors, and a small - volume csf sample [11, 14, 16 ]. in addition, low csf cell counts, variation in primers used during pcr processing, sample dilution, and location of infection in the central nervous system have all been suggested as the cause for false - negatives results [1, 14, 17 ]. recommend repeating the pcr 4 days after development of symptoms and before deciding the patient does not have hse and prematurely concluding acyclovir treatment. patients with normal diagnostic tests and mild clinical symptoms are more likely to have negative pcr tests. in our case, the patient had a repeat pcr sent 6 days after the development of symptoms, and despite suggestive mri and eeg and worsening clinical symptoms, the result was negative. attempts were made to uncover an alternative diagnosis according to the experiences highlighted by chataway.. radiographically, the most common areas of involvement are the medial temporal lobes, which are best observed with mri. sensitivity of mri is reported to be best 48 h after onset of symptoms and approaches 85%, although it can be falsely negative in very early stages on infection. eeg may serve as an occasional adjunct in the diagnosis of hse, demonstrating periodic lateralized epileptiform discharges as characteristic findings. specificity of eeg remains low in the diagnosis of hse when used as a single diagnostic exam but can be helpful when pcr is negative in patients with high clinical probability. in our case, the mri on initial presentation failed to demonstrate any significant areas of disease, although repeat mri, 6 days following onset of symptoms, revealed florid changes suggestive of hsv infection. treatment course is weight based in adults, roughly 10 mg / kg every 8 h intravenously for 10 - 21 days. suspicion of hse should remain high in all cases of acute encephalitis, especially in the absence of alternative diagnoses, even in cases when pcr examination is negative. in such cases, repeating lumbar puncture should be entertained with repeat hsv - pcr testing and possible serologic testing of the csf. in our case, the patient had clinical symptoms, mri and eeg suggestive of hse, although with two negative pcr tests separated in time. in patients with high clinical suspicion and negative hsv - pcr, repeat lumbar puncture should be considered and empirical acyclovir treatment should be considered. in our case, continuation of acyclovir was warranted and proved efficacious as the patient achieved near - complete resolution. in all such cases, the authors report that there are no conflicts of interest. no monetary compensation or grants | introductionherpes simplex virus encephalitis (hse) is an acute infection accompanied by significant morbidity and mortality with the diagnosis often made by cerebrospinal fluid (csf) polymerase chain reaction (pcr) testing.case presentationwe report a case of a healthy 35-year - old woman presenting with altered mental status. due to suspicion of herpes encephalitis, a csf pcr for herpes virus was sent for examination and acyclovir was started. the patient had an immediate response to acyclovir ; however, when the pcr returned negative she was discharged without therapy. the altered mental status returned and she was started on acyclovir therapy and a second csf pcr sample was sent and was again negative. mri performed at initial hospitalization was negative, but a repeat mri demonstrated bilateral temporal lobe involvement suggestive of herpes encephalitis. the patient was successfully treated for 21 days with acyclovir.conclusioncsf pcr for herpes virus is highly sensitive and specific and remains the standard for diagnosing herpes encephalitis. clinicians should be aware of the pitfalls of csf pcr testing, specifically false - negative results. although rare, these false negatives can result in premature termination of treatment. |
additional supporting information may be found in the online version of this article : video s1. this video runs at three frames per second : 96 sec. of running video time distal podomeres detach from tp of tc, and their presence guides the elongation and migration of tp belonging to other tc, as preliminary stages in the formation of a complex multi - cellular network. for supplementary materials on tc, please see http://www.telocytes.com. please note : wiley - blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. any queries (other than missing material) should be directed to the corresponding author for the article. | abstracttelocytes (tc) are interstitial cells with telopodes (tp). these prolongations (tp) are quite unique : very long (several tens of micrometres) and very thin (0.5 m), with moniliform aspect : thin segments (podomeres) alternating with dilations (podoms). to avoid any confusion, tc were previously named interstitial cajal - like cells (iclc). myocardial tc were repeatedly documented by electron microscopy, immunohistochemistry and immunofluorescence. tc form a network by their tp, either in situ or in vitro. cardiac tc are (completely) different of classic fibroblasts or fibrocytes. we hereby present a synopsis of monitoring, by time - lapse videomicroscopy, of tp network development in cell culture. we used a protocol that favoured interstitial cell selection from adult mouse myocardium. videomicroscopy showed dynamic interactions of neighbour tc during the network formation. during their movement, tc leave behind distal segments (podomeres) of their tp as guiding marks for the neighbouring cells to follow during network rearrangement. |
posterior instability after total shoulder arthroplasty (tsa) is a reported complication and can occur for a variety of reasons. factors related to the development of this condition include glenoid and humeral retroversion, glenoid component loosening, soft tissue imbalance, and rotator cuff tears. in a cohort of tsa 's performed at the mayo clinic, posterior instability was described in 1.8% of cases. because of the low reported incidence after anatomic tsa, the methods of treatment are likewise not well studied. treatment options for posterior instability are dependent on the causes of the instability, and the outcomes for revision surgery demonstrate a moderately high rate of failure. a thorough evaluation is required to determine the potential etiology of the instability before any revision surgical procedure is contemplated. we present guidelines to aid in the diagnosis, cause, severity, and treatment options of posterior instability. in addition, we present a novel treatment method for a case of posterior instability with glenoid retroversion and a well - fixed glenoid component treated with an arthroscopic posterior bone block procedure. a 50-year - old male house painter with longstanding shoulder pain and glenohumeral arthritis refractory to conservative management underwent a tsa with press - fit humeral stem (global unite, depuy, warsaw, in, usa) and uncemented glenoid component (anchor peg glenoid, depuy, warsaw, in, usa) in his nondominant extremity. his preoperative imaging revealed a biconcave glenoid with 20 of glenoid retroversion and posterior subluxation of the humeral head [figure 1a ]. intraoperatively, the anterior glenoid was reamed preferentially in an attempt to correct the posterior retroversion of the biconcave glenoid prior to placement of the glenoid component. postoperative imaging revealed a well - fixed glenoid component with persistent 20 of retroversion [figure 1b ]. intra - operative examination following component implantation revealed a stable glenohumeral joint and no additional corrections or modifications were performed. postoperatively, the patient had a sense of instability coupled with pain and weakness. while he did not experience frank dislocation episodes, the pain and dysfunction prevented him from returning to work. his examination revealed posterior apprehension, but normal strength of both the deltoid and rotator cuff musculature. given the clinical history, imaging and exam findings, the patient was diagnosed with posterior instability as a result of glenoid retroversion. preoperative axillary x - ray postoperative axillary x - ray with posterior subluxation and glenoid component retroversion potential options for treatment in this situation included revision of the glenoid component with bone grafting or placement of a posterior bone block with retention of the glenoid component. given the amount of morbidity involved in a revision surgery, a less invasive and alternative method of treatment was chosen. a 25 mm 10 mm 10 mm tri - cortical bone graft was harvested and prepared. verification of glenoid fixation was confirmed, and a horizontal slit in the posterior rotator cuff muscles was performed. in addition, the bone on the posterior glenoid was prepared to a flat surface with an arthroscopic burr [figure 2a ]. utilizing a custom double cannula instrument (depuy - mitek, raynham, ma, usa), the graft was advanced through the widened posterior portal [figure 2b ]. graft was then precisely placed on the posterior glenoid with the surface of the bone block placed parallel and flush with the surface of the glenoid prosthesis [figure 3a ]. fixation of the bone block was performed with two parallel, cannulated, titanium, 3.5 mm screws. given the existing anchor peg glenoid component (dupuy, raynham, ma, usa) relies heavily on the central peg, care was taken to ensure that both screws lie below the equator of the glenoid prosthesis [figure 3b ]. this was critical as damage to the central peg risks compromising the fixation of the glenoid component. by placing the 10 mm wide graft flush with the size 48 mm glenoid, the anterior to posterior articular surface area pre- and post - arthroscopic preparation of the posterior glenoid bone surface iliac crest bone graft in custom double cannula instrument (depuy - mitek, raynham, ma, usa) for insertion through the posterior portal intra - articular placement of bone block flush with the surface of glenoid component axial computed tomography scan showing orientation of screws relative to intact central peg of glenoid component at 14-month postoperatively from the bone block procedure, the patient regained near full motion and strength. he has no sense of pain or instability with resisted forward flexion with his arm in an adducted and internally rotated position [figure 4a ]. the patient 's subjective shoulder value is 80, with a 2 out of 10 pain score. imaging obtained at 14-month revealed osseous integration of the bone block to the posterior glenoid, no evidence of glenoid prosthesis loosening and an intact central peg [figure 4b ]. postoperative examination without instability postoperative x - ray and computed tomography scan demonstrating osseous integration and intact central peg instability in the setting of tsa is well described in the literature. in a large review, instability was noted in 5.2% of the cases and was the most common complication of the procedure, however, isolated posterior instability is felt to be relatively rare. the type of instability is classified by the direction (superior, inferior, anterior, and posterior), and the etiology is often multifactorial. in cases of posterior instability, causative factors are reported to be excessive glenoid component retroversion, humeral component retroversion, glenoid component loosening, continued static posterior subluxation, a smaller glenoid component, and soft tissue imbalances or insufficiency including rotator cuff tear. preoperative factors include posterior glenoid wear, bone loss, a biconcave glenoid, and static posterior subluxation. a detailed understanding of the underlying etiology is essential to determine the appropriate treatment. at the time of the arthroplasty, methods to prevent postoperative instability include eccentric reaming, posterior bone grafting, using an augmented glenoid component, and addressing posterior capsular laxity or rotator cuff pathology. a study has demonstrated that increasing the humeral component anteversion does not provide improved stability in the setting of persistent glenoid retroversion. patients with postoperative instability of the prosthesis despite the use of the aforementioned methods present a difficult clinical challenge. nonoperative measures may not be successful in alleviating the symptoms. in evaluating a patient with instability, the etiology must be determined through a clinical exam and appropriate imaging. the position of the components, loosening, static posterior subluxation and soft tissue stabilizers are all assessed. based on the underlying cause of the instability, operative options include revision of one or both of the components, soft tissue plication or repair, bone grafting procedures with or without component revision, and bone block type procedures. understanding the reasons for posterior instability is critical to determining the treatment options [figure 5 ]. in many cases, causes and treatment options for posterior instability in the case presented here, the patient was found to have clinically symptomatic posterior instability after a primary tsa with persistent subluxation and glenoid retroversion. an open revision surgery was considered, but secondary to the reported mixed results of these procedures a novel, less invasive surgery was undertaken. this included an arthroscopic posterior bone block augmentation of the glenoid with iliac crest autograft. while this procedure has recently been published for posterior instability, there are no reports to our knowledge of this technique in the setting of a tsa. endres and warner described two patients with anterior instability after tsa successfully treated with an open latarjet procedure. it can be performed through a minimally invasive approach, and excellent visualization is achieved to allow proper positioning of the graft. the graft helps to re - center the humeral head by increasing the surface area of the glenoid, thus making a dislocation more difficult. in addition, the graft is placed flush against the glenoid component, which may help stabilize this and prevent loosening from edge loading. this case report describes the management of symptomatic posterior shoulder instability following tsa and also reports a novel technique. to our knowledge, this is the first description of a posterior, arthroscopic iliac crest bone block grafting procedure in this setting. the procedure represents a treatment option for posterior instability in the setting of a stable glenoid prosthesis following tsa. | we report a case of posterior shoulder instability following anatomic total shoulder arthroplasty (tsa). in addition, we present guidelines to aid in the management of posterior instability after tsa. a 50-year - old male underwent anatomic tsa for glenohumeral osteoarthritis. postoperatively, the patient developed posterior instability secondary to glenoid retroversion. he did not improve despite conservative treatment. he underwent an arthroscopic posterior bone block procedure, 4-month after his index arthroplasty. at 14-month follow - up, the patient had regained near full motion and strength, and radiographs demonstrated osseous integration with no evidence of component loosening. posterior instability following tsa is a relatively rare complication and challenging to manage. the posterior, arthroscopic iliac crest bone block grafting procedure represents a treatment option for posterior instability in the setting of a stable glenoid prosthesis following tsa. |
supercapacitors (strictly, electric double - layer capacitors) store charge at the interface between porous carbon electrodes and an electrolyte solution (figure 1). in contrast to batteries, charge storage in supercapacitors is non - faradaic and occurs by the physical adsorption and desorption of ions inside the pores of the carbon electrodes when an external voltage is applied. as electronic charge accumulates in an electrode, it is balanced at the interface by an equal and opposite ionic charge in the electrolyte. this physical mechanism of charge storage gives rise to fast charge and discharge times and long cycle lives, characteristic properties that make supercapacitors attractive devices to complement batteries (which can store and deliver more energy but with slower charge and discharge times). today, supercapacitors are used in a range of industrial, automotive, and electric utility applications including electric buses, trains, uninterruptible power supply systems, elevators, camera flashes, cranes, and engine starters. their more widespread use could be facilitated by the development of new devices with improved energy densities, which retain the high power densities and long cycle lives that are characteristic of supercapacitors. porous carbon materials with disordered structures are used as the electrodes, and the cell is soaked with an electrolyte that may be organic, aqueous or ionic liquid - based, with some typical electrolytes shown. note, for simplicity the separator (which prevents short circuit), the binder that holds the electrode materials together and the current collectors are not shown. schematic porous carbon structure adapted from ref (17) with permission from springer. porous carbon electrode materials are generally prepared by the heat treatment and subsequent chemical activation of organic materials, such as coconut shells and wood, while a related class of materials, carbide - derived carbons (cdcs), are obtained from metal carbides by extracting the metal atoms. more exotic materials such as carbon nanotubes (cnts) and graphenes are also being developed for supercapacitor application, but here we will focus our attention on disordered porous carbons (activated carbons) as they are well - studied and widely used in commercial devices due to their cheap price, facile synthesis, and sustainability. for the electrolyte, the most widely used systems are comprised of salts dissolved in organic solvents (e.g., tetraethylammonium tetrafluoroborate in acetonitrile solvent, net4bf4/acn). such organic electrolytes offer a good balance of relatively large maximum operating voltages (2.5 v) and high ionic conductivities (2060 mscm). the stored energy, e, is given by1where c is the cell capacitance, and v is the operating voltage. thus, organic electrolytes are typically preferred to aqueous electrolytes that are limited to 1 v before water decomposes. aqueous - based systems are, however, being studied for applications where cost is a critical parameter (e.g., on the electricity grid). room - temperature ionic liquids are also emerging as alternative electrolytes for supercapacitors, with operating voltages as high as 4 v achievable. however, the increase in energy by eq 1 comes at a cost, as slower ionic transport (ionic conductivities are typically below 20 mscm) results in poorer device power performances. most efforts to increase the energy density of supercapacitors have focused on the development of new carbon materials with increased capacitances, so that more energy can be stored, as shown by eq 1. however, the success of such an approach requires an understanding of the carbon structure, and how this in turn affects the charge storage mechanism and the capacitance. this is a complex problem, as porous carbon materials lack long - range order, making their characterization challenging. these materials do, however, exhibit order on a local scale (at length scales up to 10 or 20). while analysis of the broad bragg peaks in diffraction experiments offers little information, pair distribution function (pdf) analysis and nuclear magnetic resonance (nmr) experiments show that typical activated carbons and cdcs consist of predominantly sp - hybridized carbon atoms organized in a hexagonal arrangement. recent studies have suggested that non - hexagonal rings, such as 5-membered and 7-membered rings, are also present, giving rise to curvature in the carbon sheets (see schematic structure in figure 1). the curved and defective carbon sheets and fragments do not pack together well (structures are typically non - graphitizing, with graphite not formed even on heating to temperatures as high as 3000 c), and nanometer - sized pores exist between the various carbon surfaces (see figure 1). this porosity is typically characterized by pore size distributions obtained from the analysis of gas sorption isotherms. state of the art methods use classical density functional theory (dft) to obtain the pore size distribution. the recently developed nldft (non - local dft) and qsdft (quenched solid dft) methods both rely on the assumption that the porous material is a collection of pores with identical shapes (typically slit - shaped pores), and with different pore widths. classical dft is used to determine gas adsorption profiles inside the pores, and a set of partial isotherms corresponding to different pore sizes are generated. the pore size distribution is then obtained by fitting the experimental isotherm to a sum of partial isotherms. owing to the relative ease of this technique, pore size distributions are the most frequently used metric in the characterization of carbon structure, and have been used as the primary tool in the search for relationships between carbon structure and capacitance. seminal studies in 2006 showed that the carbon capacitance could be increased by optimizing the pore size of the carbon electrodes. an anomalous increase in capacitance was observed as the carbon pore size was decreased below 1 nm. these results showed the importance of pore size, and challenged the previous view that pores smaller than the solvated electrolyte ions do not contribute significantly to the capacitance. it was hypothesized that in small pores ion desolvation allows a closer approach of charge centers at the electrode electrolyte interface, which increases the capacitance. however, the capacitance increase was also observed in experimental, and theoretical, studies of ionic liquids, with the capacitance maximized when the pore size matched the ion size. it was even shown that the capacitance varies in an oscillatory fashion as the (slit-)pore width is varied. these findings indicate that factors beyond simple ion desolvation arguments are responsible for the anomalous capacitance increase. interestingly, some experimental studies have reported that there is no correlation between pore size and capacitance, with the origin of these differences currently unclear. needless to say, the observation of pore size effects led to a wave of interest in the mechanisms of charge storage in porous carbon electrodes. in the past 10 years, pioneering experimental and theoretical studies have led to an improved understanding of the molecular mechanisms of charge storage in supercapacitors. this perspective seeks to bring together recent findings from a range of studies to provide a coherent and detailed view of modern theories of supercapacitor charging mechanisms. recent studies have shown that charging is not simply driven by adsorption of counter - ions into the electrode pores, as was previously believed (note, counter - ions are defined as having opposite charge to the electrode). nmr experiments and molecular dynamics (md) simulations show that porous carbon electrodes typically contain a large number of ions in the absence of an applied potential, such that a range of different charging mechanisms, based on different amounts of ion adsorption and desorption, are then possible when a voltage is applied. new in situ experimental techniques (nmr spectroscopy, electrochemical quartz crystal microbalance (eqcm), infrared (ir) spectroscopy, and scattering approaches) have shown that the charging mechanism is sensitive to the electrode and electrolyte materials used, as well as the polarization of the electrode. understanding and controlling the charge storage mechanism of supercapacitors may hold the key to developing next generation devices with enhanced properties, and we identify new areas of research that can facilitate this process. before embarking on the study of supercapacitor charging mechanisms, it is crucial to have a detailed understanding of the structure of the electrode electrolyte interface in the absence of an applied potential (i.e., at 0 v). as illustrated by figure 2a, the carbon nanopores may be either filled with electrolyte ions, or they may contain no ions. these two possibilities have been described as ionophilic and ionophobic together, md simulations and nmr spectroscopy experiments have shown that the pores of the carbon electrodes are generally filled at 0 v. coarse - grained md simulations of the ionic liquid butylmethylimidazolium hexafluorophosphate (bmi pf6) showed a large number of ions inside the carbon nanopores of realistic carbon (cdc) structures at zero applied potential (table 1). with the md approach, subtly different local adsorption environments could be identified for confined ions, sitting at sheet edges, on top of sheet planes, inside curved hollows, and inside small pockets. pf6 dissolved in acn to 1.5 m (i.e., an organic electrolyte) has also been studied under nanoconfinement, with a considerable number of in - pore ions again observed (figure 2b). in this case the solvent molecules replace some of the in - pore ions, leading to a lower in - pore population relative to the neat ionic liquid study, with the small solvent molecules tending to occupy the most confined pore sites. other theoretical studies based on idealized electrode geometries, such as cnts and slit pores, have also shown spontaneous pore - filling behavior. (a) schematic illustrating the concept of filled and empty carbon pores at 0 v. the carbon (slit-)pore walls are represented by black rectangles. (b) snapshot of an md simulation showing the presence of in - pore ions and solvent molecules at 0 v. red, blue, and green correspond to cations, anions, and solvent molecules, respectively, while the carbon surfaces are gray. (c) nmr (9.4 t) measurements of yp50f activated carbon soaked with a typical supercapacitor electrolyte, net4bf4/dacn (1.5 m), recorded with magic angle spinning at a frequency of 5 khz. note, deuterated acetonitrile (dacn) was used here to allow a convenient separation of the signals from the cations and the solvent molecules. md simulations and nmr experiments both reveal a significant number of in - pore ions in the absence of an applied potential. in the nmr studies the commercially activated carbon yp50f was studied in all cases, except for in ref (55), where activated carbons derived from poly(ether - ether - ketone) (peek ac) were studied. in most of the md simulations listed, idealized carbon geometries such as slit pores and multi - walled carbon nanotubes (mwcnts) were studied, with the exception of the studies of model cdc electrodes in refs (39) and (40). for the calculation of the gravimetric in - pore population from ref (41), only the inner wall of the mwcnt note : pyr13tfsi is 1-methyl-1-propylpyrrolidinium bis(trifluoromethanesulfonyl)imide, and emi tfsi is 1-ethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide. experimental results from nmr spectroscopy are in excellent accord with the findings from md simulations, also revealing the predominance of filled pores in the absence of an applied potential (see table 1). in nmr experiments, ions and solvent molecules inside carbon pores (give rise to peaks which are distinct to those from species in bulk electrolyte between the carbon particles (ex - pore). by studying different nmr nuclei, the anions, cations, and solvent molecules can be studied separately. for example, for yp50f (a commercial activated carbon) soaked with the electrolyte net4bf4 in acn (1.5 m), clear resonances can be distinguished for in - pore anions (f nmr), cations (h nmr), and solvent (h nmr) (figure 2c). in each case the in - pore peak appears at a negative chemical shift (relative to the ex - pore) due to the local magnetic field originating from the circulation of the carbon s delocalized electrons in the applied magnetic field. these effects are largely independent of the choice of nmr nucleus, such that anions, cations, and solvent molecules all experience a similar change of chemical shift upon adsorption in a given carbon. since nmr is quantitative, the number of ions inside the carbon pores can be readily determined by fitting and integration of the spectra. nmr studies have been carried out on carbons soaked with a range of organic electrolytes. for pet4bf4 in acn (1.5 m) and the carbon yp50f, there was 0.86 mmol of in - pore anions and cations per gram of carbon, with this number decreasing at lower electrolyte concentrations (table 1). nmr studies of ionic liquids and yp50f carbon have revealed larger numbers of in - pore ions at 0 v (table 1). when the ionic liquid samples were diluted with acn (to 1.8 m), the in - pore ion populations dropped as solvent molecules displaced some of the ions (table 1). this is consistent with md simulations on model cdc electrodes with and without a solvent present (table 1). the nmr approach has also recently been applied to study the confinement of aqueous electrolytes, highlighting the versatility of the method. it was shown that for a carbon with a relatively small average pore width (0.58 nm) soaked with naf (0.8 mol kg aq), the ions were unable to enter the carbon pores in the absence of an applied potential, despite the presence of in - pore water. measurements on a carbon with larger pores (average width 1.55 nm) showed there were a considerable number of in - pore ions, confirming that the absence of in - pore ions in the former carbon arose from steric factors. in the absence of any steric effects, it is currently unknown if it is possible to synthesize carbons with ionophobic pores (see later). electrolyte interface in aqueous systems is highly complex, as the chemical nature of the carbon surface will depend on the (de)protonation of the various functional groups, and oh and h3o ions are present alongside the main electrolyte ions. beyond nmr spectroscopy and md simulations, small - angle x - ray scattering (saxs) and small - angle neutron scattering (sans) have also emerged as experimental methods to probe the wetting of carbon pores in the absence of an applied potential. changes of the scattered neutron intensity after the addition of acn to activated carbon fabric suggested that the solvent had wetted the carbon nanopores. interestingly, this approach allows wetting to be studied as a function of pore size, and it was shown that the pore wetting of activated carbon fabrics was incomplete for the smallest nanopores. in contrast to nmr and md simulations, however, these methods have not an absolute quantification of the numbers of in - pore anions, cations, and solvent molecules. given the large number of ions inside the carbon pores at 0 v, a range of different charging mechanisms are possible (figure 3). a second possibility is that counter - ion adsorption is accompanied by simultaneous co - ion desorption from the pores, which we refer to as ion exchange (where co - ions are defined as having charge with the same sign as the electrode). a third possibility is that charging is driven purely by the desorption of co - ions (figure 3). in each case the excess ionic charge inside the carbon pores is equal and opposite to the electronic charge stored in the carbon. in practice, charging may involve a combination of the different mechanisms shown in figure 3. for example, ion - exchange and counter - ion adsorption could occur simultaneously. different possible charging mechanisms for carbon pores that are initially filled with electrolyte : counter - ion adsorption, ion exchange, and co - ion desorption. the different charging mechanisms may be described by the charging mechanism parameter, x, as defined in eq 2. example calculations of x (using eq 2) are shown for the three depicted charging mechanisms, with v0 taken as 0 v. a value of x = + 1 is obtained for charging solely by counter - ion adsorption, while x = 0 is obtained for ion exchange, and x = 1 for co - ion desorption. as indicated by the scale on the right, x is a continuous variable (e.g., an x value intermediate between 1 and 0 would indicate that both io - exchange and counter - ion adsorption processes occur during charging). part of this figure is adapted from ref (49) with permission from the royal society of chemistry. here we introduce the charging mechanism parameter, x(v, v0), defined as2where n(v) is the total number of in - pore ions at a charging voltage v, n(v0) is the total number of in - pore ions at some initial voltage v0 (typically 0 v), and ncounter(v) and nco(v) are the number of in - pore counter - ions and co - ions, respectively, at a voltage v. qionic(v) and qionic(v0) are then the net in - pore ionic charges at the two voltages, and e is the elementary charge. put simply, x gives a measure of the roles of counter - ion adsorption, ion exchange, and co - ion desorption in the charging mechanism. for the classical charging mechanism of counter - ion adsorption, x = + 1. for ion exchange, x = 0, while for co - ion desorption, x = 1 (see example calculations in figure 3). intermediate values of x are also possible ; for example, x = 0.3 would indicate that both ion exchange and counter - ion adsorption occur during charging, with ion exchange dominating (as 0.3 is closer to 0 than it is to 1). we stress that this parameter refers to charging ; i.e., |v| must be greater than |v0|. it is also important to realize that x can take different values in the positive and negative electrodes, such that separate calculations must be performed for each, and that x may also depend on the studied voltage range. we note that for pores that are initially empty, charging must at least initially proceed by counter - ion adsorption (x = 1), as there are no in - pore co - ions available for desorption. for such ionophobic pores, charging may also involve the net movement of pairs of ions into the carbon pores. in this unusual case, as we will see in the next section, the different charging mechanisms can be probed with computational and experimental techniques, and the mechanism depends on the electrode and electrolyte materials studied. a schematic illustrating some of the main approaches that have been developed to study the charging mechanisms of supercapacitors is shown in figure 4. computational methods have been extremely successful in this area as they allow one to probe local length scales. the simulation of supercapacitors and their charging mechanisms presents a number of challenges which have been tackled through different approaches ranging from mean - field theories to md simulations. one of the main difficulties is related to the complex structure of the carbon electrodes. in mean - field theories, electrodes are usually represented as single slit or cylindrical pores. in molecular simulations, it is also difficult to account for the complexity of the carbon structure as (i) an accurate description of the structure at the atomistic scale is not readily available and (ii) a realistic representation of the carbon structure requires the use of a large number of atoms in the simulations, which is time consuming. nevertheless, md simulations are valuable as they provide a precise description of the ion ion correlations and packing effects that are important in the context of the concentrated electrolytes used in supercapacitors. schematic showing the different approaches for studying the charging mechanisms of supercapacitors, and their advantages and challenges. experiments and simulations have shown that the materials and conditions used in the carbon synthesis have a large effect on the ordering of the carbon sheets. this ordering, as well as the presence of functional groups, impacts the electronic conductivity of the carbons and their performances as electrode materials. in molecular simulations, there are two main approaches to deal with the electrode charge : (i) constant charge and (ii) constant potential. in the constant charge approach, charges are assigned to the carbon atoms at the beginning of the simulations and kept constant throughout. as such the converse approach is to consider the electrode as metallic and include the fluctuation of carbon atom charges in the simulations. while this approach is likely to be more realistic, especially for carbons with high degrees of order, it is much more computationally expensive and has been used in only a limited number of studies. further work is needed to assess the validity of these different approaches and also to develop quantum mechanical methods to treat the carbon charge. one way of circumventing the problem of computational cost is to explore the effects of confinement and polarization through analytical expressions. indeed, while early theories considered ions as point charges and electrolytes as dilute, new advances in this field have allowed the inclusion of steric interactions and charge screening in the representation of the systems. superionic effect which states that the packing of ions of the same charge is easier in confined spaces because of an exponential screening of the electrostatic interactions by the charged pore wall. this charge screening has recently been explored in more detail by inserting atoms in gold nanotubes and cnts using dft calculations. the authors showed that the atoms were fully ionized upon insertion, turning the initially semi - conducting nanotubes into conductors. both analytical theories and quantum chemistry simulations have provided valuable insights into the understanding of charging mechanisms by investigating the impact of charge screening. nevertheless, the specific performances of different electrolytes, with or without solvent, can be understood only via techniques that explicitly account for ionic correlations, and the size and shape of the electrolyte molecules. classical md simulations are currently the method of choice here, although md - dft simulations will become increasingly important as computational power increases. ideally, md simulations of supercapacitors would include a realistic representation of the carbon electrode structure, as well as a description of its electronic conductivity. as mentioned above, this is very challenging, as it involves using a large number of atoms for the carbon porous structure and a constant potential approach, which are both computationally expensive. to date, only a few studies have included both of these characteristics. effects in nanoporous electrodes contributes to the anomalous capacitance increase observed in nanometer - sized pores. in nanoporous electrodes, only a single layer (or a few layers) of adsorbed ions this is in stark contrast to planar electrode surfaces, such as the graphite surface, where several layers of ions of alternating charge extend from the charged surface into the bulk. at planar surfaces, the first layer of adsorbed counter - ions carries a greater charge than the electrode surface (overscreening the surface charge), and subsequent layers of co - ions and counter - ions then balance the excess charge in the first layer. the lack of overscreening in nanoporous electrodes does not, however, account for all of the anomalous capacitance, and hence a more atomistic / molecular understanding of charge storage is required. to this end, md simulations of a pure ionic liquid (bmi pf6) and cdc electrodes have shown that charge storage does not occur by counter - ion adsorption alone (table 2). in the positive electrode, charging occurred by a combination of ion exchange and counter - ion adsorption (x = 0.34), while in the negative electrode charging was mainly by ion exchange, with a small amount of co - ion desorption (x = 0.11). thus, it appears that the pf6 anions are the more active species here, playing the dominant role in the charge storage process in both electrodes. similar results were found for the same disordered carbon with the organic electrolyte (bmi pf6/acn), showing that the solvent does not significantly affect the capacitance or charging mechanism (table 2). for a model supercapacitor system with slit - pore electrodes and the ionic liquid 1-ethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide (emi tfsi), charging in the positive electrode involved both ion exchange and co - ion desorption (x = 0.36), while charging in the negative electrode involved both ion exchange and counter - ion adsorption (x = 0.45) ; i.e., the emi cations are more active in this system. these observations for the different md studies indicate that ion packing effects and ion ion interaction energies influence the charging mechanism that operates. we note that, for an identical potential difference and a similar pore size, the difference between the number of anions and cations in the charged electrodes (which is proportional to the capacitance) is 4 times larger in the disordered cdc electrodes compared to the single slit pores (table 2). this is partly because both sides of the carbon sheets are accessible to ions in the disordered electrodes while only a single surface is accessible in the slit - pore system. accounting for this, the capacitance is still twice as large for the disordered electrodes, indicating that the curved and defective nature of the carbon sheets facilitates charge separation. it has been shown that more confined pocket - like ion adsorption sites allow the more effective storage of charge, than plane sites, presumably because the curved surfaces maximize favorable coulombic interactions between the carbon surfaces and the counter - ions (while also screening repulsive interactions between different counter - ions). these effects result in a much larger capacitance for disordered porous electrodes and highlight the importance of using complex porous carbon structures in simulations of supercapacitors. ncounter - ion nco - ion is proportional to the excess ionic charge (which is in turn directly proportional to gravimetric capacitance). besides providing a microscopic picture and fundamental understanding of the local processes occurring during supercapacitor charging, both mean - field theories and molecular simulations can be used to explore new ideas to improve energy storage efficiency in supercapacitors. an interesting example of these hypothetical explorations is the concept of ionophobicity and ionophilicity. simulations can be used to investigate the effects of having initially empty (ionophobic) pores, on both the energy stored and the charging rate. both mean - field theories and md simulations for single slit pores have shown that ionophobic pores perform better in terms of charging rate, and suggest that, in some cases, the quantity of energy stored is higher for ionophobic pores. one interesting aspect is that the charging of initially filled pores usually leads to an overfilling of the porosity, corresponding to a temporary state where the density of ions is higher than the final density at the end of charging. this type of phenomena raises the issues of kinetic barriers and the difference between static and dynamic charging of supercapacitors. while the effects of ionophobicity and ionophilicity are now well studied in idealized slit - pore geometries, it would be interesting to study similar ideas in realistic three - dimensional porous geometries where it was shown that charging mechanisms are different from slit pores due to local heterogeneities in the carbon structure. experiments using an eqcm were some of the first to investigate the molecular mechanisms of supercapacitor charging. levi. demonstrated that by depositing porous carbon electrodes on a quartz crystal, it was possible to measure changes of electrode mass during the operation of a supercapacitor cell. in practice, variations of the resonance frequency of the quartz crystal are measured, and these are converted into mass changes using the sauerbrey equation. these experiments have mostly been performed with dynamic charging conditions, with the voltage continuously swept between the voltage limits at a constant rate (i.e., cyclic voltammetry). the extracted mass changes are then typically compared to the predicted mass changes assuming the adsorption of a single counter - ion to balance each electronic charge in the electrode (i.e., assuming x = 1). while this assumption generally does not hold, deviations from the predicted mass changes can be used to infer the presence of ion - exchange, and qualitative studies of the supercapacitor charging mechanism are possible. initial studies of activated carbon electrodes with an aqueous cscl (0.5 m) electrolyte suggested that counter - ion adsorption processes dominated the charging mechanism. in contrast, further experiments on tetrabutylammonium chloride (nbu4cl, 0.5 m aq), with the bulky nbu4 cation, suggested that charging was brought about predominantly by the migration of chloride anions (i.e., counter - ion adsorption in the positive electrode, and co - ion desorption in the negative electrode), with these experiments providing the first evidence that the charging mechanism depends on the electrolyte. in follow - on studies on organic electrolytes, different charging mechanisms could be discerned depending on the magnitude of the charge stored in the electrode (i.e., depending on the cell voltage), see figure 5a. for low charge densities (region i), the measured mass change was smaller than that expected for pure counter - ion adsorption processes, indicating the presence of ion exchange. for larger (intermediate) charge densities (region ii), the measured mass changes were consistent with a counter - ion adsorption mechanism. finally, for high charge densities (region iii), the measured mass changes were larger than those predicted for pure counter - ion adsorption, and it was suggested the source of the additional mass was extra solvent molecules that entered the electrode pores. this is surprising given that the pores are already densely packed with ions and solvent molecules, and we note that md simulations have suggested that the in - pore solvent population does not significantly change during charging. further studies are thus needed to investigate how the in - pore solvent population varies during charging. interestingly, when electrolytes with different cations were studied with eqcm (figure 5b), it was shown that the ion - exchange processes in the negative electrode became more significant for electrolytes with bulkier cations (e.g., nbu4bf4), with the role of desorption of the smaller anions becoming more important. gives the moles of ions per surface area of quartz crystal, obtained from the measured electrode mass changes with the assumption of charge storage solely by counter - ion adsorption processes. dashed lines showing theoretical values for charging by pure counter - ion adsorption. (a) measurements for yp-17 activated carbon and net4bf4 in propylene carbonate (pc) solvent at two different concentrations. (b) further measurements for yp-17 carbon supercapacitors with different electrolyte salts (0.1 m in pc) : tea, tba, and toa are tetraethylammonium, tetrabutylammonium, and tetraoctylammonium (noc4), respectively. again, the charging mechanism was found to depend on the magnitude of the charge stored on the electrodes. in the positive electrode ion exchange was observed at low charge densities, with counter - ion adsorption processes then dominating at higher charge densities. in the negative electrode, on the other hand, counter - ion adsorption appeared to dominate over the full range of studied charge densities. interestingly, the charging mechanism was largely unaffected by the presence of solvent, though additional mass changes were detected in the negative electrode, assigned to the solvent molecules that the emi cations carried into the pores. following the approach developed by levi for aqueous electrolytes, a solvation number of 3.7 could be estimated for the emi cations by assuming a purely ion - adsorption - driven charging mechanism, and assuming that the unaccounted mass was due to acn solvent molecules. given the assumptions here, it would be beneficial if solvation numbers could be verified by a second method such as nmr spectroscopy (see later)., a significant limitation of these studies is that a single parameter is measured (the electrode mass), which depends on the number of cations, the number of anions and the number of solvent molecules. thus, the populations of the various in - pore species can not be determined. this makes it difficult to fully quantify the charging mechanism and obtain x values, such that information from other experimental techniques is crucial. as discussed in section 2, nmr experiments allow the absolute quantification of ions inside the carbon nanopores, with the separate study of anions, cations, and solvent possible. along with co - workers, we have developed the in situ nmr approach for studying supercapacitors, such that in - pore ion populations can be tracked at different charging potentials in working supercapacitor cells. these measurements are typically performed in constant voltage mode ; i.e., a fixed voltage is applied to the cell, and nmr spectra are acquired after equilibration of the system. figure 6a shows in situ nmr spectra for a supercapacitor cell with yp50f activated carbon electrodes and a pet4bf4/acn (1.5 m electrolyte), with p and f nmr allowing the study of the pet4 cations and bf4 anions, respectively. changes of in - pore chemical shift during charging arise from the changes of the carbon electronic structure as electrons are added or removed from the electrodes. crucially, changes of in - pore peak intensities relate directly to changes of the in - pore ion populations. by fitting the spectra the number of in - pore cations and anions could be determined, revealing that the charge storage mechanism is inherently different depending on the polarization of the electrode (see figure 6b). in the positive electrode, charging in this system occurs mainly by ion exchange (x = 0), with simultaneous counter - ion adsorption and co - ion desorption. in contrast, charging in the positive electrode occurs purely by counter - ion adsorption (x = 1). despite the different charging mechanisms, the excess ionic charge in the carbon pores balances the electronic charge in both electrodes (figure 6c). interestingly, the charging mechanism was also found to be invariant to the concentration of the electrolyte, with the same mechanisms observed for concentrations of 1.5, 0.75, and 0.5 m. this mirrors observations on neat and diluted ionic liquids studied by eqcm and md simulations above, and further confirms the idea that the solvent concentration does not necessarily dictate which charging mechanism operates, but simply modulates the absolute in - pore populations. in our study, the findings from the nmr measurements were corroborated by eqcm measurements, where a small negative mass change was observed in the positive electrode, as the slightly heavier cations were desorbed from the pores while the lighter anions were adsorbed. meanwhile, a positive mass change was observed in the negative electrode, in agreement with the counter - ion adsorption mechanism revealed by nmr. the mass increase in this electrode was greater than that expected due to cation adsorption, and the additional mass was ascribed to solvent molecules, with an estimated cation solvation number of 5.4. h nmr experiments were also performed to study the deuterated acetonitrile solvent, though the large peak line widths precluded the quantification of the in - pore solvent. future in situ nmr studies may allow quantitative measurement of in - pore ion solvation numbers, allowing comparison with the results from eqcm analysis. (a) in situ nmr measurements of a supercapacitor with yp50f electrodes and pet4bf4/acn (1.5 m) electrolyte. (b) in - pore ion populations at different charging voltages, obtained by fitting of the spectra in (a). (c) ionic charge stored by in - pore ions, obtained from the in - pore ion populations in (b), as well as electronic charge, obtained from electrochemistry data. reprinted with permission from ref (52). further nmr measurements on the same activated carbon (yp50f) and a range of different electrolytes have revealed that the charging mechanism can vary significantly when different electrolyte ions are studied. ex situ nmr studies of supercapacitors with the ionic liquid pyr13tfsi showed that in the positive electrode charging took place by ion exchange and counter - ion adsorption (x = 0.3), while in the negative electrode ion exchange and co - ion desorption were observed (x = 0.4). in this system the tfsi anions are more active in the charge storage than the pyr13 cations. this charging mechanism is markedly different to that observed for the pet4bf4/acn system above, where the bf4 anions played no significant role in charging in the negative electrode (see schematic in figure 7). similarly, in situf nmr measurements on yp50f with li tfsi / acn (1.5 m) and na tfsi / acn (1.5 m) electrolytes revealed significant co - ion desorption processes in the negative electrode, though the cations were not studied. the exact origin of these different charging mechanisms is currently unclear, but ion packing and ion carbon interaction energies are expected to be important. co - ion (anion) desorption was also observed in the positive electrode of a supercapacitor with the electrolyte nbu4bf4, suggesting that the large size of the nbu4 cations impeded their adsorption into the carbon pores, necessitating desorption of anions to store charge. this finding is consistent with eqcm studies on activated carbons, where the role of the anions (co - ions) in the negative electrode became more significant for electrolytes with larger cations. it should, however, be kept in mind that the eqcm measurements were performed in a dynamic mode, with the frequency response of the quartz crystal measured during cyclic voltammetry measurements. such dynamic measurements with eqcm are more likely to reveal kinetic effects (e.g., effects due to the different diffusion rates of the anions and cations) than the in situ nmr measurements that were performed at equilibrium after charging to a specific voltage. schematics showing charge storage mechanisms determined by nmr experiments. for the same activated carbon (yp50f) electrodes, the charging mechanism differs depending on the electrolyte : (a) pet4bf4/acn (1.5 m) and (b) pyr13tfsi ionic liquid. note in part (b) the x value given for the negative electrode is the experimentally measured one, whereas based on the number of ions in the schematic one obtains an x value of 0.3. other research groups have also applied nmr methods to the study of supercapacitor charging mechanisms. ex situ nmr measurements by deschamps. on net4bf4/acn showed that ion - exchange processes operated in both the positive and negative electrodes of two different activated carbons. more recently, luo. have applied the in situ nmr methodology to supercapacitors with aqueous electrolytes. they studied an activated carbon material with small pores that were inaccessible to the ions (na and f) in the absence of an applied potential. however, when a potential above 0.4 v was applied, the f anions were able to enter the pores of the positive electrode, with charging proceeding via counter - ion adsorption. following discharge to 0 v, some ions remained in the carbon pores, suggesting the presence of a hysteresis in the charging mechanism. interestingly, changes of in - pore chemical shift at 0.8 v indicated that the solvation number of the ions decreased above this voltage. the strength of the nmr approach arises from the ability to separately observe and quantify in - pore ions, as well as to separately detect anions and cations. this allows the absolute quantification of supercapacitor charging mechanisms (and x values can be determined). the technique, however, is limited to ions with nmr active nuclei, and if many experiments are to be performed at different voltages, ions containing sensitive nuclei such as h, f, p, li, b, and na must generally be studied. another limitation is that in some cases the in - pore resonances can not be clearly resolved (e.g., due to broad peaks, or small ring current shifts), and methods should thus be developed to improve spectral resolution for challenging carbon and electrolyte systems. for example, we have shown how cross - polarization experiments can be used to edit the nmr spectrum to reveal peaks solely due to ions at the carbon surface (i.e., in - pore ions). beyond eqcm and nmr, ir spectroscopy has also been demonstrated as a useful probe of supercapacitor charging mechanisms. here, changes in intensity of the absorbances from bond vibrations in the electrolyte anions and cations are monitored during charging, allowing the behavior of the two ions to be tracked separately. the ir radiation is directed on the working electrode of a supercapacitor cell (which is clamped onto the surface of a diamond attenuated total reflectance (atr) crystal), and the reflected signal intensity is recorded to obtain an absorbance spectrum. in these measurements the ir radiation penetrates to 1 m depth in the working electrode. in experiments on titanium carbide - derived carbon (tic - cdc) supercapacitors with ionic liquid (emi tfsi) electrolyte, approximately equal losses of intensity were observed for both the anion and cation absorbances during charging, suggesting that both ions penetrated deeper into the carbon particles, beyond the depth to which the ir photons were able to penetrate. this was explained by postulating that both anions and cations had entered the carbon nanopores during charging, with experiments on nonporous onion - like carbons revealing no significant changes of absorbance during charging and supporting the hypothesis. these experiments demonstrated that ions entered the carbon nanopores during charging, though the charging mechanism was not fully quantified. tfsi and nanoporous carbon nanofiber (cnf) electrodes revealed similar losses of intensity for anions and cations in the positive electrode, though the intensity loss was more significant for the anions than the cations, explaining how an excess of ionic charge could develop inside the carbon pores. experiments were also performed on modified cnfs, which had been activated with koh at 800 c and contained more oxygen - containing functional groups. in contrast to the measurements on the untreated cnfs, an increase in intensity was observed for the cation absorbances, indicating that cations were desorbed from the nanopores of the cnfs in the positive electrode. together with the observation of the loss of intensity of the anion absorbance (as anions entered the pores of the cnfs), it was shown that an ion - exchange mechanism was operating. the measurements intriguingly showed how the charging mechanism of supercapacitors is dependent on the surface chemistry of the carbon materials. ir spectroscopy experiments have revealed new insights into the charging mechanisms of supercapacitors. however, a key limitation of the ir approach is that the in - pore ions can not be directly detected, and instead one must rely on measurements of the bulk electrolyte surrounding the carbon particles. it is therefore challenging to make fully quantitative studies of the supercapacitor charging mechanism, and x values can not be readily determined. scattering - based methods have also been applied to the study of supercapacitor charging mechanisms. in situ sans experiments utilize the different scattering properties of the various atoms in the electrolyte and electrode. by measuring the scattered neutron intensity from a single (working) electrode at different charging potentials, for example, in a study of aqueous h2so4 electrolytes in activated carbon fabric electrodes, changes of scattered intensity were dominated by the migration of the hydrogen - containing ions and solvent molecules. on this basis it was inferred that ion - exchange processes bring about charging in activated carbon fabric electrodes, for example, with h3o replacing hso4 and so4 in the negative electrode. similar findings were obtained for devices with the organic electrolyte net4bf4 in dacn (1 m). here, the increase in scattered intensity in the negative electrode indicated that the strong h - containing scatterers net4 were adsorbed into the carbon pores, while neutron - absorbing bf4 were desorbed from the pores, i.e., an ion - exchange mechanism. converse effects were observed in the positive electrode, again rationalized by an ion - exchange mechanism. an interesting feature of small - angle scattering measurements is that adsorption / desorption processes can be monitored as a function of the carbon pore size. boukhalfa. found that the largest changes of scattered intensity were observed for the smallest pores (i.e., at large scattering wavevectors). these measurements have the potential to complement md simulations, where the relative ion populations in pores of different geometries and sizes can be tracked. however, in the sans measurements full quantification of the in - pore ionic species at different potentials was not possible as the scattered neutron intensity (a single measured quantity) depends on the population of anions, cations, and solvent molecules. a recent study by prehal. developed in situ x - ray transmission (xrt) and saxs methods to provide deeper insights into the charging mechanisms of supercapacitors with simple aqueous electrolytes (cscl, kcl, and nacl, all 1 m concentration) and the activated carbon yp-80. saxs measurements on evacuated and electrolyte - soaked carbon electrodes confirmed that the pores were filled with electrolyte at 0 v. variations of the xrt intensity during electrochemical cycling, taken together with the electrochemical data, then allowed semi - quantitative measurements of the changes of ion concentrations at different charging potentials. the analysis relies on the different x - ray attenuation coefficients of the cations and anions, and it is assumed that changes of the amount of solvent (water) have no significant effects on the xrt signal. this analysis showed that an ion - exchange mechanism (x = 0) occurs for all three electrolytes studied, with the charging mechanism independent of the choice of cation. in situ saxs measurements were consistent with these findings and also indicated that the counter - ions became more closely associated with the pore surfaces as the potential was increased. further work with scattering - based approaches stands to further our understanding of supercapacitor charging mechanisms. in summary, nmr spectroscopy measurements and md simulations have shown that the pores of the carbon electrodes contain a considerable number of electrolyte ions in the absence of an applied potential. larger in - pore populations are observed for more concentrated electrolytes, with the largest populations observed for ionic liquids. given the large in - pore ion population in the absence of an applied potential, different charging mechanisms can operate when a potential is applied : counter - ion adsorption, co - ion desorption, and ion exchange (and combinations of these). we have introduced the charging mechanism parameter, x, to allow a convenient comparison of different charging mechanisms. in situ characterization experiments (nmr, eqcm, ir, and scattering methods) and simulations have shown that supercapacitor charging does not generally take place by counter - ion adsorption alone, as is the traditional view, and ion exchange plays an important role in the charge storage process. as shown by table 3, in a wide range of experimental and computational studies, charging almost always involved some degree of ion exchange (x 1), and only rarely was charging driven purely by counter - ion adsorption (x = 1). experiments have shown that the exact charging mechanism depends on the polarization of the electrode, the choice of electrolyte ions, and the choice of electrode material (table 3). surprisingly, the solvent concentration does not appear to significantly influence the charging mechanism that operates (table 3), and it appears the role of the solvent is less significant than previously thought. this suggests that ion confinement effects, screening of ions by the carbon surfaces, as well as the lack of overscreening dominate the capacitance increase reported for nanoporous carbons, rather than ion desolvation, while different in - pore ion populations at 0 v and fundamentally different charging mechanisms may also play a role. the wide range of charging mechanisms (x values) presented in table 3 indicate that factors such as ion - packing energies and ion carbon interaction energies dictate which charging mechanism is the thermodynamic one. under dynamic (fast) charging conditions the charging mechanism may differ, though studies contrasting thermodynamic and kinetic charging mechanisms have not yet been carried out. when studying table 3 it should be kept in mind that only nmr and md simulations allow the absolute quantification of the different electrolyte species at different potentials, and that various assumptions must be made when inferring charging mechanisms from other in situ methods (e.g., with eqcm changes of in - pore anion, cation, and solvent populations have to be determined from a single measured parameter, the electrode mass). further in situ characterization of supercapacitors with a wide range of different electrolytes and carbon materials will help to fully elucidate the factors which control supercapacitor charging mechanisms. as discussed above, each in situ characterization method comes with its limitations, and these methods must be further developed such that they can be applied to a wide range of systems in a quantitative manner, and on a range of charging time scales. beyond experiments, advanced theoretical methods that can properly take into account the electronic structure of the ions and carbon electrodes could provide new insights. if one can properly understand the factors that influence the charging mechanism, it should be possible to tailor the mechanism by choice of the correct electrolyte - electrode combination. in particular, it would be interesting to study the relative magnitudes of ion ion and ion carbon interaction energies within the carbon pores. additionally, one could investigate packing effects by studying ions with different shapes and sizes within carbon pores of different geometries. it is clear that systematic studies in which the electrolyte and carbon structure are varied independently will allow the understanding to progress here. this leads to an important question : how does the charging mechanism affect the performance of a supercapacitor ? the mechanism should have a significant effect on the power that supercapacitors can offer, and tailoring the mechanism may allow us to improve the power performance of devices. indeed, theoretical work has indicated that initially empty pores (ionophobic pores) should charge more quickly than initially filled pores (ionophilic pores), though this effect is yet to be realized experimentally. similarly, we expect that ion adsorption, ion exchange, and ion desorption mechanisms from initially filled pores should each result in different device power performances, and work must be done to establish which mechanism is optimal for fast charging. purely counter - ion adsorption processes might be expected to allow fast charging in a front - like manner, with net migration of ions into the interior of the carbon nanoporosity, while ion exchange requires ionic migration in opposite directions. at the same time, these different mechanisms will bring about changes of in - pore ionic density, and therefore packing during charging, which will also affect the charging rate. for example, counter - ion adsorption mechanisms will increase the number of ions inside the carbon pores, and recent theoretical work has suggested that more densely packed pores result in slower ionic diffusion. as well as ion packing effects, interactions of the different ions with charged carbon surfaces will also affect in - pore transport processes. clearly, experimental measurements and simulations of diffusion and migration processes in charged carbon nanopores represents another exciting area of research. in principle, the charging mechanism will affect the capacitance, and therefore the energy density, that can be achieved in supercapacitors. under thermodynamic conditions, the charge storage mechanism which operates is the one that minimizes the increase in free energy associated with charging, thus minimizing the voltage increase per unit charge (i.e., maximizing the capacitance as c = q / v). kondrat and kornyshev point out that counter - ion adsorption is disfavored here, as there is an entropic penalty for an ion entering a pore, and there are also unfavorable electrostatic (enthalpic) terms associated with the packing of ions of the same charge inside the carbon pores (though this is alleviated to some extent by charge screening from the pore walls). in principle, the ion - exchange mechanism reduces the enthalpic penalty associated with denser ion packing because the total in - pore density remains essentially constant during charging, while the entropic penalty associated with charging will also be reduced. this may help to explain the prevalence of ion exchange mechanisms revealed by in situ characterization methods detailed in this article (table 3). charging by co - ion desorption should minimize the enthalpic penalty due to interactions between like charges, while also increasing entropy, and should thus maximize the capacitance. that said, charging by purely co - ion desorption (x = 1) has not yet been observed, indicating that other factors beyond these simple arguments are important. it is clear that additional work must be done to understand the interplay between supercapacitor charging mechanisms and capacitance. under kinetic control, the charging mechanism will depend on the relative rates of in - pore motion of the anions and cations, and we again stress the need for further experimental and theoretical studies of these effects. if the diffusion rates of the different in - pore ions can be controlled, then it should be possible to control the kinetic charging mechanism and thus improve the capacitance. finally, we stress that the structural complexity of porous carbon electrodes poses a significant challenge as we aim to design enhanced supercapacitors. ideally the electrode structure would be modified in a controlled way to study its performance, though for this to happen new tools must be developed to characterize and model amorphous carbon structures. beyond activated carbons, more ordered carbon materials based on nanotubes, graphenes, and templated materials may serve as model systems to probe structure property relationships. | supercapacitors (or electric double - layer capacitors) are high - power energy storage devices that store charge at the interface between porous carbon electrodes and an electrolyte solution. these devices are already employed in heavy electric vehicles and electronic devices, and can complement batteries in a more sustainable future. their widespread application could be facilitated by the development of devices that can store more energy, without compromising their fast charging and discharging times. in situ characterization methods and computational modeling techniques have recently been developed to study the molecular mechanisms of charge storage, with the hope that better devices can be rationally designed. in this perspective, we bring together recent findings from a range of experimental and computational studies to give a detailed picture of the charging mechanisms of supercapacitors. nuclear magnetic resonance experiments and molecular dynamics simulations have revealed that the electrode pores contain a considerable number of ions in the absence of an applied charging potential. experiments and computer simulations have shown that different charging mechanisms can then operate when a potential is applied, going beyond the traditional view of charging by counter - ion adsorption. it is shown that charging almost always involves ion exchange (swapping of co - ions for counter - ions), and rarely occurs by counter - ion adsorption alone. we introduce a charging mechanism parameter that quantifies the mechanism and allows comparisons between different systems. the mechanism is found to depend strongly on the polarization of the electrode, and the choice of the electrolyte and electrode materials. in light of these advances we identify new directions for supercapacitor research. further experimental and computational work is needed to explain the factors that control supercapacitor charging mechanisms, and to establish the links between mechanisms and performance. increased understanding and control of charging mechanisms should lead to new strategies for developing next - generation supercapacitors with improved performances. |
it is a well - entrenched dogma that microtubule- and f - actin based intracellular organelle transport is down - regulated during mitosis in order to ensure the accuracy of stochastic partitioning of the diverse organelles into daughter cells (warren and wickner, 1996). unlike the golgi apparatus, which undergoes fragmentation during mitosis, the highly interconnected er does not undergo dramatical fragmentation and is maintained during mitosis (warren, 1993). in mitosis, all microtubule - dependent movement of membranous organelles, including that of er, is inhibited (allan and vale, 1991), presumably because microtubules and microtubule - associated motors are needed for spindle formation and chromosome segregation. we considered the possibility that during mitosis the f - actin based system facilitates not only cleavage furrow formation but also the positioning and partitioning of the er. that f - actin system can generally interact with er is supported by the finding that the er in neurons is transported on f - actin by myosin v (tabb., 1998) and by the study showing that f - actin is involved in er dynamics throughout the cell cycle in yeast (prinz., 2000). a convenient system to study cell cycle dependent motility and network formation of er is interphase - arrested extract (i extract) and metaphase - arrested extract (m extract) from xenopus frog eggs (allan and vale, 1991). we use this system here to investigate whether mitotic reorganization affects er movement on f - actin and found that both myosin v driven er motility and f - actin dependent er network formation, a consequence of membrane movement and fusion, became activated in m extracts. to our knowledge, this is the first study demonstrating an up - regulation of both membrane organelle motility and fusion during mitosis. our goal here was to study organelle motility along f - actin in i and m extracts from xenopus frog eggs. for this we developed an in vitro procedure to generate stable three - dimensional f - actin networks attached to a coverslip surface under conditions where microtubules are absent. this was achieved by diluting xenopus egg extracts with a glycine buffer containing nocodazole following incubation in the presence of rhodamine - phalloidin and an atp - regenerating system. the network was stable and did not change in density for at least 60 min of observation (fig. 1 a). the density and stability of f - actin networks formed in m extracts were very similar to those observed in i extracts (fig. 1 a). reconstitution of organelle motility on f - actin in extracts isolated from xenopus eggs. (a) stability of an f - actin network in i (interphase) and m (metaphase) extracts visualized by fluorescence microscopy after 15 and 60 min of incubation with 0.5 m rhodamine - phalloidin. (b) movement of a globular vesicle on f - actin monitored by dic microscopy. selected frames of a video sequence covering 18 s are shown. (b) track diagram mapping the movement of the vesicle marked by an asterisk in b. (c) movement of a tubular organelle (asterisk) on f - actin monitored by dic microscopy. selected frames of a video sequence covering 5 s are shown. (c) track diagram mapping the movement of the tubular organelle shown in c. the numbers in the top left corner of b and c indicate the time points of image acquisition. (d) tubular organelles in i (interphase) and m (metaphase) extracts visualized by dic microscopy (a and c) and by fluorescence microscopy after er staining with dioc6 (b and d). (a and b) distribution of the velocities and average velocities (v) attained by 30 globular vesicles (vesicles) in i extracts (a) and by 30 tubular organelles (er) in meiosis ii m extracts (b). (c and d) distributions of the frequencies of run distances and average run distance (d) attained by 30 globular vesicles (vesicles) in i extracts (c) and by 30 tubular organelles (er) in meiosis ii m extracts (d). see also videos 1 and 2 available at http://www.jcb.org/cgi/content/full/jcb.200204065/dc1 in both i and meiosis ii m extracts, the motility of two morphologically different types of organelles was detected, namely that of globular membrane vesicles (fig. 1, b and b ' ; video 1 available at http://www.jcb.org/cgi/content/full/jcb.200204065/dc1) and tubular membrane structures (fig. 1, c and c ' ; video 2 available at http://www.jcb.org/cgi/content/full/jcb.200204065/dc1). the latter observations were highly reminiscent of moving er tubules observed previously in both xenopus egg extracts (allan and vale, 1991) and squid axoplasm preparations (tabb., 1998). to identify these membrane tubules, we first labeled i and m extracts with fluorescent dye dioc6, which preferentially labels er (jaffe and terasaki, 1993), and c6-nbd - cer, which labels the golgi apparatus (martin., 1993). in both types of extracts, this was further confirmed by immunofluorescence microscopy using an antibody to an er - resident protein, er calcistorin / protein disulfide isomerase (ecast / pdi) (lucero., 1994) (see below). no labeling of tubular membranes was found after treatment with c6-nbd - cer (unpublished data). the motion analysis showed that the movement of all membranous organelles on f - actin was atp dependent and unidirectional. the average velocity of moving globular vesicles was almost identical in both types of extracts (fig. 1 e, a and b ; student 's t test, p = 0.94). however, the velocity of moving er tubules in m extracts was significantly higher (30% ; student 's t test, p = 4.6 10) than in i extracts (fig. 1 e, a and b). greater differences were observed for the distribution of the measured distances of the moving organelles. the average distance moved by globular vesicles in the i extracts was 50% greater (student 's t test, p = 2.2 10) than in m extracts (fig. in contrast, the average distance moved by er tubules in i extracts was 60% shorter (student 's t test, p = 2.0 10) than in m extracts (fig. we found that the motile activity (number of organelle movements per field per min) of globular vesicles was down - regulated approximately fivefold (fig. in contrast, the motile activity of the er tubules was greatly up - regulated (about fourfold) in m extracts (fig. 2 a). together, these results demonstrate that er movement and movement of globular vesicles on f - actin is differentially regulated throughout the cell cycle. quantitative analysis of actin - dependent organelle movement. (a) motile activities of globular vesicles (vesicles) and tubular organelles (er) in i and meiosis ii m extracts. (b) motile activities of globular vesicles (vesicles) and tubular organelles (er) during transition from interphase to mitotic metaphase induced by cyc. the values at the 0-min time point indicate the motile activity in the same i extract in the absence of cyc. (c) determination of cell cycle status during transition from interphase to mitotic metaphase induced by cyc using morphology assay of added sperm nuclei. to confirm our finding that the movement of different organelles on f - actin is differently regulated by the cell cycle, we analyzed the f - actin based motility in extracts during the transition from interphase to mitotic metaphase, the latter being induced by addition of nondegradable cyclin b (cyc) to i extracts (glotzer. during the first 15 min of incubation with cyc, corresponding to the switch from interphase to mitosis, the motile activity of globular vesicles was significantly increased (fig. after 15 min of incubation, we detected a gradual inhibition of globular vesicle movement and a concomitant stimulation of er movement (fig.. the maximum rate of er motile activity was observed at mitotic prophase (6075 min of incubation, according morphology assay of added sperm chromatin ; fig. 2 c). at mitotic metaphase (7590 min of incubation), the motile activity of globular vesicles and er was almost identical to that detected in meiosis ii m extracts (fig., we detected not only activation of f - actin dependent er motility but also the formation of a reticular network (fig. this network formed after incubation of extract for 30 min was labeled with both the fluorescent dye dioc6 and an antibody to ecast / pdi (fig. 3 a, c, d, and f). furthermore, er network formation in m extracts was dynamic (video 3 available at http://www.jcb.org/cgi/content/full/jcb.200204065/dc1) and strongly dependent on the presence of f - actin, since in the presence cytochalasin d, a drug preventing actin polymerization, no er network was observed (fig. 3 b). collectively, our results argue that both f - actin dependent er network formation and er tubule movement on f - actin are activated in m extracts. notably, the microtubule - dependent er network formation and movement (allan and vale, 1991) and the er network formation by a controlled fusion reaction (dreier and rapoport, 2000) are inhibited in these m extracts. f - actin dependent er network formation in m extracts. (a) an extensive network of er was visualized with both dic microscopy (a) and fluorescence microscopy after staining with dioc6 (b) and immunofluorescence labeling with an antibody against ecast / pdi (e) in m extracts. no membrane network was detected in i extracts (c, d, and f). (b) an er network was also observed in mitotic m extract obtained by incubation of i extracts with cyc (a). an er network did not form in the presence of 5 m cytochalasin d (b). we next asked what type of myosin might be responsible for er transport on f - actin in xenopus egg extracts. the average velocity (0.7 m / s) of organelle movement on f - actin we detected in extracts was similar to that seen with myosin v 1998). furthermore, an equal amount of myosin v was found tightly associated with endogenous membrane organelles isolated from both i and m extracts (see fig. since the er is the most abundant membrane organelle in egg extracts, these data make it unlikely that myosin v dissociated from the er during mitosis and suggest that this myosin could be responsible for the er transport we observe in m extracts. to test directly whether myosin v is indeed still associated with er in m extracts, we performed indirect immunofluorescence labeling of er networks with a dil2 antibody against mouse myosin va, which strongly reacted with xenopus myosin v as shown by immunoblotting (see fig. 5 b). dil2 antibody labeling revealed a pattern very similar to the er network (fig. 4 a, a). this pattern was not observed when a control antibody against myosin ii was used (fig. 4 a, b). strikingly, the bulk of myosin v labeling colocalized with the immunosignal for ecast / pdi, the er marker protein (fig. identification of myosin v as a motor responsible for organelle motility and er network formation. (a) the dil2 antibody against myosin va (a) but not a control antibody against myosin ii (b) labeled the network in fixed preparations of m extract. the labeling of er network with dil2 antibody (c) and with an antibody against ecast / pdi (d) showed strong colocalization of myosin v immunosignal with ecast / pdi immunosignal. panels c and d represent the region marked in panel a. (b) inhibition of organelle motility on f - actin by an antibody against myosin va. motile activity in i and m extracts treated with dil2 antibody (anti - myo v) and a control antibody (anti - myo ii). (c) in m extracts er network formation occurred in the presence of anti myosin ii (a) but not in the presence of dil2 antibody (b). to show directly that myosin v is involved in organelle transport, the extracts were treated with a dil2 antibody and, as a control, the antibody against myosin ii. motile activity of all visible organelles on f - actin was inhibited in both types of extracts by > 90% in the presence of dil2 antibody (fig. 4 b). however, the antibody against myosin ii showed no effect on the motile activities in both types of extracts (fig. 4 b). moreover, the treatment of m extracts with the antibody against myosin va but not against myosin ii fully blocked the f - actin dependent er network formation (fig. 4 c). thus, these experiments are consistent with the notion that myosin v is the motor protein responsible for the f - actin based movement of both globular vesicles and er in i and m extracts and for f - actin dependent er network formation in m extracts. because myosin v is more strongly phosphorylated in metaphase compared with interphase (rogers., 1999) and the phosphorylation of myosin v by calcium / calmodulin - dependent kinase ii (camkii) resulted in the release of the motor from pigment granules (karcher., 2001), we tested the involvement of protein phosphorylation in the movement of membrane organelles on f - actin. we first treated extracts with 2 m staurosporine, a broad spectrum inhibitor of protein kinases (ruegg and burgess, 1989). this treatment blocked strongly both vesicle and er motility in i and m extracts (fig. surprisingly, the addition of 2 m camkii (281309) inhibitor peptide, which completely inhibits camkii activity in xenopus egg extracts (matsumoto and maller, 2002), also blocked an activation of both er movement in m extracts and vesicle movement in i extracts (fig. 5 a). (a) 2 m of staurosporine (stauro) or 2 m of camkii inhibitor (camkiii) inhibited organelle motility in both i and meiosis ii m extracts. the same effect was observed in mitotic m extract (data not depicted). (b) immunoblotting of membrane fractions (a) and membrane free fractions (b) isolated from i extract (i), m extract (m), and m extract treated with camkii inhibitor (mi) with dil2 antibody (myov) and er marker ecast / pdi (pdi). the equal amount of myosin v and ecast / pdi associated with membrane fractions. (c) phosphoimaging of membranes isolated from p - labeled mitotic m extract (m) and p - labeled mitotic m extract treated with camkii inhibitor (mi) and myosin v immunoprecipitates (mp and mip) from the respective membrane fractions (a). (b) ponceau s staining (top) and immunoblotting with dil2 antibody (myov) (bottom) as in panel a to demonstrate approximately equal protein load and the presence of myosin v in membrane fractions and immunoprecipitates. (d) immunoblotting (a) and phosphoimaging (b) of myosin v immnunoprecipitates from the nonfractionated i extract (ie) and m extract (me). to address whether motility inhibition reflects dissociation of myosin v from organelles, we isolated membrane fractions by flotation from m extracts preincubated with and without the camkii inhibitor. quantitative analysis showed that this method yielded a distinct membrane fraction that contained 80% of the total er and 5% of total myosin v (fig. we found that inhibition of camkii activity did not affect the amount of myosin v that copurified with the membrane fraction (fig. in addition, the camkii inhibitor did not change the level of protein phosphorylation in membrane fractions isolated from m extracts prelabeled with p (fig. in contrast to the immunoprecipitation of myosin v from the nonfractionated p - labeled m extract (fig. 5 d), the immunoprecipitation of myosin v from membrane fractions isolated from p - labeled m extract did not reveal any myosin v phosphorylation either in the absence or presence of the camkii inhibitor (fig. thus, our results indicate that camkii activity is required for activation of myosin v based movements of both er in m extracts and of vesicles in i extracts. however, camkii activity alone is evidently not sufficient to mediate the regulatory mechanism responsible for controlling the myosin v driven motility of different organelle types in the cell cycle. since myosin v associated with membrane organelles does not undergo additional phosphorylation during mitosis, we suggest that camkii has additional targets of phosphorylation, which could, for example, modify the atpase activity or processivity of myosin v during the cell cycle. these putative target proteins and the precise mechanisms regulating organelle motility on f - actin throughout the cell cycle remain to be determined. since the number of myosin v isoforms in xenopus is not yet known, we also can not exclude the possibility that different isoforms undergo differential regulation relative to each other and to the cell cycle. our data provide strong evidence that er membranes can move along f - actin and fuse during mitosis. driven motility that we have observed could also explain the increase of er motility seen during meiotic maturation of xenopus oocytes (kume., 1997) and the accumulation of er in the f - actin rich cortex of frog, mouse, and hamster eggs in vivo (houliston and elinson, 1991 ; mehlmann., 1995 ; it thus appears that the role of microtubules in moving the er during interphase is taken over during mitosis by the actin system. i and m extracts were isolated from xenopus eggs according to murray (1991) with some modifications. i extracts were obtained by activation in the presence of 0.4 g / ml ionophore a23187 for 5 min followed by washing in mmr/4 buffer containing 100 g / ml cyclohexemide and incubation at rt for 30 min. the eggs were washed in ice - cold tris - buffer (25 mm tris - hcl, ph 7.4, 5 mm mgcl2, 1 mm dithiothreitol, 50 mm sucrose). protease inhibitors (10 g / ml leupeptin, 10 g / ml aprotinin, and 10 g / ml pepstatin, 10 g / ml tpck) were added to the last washing step. after egg crushing by centrifugation, the cytoplasmic extract was removed and protease inhibitors and 60 m nocodazole were added. meiosis ii m extracts were prepared without egg activation and cycloheximide addition, and the last washing step of the eggs was performed with tris buffer containing 15 mm egta. for some experiments, the transition from i to mitotic m extracts was induced by 1:0.75 (vol : vol) dilution of i extract in glycine buffer (300 mm glycine, 120 mm d - gluconic acid, 100 mm taurine, 20 mm kcl, 5 mm mgcl2, 5 mm egta, 30 m nocodazole, ph 7.2) supplemented with 0.1 mg / ml bacterially expressed cyc (glotzer., 1991) and after incubation at rt in the presence of 0.5 m rhodamine - phalloidin and atp - regenerating system (1 mm atp, 0.5 mg / ml phosphocreatine kinase, 10 mm creatine phosphate). the morphology of added sperm chromatin (murray, 1991) and the histone h1 kinase assays were used to monitor the status of the cell cycle and the activity of p34kinase (felix., 1993). i or m extracts were diluted by 1:0.75 (vol : vol) in glycine buffer. diluted extracts were supplemented with an atp - regenerating system and 0.5 m rhodamine - phalloidin and incubated for 15, 30, 45, and 60 min at rt. at each time point organelle motility was monitored by video - enhanced contrast differential interference (dic) microscopy, and the presence of rhodamine - phalloidin labeled f - actin was confirmed by fluorescence microscopy. at each time point of incubation, three random fields (23 m 22 m) were recorded for 3 min. the myosin - based motile activity was determined by counting moving organelles on tracks invisible with dic microscopy and averaging the numbers obtained from 12 random fields after all time points of extract incubation. for organelle motility during transition from interphase to metaphase, the extract was examined by microscopy immediately after dilution, and continuous recording of 30 random fields for 3 min during 90 min of observation was performed. er network formation was monitored by both dic microscopy and fluorescence microscopy after staining with 1 g / ml dioc6 (molecular probes). to stain the golgi apparatus, the extracts were incubated with 5 m c6-nbd - cer (n-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]-6-aminocaproyl d - erythro - sphingosine) (molecular probes) and examined by fluorescence microscopy. for immunofluorescence labeling of er, 510 l of extracts prepared as for dic microscopy were placed on a poly - l - lysine coated coverslip followed by incubation for 30 min at rt and then fixed by adding carefully 200 l of 4% pfa with 4% sucrose in pbs. after fixation for 30 min, immunofluorescence labeling of the er was performed as previously described (kaether., 1997) using an mab against rat ecast / pdi (stressgen biotechnologies) and dil2 polyclonal rabbit antibody against a gst fusion protein containing heavy chain residues 9101,106 of mouse myosin - va (wu., 1997). the effect of the following reagents on organelle motile activity and er network formation was tested : a dil2 antibody at 1:25 dilution, a polyclonal rabbit antibody raised against skeletal myosin ii (m7648 ; sigma - aldrich) at 1:25 dilution, and 5 m cytochalasin d (sigma - aldrich), 2 m staurosporine (calbiochem), and 2 m camkii (281309) inhibitor peptide (calbiochem). for this, the diluted extracts were preincubated with the agents mentioned above for 30 min at rt. membrane fractions were prepared by flotation as described in lane and allan (1999) which was slightly modified. 300 l of i extracts were diluted 1:0.75 (vol : vol) in glycine buffer containing an atp - regenerating system. for obtaining the membrane fraction from mitotic m extracts, diluted i extracts were supplemented with cyc. to study the effect of camkii activity on membrane protein phosphorylation, 2 m camkii inhibitor and 50 ci [-p]atp (amersham biosciences) were added to diluted extracts. all samples were incubated for 60 min at rt and then were diluted once again with 2.75 ml of ice - cold glycine buffer containing 65% sucrose and 10 mm -glycerophosphate. after centrifugation, the membrane fractions floated through 0.8 ml of glycine buffer with 50% sucrose were subjected to sds - page followed by western blot and phosphoimaging. the immunoprecipitation of myosin v from the membrane fractions using affinity - purified dil2 antibody was performed as described (kromer., 1998). the video illustrates the movement of globular organelles along f - actin observed with dic microscopy and described in fig. 1 b. video 2 shows movement of tubular membrane structures (er) on f - actin in meiosis ii m extract. the video illustrates the movement of tubular membrane structures (er) along f - actin observed with dic microscopy and described in fig. 1 c. video 3 shows f - actin dependent dynamics of er network in meiosis ii m extract. the video illustrates the movement along f - actin of tubular extensions of er network observed with dic microscopy and shown in fig. i and m extracts were isolated from xenopus eggs according to murray (1991) with some modifications. i extracts were obtained by activation in the presence of 0.4 g / ml ionophore a23187 for 5 min followed by washing in mmr/4 buffer containing 100 g / ml cyclohexemide and incubation at rt for 30 min. the eggs were washed in ice - cold tris - buffer (25 mm tris - hcl, ph 7.4, 5 mm mgcl2, 1 mm dithiothreitol, 50 mm sucrose). protease inhibitors (10 g / ml leupeptin, 10 g / ml aprotinin, and 10 g / ml pepstatin, 10 g / ml tpck) were added to the last washing step. after egg crushing by centrifugation, the cytoplasmic extract was removed and protease inhibitors and 60 m nocodazole were added. meiosis ii m extracts were prepared without egg activation and cycloheximide addition, and the last washing step of the eggs was performed with tris buffer containing 15 mm egta. for some experiments, the transition from i to mitotic m extracts was induced by 1:0.75 (vol : vol) dilution of i extract in glycine buffer (300 mm glycine, 120 mm d - gluconic acid, 100 mm taurine, 20 mm kcl, 5 mm mgcl2, 5 mm egta, 30 m nocodazole, ph 7.2) supplemented with 0.1 mg / ml bacterially expressed cyc (glotzer., 1991) and after incubation at rt in the presence of 0.5 m rhodamine - phalloidin and atp - regenerating system (1 mm atp, 0.5 mg / ml phosphocreatine kinase, 10 mm creatine phosphate). the morphology of added sperm chromatin (murray, 1991) and the histone h1 kinase assays were used to monitor the status of the cell cycle and the activity of p34kinase (felix., 1993). i or m extracts were diluted by 1:0.75 (vol : vol) in glycine buffer. diluted extracts were supplemented with an atp - regenerating system and 0.5 m rhodamine - phalloidin and incubated for 15, 30, 45, and 60 min at rt. at each time point organelle motility was monitored by video - enhanced contrast differential interference (dic) microscopy, and the presence of rhodamine - phalloidin labeled f - actin was confirmed by fluorescence microscopy. at each time point of incubation, three random fields (23 m 22 m) were recorded for 3 min. the myosin - based motile activity was determined by counting moving organelles on tracks invisible with dic microscopy and averaging the numbers obtained from 12 random fields after all time points of extract incubation. for organelle motility during transition from interphase to metaphase, the extract was examined by microscopy immediately after dilution, and continuous recording of 30 random fields for 3 min during 90 min of observation was performed. er network formation was monitored by both dic microscopy and fluorescence microscopy after staining with 1 g / ml dioc6 (molecular probes). to stain the golgi apparatus, the extracts were incubated with 5 m c6-nbd - cer (n-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]-6-aminocaproyl d - erythro - sphingosine) (molecular probes) and examined by fluorescence microscopy. for immunofluorescence labeling of er, 510 l of extracts prepared as for dic microscopy were placed on a poly - l - lysine coated coverslip followed by incubation for 30 min at rt and then fixed by adding carefully 200 l of 4% pfa with 4% sucrose in pbs. after fixation for 30 min, immunofluorescence labeling of the er was performed as previously described (kaether., 1997) using an mab against rat ecast / pdi (stressgen biotechnologies) and dil2 polyclonal rabbit antibody against a gst fusion protein containing heavy chain residues 9101,106 of mouse myosin - va (wu., 1997). the effect of the following reagents on organelle motile activity and er network formation was tested : a dil2 antibody at 1:25 dilution, a polyclonal rabbit antibody raised against skeletal myosin ii (m7648 ; sigma - aldrich) at 1:25 dilution, and 5 m cytochalasin d (sigma - aldrich), 2 m staurosporine (calbiochem), and 2 m camkii (281309) inhibitor peptide (calbiochem). for this, the diluted extracts were preincubated with the agents mentioned above for 30 min at rt. membrane fractions were prepared by flotation as described in lane and allan (1999) which was slightly modified. 300 l of i extracts were diluted 1:0.75 (vol : vol) in glycine buffer containing an atp - regenerating system. for obtaining the membrane fraction from mitotic m extracts, diluted i extracts were supplemented with cyc. to study the effect of camkii activity on membrane protein phosphorylation, 2 m camkii inhibitor and 50 ci [-p]atp (amersham biosciences) were added to diluted extracts. all samples were incubated for 60 min at rt and then were diluted once again with 2.75 ml of ice - cold glycine buffer containing 65% sucrose and 10 mm -glycerophosphate. after centrifugation, the membrane fractions floated through 0.8 ml of glycine buffer with 50% sucrose were subjected to sds - page followed by western blot and phosphoimaging. the immunoprecipitation of myosin v from the membrane fractions using affinity - purified dil2 antibody was performed as described (kromer., 1998). the video illustrates the movement of globular organelles along f - actin observed with dic microscopy and described in fig. 1 b. video 2 shows movement of tubular membrane structures (er) on f - actin in meiosis ii m extract. the video illustrates the movement of tubular membrane structures (er) along f - actin observed with dic microscopy and described in fig. 1 c. video 3 shows f - actin dependent dynamics of er network in meiosis ii m extract. the video illustrates the movement along f - actin of tubular extensions of er network observed with dic microscopy and shown in fig. | it is widely believed that microtubule- and f - actin based transport of cytoplasmic organelles and membrane fusion is down - regulated during mitosis. here we show that during the transition of xenopus egg extracts from interphase to metaphase myosin v driven movement of small globular vesicles along f - actin is strongly inhibited. in contrast, the movement of er and er network formation on f - actin is up - regulated in metaphase extracts. our data demonstrate that myosin v driven motility of distinct organelles is differently controlled during the cell cycle and suggest an active role of f - actin in partitioning, positioning, and membrane fusion of the er during cell division. |
tennis elbow or lateral epicondylitis is the commonest cause of chronic pain on the lateral side of the elbow and wrist extensor dysfunction. the chief complaints in lateral epicondylitis are decreased grip strength, decreased functional activities and increased pain, which may impart significant disability in activities of daily living. historically, the primary lesion in lateral epicondylitis was considered to be inflammatory granulation tissue in the tendinous portion of the common extensor origin. recent studies of chronic tennis elbow have not found any significant evidence of inflammatory processes and the term epicondylosis or tendinosis has been suggested as a more appropriate term than epicondylitis. diagnosis of lateral epicondylitis is straightforward but there is no consensus on treatment while efficacy of existing treatments is poor. to reduce the need for surgery, recent studies show a beneficial role of locally delivered biological growth factors in form of platelet rich plasma (prp) and autologous blood in healing of various tendinopathies. according to a recent report by pascual - garrido., inoculation of bone marrow mononuclear stem cells (bm - mncs) aspirated from iliac crest could be considered as a potential therapy for those patients with chronic patellar tendinopathy refractory to non - operative treatments. a combination of bm - mncs and anabolic growth factors would seem as an ideal approach for managing tendinopathy of tennis elbow. bone marrow aspirated (bma) from iliac crest contains both prp and bm - mncs. the objective of this study is to evaluate the outcome of single injection of bma in tennis elbow. adult patients, 18 - 65 years old, were recruited from orthopedic and physiotherapy out - patient department of a tertiary medical college. the study was approved by the ethical committee of the medical college and attached hospital. a total of 30, both male (n = 18) and female (n = 12) patients of previously untreated tennis elbow were included in the study. a detailed clinical history and clinical examination along with standard anteroposterior and lateral radiographs of involved elbow only previously untreated patients of tennis elbow and having no other identifiable cause of lateral elbow pain were included in the study. informed written consent bone marrow plasma was aspirated from anterior - superior iliac spine of pelvis in 10 ml syringe containing 1 ml of heparin. 10 ml of bone marrow was centrifuged for approximately 20 - 30 min at 2000 rpm. of the centrifuged plasma, only the clear upper layer (containing plasma) and the buffy coat layer that contained mononuclear cells was used for injection and approximately 4 - 5 ml was obtained from each patient. all injections were administered taking aseptic precautions into the point of maximal tenderness at the extensor origin of the lateral epicondyle of the humerus by single author in all the cases. all subjects were advised to rest and moderate their activities to avoid aggravation of their symptoms. primary outcome measure was patient - rated tennis elbow evaluation (prtee), (100 points) assessed at baseline, 2, 6 and 12 weeks. it measures three dimensions : pain, function with the affected arm and usual activities. this differs from the visual analogue scale in that it is an ordinal scale as opposed to a continuous one. respondents are asked to circle the number that best describes the situation or condition stated in the question. the range of possible values is from 0 to 10, where 0 represents no pain or no difficulty and 10 represents worst pain imaginable or unable to do, depending on the subscale (pain vs. function / activities). sub scores for each dimension are scored as the mean of all the items in each particular dimension. the prtee is a reliable, reproducible and sensitive instrument for assessment of tennis elbow. the data was analyzed using the open epi version 3.01 software for windows (www.openepi.com, copyright (c) 2003, 2008 andrew g. dean and kevin m. sullivan, atlanta, ga, usa) and p < 0.05 were considered to be significant. statistical analysis of differences between pre- and post - injection evaluation was performed using the paired t - test. the minimum sample size required for this study minimum expected difference post - treatment was chosen to be 10 on the prtee scale and standard deviation was taken to be 15. adult patients, 18 - 65 years old, were recruited from orthopedic and physiotherapy out - patient department of a tertiary medical college. the study was approved by the ethical committee of the medical college and attached hospital. a total of 30, both male (n = 18) and female (n = 12) patients of previously untreated tennis elbow were included in the study. a detailed clinical history and clinical examination along with standard anteroposterior and lateral radiographs of involved elbow only previously untreated patients of tennis elbow and having no other identifiable cause of lateral elbow pain were included in the study. informed written consent bone marrow plasma was aspirated from anterior - superior iliac spine of pelvis in 10 ml syringe containing 1 ml of heparin. 10 ml of bone marrow was centrifuged for approximately 20 - 30 min at 2000 rpm. of the centrifuged plasma, only the clear upper layer (containing plasma) and the buffy coat layer that contained mononuclear cells was used for injection and approximately 4 - 5 ml was obtained from each patient. all injections were administered taking aseptic precautions into the point of maximal tenderness at the extensor origin of the lateral epicondyle of the humerus by single author in all the cases. all subjects were advised to rest and moderate their activities to avoid aggravation of their symptoms. primary outcome measure was patient - rated tennis elbow evaluation (prtee), (100 points) assessed at baseline, 2, 6 and 12 weeks. it measures three dimensions : pain, function with the affected arm and usual activities. this differs from the visual analogue scale in that it is an ordinal scale as opposed to a continuous one. respondents are asked to circle the number that best describes the situation or condition stated in the question. the range of possible values is from 0 to 10, where 0 represents no pain or no difficulty and 10 represents worst pain imaginable or unable to do, depending on the subscale (pain vs. function / activities). sub scores for each dimension are scored as the mean of all the items in each particular dimension. the prtee is a reliable, reproducible and sensitive instrument for assessment of tennis elbow. the data was analyzed using the open epi version 3.01 software for windows (www.openepi.com, copyright (c) 2003, 2008 andrew g. dean and kevin m. sullivan, atlanta, ga, usa) and p < 0.05 were considered to be significant. statistical analysis of differences between pre- and post - injection evaluation was performed using the paired t - test. the minimum sample size required for this study minimum expected difference post - treatment was chosen to be 10 on the prtee scale and standard deviation was taken to be 15. a total of 30 patients of previously untreated tennis elbow were recruited for this study out of which four were lost in follow - up. right elbow (n = 15) was involved more commonly than left elbow (n = 11). mean age of patients was 35.2 6.84 years and mean duration of symptoms was 7.33 2.49 weeks. baseline pre - injection mean prtee score was 72.8 6.97 which decreased to a mean prtee score of 40.93 5.94 after 2 weeks of injection which was highly significant (p < 0.0001). the mean prtee score at 6 week and 12 week follow - up was 24.46 4.58 and 14.86 3.48 respectively showing a highly significant decrease from baseline scores (p < 0.0001). tennis elbow is a tendinopathy which is a common and often debilitating condition that can be quite difficult to treat. conservative management consisting of activity restriction, splints and orthotics, non - steroidal anti - inflammatory drugs and physiotherapy are the first line of management. local corticosteroid injection is the most common treatment given in cases where conservative management fails. a recent cochrane review has concluded that glucocorticoid injection has only short term effect and it yielded poor results in long - term. other modalities such as prolotherapy, topical nitroglycerin, iontophoresis, phonophoresis, therapeutic ultrasound, extracorporeal shock wave therapy and low - level laser therapy have less evidence of effectiveness in treatment of tendinopathies. surgical debridement remains a last option for the treatment of tendinopathy because this has considerable cost and morbidity and modest success in treating chronic tendinopathy. growth factors have drawn a lot of interest in the field of tendon injury and repair. these humoral growth factors can be given in form of whole blood or prp injection. a recent review of such studies showed that injections of autologous growth factors (whole blood and prp) in patients with chronic tendinopathy had a significant improvement with prp being more effective. recently stem cell technology is being applied to the treatment of degenerative conditions of the musculoskeletal system such as tendinopathy, creation of tendon and ligament grafts and in enhancing graft incorporation. according to a recent report by pascual - garrido., inoculation of bm - mncs aspirated from iliac crest showed marked improvement in patients with chronic refractory patellar tendinopathy. once the stem cells are in the desired location, either local signaling or the addition of exogenous factors can drive the pluripotent cells to differentiate into the needed cell line. prp has been shown to influence the behavior of stem cells. using tendon stem cells derived from rabbit patellar tendons, zhang and wang demonstrated that prp releasate increased tendon stem cell proliferation, induced tendon stem cell differentiation into tenocytes and increased protein expression and collagen type i and type iii production. this explains the results of our study which show a highly significant improvement in terms of pain relief and down staging of the disease following a single injection of bma. another advantage of our method is simplicity and ease of application ; the time required for centrifugal separation is < 1 hour and does nt requires any special instrument and can be carried out in most clinics without any specialized staff. the major limitation of this study is the lack of control group, resulting in a low level of evidence study (level 4). long - term follow - up is required to see the sustained effect of bone marrow concentrate (bmc) injection in terms of pain relief and healing of disease. we have chosen a follow - up only up to 12 weeks as improvement in symptoms after this period may be a result of natural healing process and activity modification by patients. studies with longer follow - up are also required to see any adverse effects like calcification in tendon or tumorogenesis. no hematological analysis was performed to determine the numbers of nucleated cells or platelets in bmc. we did nt perform any pre- or post - injection radiological assessment of tendon healing by magnetic resonance imaging or ultrasonography. further studies are required to standardize the dose, number and timing of autologous bmc injection for treating refractory tennis elbow. we believe that bma injection could be considered as an alternative treatment for those patients who have failed non - operative treatment before surgical intervention is considered. in the future, growth factors and/or stem cells based injection can be developed as second line conservative treatment in chronic tendinopathy as they could potentially reverse the degenerative process and encourage the regeneration of healthy tendon. | objective : the objective of this prospective study was assessment of efficacy of bone marrow aspirate (bma) (containing plasma rich in growth factors and mesenchymal stem cells) injection in treatment of tennis elbow.materials and methods : a total of 30 adult patients of previously untreated tennis elbow were administered single injection of bma. this concentrate was made by centrifugation of iliac bma at 2000 rpm for 20 - 30 min and only upper layer containing platelet rich plasma and mononuclear cells was injected. assessment was performed at baseline, 2 weeks, 6 weeks and 12 weeks using patient - rated tennis elbow evaluation (prtee) score.results:baseline pre - injection mean prtee score was 72.8 6.97 which decreased to a mean prtee score of 40.93 5.94 after 2 weeks of injection which was highly significant (p < 0.0001). the mean prtee score at 6 week and 12 week follow - up was 24.46 4.58 and 14.86 3.48 respectively showing a highly significant decrease from baseline scores (p < 0.0001).conclusion : treatment of tennis elbow patients with single injection of bma showed a significant improvement in short to medium term follow - up. in future, such growth factors and/or stem cells based injection therapy can be developed as an alternative conservative treatment for patients of tennis elbow, especially who have failed non - operative treatment before surgical intervention is taken. |
cat - scratch disease (csd) is a zoonotic infection caused by the bacillus bartonella henselae, which can be transmitted to humans after contact with infected cats. typical presentation is characterized by a primary lesion (papule) at the site of inoculation (cat scratch or bite) followed by the development of regional painful and/or suppurative lymphadenopathy that is, occasionally, associated to systemic symptoms (fever, discomfort, etc.) and tends to have spontaneous resolution in a few weeks [1, 2 ]. atypical presentations or disseminated infection, except for immunosuppression, are exceptional, even though cases showing neurological manifestations (encephalitis, facial paralysis, transverse myelitis, etc.), ophthalmologic complications, hepatosplenic granuloma, osteomyelitis, endocarditis, prolonged fever, and so forth, have been described ; in these cases, diagnostic suspicion and immediate antibiotic therapy are fundamental [38 ]. we present a case of unilateral neuroretinitis associated to bartonella henselae infection in a teenager with optimal response to antibiotic treatment. a 13.7-year - old patient was admitted to the emergency department because of vision loss starting seven days before admission, ocular pain, and photopsia in the left eye. symptoms were sudden (at the time she was doing a school test) and coincided with an upper respiratory tract infection with fever in the previous 2 - 3 days. personal history : asthma (allergy to dust mites, pollen, and animal epithelium) and strabismus / astigmatismus. familiar history : she has a sister suffering from idiopathic partial epilepsy, a paternal uncle who suffered a cerebral infarction (cerebral aneurysm), a paternal aunt suffering from multiple sclerosis, and a maternal grandfather who suffered a cerebral stroke. ophthalmologic examination revealed a decrease of visual acuity in the left eye (right eye = 0.9, left eye = 0.3), disc swelling and macular star exudates with detachment of neuroepithelium (figure 1), and central scotoma in campimetry. blood analysis showed leukocytosis and neutrophilia and an increase of erythrocyte sedimentation rate (esr = 72 mm) and c - reactive protein (crp = 12 mg / dl). biochemistry, proteinogram, coagulation, immunological, antinuclear antibodies, and rheumatoid factor studies were normal. pressure (170 mmh2o), leukocyte count, and protein and glucose in cerebrospinal fluid (csf) results were normal, and monoclonal bands were not detected. serological studies (hsv - i, hsv - ii, adenovirus, cmv, ebv, vih, vdrl, mycoplasma pneumoniae, rickettsia conorii, borrelia burdoferi, and toxoplasma) were also negative, except for antibodies titer to bartonella henselae (indirect immunofluorescence, (ifi)) : igm 1/80 (positive > 1/10) and igg 1/800 (positive > 1/100). visual evoked potential test showed an increase in latency and a decrease in amplitude in the left eye. auditory and somatosensory evoked potential tests were normal. progression. on initial findings (neutrophilia and increase in acute - phase reactants) antibiotic therapy with cefotaxime several days later (negative bacteriology, visual evoked potential alteration, and normal neuroimaging) oral prednisone was added to her treatment (80 mg/24 hours, 10 days), and after clinical and epidemiological suspicion of neuroretinitis associated to cat - scratch disease (serology was still unknown), treatment with cefotaxime was ended, and rifampicin (300 mg/12 hours) and doxycycline (100 mg/12 hours) were prescribed. the patient remained hospitalized for 15 days, and, by the time of discharge, retinography image and visual acuity had improved (left eye = 0.6), visual evoked potential tests were normal, and antibody titers to bartonella henselae had increased (igg = 1/1600). after six weeks of antibiotic therapy, ophthalmologic exploration was completely normal, and visual acuity had recovered (left eye = 0.9, right eye = 0.9). bartonella henselae infection is usually a self - limited and oligosymptomatic disease in immunocompromised patients. it usually manifests as a regional painful lymphadenopathy preceded by an erythematous papule and/or pustule located in the place of a cat scratch or bite. atypical presentations are quite variable in patients likely to be immunocompromised, and neuroretinitis associated with cat - scratch disease is not frequently seen [1, 5, 9, 10 ]. neuroretinitis usually appears a few weeks after typical symptoms of this pathology manifest, which many times goes unnoticed. the main symptom is the abrupt unilateral loss of visual acuity although cases with bilateral affectation have been described [1113 ]. the finding of disc swelling associated to macular star exudates, as it happened in this patient, is considered as a predictable sign of an ocular manifestation of cat - scratch disease [9, 14, 15 ]. however, other etiologies should be discarded as a cause of optic neuropathy, such as arterial hypertension, diabetes mellitus, pseudotumor cerebri, syphilis, tuberculosis, toxoplasmosis, lyme disease, hiv, leptospirosis, and even multiple sclerosis and/or acute disseminated encephalomyelitis (although the characteristic star exudates are not observed in demyelinating diseases [5, 9, 16 ]). other ophthalmologic findings related to bartonella henselae infection have been described, such as massive subretinal exudates, multifocal retinochoroiditis, diffuse retinal hemorrhages, vascular occlusive episodes, and necrotizing retinitis. periodic ophthalmologic examination is mandatory in these cases [9, 15, 17 ]. diagnostic suspicion in atypical presentations is difficult, above all in those cases in which there are not any presentations of skin lesions and/or lymphadenopathy, as did occur in this adolescent ; this could explain the possibility of delay in diagnosis and treatment [8, 10, 11, 18 ]. diagnosis is mainly serological through enzyme immunoassays or indirect immunofluorescence with a high sensibility and specificity [10, 11, 15, 1820 ]. dna sequencing from gland tissue samples and visceral granulomas require invasive and expensive procedures. in this case, diagnosis was accomplished through serological confirmation by indirect immunofluorescence of an increased igm antibody titer to bartonella henselae although a significant increase in igg antibodies titer was observed, enough to consider an acute infection by bartonella sp. since the disease is fairly benign, azithromycin prescription has been proposed exclusively in those patients with general symptoms or big and/or painful lymphadenopathies as well as immunocompromised patients. nevertheless, antibiotic therapy is recommended in every patient with an atypical or disseminated presentation of neuroretinitis caused by bartonella henselae despite its evolution is usually benign. the possibility of ophthalmic irreversible structural lesions [5, 17, 18, 21, 22 ] suggests prescription of combined antibiotic therapy : rifampicin and doxycycline in patients older than eight and rifampicin with azithromycin or co - trimoxazole for patients below that age of four to six weeks. in this patient, antibiotic prescription was based upon clinical and epidemiological suspicion, and steroids were prescribed despite questionable usefulness [9, 15, 16, 22 ] after the visual evoked potential test findings. quick and complete recovery of ophthalmic symptoms experienced by this patient, in contrast to other cases referred to by the literature [11, 12, 18 ], could be in relation to the prompt beginning of treatment. as a conclusion, cat - scratch disease with associated neuroretinitis is quite rare. it should be suspected in any patient that manifests an abrupt loss of visual acuity together with the finding of disc swelling and macular star exudates. | cat - scratch disease - related neuroretinitis is a relatively unusual pathology, with suspicious clinical epidemiological and serological diagnosis. we present a case of an adolescent suffering from unilateral neuroretinitis associated with bartonella henselae infection characterized by abrupt loss of vision, optic disc swelling, and macular star exudates with optimal response to antibiotic treatment. |
the cerebral cortex (which includes the hippocampus, the entorhinal cortex, the piriform cortex, and the neocortex) is the origin of the most sophisticated cognitive functions and complex behaviors. indeed, the constant computation of incoming sensory information is dynamically integrated to provide a coherent representation of the world, elaborate the past, predict the future, and ultimately develop a consciousness and the self. in particular, the specific activity states of intricate cortical networks often produce a wide range of rhythmic activities, believed to provide the computational substrate for different aspects of cognition and various behaviors [1, 2 ]. cortical oscillations range from slow - wave activity (100 hz), with several intermediate rhythms (e.g., theta, beta gamma), each of which is considered to underlie specific cognitive aspects, such as non - rem sleep (slow - waves), sensory integration (gamma), working memory (theta), and motor planning (beta). importantly, inhibitory neurons were proposed to play a fundamental role in the genesis of most of these rhythms [313 ] through the specialized activity of their gabaergic synapses [710 ]. in fact, it is noteworthy that malfunctioning of specific gabaergic circuits is often indicated as a leading pathophysiological mechanism (among others) of psychiatric diseases, such as schizophrenia and autism [1418 ]. one of the hallmarks of synaptic transmission is its ability to be modified by certain activities or specific modulators. modifications of synaptic strength can occur in a short- (seconds) or long - term (from hours to days) fashion. in the last decades, the plasticity of excitatory glutamatergic synapses was extensively studied as it has been proposed to be the synaptic correlate of learning and memory [1921 ]. in contrast, plasticity of gabaergic synapses received less attention until recently, when it became clear that also inhibitory synapses can undergo short- and long - term plasticity. however, the underlying mechanisms for gabaergic plasticity are not completely understood, given also the staggering diversity of inhibitory neurons embedded in cortical circuits and their equal heterogeneity of synaptic properties [3, 9, 2338 ]. here, we review some aspects of gabaergic synaptic plasticity in the context of the great disparity of gabaergic interneuron classes and the putative roles of specific changes of gabaergic synaptic strength during cortical operations. covered several aspects of gabaergic synaptic plasticity, focusing on the pre- versus postsynaptic induction and expression mechanisms (see in table 1 in). in the mammalian cerebral cortex, the stereotyped interactions of multiple neuron types arranged in layers result in complex networks composed by excitatory (glutamatergic) and inhibitory (gabaergic) neurons. although some heterogeneity of cortical excitatory neurons exists in terms of anatomy, electrophysiology, and connectivity patterns [4046 ], the morphological and physiological properties of excitatory neurons are relatively homogeneous. in contrast, inhibitory neurons of cortical structures encompass a vast number of different cell types [3, 23, 3438 ]. for example, in ca1 region of the hippocampus, 16 different types of interneurons have been identified so far. inhibitory neurons release gaba and are locally projecting cells, hence their interneuron denomination, indicating that cell body, dendrites, and axonal projections, are confined within the same anatomical area. the vast majority of interneurons show aspiny dendrites, or a relatively small spine density, and, unlike glutamatergic cells, they can be contacted by both glutamatergic and gabaergic synapses at the soma. the classification of interneurons is based on the expression of certain calcium binding proteins and/or neuropeptides, specific electrophysiological signatures (action potential waveform and dynamic range), and functional characteristics of synapses that they form and receive, as well as specific anatomical and morphological properties [7, 25, 27, 3438, 49, 50 ]. overall, interneurons provide inhibition to neuronal networks and dictate the temporal pattern of activity of principal pyramidal and other inhibitory neurons. in this context, the rich diversity of gabaergic cells operates a division of labor during cortical activities (oversimplified in figure 1) [11, 13 ], and the specific roles played by each interneuron subtype in the functional organization of cortical networks has only recently begun to be elucidated. whereas interneuron dendritic morphology is highly variable, the axonal arborization can reveal specific functional features (figures 2(a) and 2(b)). indeed gabaergic interneurons are specialized in targeting specific domains of excitatory principal cells, and specific patterns of axonal projection result in one of the most relevant functional classifications of interneurons. for instance, oriens to lacunosum - moleculare (ol - m) neurons in the hippocampus and their neocortical counterpart, the martinotti cells, represent a prominent type of dendrite - targeting interneurons [28, 37, 52, 53 ]. other dendrite - targeting interneurons include the neurogliaform cells [35, 5457 ], the bi - stratified and tri - stratified interneurons [3, 38, 58 ], and ivy cells in the hippocampus. all these cell types target the dendrites of pyramidal neurons (at different distances) and are thus optimally predisposed to filter synaptic glutamatergic inputs that are exclusively present on pyramidal cell dendrites (figure 1) [41, 60 ]. on the other hand, basket cells (bcs, representing ~50% of all inhibitory neurons) are specialized in targeting the soma and proximal dendrites of pyramidal cells. by setting the timing of action potentials of many pyramidal neurons, bcs crucially regulate the neuronal output and promote synchronous discharge of a large population of principal cells (figure 1) [5, 6, 10 ]. moving along the dendro - soma - axon line of pyramidal neurons, another type of interneuron is specialized in targeting the axonal initial segment of principal cells : the axo - axonic or chandelier cells [23, 35, 37, 61 ]. gabaergic synapses formed by these cells on axons of pyramidal neuron suggest a powerful role as controllers of their output (figure 1). a clear functional distinction of the division of tasks between axo - axonic and perisomatic targeting interneurons is still unclear, as both cell types target the output region of pyramidal neurons. interestingly, gabaergic synapses from neocortical axo - axonic cells were recently found to exert a paradoxical excitatory role, promoting action - potential generation in pyramidal neurons [6264 ], although this is still matter of debate [64, 65 ]. in the hippocampus and cortex, parvalbumin (pv) expressing basket cells can sustain high - frequency firing (hence their fast - spiking or fs denomination) and receive strong and fast glutamatergic input that relies mainly on ampa receptors and efficiently recruits them during cortical activity [7, 25, 66, 67 ]. pv+ bcs are selectively surrounded by polyanionic chondroitin sulfate - rich perineuronal nets, which seem to play an important role in controlling ocular dominant plasticity in the neocortex [69, 70 ] and protect erasure of fear memories in the amygdala. fs bcs release gaba very reliably due to the tight coupling between ca channels and ca sensors at their terminals [72, 73 ] and are extensively interconnected through chemical and electrical synapses [49, 7478 ]. in particular, in the neocortex, fs bcs make a large number of synaptic contacts with themselves (autapses) [7982 ] that modulate their own spike frequency and greatly contribute to improve precise spike - timing. all these features allow pv+ bcs to synchronize a large population of principal cells and are thus believed to be the clockwork of cortical networks as they entrain oscillations that underlie several complex cognitive functions, including sensory integration, attention, exploratory behavior, sleep, and several forms of memory. remarkably, fs interneurons might promote network desynchronization in response to certain pattern of intense activity. this effect is mediated by massive asynchronous release of gaba from fs interneurons both at autapses and synapses with pyramidal cells resulting in reduced spike - timing precision. in contrast, interneurons belonging to another perisomatic targeting interneuron subclass express cannabinoid receptor type 1 (cb1rs) and the neuropeptide cholecystokinin (cck), can not sustain high - frequency firing, are contacted by less glutamatergic synapses, and their soma - targeted synapses tend to release gaba asynchronously and unreliably, often resulting in prolonged inhibition of target cells [30, 31 ]. remarkably, gabaergic synapses formed by cck+ bcs are negatively modulated by endocannabinoids yielding to both short- and long - term synaptic plasticity [8486 ] (see below). importantly, alterations of cortical inhibition were implicated in several neuropsychiatric (e.g., schizophrenia, autism, mood disorders) [14, 1618, 8789 ] and neurological (e.g., epilepsy, and rett syndrome) diseases [90, 91 ]. several lines of evidence indicate that the pathological mechanisms leading to the development of these diseases do not affect inhibitory circuits globally, but they seem to be restricted to specific interneurons types. indeed, animal model of these diseases and postmortem analysis of human tissue [93, 94 ] indicate a decreased number and function of pv+ bcs. in line with these anatomical results, abnormal oscillatory activity was associated to schizophrenia, autism, and epilepsy [95, 96 ]. conversely, the prominent subcortical aminergic input to cck basket cell [97, 98 ] has prompted the hypothesis that this particular bc subtype is the substrate of plastic changes that control mood and its disorders. however, an increasing amount of evidence suggests that pv+ basket cells are indeed the target of several neuromodulators such as cck, opioids, and serotonin [99101 ] and could be affected by hormones and stress that has a facilitating role towards the development of depressive disorders [102, 103 ]. since the discovery of activity - dependent potentiation of synaptic strength in the hippocampus, considerable effort has been done to elucidate the mechanisms underlying the plasticity of glutamatergic transmission as it is supposed to rule the functional and structural refinement of synaptic contacts and be the neuronal correlate of learning and memory. conversely, plasticity of gabaergic synaptic transmission has received much less attention, but an increasing effort made during the last two decades is starting to give us some cues about the mechanisms and roles of inhibitory plasticity. today, there are examples of gabaergic plasticity in many different brain areas such as cerebellum, brain stem, deep cerebellar nuclei, vta, thalamus, lateral superior olive, and amygdala. in the cortex and hippocampus, both long- and short - term changes in gaba transmission were described. retrograde synaptic signaling has emerged as one of the major mechanisms for gabaergic synaptic plasticity. indeed, postsynaptic depolarization- or activity - dependent short - term suppression of presynaptic gaba release was described in the early 90s in the hippocampus and cerebellum and termed depolarization - induced suppression of inhibition (dsi) [108, 109 ]. in 2001, it was shown that endogenous cannabinoids (or endocannabinoids ; ecbs) are the actual retrograde messengers mediating this post- to presynaptic communication (figure 3(a)) [105, 106, 110113 ]. ecbs are ubiquitous signaling molecules through the cns. in the cortex and hippocampus, 2ag and anadamide, the two major endogenously produced cannabinoids [106, 114116 ], are responsible for different forms of plasticity of gabaergic neurotransmission, including short- and long - term modification of synaptic strength and homo- and heterosynaptic forms of plasticity [85, 107, 111 ]. ecbs can be synthesized on demand, in response to many stimuli such as postsynaptic depolarizations, increased ca concentrations, action potential trains and metabotropic glutamate (mglu), dopamine, and acetylcholine receptor activation. after their synthesis, ecbs travel backwards from the postsynaptic cell where they are produced to presynaptic terminals and generate a short - term (seconds to minutes) and/or long - term (minutes to hours) suppression of gaba release through activation of cb1 receptors, g - protein coupled receptors, located mainly on presynaptic terminals [85, 106, 114 ]. distinct stimuli set the duration of cb1r - mediated plasticity by activating different downstream signaling mechanism. short - term postsynaptic depolarization results in short - term gabaergic transmission inhibition, (dsi, figure 3(a)) that occurs through inhibition of voltage - dependent calcium channels by cb1rs [106, 107 ]. intense high - frequency synaptic stimulations of afferent fibers induce a long - term disinhibition of pyramidal cells in ca1 area of the hippocampus (figure 3(b)) [86, 107, 111 ]. this form of long - lasting plasticity of gabaergic transmission, termed ecb - dependent long - term depression (ecb - ltd), depends on cb1r - mediated regulation of presynaptic protein kinase a (pka) and the phosphatase calcineurin [117, 118 ]. these two signaling proteins control a cascade that results in long - term inhibition of the presynaptic release machinery. another form of ecb - independent retrograde signaling has been described in cortical gabaergic synapses formed by nonaccommodating fs cells and pyramidal cells in layer 2/3 of the cortex. zilberter showed that increase of postsynaptic pyramidal - cell ca concentrations induced by trains of action potentials results in a short - term decrease of gabaergic transmission between these two cell types. pair - pulse ratio analysis indicated a presynaptic locus for this phenomenon and suggested the involvement a retrograde signal. although increases in pyramidal neuron dendritic ca levels are a triggering signal for the synthesis of ecbs, fs cells in l2/3 of the cortex do not express detectable cb1rs, therefore ruling out the participation of ecbs in this form of plasticity. further investigations have shown that this form of disinhibition is likely mediated by somatodendritic release of glutamate - filled vesicles expressing the vesicular glutamate transporter vglut3 with consequent activation of presynaptic metabotropic glutamate receptors (figures 4(a) and 4(b)) [119, 120 ]. spike timing - dependent plasticity (stdp) is a form of synaptic plasticity that requires both pre- and postsynaptic firing, inducing changes in synaptic strength whose polarity (potentiation or depression) depends on the temporal order of pre- and postsynaptic spiking. glutamatergic stdp has been shown to follow precise general rules : long - term potentiation (ltp) of synaptic transmission is produced when presynaptic spiking precedes (in a millisecond time window) postsynaptic action potential, whereas ltd is induced when postsynaptic spikes precede presynaptic action potentials [122124 ]. stdp of gabaergic synapses (and of glutamatergic synapses onto inhibitory cells) has only recently been investigated and seems a bit more complex than glutamatergic stdp. indeed, in the hippocampus, a symmetric dependency was found : ltp of gabaergic connections was induced when pre- and poststimuli where paired at 20 milliseconds whereas longer intervals led to ltd. conversely, in the entorhinal cortex, gabaergic stdp follows the same temporal dependency as glutamatergic stdp. both hippocampal and entorhinal cortex spike - timing ltps depend on postsynaptic ca rises induced by back - propagating action potentials and were proposed to have a postsynaptic origin [126, 127 ]. interestingly, in hippocampal neurons (both cultured and in slices), it has been shown that coincident pre- and postsynaptic firing that results in ltp of gabaergic transmission produced a shift of the reversal potential for gaba - mediated (egaba) responses at this particular synapse. indeed, the coincident activity resulted in the inhibition of the cl cotransporter kcc2 resulting in a more depolarized egaba. given the rich heterogeneity of gabaergic interneuron subtypes, one key question is whether plasticity of gabaergic neurotransmission follows some general rules regardless of the gabaergic cell subtype or if specific inhibitory cell subclasses are more susceptible to develop certain forms of plasticity. remarkably, holmgren and zilberter demonstrated that in neocortical layer 2/3 unitary connections between fs interneurons and pyramidal neurons are substrate for long - term modification of synaptic strength induced by pairing pre- and postsynaptic action potentials. indeed, this study showed that ltp of gabaergic responses was induced when the presynaptic fs cell fires at least 400 ms after the postsynaptic pyramidal did. interestingly, the plasticity of this particular gabaergic synapse is bidirectional and ltd was induced if presynaptic fs fires during or shortly after a train of action potentials in a pyramidal cell (figures 4(c) and 4(d)). in contrast with the results observed in hippocampal cells, stdp of fs to pyramidal neurons did not alter the reversal potential for synaptic responses, suggesting an alternative mechanism for this form of plasticity. although the exact mechanism leading to stdp of fs to pyramidal cell gabaergic transmission is still unknown, the dependency on intact calcium signaling and unchanged pair - pulse ratio of unitary postsynaptic responses after conditioning does not favor a presynaptic origin. in line with a postsynaptic expression of gaba - mediated synaptic plasticity onto neocortical pyramidal neurons, recent evidence indicated the role of postsynaptic l- and r - type ca channels in activity state - dependent ltd and ltp of gabaergic inhibition in layer 5 pyramidal neurons. activity - dependent plasticity of gabaergic synapses has been demonstrated in adult cortex and hippocampus. both ltp and ltd of gabaergic transmission can be triggered by different forms of stimuli that consist mostly in high - frequency afferent stimulations [86, 129132 ]. several forms of heterosynaptic long - term changes of gabaergic responses were shown in adult hippocampus and have the activation of glutamatergic fibers as a common origin [86, 129 ]. although induction is invariably postsynaptic, the expression locus can be either pre- or postsynaptic. in ca1 region of the hippocampus, glutamate released by schaffer - collaterals activates mglurs, triggering the synthesis of ecbs that act presynaptically to reduce gaba release (see above) [86, 107, 111 ]. notably, a different study reported that glutamate induces postsynaptic ca increases through nmda receptors that, in turn, activate postsynaptic calcineurin. importantly, this calcium - sensitive phosphatase has been involved in the negative regulation of gabaa receptors activity resulting in a postsynaptic locus of expression for this form of gabaergic ltd. as a common theme, it seems that the induction of all these forms of gabaergic plasticity requires the sustained firing of the gabaergic cell that produced gabaergic ltd. this suggests a dual role of gabaergic interneurons : promoting synaptic plasticity and conferring synapse specificity [117, 133136 ]. another form of activity - dependent potentiation of inhibitory synaptic transmission is mediated by astrocytic calcium signaling in the hippocampus. in synaptically coupled pairs of interneurons and pyramidal cells, a train of high - frequency action potentials in the presynaptic inhibitory cell produces an increase in the probability of gaba release that lasted for 1520 minutes. indeed, interneuron firing and consequent release of gaba triggered gabab - mediated calcium signaling in astrocytes adjacently located to the inhibitory neuron. upon gabab receptor activation and through a mechanism dependent on ampa and nmda receptors, astrocytes induced potentiation of inhibitory transmission between interneuron and pyramidal cells. another form of gabaergic synaptic potentiation has been described in fs to stellate cells connections in layer 4 of mouse visual cortex. at this synapse, paring of presynaptic fs spikes with subthreshold depolarization of postsynaptic stellate cells resulted in a significant potentiation of the gabaergic synapses that lasted for at least 30 minutes. in this study, no changes in the ppr were detected and the reversal potential of synaptic responses remained unaltered. interestingly, this form of plasticity is prevented by coupling pre- and postsynaptic spikes suggesting that stdp at neocortical fs to principal cell connections is layer dependent. many examples of gabaergic synaptic plasticity come from studies focused on the development of cortical inhibitory circuits. indeed, in the developing mouse neocortex, gaba levels are modulated by neuronal activity and sensory experience through the regulation of the gad1 gene [139, 140 ], which codes for gad67, a glutamic acid decarboxylase that is the rate - limiting enzyme responsible for gaba synthesis. in turn, modified gaba transmission increases the number of synaptic contacts, axon branching, and innervation field of single perisomatic interneurons [142, 143 ]. in the dentate gyrus, both pre- and postsynaptic changes occur during development of gabaergic synapses originating from pv+ bcs, including increased amplitude, decreased failure rate, and decay constant of unitary inhibitory responses. these changes reflect a developmentally regulated plasticity of fs cell - mediated gabaergic transmission transforming this cellular element into the well - known precise synaptic metronome and fast signaling unit. despite the growing evidence in favor of gabaergic transmission as a pivotal mechanism for several functions of neuronal circuits, little is know about the actual role of activity - dependent modifications of inhibitory synapses in altering network activities that are strongly dependent on specific gabaergic circuits. in fact, functional consequences of changes in inhibitory synapse strength can vary dramatically depending on the interneurons subtype involved. indeed, different interneuron subclasses possess different mechanisms underlying basic gabaergic transmission, such as, for example, different expression of presynaptic voltage - gated ca channels and/or metabotropic receptors that modulate gaba release [10, 52 ]. since these differences result in specific modes of gabaergic transmission, it is likely that specific gabaergic synapses originating from specific interneuron types will generate different forms of plasticity in response to similar activity patterns. to complicate things even further, different classes of inhibitory interneurons are activated by glutamatergic synapses exhibiting peculiar properties, including short- and long - term plasticity and expression of specific ionotropic and metabotropic glutamate receptors [2629, 33, 37, 145 ]. this diversity of excitatory properties onto different interneuron classes was shown to underlie differential temporal recruitment of different gabaergic cell types during cortical activities, therefore limiting or promoting induction of downstream gabaergic plasticity in selective cell types. some indirect evidences for plasticity of gabaergic transmission arising from specific interneuron types were found in development when sensory activity is a critical regulator of gabaergic plasticity. for example, fs cell - mediated transmission in visual cortex was shown to develop an ltp at these inhibitory synapses in mice that were visually deprived (see above). in neocortical low - threshold spiking interneurons (including dendrite targeting martinotti cells) similar sensory deprivation (whisker trimming) induced a change in the pattern of inhibitory transmission, with increased amplitude and decay kinetics. on this line, sensory deprivation induces a decrease in the number of dendrite targeting gabaergic synapses in l4 and somatic targeting interneurons. the induction of plastic changes in gabaergic synapses may have different outcomes depending not only on the polarity or duration of the change, but also on the location and origin of these gabaergic synapses. in the hippocampus, stimuli that induce ltp of glutamatergic transmission this form of ltd is likely restricted to interneuron types expressing cb1 receptors that include cck - positive basket cells and schaffer collateral - associated (sca) interneurons [85, 150 ]. the plasticity of this gabaergic input has been shown to be responsible for the increased excitability of pyramidal cells after ecb signaling activation and for the epsp - to - spike (e - s) coupling, that is, an important component of ltp of glutamatergic transmission. the increased strength of gabaergic transmission between pv+ bcs and pyramidal neurons would decrease the excitation - to - inhibition ratio in the somatic compartment of principal cells and limit their time window for spike generation. since each pv+ bc contact a large number of pyramidal neurons, the plasticity of its gabaergic connections will influence a large portion of the network, and therefore change some global properties of network activities. this applies if plasticity of gabaergic synapses results from a broad change of presynaptic neurotransmitter release, regardless of postsynaptic activity. on the other hand, combined presynaptic and single pyramidal neuron firing might induce stdp modifying a small portion of gabaergic synapses. this can happen during theta and gamma activities, when the firing of pyramidal neurons and fs cells are temporally displaced as they are locked to different components of the oscillation phase [3, 13 ]. another form of fine regulation of few components of a network is represented by ecb - dependent synaptic plasticity. indeed, ecb - mediated decrease of perisomatic inhibition arising from cck+ interneurons will likely disinhibit and thus increase excitability of those single pyramidal cells that retrogradely delivered ecbs. in addition, since these signaling molecules are produced by highly active principal cells, ecbs are ideally placed to organize cell assemblies that fire in close relation during certain behavioral states, although the role of ecb - mediated retrograde signaling onto cck+ cells during oscillations and network activities is far from being clear [151, 152 ]. in this scenario, it is possible that sustained firing activities of pyramidal cells will induce an ecb - dependent overall depression of gabaergic transmission originating from cck+ interneurons. this will likely shift the balance of perisomatic inhibition towards the fast, precise, and reliable inhibition from pv+ basket cells, which are insensitive to ecbs. since these two types of interneurons differentially contribute to feed forward and feed back inhibition onto ca1 cells, retrograde ecb signaling has the potential of changing the integration properties of principal cells by narrowing the time window for spike generation and allowing increased temporal resolution [10, 146 ]. as detailed above, neocortical pyramidal cells use different mechanisms to selectively modulate specific sources of perisomatic gabaergic transmission in a retrograde fashion (ecbs in cck+ basket cellls versus glutamate in fs interneurons). it is still unclear, however, if these two modulation mechanisms can be uncoupled, thus leading to a change in the perisomatic inhibition balance originating from pv+ and cck+ basket cells. it has been shown that ecb - mediated suppression of gaba transmission is present at gabaergic synapses on pyramidal neurons but not on interneurons in layer 2/3 of the mouse neocortex [153, 154 ]. in the hippocampus, however, both gabaergic synapses on interneurons and pyramidal cells can be modulated by retrograde ecb signaling. in addition, gabaergic inputs to layer 5 pyramidal cells in the neocortex is cannabinoid - insensitive, whereas gabaergic synapses onto layer 2/3 principal cells are strongly modulated by retrograde ecb signaling [155, 156 ]. these observations raise the possibility that certain forms of ecb - mediated plasticity may rely on the identity and location of both pre- and postsynaptic neurons. therefore, specific activities can differentially suppress inhibition in distinct cortical layers and specific cell types (glutamatergic versus gabaergic). there is little (if any) direct evidence for plasticity of gabaergic transmission at distal dendritic sites, such as that provided by o - lm interneurons and martinotti cells in the hippocampus and neocortex, respectively. importantly, martinotti cells mediate a prominent disynaptic dendritic inhibition triggered by high - frequency firing of pyramidal neurons [157159 ]. plasticity of these gabaergic connections will, therefore, be crucial for information filtering by these dendrite - targeting interneurons. interestingly, the polarity of stdp of glutamatergic synapses depends on the location of the synapses within the dendritic arbor. the same timing of pre- and postspiking gives rise to ltd at most distal synapses, but ltp at more proximal dendritic synapses. it will be interesting to investigate whether interneurons targeting different compartments of principal cells, for example, dendritic versus somatic, have different plasticity rules and whether specific patterns of network activation have differential effects on inhibition arising from specific sources. in this paper we emphasized how the great diversity of interneuron types gives rise to an even greater diversity of gabaergic transmission and plasticity. indeed, the specific key role of each gabaergic circuit in sculpting different forms of cortical activity has only recently begun to be elucidated. since it has been shown that gabaergic synapses exhibit plasticity, it will be fundamental to reveal the governing rules of gabaergic transmission originating from different neuron subclasses. in addition to the interneuron type - specific forms of synaptic plasticity, several open questions remain, such as, for example : (i) what are the physiological activities (single neuron and/or network activities) necessary to induce plasticity of gabaergic synapses ? (ii) is there a heterogeneity or bidirectional plasticity of gabaergic synapses in different cortical areas ? (iii) what is the functional role of gabaergic transmission during different cortical activities ? (iv) what other neuromodulators, in addition to endocannabinoids and glutamate, can induce activity - dependent changes of gabaergic synaptic strength ? (v) could gabaergic plasticity lead to complex cl gradients inside a principal neuron, such that the direction (inhibition versus excitation) of gaba - mediated responses might, in some cases, contribute to some forms of hyperexcitability ? (vi) is plasticity of inhibitory synapses altered in pathological situations ? addressing these questions will help define the fundamental molecular, cellular, and synaptic mechanisms governing several core functions of cortical activities, therefore advancing our knowledge on the basic rules underlying complex cognitive and behavioral functions, with likely important implications for neurological and psychiatric diseases. | cortical structures of the adult mammalian brain are characterized by a spectacular diversity of inhibitory interneurons, which use gaba as neurotransmitter. gabaergic neurotransmission is fundamental for integrating and filtering incoming information and dictating postsynaptic neuronal spike timing, therefore providing a tight temporal code used by each neuron, or ensemble of neurons, to perform sophisticated computational operations. however, the heterogeneity of cortical gabaergic cells is associated to equally diverse properties governing intrinsic excitability as well as strength, dynamic range, spatial extent, anatomical localization, and molecular components of inhibitory synaptic connections that they form with pyramidal neurons. recent studies showed that similarly to their excitatory (glutamatergic) counterparts, also inhibitory synapses can undergo activity - dependent changes in their strength. here, some aspects related to plasticity and modulation of adult cortical and hippocampal gabaergic synaptic transmission will be reviewed, aiming at providing a fresh perspective towards the elucidation of the role played by specific cellular elements of cortical microcircuits during both physiological and pathological operations. |
this study is designed to show an evaluation of prospectively collected data of all consecutive adult patients admitted to our department of thoracic surgery after chest injures and opiate analgesia treatment between 1st october 2013 and 31 st march 2014 to analyze development of early inflammatory response. inadequately controlled pain after severe chest injures may still extend the length of hospital stay and predispose to expensive, time - consuming complications such as pneumonia (1). experimental and clinical investigations suggested that acute pain causes chronic pain very fast and has impact on long - term neurophysiological changes and quality of life with reduced morbidity and mortality (2). effective management of pain decreased intra hospital complications incidence, length of hospital stay and chronic pain syndrome occurrence (3). it has been concluded that good controlled pain management of severe chest injures will improve the outcome with reduced morbidity, need for repeated hospitalization and convalescence, and there is a common consensus that optimal pain relief is a prerequisite for early postoperative recovery (4). sixty patients were included consecutively between 1st october 2013 and 31 st march 2014 in prospective study with university clinical centre tuzla ethical committee approval. radial immunodiffusion method on dimension rxl- max sysmex - simens device was used to determine serum level of acute phase proteins fibrinogen (1.8 - 3.5 leukocytes and neutrophils cell by cell analyses were performed by the cel - dyn ruby system instrument. patients were divided in two homogeneous groups, 30 patients each, which were of the same age and sex. non - opiate intermittent analgesia of metamizol sodium was applied to the first group of patients and continuous tramadol hydrochloride opiate analgesia to the second group. all the patients had three measurements : the first - at time of admission, the second - 24 hour after and the third - 48 hours after admission. metamizol sodium was applied intramuscularly or intravenously, intermittently, in ampules of 5ml which contains 2,5 g. the maximum dose was 10g/24h. tramadol 50mg / ml solution was administered in ampule which contains 100 mg tramadol hydrochloride in 2ml solution (50mg / ml) for injection or infusion. the usual dose was 50 mg or 100 mg, 4 to 6 hourly by either intramuscular or intravenous routes, maximum 400mg/24h. the patients with polytrauma, diabetes mellitus, systemic chronic diseases and respiratory support were excluded from study. data were compared by the wilcoxon signed -rank test so that value of p<0.05 was considered significant in all tests. after the administration of analgesia, mean neutrophils serum levels were lower in the examined group, to which opiate analgesia was administered, in the second and third measurement with statistically significant difference (p=0.026 and p=0.03) as seen in table 1. mean serum sd neutrophiles values (109 /l) in two groups. p= 0.16 p = 0.026 p = 0.03 the dynamics of decreasing neutrophils serum values was slower among the patients in the controlled group. in the second measurement mean serum values in the controlled group were 5.421.36 and in the examined group they were 3.921.56, with statistically significant difference p=0.05. in the third measurement mean crp serum values were 4.80.78 in the controlled group whereas in the examined group they were 3.60.69 with the statistically significant difference p=0.025. in the controlled group, during the second measurement, patients showed increased crp serum values compared to the initial value, while crp serum value in the examined group were continually decreasing during each of three measurements in the 48-hour period. crp serum values in both groups during all measurements were above the referential serum values with tendency of returning within the normal range of values (table 2). p= 0.31 p=0.05 p=0.025 during the measurements in both groups of patients, mean serum values of fibrinogen have increased compared to the initial measurement, having in mind that the level of increase was much higher in group i. during each measurement serum values of fibrinogen were within referential values (table 3). p= 0.25 p= 0.82 p= 0.06 mean serum sd leucocytes values (109 /l) in two groups. p= 0.18 p= 0.23 p= 0.016 in the third measurement serum values of leukocytes in the controlled group were 5.470.90 and in the examined group they were 4.750.77 with statistically significant difference p=0.016 during the measurements, leukocyte serum values were decreasing compared to the initial values, taking into consideration that the values were lower in the examined group of patients compared to the initial values, than in the controlled group of patients. in all measurements mean serum values were within reverential range of values. of the total number of patients, 17 patients or 28.3% had fractures of one or two ribs and 13 patients or 21.6% had fractures of three or more ribs. in the group of patients administered by metamizole (group i / controlled group) were 9 patients or 30% who had up to 2 fractures of ribs whereas 8 patients or 26.6% were in the group administered by tramadol (group ii / examined group). 27 patients (45%) after x - ray and ct diagnostics had pulmonary parenchyma contusions of different degree. nine patients or 15% were diagnosed with initial stage of pneumonia, 6 patients or 20% were from the controlled group and 3 patients or 10% were from the examined group. i there were 13 patients with atelectasis or 43.3% and in group ii there were just 6 patients or 20% with diagnosed atelectasis. 11 patients or 18.35% were diagnosed with pleural effusion, 7 patients or 23% from group i and 4 patients or 13.3% from group ii. the length of hospital stay for the patients in group i was 7.31.15 days and for the patients in group ii it was 6.10.87 days with statistically significant difference p=0.017. suppressing the pain caused by chest trauma is the primary therapeutic procedure. by reducing the pain, patient has no restrictions in respiratory process and lowering the secretion of inflammation mediators results in lower tissue injury (5). as a result, faster better recovery of the patient is evident and the incidents of chronic pain syndrome occurrence, preventing the patient, after healing, to return to normal everyday life or causing repeated hospitalization or respiratory complications, are much more rare. the releasing of histamine and inflammatory mediators like bradykinin, prostaglandin, neurotransmitters or neutrophils, are conditioned by tissue injuries. the secretion of these mediators activate peripheral nociceptors and by transduction and transmission, nociceptive information are communicated to cns (6). in acute phase it responds to trauma and stress, initiate the secretion and the increase of serum values of acute phase proteins (7). all inflammatory mediators, at certain point, have protective role, although in the case of massive tissue injuries, their secretion can have harmful impact on the tissue. opioids are one of the most frequent options when it comes to the treatment of postoperative and post traumatic pain (8). this agent acts through receptors in cns even though there are proofs of existence of peripheral opioid receptors (9). analgesic activity of opioids is limited by development of tolerance and secondary effects like nausea, vomiting, sedation and respiratory depression. the advantage of tramadol use as postoperative analgesia is the absence of respiratory depression, systemic toxicity, gastrointestinal motility and low percentage of drug abuse. the administration of tramadol has lowered the number of leukocytes after 24 hours and 48 hours more efficiently than the administration of metamizole sodium (table 1). decreasing of crp serum values during the second measurement (after 24 hours) among the patients in the controlled group after the administration of tramadol was more than evident. taking into consideration crp synthesis during the tissue injury, it is essential to suppress it to avoid more serious tissue injuries and prevent any further complications (10). the administration of tramadol decreased mean crp serum values more efficiently than the use of metamizole sodium. the efficiency of tramadol in pain suppression resulted in better respiratory functions and less frequent occurrence of pneumonia. pneumonia can extend the length of hospital stay and cause other complications to patients with co morbidity (the occurrence of other diseases). in group i, 6 patients (20%) had pneumonia and in group ii 3 patients (10%) had pneumonia. of the total number of patients, 19 patients or 31.6% had atelectasis. in group i 13 patients or 43.3% had atelectasis and in group ii just 6 patients or 20% had atelectasis. therefore, in the group of patients administered by tramadol after the injury, the incidents of atelectasis occurrence were less frequent. these patients had shorter period of hospital stay, namely for group i it was 7.31.15 days and for group ii it was 6.10.87 days with statistically significant difference p=0.01. the level of early inflammatory response is lower among the patients with severe chest injuries treated with opiate analgesia treatment than those treated with non - opiate analgesia. considering that tramadol is a semi - synthetic opioid and its efficiency as analgesia is more intense than metamizole sodium, its use in treatments of pain caused by chest trauma has significantly better results in suppressing neutrophils and crp activation thereby lowering the occurrence of secondary injuries caused by inflammatory mediators to minimum with a lower incidence of complications and shorter hospital stay. | background : the frequency of severe chest injuries are increased. their high morbidity is followed by systemic inflammatory response. the efficacy of pharmacological blockade of the response could prevent complications after chest injures.aim:the aim of the study was to show an inflammatory response level, its prognostic significant and length of hospital stay after chest injures opiate analgesia treatment.methods:sixty patients from department of thoracic surgery with severe chest injures were included in the prospective study. with respect of non opiate or opiate analgesia treatment, the patients were divided in two groups consisted of 30 patients. as a inflammatory markers, serum values of leukocytes, neutrophils, c - reactive protein (crp) and fibrinogen in three measurements : at the time of admission, 24hours and 48 hours after admission, were followed.results:statistically significant differences were found between the examined groups in mean serum values of neutrophils (p=0.026 and p=0.03) in the second and the third measurement, crp (p=0.05 and 0.25) in the second and the third measurement and leukocytes in the third measurement (p=0.016). 6 patients in group i and 3 in group ii had initial stage of pneumonia, 13 patients in group i and 6 in group ii had atelectasis and 7 patients from group i and 4 from group ii had pleural effusion. the rate of complications was lower in group of patient who were under opiate analgesia treatment but without significant difference. the length of hospital stay for the patients in group i was 7.31.15 days and for the patients in group ii it was 6.10.87 days with statistically significant difference p=0.017.conclusion : the opiate analgesia in patients with severe chest injures reduced level of early inflammatory response, rate of intra hospital complications and length of hospital stay. |
natural and unexpected death that happens within less than one hour of first symptom occurrence is called sudden death (1 - 4). sudden death is more common among men and the black race (5, 6). in a systematic review based on data related to 1980 - 2007, annual incidence of sudden cardiac death (scd) in the united states varied between 180000 and 450000 (7). in some other studies, scd rate range was reported 50 - 100 per 100000 of general population in countries such as netherlands, ireland, and china (8). interestingly, in about 25% of patients who die of coronary artery atherosclerosis, the first sign is sudden death. after coronary artery diseases, cardiomyopathies including hypertrophic and dilated types are among the second cardiac causes of sudden death (1). dysfunctions of the heart valves, especially aortic stenosis, are considered as other underlying cardiac causes of sudden death (9). hypertrophic cardiomyopathy is the most common cause of sudden death in people under thirty years of age (10). most patients with hypertrophic cardiomyopathy are asymptomatic and death can occur at rest or with mild physical activity, however, sudden death often occurs during intense physical activity (10, 11). in many cases, hypertrophic cardiomyopathy is the only autopsy finding in young victims of sudden death and is the most common reason of sudden death during exercise (12). drug abuse (such as stimulants, anabolic steroids, opioids, and etc.), bronchoconstriction, heat stroke, and drowning can be mentioned as non - cardiac causes of sudden death in athletes but cardiac causes are more important (9, 13 - 15). about 69% of sudden deaths in general population are attributed to cardiovascular diseases, while more than 75% of sudden deaths in athletes are assigned to it (13, 16). to emphasize the importance of this topic, here we review the forensic reports of all cases of sudden death following exercise, referred to forensic medicine center of tehran, iran, from 2009 to 2014. in this cross sectional study, all subjects who were registered to forensic medicine center of tehran, iran, from 2009 to 2014, as a case of sudden death following exercise were evaluated. demographic data (age, sex) and medical history as well as autopsy and toxicology findings were gathered using deceased profiles, retrospectively. the protocol of study was approved by the ethics committee of shahid beheshti university of medical sciences. 14 cases were registered as sudden death following exercise in forensic medicine profiles during the study period. exploring the files of the mentioned deceased, revealed five non - compatible cases in this regard. the cause of death was homicide in one case (4.5 years old boy), drowning in another (38 year - old female), and myocardial infarction in the last three cases (all above 40 years old). the mean age of the deceased was 28.66 10.86 years (range : 7 40). toxicological tests were available for 7 cases, which was positive for tramadol in one case. only 3 (33.3%) cases had herald signs such as chest pain, syncope, or loss of consciousness. baseline characteristics and autopsy findings of the studied deceased : year ; na : not available. in recent years, concerns and interests of the people and physicians regarding causes of sudden death in athletes have impressively increased. congenital heart defects, hypertrophic cardiomyopathy, coronary artery disease, and myocarditis are among the most prevalent causes of scd in patients under 35 years of age. on the other hand, acquired coronary artery disease is a common cause of sudden death in over 35 year old population (13 - 16). sudden death can occur in approximately all types of sports but it has been more frequently reported in sports like football and basketball. in the current study, most deaths had occurred after football and swimming. in some studies, the risk of sudden death following exercise in men has been reported to be 5 times more than women and the findings of this study has demonstrated this matter (16). baggish and colleagues studied cardiovascular screening effects with and without electrocardiography (ecg) in the 510 collegiate athletes of united states. involving ecg, compared to its non - use in the screening, increased identification of cardiomyopathy cases, sensitivity of screening program (from 45.5% to 90.9%), and negative predictive value (from 98.7% to 99.8 %) (17). mild symptoms such as slight chest pain and other symptoms that indicate heart problems such as exertional dyspnea should be seriously regarded and taught to all people, so that similar cases can be prevented. the authors of this article recommend asking the necessary questions listed in table 2 for primary screening of at risk patients for sudden death following exercise. in case of any positive answer to the mentioned questions it should be mentioned that not all cases of sudden death are referred to forensic medicine center for autopsy, especially in cases of clear diagnosis such as acute myocardial infarction or brain insults (hemorrhagic, ischemic, etc.). although most sudden death victims are asymptomatic until the event, all those who suffer from symptoms such as chest pain, shortness of breath, dizziness, fatigue and irregular heart rate during physical activities, should be screened regarding common probable causes of sudden death. all authors passed four criteria for authorship contribution based on recommendations of the international committee of medical journal editors. | introduction : natural and unexpected death that happens within less than one hour of first symptom occurrence is called sudden death. cardiovascular diseases are the main known reason of sudden death and more than 75% of sudden deaths in athletes are assigned to it. here we reported the autopsy results of all cases with sudden death following exercise that were referred to forensic center of tehran, iran, from 2009 to 2014. methods : in this cross sectional study all subjects who were registered to forensic medicine center of tehran, iran, from 2009 to 2014, as a case of sudden death following exercise were evaluated. demographic data and medical history as well as autopsy and toxicology findings were retrospectively gathered using profiles of the deceased. results were reported using descriptive analysis. results:14 cases were registered as sudden death following exercise in forensic medicine profiles during the study period. exploring the files of the mentioned deceased, revealed five non - compatible cases in this regard. finally, 9 eligible cases were enrolled (88.9% male). the mean age of the deceased was 28.66 10.86 years (range : 7 40). toxicological tests were available for 7 cases, one of which was positive for tramadol. sudden death following football was reported most frequently (44.4%). only 3 (33.3%) cases had herald signs such as chest pain, syncope, or loss of consciousness. 1 case (11.11%) had a positive history of sudden death in relatives. conclusion : although most sudden death victims are asymptomatic until the event, all those who suffer from symptoms such as chest pain, shortness of breath, dizziness, fatigue and irregular heart rate during physical activities, should be screened regarding common probable causes of sudden death. |
to measure the soft and hard - tissue thickness of the areas mostly interested by miniscrews insertion, such as buccopalatal interdental bone and maxillary mid - palatal suture, 12 cadavers were selected with all maxillary premolars and molars still intact. to measure the incisal areas, 12 cadavers with intact maxillary occlusal planes (at least incisors and first molars remaining) were selected. cross - sectional specimens were made at three areas : the interdental area between the first and second premolars (group 1), the interdental area between the second premolar and the first molar (group 2), and the interdental area between the first and second molars (group 3). the thickness of soft and hard - tissues were measured at five sectional areas parallel to the buccopalatal cementoenamel junction (cej) line at 2 mm intervals [figure 1 ] for each group. measurement of the thickness of the soft- and hard - tissues on the buccal side and palatal side of the sectioned specimen on parallel lines drawn 2, 4, 6, 8, and 10 mm, from cementoenamel junction outer point of the buccal side of the sectioned specimen on a parallel line drawn 2 mm superior to the cej lineouter point of the buccal side of the sectioned specimen on a parallel line drawn 4 mm superior to the cej lineouter point of the buccal side of the sectioned specimen on a parallel line drawn 6 mm superior to the cej lineouter point of the buccal side of the sectioned specimen on a parallel line drawn 8 mm superior to the cej lineouter point of the buccal side of the sectioned specimen on a parallel line drawn 10 mm superior to the cej lineouter point of the palatal side of the sectioned specimen on a parallel line drawn 2 mm superior to the cej lineouter point of the palatal side of the sectioned specimen on a parallel line drawn 4 mm superior to the cej lineouter point of the palatal side of the sectioned specimen on a parallel line drawn 6 mm superior to the cej lineouter point of the palatal side of the sectioned specimen on a parallel line drawn 8 mm superior to the cej lineouter point of the palatal side of the sectioned specimen on a parallel line drawn 10 mm superior to the cej line. outer point of the buccal side of the sectioned specimen on a parallel line drawn 2 mm superior to the cej line outer point of the buccal side of the sectioned specimen on a parallel line drawn 4 mm superior to the cej line outer point of the buccal side of the sectioned specimen on a parallel line drawn 6 mm superior to the cej line outer point of the buccal side of the sectioned specimen on a parallel line drawn 8 mm superior to the cej line outer point of the buccal side of the sectioned specimen on a parallel line drawn 10 mm superior to the cej line outer point of the palatal side of the sectioned specimen on a parallel line drawn 2 mm superior to the cej line outer point of the palatal side of the sectioned specimen on a parallel line drawn 4 mm superior to the cej line outer point of the palatal side of the sectioned specimen on a parallel line drawn 6 mm superior to the cej line outer point of the palatal side of the sectioned specimen on a parallel line drawn 8 mm superior to the cej line outer point of the palatal side of the sectioned specimen on a parallel line drawn 10 mm superior to the cej line. the thickness of soft tissue were also measured at 6 landmarks mentioned below, including the incisive papilla (ip) on the palate, which meet with the line perpendicular to the occlusal plane and passing through closest five points from the ip at 4-mm intervals [figure 2 ]. measurement of the thickness of the soft tissue at six points on the palate of the sectioned specimen, which were drawn at 4-mm intervals from the incisive papilla and perpendicular to the occlusal plane outer point of the sectioned specimen at the ip of the palateouter point of the sectioned specimen contacted with a parallel line drawn 4 mm posterior to the line passing through ip perpendicular to the occlusal planeouter point of the sectioned specimen contacted with a parallel line drawn 8 mm posterior to the line passing through ip perpendicular to the occlusal planeouter point of the sectioned specimen contacted with a parallel line drawn 12 mm posterior to the line passing through ip perpendicular to the occlusal planeouter point of the sectioned specimen contacted with a parallel line drawn 16 mm posterior to the line passing through ip perpendicular to the occlusal planeouter point of the sectioned specimen contacted with a parallel line drawn 20 mm posterior to the line passing through ip perpendicular to the occlusal plane. outer point of the sectioned specimen at the ip of the palate outer point of the sectioned specimen contacted with a parallel line drawn 4 mm posterior to the line passing through ip perpendicular to the occlusal plane outer point of the sectioned specimen contacted with a parallel line drawn 8 mm posterior to the line passing through ip perpendicular to the occlusal plane outer point of the sectioned specimen contacted with a parallel line drawn 12 mm posterior to the line passing through ip perpendicular to the occlusal plane outer point of the sectioned specimen contacted with a parallel line drawn 16 mm posterior to the line passing through ip perpendicular to the occlusal plane outer point of the sectioned specimen contacted with a parallel line drawn 20 mm posterior to the line passing through ip perpendicular to the occlusal plane. sectioned samples along with reference rulers were digitally scanned (umax flatbed scanner [umax. scanned images were calibrated and measurements were made with image - analysis software (autocad 2004 [autocad is the industry software that sets the standard in cad design.). the buccal soft tissue is thickest at 10 mm (1.73742) and thinnest at 4 mm (1.50317) from the cej the palatal soft tissue is thickest at 8 mm (3.14033) and thinnest at 2 mm (2.44308) from the cejthe buccal hard tissue is thickest at 10 mm (1.43733) and thinnest at 2 mm (1.25350) from the cejthe palatal hard tissue is thickest at 2 mm (1.55575) and thinnest at 10 mm (1.36567) from the cej. the palatal soft tissue is thickest at 8 mm (3.14033) and thinnest at 2 mm (2.44308) from the cej the buccal hard tissue is thickest at 10 mm (1.43733) and thinnest at 2 mm (1.25350) from the cej the palatal hard tissue is thickest at 2 mm (1.55575) and thinnest at 10 mm (1.36567) from the cej. the hard tissue is thickest at 10 mm (1.43733) and soft tissue at 10 mm (1.73742) from the cejthe soft tissue is the thinnest at 4 mm (1.50317) and hard tissue at 4 mm (1.28233) from the cejaverage thickness of soft tissue at 6 mm is (1.62042) and hard tissue at 6 mm is (1.39650) from cej. the hard tissue is thickest at 10 mm (1.43733) and soft tissue at 10 mm (1.73742) from the cej the soft tissue is the thinnest at 4 mm (1.50317) and hard tissue at 4 mm (1.28233) from the cej average thickness of soft tissue at 6 mm is (1.62042) and hard tissue at 6 mm is (1.39650) from cej. the hard tissue is thickest at 2 mm (1.55575) and soft tissue at 2 mm (2.44308) from the cejthe soft tissue is the thinnest at 2 mm (2.44308) and hard tissue at 2 mm (1.55575) from the cejaverage thickness of soft tissue at 6 mm is (3.00008) and hard tissue at 6 mm is (1.47767) from cej [table 1 ]. the hard tissue is thickest at 2 mm (1.55575) and soft tissue at 2 mm (2.44308) from the cej the soft tissue is the thinnest at 2 mm (2.44308) and hard tissue at 2 mm (1.55575) from the cej average thickness of soft tissue at 6 mm is (3.00008) and hard tissue at 6 mm is (1.47767) from cej [table 1 ]. comparison of measurements (in mm) of sectioned specimens in group 1 the buccal soft tissue is thickest at 10 mm (2.04292) and thinnest at 4 mm (1.85242) from the cejthe palatal soft tissue is thickest at 10 mm (3.37350) and thinnest at 2 mm (2.75925) from the cejthe buccal hard tissue is thickest at 8 mm (1.58342) and thinnest at 6 mm (1.49608) from the cejthe palatal hard tissue is thickest at 4 mm (1.70542) and thinnest at 2 mm (1.53958) from the cej. the buccal soft tissue is thickest at 10 mm (2.04292) and thinnest at 4 mm (1.85242) from the cej the palatal soft tissue is thickest at 10 mm (3.37350) and thinnest at 2 mm (2.75925) from the cej the buccal hard tissue is thickest at 8 mm (1.58342) and thinnest at 6 mm (1.49608) from the cej the palatal hard tissue is thickest at 4 mm (1.70542) and thinnest at 2 mm (1.53958) from the cej. the hard tissue is thickest at 8 mm (1.58342) and soft tissue at 8 mm (1.86558) from the cejthe soft tissue is the thinnest at 4 mm (1.85242) and hard tissue at 4 mm (1.54075) from the cejaverage thickness of soft tissue at 6 mm is (1.96000) and hard tissue at 2 mm is (1.51608) from cej. the hard tissue is thickest at 8 mm (1.58342) and soft tissue at 8 mm (1.86558) from the cej the soft tissue is the thinnest at 4 mm (1.85242) and hard tissue at 4 mm (1.54075) from the cej average thickness of soft tissue at 6 mm is (1.96000) and hard tissue at 2 mm is (1.51608) from cej. the hard tissue is thickest at 4 mm (1.70542) and soft tissue at 4 mm (2.84675) from the cejthe soft tissue is thinnest at 2 mm (2.75925) and hard tissue at 2 mm (1.53958) from the cejaverage thickness of soft tissue at 6 mm is (3.12100) and hard tissue at 6 mm is (1.67742) from cej [table 2 ]. the hard tissue is thickest at 4 mm (1.70542) and soft tissue at 4 mm (2.84675) from the cej the soft tissue is thinnest at 2 mm (2.75925) and hard tissue at 2 mm (1.53958) from the cej average thickness of soft tissue at 6 mm is (3.12100) and hard tissue at 6 mm is (1.67742) from cej [table 2 ]. comparison of measurements (in mm) of sectioned specimens in group 2 the buccal soft tissue is thickest at 2 mm (2.0055) and thinnest at 6 mm (1.5177) from the cejthe palatal soft tissue is thickest at 10 mm (3.05708) and thinnest at 6 mm (2.48025) from the cejthe buccal hard tissue is thickest at 8 mm (1.61108) and thinnest at 2 mm (1.53433) from the cejthe palatal hard tissue is thickest at 2 mm (1.5494) and thinnest at 6 mm (1.3058) from the cej. the buccal soft tissue is thickest at 2 mm (2.0055) and thinnest at 6 mm (1.5177) from the cej the palatal soft tissue is thickest at 10 mm (3.05708) and thinnest at 6 mm (2.48025) from the cej the buccal hard tissue is thickest at 8 mm (1.61108) and thinnest at 2 mm (1.53433) from the cej the palatal hard tissue is thickest at 2 mm (1.5494) and thinnest at 6 mm (1.3058) from the cej. the hard tissue is thickest at 8 mm (1.61108) and soft tissue at 8 mm (1.6779) from the cejthe soft tissue is the thinnest at 6 mm (1.5177) and hard tissue at 6 mm (1.54778) from the cejaverage thickness of soft tissue at 4 mm is (1.7431) and hard tissue at 6 mm is (1.54778) from the cej [table 3 ]. the hard tissue is thickest at 8 mm (1.61108) and soft tissue at 8 mm (1.6779) from the cej the soft tissue is the thinnest at 6 mm (1.5177) and hard tissue at 6 mm (1.54778) from the cej average thickness of soft tissue at 4 mm is (1.7431) and hard tissue at 6 mm is (1.54778) from the cej [table 3 ]. comparison of measurements (in mm) of sectioned specimens in group 3 the hard tissue is thickest at 2 mm (1.5494) and soft tissue at 2 mm (2.69117) from the cejthe soft tissue is thinnest at 6 mm (2.48025) and hard tissue at 6 mm (1.3058) from the cejaverage thickness of soft tissue at 8 mm is (2.66167) and hard tissue at 10 mm is (1.4190) from cej. measurement of thickness of soft tissue at 6 landmarks (on the ip and at 4-mm intervals from the ip) showed that the soft tissue is thickest at 12 mm (1.92079) and thinnest at 20 mm (1.65256) from ip [table 4 ]. the hard tissue is thickest at 2 mm (1.5494) and soft tissue at 2 mm (2.69117) from the cej the soft tissue is thinnest at 6 mm (2.48025) and hard tissue at 6 mm (1.3058) from the cej average thickness of soft tissue at 8 mm is (2.66167) and hard tissue at 10 mm is (1.4190) from cej. measurement of thickness of soft tissue at 6 landmarks (on the ip and at 4-mm intervals from the ip) showed that the soft tissue is thickest at 12 mm (1.92079) and thinnest at 20 mm (1.65256) from ip [table 4 ]. the buccal soft tissue is thickest at 10 mm (1.73742) and thinnest at 4 mm (1.50317) from the cej the palatal soft tissue is thickest at 8 mm (3.14033) and thinnest at 2 mm (2.44308) from the cejthe buccal hard tissue is thickest at 10 mm (1.43733) and thinnest at 2 mm (1.25350) from the cejthe palatal hard tissue is thickest at 2 mm (1.55575) and thinnest at 10 mm (1.36567) from the cej. the palatal soft tissue is thickest at 8 mm (3.14033) and thinnest at 2 mm (2.44308) from the cej the buccal hard tissue is thickest at 10 mm (1.43733) and thinnest at 2 mm (1.25350) from the cej the palatal hard tissue is thickest at 2 mm (1.55575) and thinnest at 10 mm (1.36567) from the cej. the hard tissue is thickest at 10 mm (1.43733) and soft tissue at 10 mm (1.73742) from the cejthe soft tissue is the thinnest at 4 mm (1.50317) and hard tissue at 4 mm (1.28233) from the cejaverage thickness of soft tissue at 6 mm is (1.62042) and hard tissue at 6 mm is (1.39650) from cej. the hard tissue is thickest at 10 mm (1.43733) and soft tissue at 10 mm (1.73742) from the cej the soft tissue is the thinnest at 4 mm (1.50317) and hard tissue at 4 mm (1.28233) from the cej average thickness of soft tissue at 6 mm is (1.62042) and hard tissue at 6 mm is (1.39650) from cej. the hard tissue is thickest at 2 mm (1.55575) and soft tissue at 2 mm (2.44308) from the cejthe soft tissue is the thinnest at 2 mm (2.44308) and hard tissue at 2 mm (1.55575) from the cejaverage thickness of soft tissue at 6 mm is (3.00008) and hard tissue at 6 mm is (1.47767) from cej [table 1 ]. the hard tissue is thickest at 2 mm (1.55575) and soft tissue at 2 mm (2.44308) from the cej the soft tissue is the thinnest at 2 mm (2.44308) and hard tissue at 2 mm (1.55575) from the cej average thickness of soft tissue at 6 mm is (3.00008) and hard tissue at 6 mm is (1.47767) from cej [table 1 ]. the hard tissue is thickest at 10 mm (1.43733) and soft tissue at 10 mm (1.73742) from the cejthe soft tissue is the thinnest at 4 mm (1.50317) and hard tissue at 4 mm (1.28233) from the cejaverage thickness of soft tissue at 6 mm is (1.62042) and hard tissue at 6 mm is (1.39650) from cej. the hard tissue is thickest at 10 mm (1.43733) and soft tissue at 10 mm (1.73742) from the cej the soft tissue is the thinnest at 4 mm (1.50317) and hard tissue at 4 mm (1.28233) from the cej average thickness of soft tissue at 6 mm is (1.62042) and hard tissue at 6 mm is (1.39650) from cej. the hard tissue is thickest at 2 mm (1.55575) and soft tissue at 2 mm (2.44308) from the cejthe soft tissue is the thinnest at 2 mm (2.44308) and hard tissue at 2 mm (1.55575) from the cejaverage thickness of soft tissue at 6 mm is (3.00008) and hard tissue at 6 mm is (1.47767) from cej [table 1 ]. the hard tissue is thickest at 2 mm (1.55575) and soft tissue at 2 mm (2.44308) from the cej the soft tissue is the thinnest at 2 mm (2.44308) and hard tissue at 2 mm (1.55575) from the cej average thickness of soft tissue at 6 mm is (3.00008) and hard tissue at 6 mm is (1.47767) from cej [table 1 ]. the buccal soft tissue is thickest at 10 mm (2.04292) and thinnest at 4 mm (1.85242) from the cejthe palatal soft tissue is thickest at 10 mm (3.37350) and thinnest at 2 mm (2.75925) from the cejthe buccal hard tissue is thickest at 8 mm (1.58342) and thinnest at 6 mm (1.49608) from the cejthe palatal hard tissue is thickest at 4 mm (1.70542) and thinnest at 2 mm (1.53958) from the cej. the buccal soft tissue is thickest at 10 mm (2.04292) and thinnest at 4 mm (1.85242) from the cej the palatal soft tissue is thickest at 10 mm (3.37350) and thinnest at 2 mm (2.75925) from the cej the buccal hard tissue is thickest at 8 mm (1.58342) and thinnest at 6 mm (1.49608) from the cej the palatal hard tissue is thickest at 4 mm (1.70542) and thinnest at 2 mm (1.53958) from the cej. the hard tissue is thickest at 8 mm (1.58342) and soft tissue at 8 mm (1.86558) from the cejthe soft tissue is the thinnest at 4 mm (1.85242) and hard tissue at 4 mm (1.54075) from the cejaverage thickness of soft tissue at 6 mm is (1.96000) and hard tissue at 2 mm is (1.51608) from cej. the hard tissue is thickest at 8 mm (1.58342) and soft tissue at 8 mm (1.86558) from the cej the soft tissue is the thinnest at 4 mm (1.85242) and hard tissue at 4 mm (1.54075) from the cej average thickness of soft tissue at 6 mm is (1.96000) and hard tissue at 2 mm is (1.51608) from cej. the hard tissue is thickest at 4 mm (1.70542) and soft tissue at 4 mm (2.84675) from the cejthe soft tissue is thinnest at 2 mm (2.75925) and hard tissue at 2 mm (1.53958) from the cejaverage thickness of soft tissue at 6 mm is (3.12100) and hard tissue at 6 mm is (1.67742) from cej [table 2 ]. the hard tissue is thickest at 4 mm (1.70542) and soft tissue at 4 mm (2.84675) from the cej the soft tissue is thinnest at 2 mm (2.75925) and hard tissue at 2 mm (1.53958) from the cej average thickness of soft tissue at 6 mm is (3.12100) and hard tissue at 6 mm is (1.67742) from cej [table 2 ]. the hard tissue is thickest at 8 mm (1.58342) and soft tissue at 8 mm (1.86558) from the cejthe soft tissue is the thinnest at 4 mm (1.85242) and hard tissue at 4 mm (1.54075) from the cejaverage thickness of soft tissue at 6 mm is (1.96000) and hard tissue at 2 mm is (1.51608) from cej. the hard tissue is thickest at 8 mm (1.58342) and soft tissue at 8 mm (1.86558) from the cej the soft tissue is the thinnest at 4 mm (1.85242) and hard tissue at 4 mm (1.54075) from the cej average thickness of soft tissue at 6 mm is (1.96000) and hard tissue at 2 mm is (1.51608) from cej. the hard tissue is thickest at 4 mm (1.70542) and soft tissue at 4 mm (2.84675) from the cejthe soft tissue is thinnest at 2 mm (2.75925) and hard tissue at 2 mm (1.53958) from the cejaverage thickness of soft tissue at 6 mm is (3.12100) and hard tissue at 6 mm is (1.67742) from cej [table 2 ]. the hard tissue is thickest at 4 mm (1.70542) and soft tissue at 4 mm (2.84675) from the cej the soft tissue is thinnest at 2 mm (2.75925) and hard tissue at 2 mm (1.53958) from the cej average thickness of soft tissue at 6 mm is (3.12100) and hard tissue at 6 mm is (1.67742) from cej [table 2 ]. the buccal soft tissue is thickest at 2 mm (2.0055) and thinnest at 6 mm (1.5177) from the cejthe palatal soft tissue is thickest at 10 mm (3.05708) and thinnest at 6 mm (2.48025) from the cejthe buccal hard tissue is thickest at 8 mm (1.61108) and thinnest at 2 mm (1.53433) from the cejthe palatal hard tissue is thickest at 2 mm (1.5494) and thinnest at 6 mm (1.3058) from the cej. the buccal soft tissue is thickest at 2 mm (2.0055) and thinnest at 6 mm (1.5177) from the cej the palatal soft tissue is thickest at 10 mm (3.05708) and thinnest at 6 mm (2.48025) from the cej the buccal hard tissue is thickest at 8 mm (1.61108) and thinnest at 2 mm (1.53433) from the cej the palatal hard tissue is thickest at 2 mm (1.5494) and thinnest at 6 mm (1.3058) from the cej. the hard tissue is thickest at 8 mm (1.61108) and soft tissue at 8 mm (1.6779) from the cejthe soft tissue is the thinnest at 6 mm (1.5177) and hard tissue at 6 mm (1.54778) from the cejaverage thickness of soft tissue at 4 mm is (1.7431) and hard tissue at 6 mm is (1.54778) from the cej [table 3 ]. the hard tissue is thickest at 8 mm (1.61108) and soft tissue at 8 mm (1.6779) from the cej the soft tissue is the thinnest at 6 mm (1.5177) and hard tissue at 6 mm (1.54778) from the cej average thickness of soft tissue at 4 mm is (1.7431) and hard tissue at 6 mm is (1.54778) from the cej [table 3 ]. comparison of measurements (in mm) of sectioned specimens in group 3 the hard tissue is thickest at 2 mm (1.5494) and soft tissue at 2 mm (2.69117) from the cejthe soft tissue is thinnest at 6 mm (2.48025) and hard tissue at 6 mm (1.3058) from the cejaverage thickness of soft tissue at 8 mm is (2.66167) and hard tissue at 10 mm is (1.4190) from cej. measurement of thickness of soft tissue at 6 landmarks (on the ip and at 4-mm intervals from the ip) showed that the soft tissue is thickest at 12 mm (1.92079) and thinnest at 20 mm (1.65256) from ip [table 4 ]. the hard tissue is thickest at 2 mm (1.5494) and soft tissue at 2 mm (2.69117) from the cej the soft tissue is thinnest at 6 mm (2.48025) and hard tissue at 6 mm (1.3058) from the cej average thickness of soft tissue at 8 mm is (2.66167) and hard tissue at 10 mm is (1.4190) from cej. measurement of thickness of soft tissue at 6 landmarks (on the ip and at 4-mm intervals from the ip) showed that the soft tissue is thickest at 12 mm (1.92079) and thinnest at 20 mm (1.65256) from ip [table 4 ]. the hard tissue is thickest at 8 mm (1.61108) and soft tissue at 8 mm (1.6779) from the cejthe soft tissue is the thinnest at 6 mm (1.5177) and hard tissue at 6 mm (1.54778) from the cejaverage thickness of soft tissue at 4 mm is (1.7431) and hard tissue at 6 mm is (1.54778) from the cej [table 3 ]. the hard tissue is thickest at 8 mm (1.61108) and soft tissue at 8 mm (1.6779) from the cej the soft tissue is the thinnest at 6 mm (1.5177) and hard tissue at 6 mm (1.54778) from the cej average thickness of soft tissue at 4 mm is (1.7431) and hard tissue at 6 mm is (1.54778) from the cej [table 3 ]. the hard tissue is thickest at 2 mm (1.5494) and soft tissue at 2 mm (2.69117) from the cejthe soft tissue is thinnest at 6 mm (2.48025) and hard tissue at 6 mm (1.3058) from the cejaverage thickness of soft tissue at 8 mm is (2.66167) and hard tissue at 10 mm is (1.4190) from cej. measurement of thickness of soft tissue at 6 landmarks (on the ip and at 4-mm intervals from the ip) showed that the soft tissue is thickest at 12 mm (1.92079) and thinnest at 20 mm (1.65256) from ip [table 4 ]. the hard tissue is thickest at 2 mm (1.5494) and soft tissue at 2 mm (2.69117) from the cej the soft tissue is thinnest at 6 mm (2.48025) and hard tissue at 6 mm (1.3058) from the cej average thickness of soft tissue at 8 mm is (2.66167) and hard tissue at 10 mm is (1.4190) from cej. measurement of thickness of soft tissue at 6 landmarks (on the ip and at 4-mm intervals from the ip) showed that the soft tissue is thickest at 12 mm (1.92079) and thinnest at 20 mm (1.65256) from ip [table 4 ]. orthodontic anchorage is the ability to limit the movement of some teeth while achieving the desired movement of other teeth. for this reason, orthodontists have been searching for the most appropriate methods and appliances to achieve this goal. the age old problem in orthodontics is essentially the application of newton 's third law of motion : for every action, there is an equal and opposite reaction. orthodontists often have inadequate mechanical systems with which to control anchorage, which leads to a loss of anchorage in the reactive unit and thus incomplete correction of intra- and inter - arch alignment problems. moreover, in an attempt to overcome these limitations, clinicians often incorporate bulky acrylic appliances or extraoral appliances, which when combined with the ever challenging problem of uncooperative patients, are often a futile attempt at best. the stability of these microimplants depends on the placement site, thickness of the bone and soft tissue. poggio., conducted a study using the volumetric tomographic images of 25 maxillae and 25 mandibles taken with the newtom system t (newtom is a radiographic methoid used in other study by a different authors mentioned in my reference). in the maxilla, the greatest amount of mesiodistal bone was on the palatal side between the second premolar and the first molar. the greatest thickness of bone in the buccopalatal dimension was between the first and second molars, whereas the lowest was found in the tuberosity. bone stock for placement of screws was found to exist primarily in the maxillary (mesial to first molars) and mandibular (mesial and distal to first molars) posterior regions. typically, adequate bone was located more than halfway down the root length, which is likely to be covered by movable mucosa. vertical bone depth was measured at nine unilateral locations in the pp of each subject. male subjects had significantly greater mean bone thickness in six of the nine locations measured, showing a mean of 1.22 mm more vertical bone than females showed at these locations. costa., conducted a study using volumetric computed tomography for bone depth and needle with a rubber stop for mucosal depth. the results indicate that bone thickness will allow temporary anchorage devices (tads) 10 mm in length only in the symphysis, retromolar, and palatal premaxillary regions. tads 6 - 8 mm in length can be placed in the incisive fossa, in the upper and lower canine fossae. their results indicated that in all groups, buccal soft tissues were thickest closest to and far away from the cej and thinnest in the middle. palatal soft tissue thickness increased gradually from the cej toward the apical region in all groups. buccal cortical - bone was thickest closest and far away from the cej and thinnest in the middle in groups 1 and 2. in group 3, unlike groups 1 and 2, thickness was greatest at the middle. palatal cortical - bone thickness was greatest 6 mm apical to the cej in groups 1 and 3, and 2 mm apical to the cej in group 2. along the mid - palatal suture, palatal mucosa remained uniformly 1 mm thick posterior to the ip. however, in our study, we found different findings with regard to hard- and soft - tissue thickness at the various sites, and these differences may be attributed to racial differences between the two populations. according to our study, the best site for placement of implant considering the thinnest soft tissue and thickest hard tissue is group 1 : 6 mm from cej in buccal side and 2 mm from cej in palatal sidegroup 2 : 8 mm from cej in buccal side and 4 mm from cej in palatal sidegroup 3 : 8 mm from cej in buccal side and 8 mm from cej in palatal side. group 1 : 6 mm from cej in buccal side and 2 mm from cej in palatal side group 2 : 8 mm from cej in buccal side and 4 mm from cej in palatal side group 3 : 8 mm from cej in buccal side and 8 mm from cej in palatal side. implant passes through the soft tissue and bone, and therefore the thickness of the soft tissue and cortical - bone at the surgical site are critical factors for success of the implant. in terms of soft- and hard - tissues, the stability of miniscrew implants depends on the quality and quantity of the cortical - bone. the main objective of an orthodontic screw is to gain maximum retention by placing the screw in an area with the thinnest soft tissue and the thickest cortical - bone. surgical placement of miniscrew implants for orthodontic anchorage in the maxillary molar region requires consideration of the placement site and angle based on anatomical characteristics. the best site for placement of implant is with thinnest soft tissue and thickest hard tissue, which is in the middle from cej in buccal side and closest from cej in palatal side in group 1 and faraway from cej in buccal side and closest from cej in palatal side in group 2 and faraway from cej in buccal side and faraway from cej in palatal side in group 3. | introduction : anchorage control is a critical consideration when planning treatment for patients with dental and skeletal malocclusions. to obtain sufficient stability of implants, the thickness of the soft tissue and the cortical - bone in the placement site must be considered ; so as to provide an anatomical map in order to assist the clinician in the placement of the implants.objective:the aim of this study is to evaluate the thickness of soft- and hard-tissue.materials and methods : to measure soft tissue and cortical - bone thicknesses, 12 maxillary cross - sectional specimens were obtained from the cadavers, which were made at three maxillary mid - palatal suture areas : the interdental area between the first and second premolars (group 1), the second premolar and the first molar (group 2), and the first and second molars (group 3). sectioned samples along with reference rulers were digitally scanned. scanned images were calibrated and measurements were made with image - analysis software. we measured the thickness of soft and hard - tissues at five sectional areas parallel to the buccopalatal cementoenamel junction (cej) line at 2-mm intervals and also thickness of soft tissue at the six landmarks including the incisive papilla (ip) on the palate. the line perpendicular to the occlusal plane was made and measurement was taken at 4-mm intervals from the closest five points to ip.results:(1) group 1:6 mm from cej in buccal side and 2 mm from cej in palatal side. (2) group 2:8 mm from cej in buccal side and 4 mm from cej in palatal side. (3) group 3:8 mm from cej in buccal side and 8 mm from cej in palatal side.conclusions:the best site for placement of implant is with thinnest soft tissue and thickest hard tissue, which is in the middle from cej in buccal side and closest from cej in palatal side in group 1 and faraway from cej in buccal side and closest from cej in palatal side in group 2 and faraway from cej in buccal side and faraway from cej in palatal side in group 3. |
it is estimated that up to 50% of depressed patients either do not respond or only partially respond to an adequate course of antidepressant treatment. although the first generation antipsychotics (fga) have been in use for treating major depression for a long time, their extrapyramidal side effects (eps) have discouraged their use. second generation antipsychotics (sga) have become widespread in depression in the past decade due to the lower risk for eps. a meta - analysis of sixteen trials with 3480 patients has found that sgas are an effective augmentation treatment for major depression, but they are frequently discontinued due to adverse events. the us food and drug administration have already approved the use of olanzapine (in combination with fluoxetine), aripiprazole (510 mg / d, maximum dosage 15 mg / d), and quetiapine extended release (50300 mg / d) as adjunctive agents for depression. regular cross - sectional surveys of prescription patterns are an efficient and fast way of obtaining a global picture of pharmacotherapy practice in a given setting. however, little is known about how antipsychotic medications are used over time in the treatment of depression in china where the mental health system is not only fundamentally different from other countries but has undergone rapid changes in the last decade. in a recent study of misdiagnosed bipolar patients receiving treatment for major depression as the study only involved 13 major psychiatric hospitals or units, its findings can not be generalized. furthermore, the impact of antipsychotics on important outcomes such as patients and their families satisfaction with treatment and quality of life (qol) was not explored. thus, this study aimed to : (1) examine the prescription patterns of antipsychotic medications for depression across china nationally from 2002 to 2012 ; and (2) to explore the demographic and clinical correlates of adjunctive antipsychotic treatment and the association with treatment satisfaction and qol. this was a cross - sectional nationwide pharmaco - epidemiological survey which was conducted in china initiated by the chinese society of psychiatry. the first survey took place in may 2002 followed by another two surveys in may 2006 and july - august 2012 using the same design and standardized protocol. consensus meetings on data collection and uniformity of data entry were held prior to each survey. a total of 45 psychiatric centers / units located in 10 provinces and municipalities including beijing, guangdong, hebei, hubei, jiangxi, jiangsu, jilin, shaanxi, shanxi, and sichuan participated in each survey. the same 45 mental health centers / units were included in all the three surveys. one for every 14 inpatients and outpatients receiving treatment in the participating hospitals / units during the data collection period of 4 weeks were consecutively referred by their treating psychiatrists to the research team to be screened for eligibility. all members of the research team were attending psychiatrist with at least 3-year working experience. inclusion criteria included : (1) dsm - iv or icd-10 diagnosis of major depressive disorder (mdd) based on a clinical interview and review of medical records ; (2) age 15 years or older ; (3) ability to understand the aims and the contents of the survey. basic socio - demographic and clinical characteristics were collected using a form designed for the study. information about the types and doses of psychotropic drugs were collected from the medical records. following the same procedures as in the research on asian psychotropic prescription project, delusions, hallucinations, and suicidality in the past month were evaluated during the diagnostic interview. global illness severity was evaluated with the chinese version of the clinical global impressions - severity scale (cgi - s). patients and their families satisfaction with the current treatment was evaluated with a self - rated, 7-point likert scale, scoring from 1 (extreme dissatisfaction) to 7 (extreme satisfaction). qol was assessed with the chinese version of the medical outcomes study short form 12 (sf-12). the sf-12 is a multidimensional generic instrument with 12 items on physical and mental qol domains., hospitals are classified into three levels according to the degree of specialization in clinical care and research. level - iii hospitals have the highest staff - patient ratio and medical resources while level - ii hospitals are regional medical centers that treat patients with severe diseases, and level - i hospitals are small, community level hospitals providing basic medical care. there was no level - i psychiatric hospital at the study time in the areas included, thus only level - iii / ii medical facilities were included in this study. all the 135 raters were trained in the use of the above - mentioned instruments. the inter - rater reliability of the instruments conducted in 20 mdd patients prior to the study yielded satisfactory to good agreement (> 0.75). the statistical analysis was performed using the statistical package for the social sciences (spss), version 20.0 (spss inc., chicago, il, usa). in the pooled sample, comparisons between the antipsychotic and nonantipsychotic groups with respect to demographic and clinical variables were conducted with chi - square tests, independent - samples t - test or mann whitney u - test, as appropriate. method (i.e., all specified independent variables were entered at one time) was used to determine the demographic and clinical variables that were independently and significantly associated with antipsychotic use. antipsychotic prescription was the dependent variable, while variables that significantly differed between the two groups in univariate analyses were entered as independent variables. patients and their families satisfaction with the current treatment and qol were only measured in the 2012 survey. in the 2012 sample, satisfaction with the current treatment and qol were compared between the antipsychotic and nonantipsychotic groups after controlling for the potential confounding effects of variables that significantly differed between the two groups in univariate analyses using analysis of covariance (ancova). this was a cross - sectional nationwide pharmaco - epidemiological survey which was conducted in china initiated by the chinese society of psychiatry. the first survey took place in may 2002 followed by another two surveys in may 2006 and july - august 2012 using the same design and standardized protocol. consensus meetings on data collection and uniformity of data entry were held prior to each survey. a total of 45 psychiatric centers / units located in 10 provinces and municipalities including beijing, guangdong, hebei, hubei, jiangxi, jiangsu, jilin, shaanxi, shanxi, and sichuan participated in each survey. the same 45 mental health centers / units were included in all the three surveys. one for every 14 inpatients and outpatients receiving treatment in the participating hospitals / units during the data collection period of 4 weeks were consecutively referred by their treating psychiatrists to the research team to be screened for eligibility. all members of the research team were attending psychiatrist with at least 3-year working experience. inclusion criteria included : (1) dsm - iv or icd-10 diagnosis of major depressive disorder (mdd) based on a clinical interview and review of medical records ; (2) age 15 years or older ; (3) ability to understand the aims and the contents of the survey. basic socio - demographic and clinical characteristics were collected using a form designed for the study. information about the types and doses of psychotropic drugs were collected from the medical records. following the same procedures as in the research on asian psychotropic prescription project, delusions, hallucinations, and suicidality in the past month were evaluated during the diagnostic interview. global illness severity was evaluated with the chinese version of the clinical global impressions - severity scale (cgi - s). patients and their families satisfaction with the current treatment was evaluated with a self - rated, 7-point likert scale, scoring from 1 (extreme dissatisfaction) to 7 (extreme satisfaction). qol was assessed with the chinese version of the medical outcomes study short form 12 (sf-12). the sf-12 is a multidimensional generic instrument with 12 items on physical and mental qol domains., hospitals are classified into three levels according to the degree of specialization in clinical care and research. level - iii hospitals have the highest staff - patient ratio and medical resources while level - ii hospitals are regional medical centers that treat patients with severe diseases, and level - i hospitals are small, community level hospitals providing basic medical care. there was no level - i psychiatric hospital at the study time in the areas included, thus only level - iii / ii medical facilities were included in this study. all the 135 raters were trained in the use of the above - mentioned instruments. the inter - rater reliability of the instruments conducted in 20 mdd patients prior to the study yielded satisfactory to good agreement (> 0.75). the statistical analysis was performed using the statistical package for the social sciences (spss), version 20.0 (spss inc., chicago, il, usa). in the pooled sample, comparisons between the antipsychotic and nonantipsychotic groups with respect to demographic and clinical variables were conducted with chi - square tests, independent - samples t - test or mann whitney u - test, as appropriate. multiple logistic regression analysis with the enter method (i.e., all specified independent variables were entered at one time) was used to determine the demographic and clinical variables that were independently and significantly associated with antipsychotic use. antipsychotic prescription was the dependent variable, while variables that significantly differed between the two groups in univariate analyses were entered as independent variables. patients and their families satisfaction with the current treatment and qol were only measured in the 2012 survey. in the 2012 sample, satisfaction with the current treatment and qol were compared between the antipsychotic and nonantipsychotic groups after controlling for the potential confounding effects of variables that significantly differed between the two groups in univariate analyses using analysis of covariance (ancova). a total of 4356 patients from 45 psychiatric hospitals / centers were screened for study entry ; 3655 met the study criteria : 735 in 2002, 1389 in 2006, and 1531 in 2012, resulting in an overall participation rate of 83.9%. the frequency of antipsychotic prescriptions was 24.9% (910/3655) in the whole sample ; the corresponding figures were 17.1% (126/735) in 2002, 20.3% (282/1389) in 2006, and 32.8% (502/1531) in 2012 (= 90.3, df = 2, p < 0.001). in the whole sample, the proportion of fgas and sgas was 6.1% (222/3655) and 19.4% (708/3655), respectively, with these figures being 12.4% (91/735) and 5.2% (38/735) in 2002, 6.9% (96/1389) and 14.2% (197/1389) in 2006, and 2.3% (35/1531) and 30.9% (473/1531) in 2012. table 1 shows the socio - demographic and clinical characteristics of the whole sample and those of the antipsychotic and nonantipsychotic groups. compared to the nonantipsychotic group, patients on antipsychotics had more delusions or hallucinations, significantly longer illness duration and higher tess total score ; were more likely to be inpatients and receive treatment in level - iii hospitals. these variables totally account for 26.9% of the variance of antipsychotic use (p < 0.001). comparison between patients with or without adjunctive antipsychotic medication with respect to basic demographic and clinical variables during 20022012 mann whitney u - test ; chi - square values. cgi - s : clinical global impressions - severity scale ; fgas : first generation antipsychotics ; sgas : second generation antipsychotics ; tess : treatment emergent symptom scale ; sd : standard deviation. factors associated with antipsychotic prescription in mdd (n = 910) multiple logistic regression analysis with the nonantipsychotic group as the reference. cgi - s : clinical global impressions - severity scale ; tess : treatment emergent symptom scale ; or : odds ratio ; ci : confidence interval ; mdd : major depressive disorder. table 3 shows the socio - demographic and clinical characteristics, treatment satisfaction, and qol in the 2012 sample, and separately according to antipsychotic prescription. after controlling for the potential confounding effects of variables that significantly differed between the two groups (inpatient status, presence of delusions or hallucinations and suicidality in the past month, hospital level, age of onset, duration of illness, and scores of cgi - s and tess) using ancova, antipsychotic use was associated with lower patients (f (9,1521) = 12.4, p < 0.001) and family 's treatment satisfaction (f (9,1521) = 11.1, p = 0.001), while there was no significant difference with respect to physical (f (9,1521) = 1.7, p = 0.18) and mental qol (f (9,1521) = 0.6, p = 0.42). comparison between the antipsychotic and nonantipsychotic groups with respect to basic demographic and clinical variables in 2012 mann whitney u - test ; chi - square values ; t values in independent - samples t - test or z value in mann cgi - s : clinical global impressions - severity scale ; sf-12 : medical outcomes study short form 12 ; tess : treatment emergent symptom scale ; sd : standard deviation. the first three most commonly prescribed antipsychotic drugs were sulpiride, perphenazine, and risperidone in 2002, olanzapine, risperidone, and quetiapine in 2006 and olanzapine, quetiapine, and risperidone in 2012. the five most commonly prescribed antipsychotic drugs for mdd in china in year 2002, 2006, and 2012 (n (%)) percentage of all patients on antipsychotic medications. the major finding of this study is that the overall frequency of antipsychotic use in treated mdd patients in china was 24.9% in the 20022012 period, which is slightly higher than found in another multicenter survey (19.2%) in chinese depressed patients. the difference between the two studies may be due to the discrepancy in the number and types of institutions involved and diagnostic methods (a review of medical records and clinical interview versus the mini international neuropsychiatric interview). another significant finding is the increase of adjunctive antipsychotic drugs from 17.1% in 2002, 20.3% in 2006 and to 32.8% in 2012. this increase might possibly be explained by the increasing use of sgas and decreasing use of fgas at the same time, the recommendations of sgas in treating depression in clinical guidelines and the growing impact of pharmaceutical companies over the past decade. as expected, one fifth of patients received sgas in the sample with increasing proportion over time (5.2% in 2002, 14.2% in 2006, and 30.9% in 2012) while the proportion of fga use decreased over time : 6.1% in the whole sample and 12.4% in 2002, 6.9% in 2006, and 2.3% in 2012. we assume that a number of socio - economic factors including local psychopharmacological practice, prior antipsychotic treatment, uneven access to antipsychotic drugs, insurance coverage, and costs might all contribute to such use in chinese mdd patients. considering the risk of antipsychotic - induced eps and the absence of a clearly documented rationale, the use of fgas should be discouraged in this population. olanzapine was the most commonly used sga in chinese mdd patients (7.7%), followed by quetiapine (5.7%), risperidone (3.4%), and aripiprazole (0.8%) in the pooled sample during the period between 2002 and 2012. such practice appears consistent with the us fda approval of two sgas aripiprazole and extended release quetiapine - as adjunctive agents in antidepressant - resistant mdd in addition to olanzapine plus fluoxetine. however, the response to antidepressants could not be measured in this cross - sectional survey. a meta - analysis has found that there were no significant differences in efficacy between adjunctive olanzapine, risperidone, quetiapine, and aripiprazole in the treatment of mdd. the american psychiatric association guideline also does not differentiate between individual sgas in its recommendations as add - on drugs for mdd. in this study, concurrent antipsychotic use in mdd was associated with a number of clinical variables. this recommendation could explain the association of antipsychotic use with inpatient treatment, level - iii hospitals and longer duration of illness because these factors are more likely to be linked with treatment - resistance. the chinese medical association recommended the concurrent use of antipsychotics in treating mdd with psychotic symptoms, which probably explains the association between antipsychotic prescriptions and delusions or hallucinations. as expected, concurrent use of antipsychotics was associated with more side effects as measured by the tess. multivariate analyses revealed that compared to the nonantipsychotic group, mdd patients on antipsychotics was associated with poorer patients and families satisfaction with treatment. this may be attributed to more frequent adverse effects, higher cost of treatment, and reduced treatment adherence associated with antipsychotics. this association between severity of illness and dissatisfaction with the efficacy of the treatment could not be excluded. surprisingly, however, there was no association between qol and antipsychotic use, which could be partly attributed to the uncontrolled patients and their families economic level. first, due to its cross - sectional design, the causal relationship between concurrent antipsychotic use and other variables could not be examined. there are differences in prescribing practices and treatment guidelines between institutions even within one province or municipality. finally, the diagnosis of depression was established by a review of medical records and a clinical interview, rather than using a structured diagnostic interview schedule. these limitations are partly offset by the study 's strengths including its large, homogeneous, and representative sample. in conclusion, about one in four depressed patients received antipsychotic medication in this study, which has increased over time. considering the increased risk of antipsychotic - induced side effects and patients and their families lack of satisfaction with psychiatric treatment in general, further examination of the rationale and appropriateness of antipsychotic prescription for depression and its alternatives is warranted. | background : optimizing treatment outcomes for depression requires understanding of how evidence - based treatments are utilized in clinical practice. antipsychotic medications concurrent with antidepressant treatment are frequently used in major depression, but few studies have investigated trends and patterns of their use over time. this study aimed to examine the prescription patterns of antipsychotic medications for major depression in china from 2002 to 2012 and their association with treatment satisfaction and quality of life (qol).methods : a total of 3655 subjects with major depression treated in 45 chinese psychiatric hospitals / centers nationwide were interviewed between 2002 and 2012. patients socio - demographic and clinical characteristics including psychopathology, medication side effects, satisfaction with treatment and qol were recorded using a standardized protocol and data collection.results:the frequency of antipsychotic use was 24.9% in the whole sample ; the corresponding figures were 17.1%, 20.3%, and 32.8% in 2002, 2006, and 2012, respectively (2 = 90.3, df = 2, p < 0.001). multiple logistic regression analyses revealed that patients on concurrent antipsychotics had significantly more delusions or hallucinations, longer illness duration, greater side effects, and more likely to be treated as inpatients and in major hospitals (i.e., level - iii hospital). antipsychotic use was associated with lower treatment satisfaction while there was no significant difference with respect to physical and mental qol between the antipsychotic and nonantipsychotic groups.conclusions:concurrent antipsychotic use was found in about one in four treated depressed patients in china, which has increased over a 10-year period. considering the association of drug - induced side effects and the lack of patients and relatives satisfaction with antipsychotic treatment, further examination of the rationale and appropriateness of the use of antipsychotics in depression is needed. |
rictor mice (10) were crossed with ap2-cre transgenic mice [strain, b6-cg - tg(fabp4-cre)1rev / j ; the jackson laboratory, bar harbor, me ] to obtain heterozygous ap2-cre;rictor offspring (where wt refers to wild type) in the f1 generation. these heterozygous mice were crossed with rictor mice to obtain the fric mice with genotype ap2-cre;rictor and their rictor littermates lacking ap2-cre expression (referred to here as fric). age - matched fric and fric mice of both sexes were studied at 35 months of age (young) and when they were aged > 9 months (old). for all mice used, the genotypes were determined by pcr analysis of tail genomic dna as described previously (10). both rictor mice as well as ap2-cre transgenic mice used for breeding had been backcrossed for 6 and 9 generations to the c57bl/6 strain, respectively. the mice were maintained under temperature- and humidity - controlled conditions with a 12-h light / dark cycle and were allowed food (total calories from fat 17%, lm-485 ; harlan - teklad) and water ad libitum. all animal studies performed in this investigation were approved by the university of virginia animal care and use committee. following an overnight fast (15 h), a 2-h hyperinsulinemic (insulin at 150 mu / kg body wt priming followed by 2.5 mu / kg / min)-euglycemic clamp was conducted in awake mice using [3-h]-glucose and 2-deoxy - d-[1-c]-glucose to assess glucose metabolism in individual tissues as described in kim. other experimental protocols are described in detail in the online appendix (available at http://diabetes.diabetesjournals.org/cgi/content/full/dc09-1061/dc1). values given are means se for the numbers of animals indicated in the figure legends. rictor mice (10) were crossed with ap2-cre transgenic mice [strain, b6-cg - tg(fabp4-cre)1rev / j ; the jackson laboratory, bar harbor, me ] to obtain heterozygous ap2-cre;rictor offspring (where wt refers to wild type) in the f1 generation. these heterozygous mice were crossed with rictor mice to obtain the fric mice with genotype ap2-cre;rictor and their rictor littermates lacking ap2-cre expression (referred to here as fric). age - matched fric and fric mice of both sexes were studied at 35 months of age (young) and when they were aged > 9 months (old). for all mice used, the genotypes were determined by pcr analysis of tail genomic dna as described previously (10). both rictor mice as well as ap2-cre transgenic mice used for breeding had been backcrossed for 6 and 9 generations to the c57bl/6 strain, respectively. the mice were maintained under temperature- and humidity - controlled conditions with a 12-h light / dark cycle and were allowed food (total calories from fat 17%, lm-485 ; harlan - teklad) and water ad libitum. all animal studies performed in this investigation were approved by the university of virginia animal care and use committee. following an overnight fast (15 h), a 2-h hyperinsulinemic (insulin at 150 mu / kg body wt priming followed by 2.5 mu / kg / min)-euglycemic clamp was conducted in awake mice using [3-h]-glucose and 2-deoxy - d-[1-c]-glucose to assess glucose metabolism in individual tissues as described in kim. other experimental protocols are described in detail in the online appendix (available at http://diabetes.diabetesjournals.org/cgi/content/full/dc09-1061/dc1). values given are means se for the numbers of animals indicated in the figure legends. fric mice were generated by breeding rictor mice with mice expressing cre recombinase under the control of the ap2 promoter. rictor protein levels were measured in isolated fat cells, liver, and skeletal muscle from fric and fric littermates. while fat cells from fric mice showed an 90% reduction (p 9 months old) showed significantly higher blood glucose levels after an overnight fast and a dramatic impairment in glucose clearance after an intraperitoneal glucose load (fig. 4a and supplemental fig. these data indicate the presence of severe insulin resistance in young and old fric mice. glucose homeostasis in male fric mice. a : intraperitoneal glucose tolerance tests in old male fric and fric mice (> 9 months old, n = 4, p 9 months old) female fric or fric mice liver sections stained with hematoxylin and eosin (h & e) (upper panels), or oil red o (lower panels) (representative images are shown out of four to five for each age - group). b : triglyceride concentration in liver homogenates of fric and fric mice (> 6-month - old female, n = 89, p c : l - type pyruvate kinase gene expression (> 6-month - old male mice, n = 5, p 6-month - old male mice, n = 5, p 9 months old) showed significantly higher blood glucose levels after an overnight fast and a dramatic impairment in glucose clearance after an intraperitoneal glucose load (fig. 4a and supplemental fig. these data indicate the presence of severe insulin resistance in young and old fric mice. glucose homeostasis in male fric mice. a : intraperitoneal glucose tolerance tests in old male fric and fric mice (> 9 months old, n = 4, p 9 months old) female fric or fric mice liver sections stained with hematoxylin and eosin (h & e) (upper panels), or oil red o (lower panels) (representative images are shown out of four to five for each age - group). b : triglyceride concentration in liver homogenates of fric and fric mice (> 6-month - old female, n = 89, p 6-month - old male mice, n = 5, p 6-month - old male mice, n = 5, p < 0.02). data shown (a high - quality digital representation of this figure is available in the online issue.) the loss of fat cell rictor expression in mice results in a metabolic phenotype similar to type 2 diabetes with impaired fat cell function associated with severe insulin resistance in adipose tissue, skeletal muscle, and liver. this suggests that rictor plays an important role in regulating fat cell metabolism and, consequently, whole - body glucose and lipid homeostasis and insulin sensitivity. fric fat cells showed impaired regulation of glucose transport and lipolysis in response to insulin. defective insulin signaling in fric fat cells most likely causes these metabolic abnormalities. our analyses of insulin signaling events that lie downstream of mtorc2 and mediate metabolic responses revealed a dramatic reduction in phosphorylation of akt at s473 and, consequently, foxo3a at t32 and as160 at t642. at this time, it is not clear whether foxo3a has a role in the observed metabolic defects in fric fat cells. however, the akt - mediated as160 phosphorylation at t642 and consequent inhibition of the rab - gap (gtpase activating protein) activity of as160 is essential for insulin - stimulated glut4 translocation to the plasma membrane (28,29). consistent with decreased phosphorylation of as160, glut4 translocation to the plasma membrane in response to insulin is impaired and glucose uptake is decreased in fric fat cells. rictor / mtorc2 activity has not been linked to regulation of either basal lipolysis or insulin - mediated suppression of lipolysis. however, our observations that in fric fat cells increased phosphorylation of hsl at s563 concomitant with upregulated pka activity and that insulin failed to inhibit these events suggests a role for mtorc2 in the regulation of lipolysis by modifying pka and hsl activities. it is not clear how mtorc2 regulates basal pka activity. in response to insulin, akt directly phosphorylates and activates phosphodiesterase 3b (pde3b) (30) increasing camp to amp conversion and thereby decreasing pka activity (31). since pka activates lipolysis via phosphorylation of hsl, a defect in insulin activation of pde3b would impair downregulation of lipolysis. in fact, defective regulation of lipolysis in response to insulin is reported in pde3b knockout mice (32). while our data supports a model whereby mtorc2 activity controls akt - mediated activation of pde3b by insulin, this remains to be established. rapamycin - mediated inhibition of mtor has implicated mtorc1 in the regulation of lipolysis (14,15) ; however, eliminating mtorc1 by ablating raptor in fat cells does not affect lipolysis (33). considering that prolonged rapamycin treatment inhibits the formation of mtorc2 in some cell types (4), our studies suggest that clinical observations of elevated nefa levels during rapamycin treatment (14,15) could be due to inhibition of mtorc2. how do fat cell specific genetic alterations in the insulin signaling pathway lead to impairment of whole - body glucose homeostasis ? although fric mice show a profound reduction in glucose transport in fat cells, this defect is likely to be only partly responsible for impaired glucose homeostasis in the fric mice. the major contributors to impaired glucose homeostasis in fric mice are reduced insulin - stimulated skeletal muscle glucose transport and impaired suppression of hepatic glucose production by insulin. we propose that elevated circulating nefas due to upregulation of lipolysis in fat cells cause the insulin resistance in skeletal muscle and liver observed in the fric mice. the increased serum nefa levels in fric mice are most likely responsible for the elevated intramuscular accumulation of tag, which in turn may be mediated by increased lipin 1 levels in fric skeletal muscles. increased nefas flux into skeletal muscle leads to elevated intramuscular levels of lipid metabolites, such as fatty acyl coa and diacylglycerol (35). these lipids induce insulin resistance in skeletal muscle by causing defects in insulin signaling (36). previously, nefas have been shown to activate serine / threonine kinases such as ib kinase-2 (37), jun nh2-terminal kinase (38), and protein kinase c (39). all of these kinases can phosphorylate irs1 on critical serine residues, thereby blocking phosphorylation of irs1 by ir kinase on tyrosine sites that are required for pi3k association and activation (40). irs1 s302 phosphorylation through ib kinase-2 (41) or jun nh2-terminal kinase (42) can lead to irs1 degradation. since we see an increase in phosphorylation of irs1 at s302 in fric muscle, a decrease in irs1 y896 phosphorylation, as well as a reduction in total irs1 levels, we suggest that elevated lipids are responsible for the insulin resistance observed in vivo and ex vivo in the skeletal muscles of fric mice. the development of hepatic steatosis in fric mice may be due to the prolonged elevation of nefas and glycerol in serum, as both are used for synthesis of tag in the liver. furthermore, under hyperglycemic conditions, an increased glucose flux into the liver in fric mice could compensate for skeletal muscle insulin resistance to maintain normoglycemia (43). the surplus glucose is converted to tag and contributes to the development of hepatic steatosis. thus, liver function also becomes affected in fric animals after chronic exposure to elevated circulating nefas, glycerol, and glucose. a different line of fat cell specific rictor knockout mice (referred to as rictor) has recently been described (21). rictor mice showed that visceral organs such as liver, pancreas, kidney, fat pads, spleen, and heart increased in size with concomitantly increased lean mass that was more apparent on a high - fat diet. this finding suggests that rictor / mtorc2 in fat cells is important for organismal growth (21). the rictor mice were hyperinsulinemic but were normoglycemic in the fed and fasting state, similar to young fric mice. increased pancreatic -cell mass could contribute to hyperinsulinemia in fric mice, as suggested for the rictor mice. however, increased -cell mass alone is not sufficient to induce hyperinsulinemia (44) but requires a factor that would induce insulin secretion. it is well known that insulin resistance is compensated by increased -cell mass and insulin secretion (45). our results from hyperinsulinemic - euglycemic clamp studies in fric mice show severe whole - body insulin resistance including adipose tissue, skeletal muscle, and liver. while the insulin sensitivity in rictor mice was not directly measured, results from insulin tolerance tests suggest that these mice are also insulin resistant. (21) ascribe the insulin resistance in the rictormice to decreased circulating adiponectin levels. there was no change in adiponectin levels in the fric animals on a standard diet. the reason for this discrepancy is not clear, as both fat cell specific rictor knockout models have very similar genetic backgrounds of 129 crossed to c57bl/6j, although the 129 substrains differ (129s6 for fric mice and 129s1/svlmj for rictor mice). however, since the genetic loss of adiponectin does not affect insulin sensitivity of standard diet fed mice (46), it is unlikely that decreased adiponectin is the cause for decreased insulin sensitivity arising from fat cell specific loss of rictor in mice. the elevated circulating nefa levels that we observe in the fric mice could not only contribute to peripheral insulin resistance but also directly stimulate insulin secretion from -cells (data not shown) as well as increase pancreatic -cell mass (47). therefore, we propose that hyperinsulinemia and increased -cell mass in fric mice is predominantly a consequence of insulin resistance and increased nefas. in conclusion, we demonstrate that mtorc2 is essential for insulin action on glucose transport and lipolysis in fat cells. our studies further demonstrate the important role for mtorc2 in the regulation of fat cell function and control of whole - body glucose and lipid homeostasis and insulin sensitivity. thus, mtorc2 may serve as a drug target for the treatment of obesity and type 2 diabetes. | objectiverictor is an essential component of mammalian target of rapamycin (mtor) complex (mtorc) 2, a kinase that phosphorylates and activates akt, an insulin signaling intermediary that regulates glucose and lipid metabolism in adipose tissue, skeletal muscle, and liver. to determine the physiological role of rictor / mtorc2 in insulin signaling and action in fat cells, we developed fat cell specific rictor knockout (fric/) mice.research design and methodsinsulin signaling and glucose and lipid metabolism were studied in fric/ fat cells. in vivo glucose metabolism was evaluated by hyperinsulinemic - euglycemic clamp.resultsloss of rictor in fat cells prevents insulin - stimulated phosphorylation of akt at s473, which, in turn, impairs the phosphorylation of downstream targets such as foxo3a at t32 and as160 at t642. however, glycogen synthase kinase-3 phosphorylation at s9 is not affected. the signaling defects in fric/ fat cells lead to impaired insulin - stimulated glut4 translocation to the plasma membrane and decreased glucose transport. furthermore, rictor - null fat cells are unable to suppress lipolysis in response to insulin, leading to elevated circulating free fatty acids and glycerol. these metabolic perturbations are likely to account for defects observed at the whole - body level of fric/ mice, including glucose intolerance, marked hyperinsulinemia, insulin resistance in skeletal muscle and liver, and hepatic steatosis.conclusionsrictor/mtorc2 in fat cells plays an important role in whole - body energy homeostasis by mediating signaling necessary for the regulation of glucose and lipid metabolism in fat cells. |
a 44-year - old woman presented to the emergency room with shortness of breath and thoracic pain radiating to the left arm and shoulder. her vital signs showed a blood pressure of 158/102 mmhg, a pulse rate of 135 beats / min, a normal temperature and oxygen saturation of 100% on room air and no signs of heart failure. laboratory research revealed an elevated troponin t of 0.18 g / l, a creatinine kinase of 170 u / l and ck - mb of 17.9 non - significant coronary abnormalities were found by coronary angiogram. left ventriculogram showed akinetic wall motion abnormality in posterolateral and diaphragmatic wall segments, and a hypokinetic apex (fig. 1). medical treatment with furosemide, anticoagulants and an ace inhibitor was started. on the ecg, negative t waves evolved combined with prolonged qt duration (fig. 2). 1left ventriculogram with wall motion abnormalities in the distal segments and normal wall motion in basal segments comparative to stress - induced cardiomyopathy. a systolic phase, b diastolic phasefig. 2ecg at admission (a) with slight st elevation in v2 and 16 h after admission (b) with negative t waves in the right precordial leads, i and avl with prolonged qt duration left ventriculogram with wall motion abnormalities in the distal segments and normal wall motion in basal segments comparative to stress - induced cardiomyopathy. a systolic phase, b diastolic phase ecg at admission (a) with slight st elevation in v2 and 16 h after admission (b) with negative t waves in the right precordial leads, i and avl with prolonged qt duration the patient appeared to have had a previous incidence of thyrotoxicosis (graves disease) in 2002. her current thyroid function confirmed a toxic state with a thyroid - stimulating hormone (tsh) of 100 pmol / l and tri - iodothyronine (t3) of 26 pmol / l. six days after treatment with propylthiouracil and propanolol, her heart rate reduced to 80 beats / min and the sequential echocardiogram showed an improvement of her systolic left ventricular function (fig. 3). magnetic resonance imaging measured a left ventricular ejection fraction of 52%. 3transthoracic echocardiogram with return of normal systolic function after initiating medical treatment transthoracic echocardiogram with return of normal systolic function after initiating medical treatment hyperthyroid patients can present with angina symptoms and abnormal ecgs in the absence of coronary artery disease. a myocardial infarction can be induced due to increased demand. a hypertrophic heart due to an increase in cardiac protein synthesis is also seen. forfar. discovered that hyperthyroid patients have an elevated ejection fraction at rest but experience significant declines during exercise, also known as hyperthyroid cardiomyopathy. we speculate that a surge of catecholamine release during physical stress in combination with a reduced ejection fraction caused by the thyrotoxicosis state resulted in severe left ventricular dysfunction. after initialising medical treatment the symptoms were reduced and the left ventricular function returned to normal with borderline left ventricular hypertrophy remaining. in this case report we demonstrate a combination of reversible cardiomyopathy related to stress (tako tsubo) and thyrotoxicosis that resolved completely with medical treatment. | we report on a 44-year - old woman presenting with chest pain and dyspnoea without previous stress - related events. by means of echocardiography severe left ventricular dysfunction and wall motion abnormalities resembling stress - induced cardiomyopathy (tako tsubo) were seen. laboratory investigation revealed thyrotoxicosis and elevated cardiac markers. six days after starting medical treatment, complete restoration of the left ventricular function was observed. the transient left ventricular dysfunction was induced by thyrotoxicosis resembling stress - induced cardiomyopathy that resolved completely after medical treatment. |
healthcare - associated infections (hais) are those infections in patients receiving medical treatment in a healthcare facility. hais include hospital - acquired infections and some patients receiving healthcare treatment on an outpatient basis. these 2 types of infections have different microbiological patterns and report higher resistance rates than community - acquired infections. finally, a worrisome aspect is the increasing isolation of enterobacteriaceae producing extended - spectrum beta - lactamases (esbl) and others multidrug - resistant microorganisms. patients hospitalized in a urology ward showed a higher prevalence of urinary catheterization in comparison with those in other department and urologic surgery is performed in most of them. therefore, infections in patients hospitalized in a urology ward may show specif ic characteristics regarding type of infections, microbiological patterns, and resistance rates. our purpose was to analyze the incidence, risk factors and characteristics of hais in patients hospitalized in a urology ward. we carried out a prospective observational study for four years (2012 to 2015) to review hais. the database included comorbidities, risk factors and microbiological characteristics of all patients admitted in the urology ward. our objectives were stated at the beginning of the study and the research was initiated in 2012. the study excludes patients younger than 16 years old and those admitted for a renal transplantation because they are hospitalized in the pediatric and nephrology ward, respectively. the study analyzed the incidence, type and risk factors for hais, the microbiological characteristics and resistance to antibiotics. we used the definition of hais according to friedman criteria as an infection that occurs 48 hours after hospital admission and those in patients receiving dialysis, chemotherapy, outpatient intravenous therapy, resided in a long - term care facility or, hospitalized in the preceding 90 days. both hais and hospital - associated infections differ to community - associated infections in term of microbiological data and susceptibility to antibiotics. we also included in our study patients admitted from emergency with an infection related to a previous hospitalization in urology. patients with negative or contaminated cultures were considered as hais if there were symptoms compatible with infection such as fever, dysuria, lumbar or suprapubic tenderness for urinary infections. surgical site infections (ssi) were defined as those infections that occurred after surgery in the place where the surgery took place and included superficial incisional, deep incisional or organ space ssi according to ccd criteria. pathogen identification and antimicrobial susceptibility testing antibiotic prophylaxis was routinely used before surgical procedure according to a protocol approved by our center. in brief, we used cephalosporins in the induction of anesthesia before open, laparoscopic or endourologic clean - contaminated surgery in the urinary tract such as transurethral resections and prostatectomies. in patients with an indwelling urinary catheter or risk factors, we recommended perioperative treatment for one day. when the surgery required bowel management, we used gentamicin and vancomycin. in case implantation of prosthetic devices such as artificial urinary sphincter or penile prosthesis, we recommend antibiotic treatment during at least 3 days. we collected demographic characteristic and risk factors for infections such as age, sex, hypertension, diabetes mellitus, heart disease, liver disease and immunosuppression and american society of anesthesiologists (asa) physical status classification. we also reviewed urological factors such as prior urinary infection, urinary catheterization, and surgery during the hospitalization. the research was a prospective investigation, and it does not affect the daily management. therefore, informed consent was not required the study was approved by the ethic committee of instituto de investigacin hospital 12 de octubre. the association between demographic variables and risk factors for hais was assessed using chi - square test or fisher exact test for qualitative variables. continuous variables were compared using the student t - test or analysis of variance with bonferroni as a posthoc test. a multivariable analysis using a binary logistic regression was performed to analyze factors associated with a higher risk of hais, including those variables with a p - value less than 0.05 in the univariate analysis. the results are shown as odds ratio (or) and 95% confidence intervals (95% ci). among 6,546 patients hospitalized in our urology ward, 415 (6.3%) reported some type hai. the incidence of hais decreased from 7.3%6.5% in 20122014 to 4.8% in 2015. the percentage of urinary infections among hais increased from 66% in 2012 and 2013 to 80% and 71.2% in 2014 and 2015, respectively. conversely, ssi decreased from 30 and 24% in 2012 and 2013 to 14.2% and 18% in 2014 and 2015. four hundered twenty - eight hais were reported in 415 patients, more than one type of hais was more commonly found after cystectomy (the evolution of each type of hais over the time is summarized in table 2). the univariate analysis showed that higher incidence of infections among patients with older age (or, 1.01 ; 95% ci, 1.0041.016 ; p=0.001), asa physical status classification iii iv (or, 1.45 ; 95% ci, 1.251.69 ; p<0.001), arterial hypertension (or, 1.26 ; 95% ci, 1.031.54 ; p=0.020), immunosuppression (or, 2.08 ; 95% ci, 1.443.00 ; p<0.001), previous urinary infection (or, 3.44 ; 95% ci, 2.325.12 ; p<0.001), and indwelling urinary catheter before admission (or, 2.31 ; 95% ci, 1.872.86 ; p<0.001) or during the hospitalization period (or, 1.62 ; 95% ci, 1.252.10 ; p<0.001). multivariate logistic regression analysis showed that higher asa physical status classification (or, 1.39 ; 95% ci, 1.151.67 ; p<0.001), immunosuppression (or, 1.80 ; 95% ci, 1.132.88 ; p=0.013), prior urinary tract infection (or, 4.465 ; 95% ci, 2.328.57 ; p<0,001), and indwelling urinary catheter prior admission (or, 1.74 ; 95% ci, 1.322.30 ; p<0.001) were independent risk factors for hais (table 3). a surgical procedure was performed in 4,952 (75.6%) of patients. among them, the incidence of hais was 4% (110 out of 2,737) after transurethral surgery, 7.4% (23/312) after open prostatic surgery, 8.7% (33/318) renal surgery and 54.2% (39/72) following radical cystectomy. the highest incidence of hais after cystectomy was reported in 2012 and 2013 (78.6%68.7%) in comparison with 50%31.8% in 20142015. the most commonly isolated pathogens were escherichia coli (25.1%), followed by species of enterococcus (17.5%), klebsiella (13.5%) and pseudomonas (12.3%). staphylococcus aureus and candida were isolated in a 6.7% and 5.6% of positive cultures, respectively (fig. the isolation of klebsiella spp. increased over the time, representing 17% of positive cultures in 2015. were the most frequently isolated microorganism in patients with diabetes mellitus, liver disease, immunosuppression and prior urinary infection. in patients with ssi following abdominal or renal surgery, e. coli showed resistance rates of 34.9% for amoxicillin+betalactamase inhibitor, 53.5% for fluoroquinolones, 9.3% for amikacin.. showed resistance rates of 39.3% for third generation cephalosporins and 4.4% for carbapenems. among cultures where e. coli was isolated, 24.7% were esbl - producing bacteria and 34.7% whether klebsiella was isolated.. showed resistance rates of 26.7% for amoxicillin / ampicillin, 47.8% for fluoroquinolones and 1.7% for vancomycin. pseudomonas aeruginosa showed resistance rate of 40.5 for piperacillin / tazobactam, 33.3% for carbapenems and 26.2% for amikacin. however, if esbl - producing bacteria were isolated, the appropriateness of empirical treatment decreased to 81.2%. isolation of esbl - producing bacteria or enterococcus and patients who underwent radical cystectomy showed a higher rate of inappropriate empirical antibiotic treatment. the mortality rate among patients who suffered from hais was 2.4% in comparison with 0.3% in those who no suffer from hais. risk factors for mortality were immunosuppression (or, 6.7) and inadequate empirical antibiotic treatment (or, 2.2). mortality causes in patients who suffered from hais were related to infection in 8 out of 10.. however, few articles evaluated this topic in urological patients. among these, it should be pointed of the european society of infections in urology (esiu - eau) which reviewed hais in urology departments since more than ten years ago. such studies are essential as hais in urological patients have some special features such as higher incidence of urolithiasis, lower urinary tract symptoms, higher prevalence of previous urinary infections. furthermore, patients admitted in urology ward frequently had an indwelling urinary catheter. in patients hospitalized in a general ward on the other hand, among patients in a urology ward, a surgical procedure is frequently carried out, 75% according to our data. endourological transurethral procedures constitute 55% of surgeries performed. therefore, urinary infections are the most common type of hais (70%). data from non - urology departments showed that urinary infections represent 15%57% of hais. both, urinary catheter, and surgical procedure are well - established risk factors for hais. surgery of the urinary tract and urinary catheter must be evaluated together. furthermore, 19% had an indwelling catheter before admission. indwelling urinary catheter and the duration of catheterization are the main risk factors for healthcare - associated urinary tract infections with the higher incidence among patients with urinary catheter before admission (or, 1.74). although antimicrobial treatment is not required in case of asymptomatic catheter - bacteriuria, adequate perioperative prophylaxis is needed before surgery. prior transurethral surgery, a urinary culture should be advised to tailor the antibiotic prophylaxis or treatment before surgery. apart from, urinary catheter and urological surgery, some characteristics increased the risk of hais. our results are in concordance with other studies that demonstrated that comorbidities such as immunosuppression, diabetes, obesity, liver dysfunction, malnutrition are related to an increased risk of hais. in our series, higher asa physical status classification is associated with higher incidence of hais, 4.5% among asa i ii and 8% in asa iii iv. moreover, previous antibiotic treatment and urinary infections are also risk factors to focus due to a higher incidence of infection and a different microbiological pattern. according to our data, in patients with previous urinary infection, it has been described that when several risk factors are associated, there is an increased risk of isolation pathogens with higher resistance rate such as klesiella spp. or pseudomonas spp. or candida spp.. our study demonstrated a reduction in the incidence of infection from 7.3% in 2012 to 4.8% in 2015. several reasons may explain the evolution of the incidence of infections. in our opinion, doctors, nurses and all personnel working with patients are aware of infections, and different measures are implemented to prevent infections. among them, a correct hand hygiene and avoid rings or watches. on the other hand, urinary catheter control has improved with prompt removal and reducing unnecessary placement. a program reviewing the appropriateness of urinary catheter has demonstrated that decrease the percentage of catheterization and the prevalence of infections. otherwise, we review our results periodically, so we are informed about our infection rates and we can design strategies to prevent them. moreover, we identify some risk factors for infections that they are also related to specific microbiological patterns. the effect of our research monitoring hais is demonstrated with the evolution of patients with suspicion of infection where cultures were not taken. the percentage of patients without culture decreased from 6.1% in 2012 to 0.0% and 3.5% in 2014 and 2015, respectively. finally, antimicrobial prophylaxis is a key point as is used to prevent infections after diagnostic or therapeutics procedures. perioperative prophylaxis must be chosen to take into account the type of surgery, the contamination burden, and some aspects related to specific risk factors of the patient, such as comorbidities, immunosuppression, indwelling catheters or a previous urinary infection. among the different types of hais, the total number of ssis were 35 in 2012, 28 in 2013, 15 in 2014 and, 17 in 2015. apart from monitoring and be aware of infections, others reason could explain the reduction in ssis. several studies demonstrated that laparoscopic nephrectomy and cystectomy are associated with lower rates of ssis. secondly, hand antisepsis using alcohol - based solutions may reduce the incidence of ssis. radical cystectomy reported the highest incidence of hais, up to 50% in our series, and ssi is the most common type of hais. demonstrated a reduction in the incidence of ssi from 32% to 18% with the standardization in suturing, drain removal and surgical dressing management. finally, it should be pointed out that there is a higher incidence of infections after renal surgery in comparison with transurethral surgery. although a laparoscopy approach is frequently performed, open surgery is required in some cases. currently, open approach is indicated in patients with a complex surgery, and it is associated with risk factors for hais such as higher amount of bleeding, longer operating time, longer hospitalization and in some cases, a contaminated surgical filed. moreover, although urinary catheter is removed earlier after renal surgery than in transurethral surgery, drainage is usually used. therefore, it is necessary to remove all drainages as soon as possible, and the management of the wound must be optimized. the microorganism most commonly isolated is entero - bacteriaceae, 59% in our series. among them, e. coli is the most frequent, 40% according to global prevalence study on infections in urology and 25% in our department. data from study on the prevalence of nosocomial infections in spain study carried out in several hospitalization wards, showed that e. coli accounted 15%17% of positive cultures and enterobacteria 33%. enterococcus is isolated in 17.5% and is the most common microorganism in patients with ssis after abdominal surgery. when enterococcus is suspected antibiotics such as vancomycin are recommended as high susceptibility pseudomonas are commonly isolated, 13.5% and 12.3% in our series, respectively. according to gpiu data, klebsiella spp. and pseudomonas accounted 11.1% and 10.8%. the main awareness related to the isolation of non - e. resistant to carbapenems were found. moreover, if previous urinary infection, diabetes mellitus, liver disease or immunosuppression ; klebsiella was the most frequently isolated pathogen. both, our data, and gpiu reported resistance to quinolones and third generation cephalosporins up to 50%. the highest resistance rate is found in south europe, asia, and south america. the resistance rate is related to the prescription of antibiotics, and an appropriate selection of antibiotics may reduce resistances. this point may be demonstrated with lower resistance to aminoglycosides in our department in comparison with gpiu. in our center, aminoglycosides are not usually prescribed as prophylaxis before surgery. multiresistant pathogens are frequently isolated in a urology ward ; esbl - producing bacteria were found in 34.3% of cultures where enterobacteriaceae were isolated. isolating of esbl - producing bacteria is a key point in prevention of hais as frequently cross - resistance is found. risk factors described for esbl - producing bacteria are older age, male sex, diabetes mellitus, urinary catheter and prior urinary infections. therefore, rationalization of antibiotic usage and select empirical antibiotics adequately is essential to achieve better outcomes. observational studies are useful to improve outcomes and reduce the incidence of hais. for instance, in our department, the incidence of hais was 7.3% at the beginning of the research and 4.8% in 2015. one of the strengths of the study is that we used a clinical definition of hais, including patients with sterile cultures. lower incidences are reported if only positive cultures are taking into account, and higher incidence is found in studies carried out evaluating hais in one day due to patients with and infections have longer hospitalization period. however, it included patients during a period of 4 years, and the results may be extrapolated to similar hospitals in the area. on the other hand, patients who suffer hais after discharge may be not included. however, hais is developed after discharge and the patient required admission to our hospital ; the hais was associated with the previous hospitalization in the urology ward. prospective monitoring of hais and an adequate knowledge of the microbiological patterns may decrease the incidence of infections in patients hospitalized in a urology ward. comorbidities, higher asa physical status classification, previous urinary infections a urinary catheter are risk factors for hais. were the most frequently isolated microorganism in patients with diabetes mellitus, liver disease, immunosuppression and prior urinary infection. | purposehealthcare - associated infections (hais) in urological patients have special features due to specific risk factors. our objective was to evaluate the characteristics and risk factors for hais in patients hospitalized in a urology ward.materials and methodswe evaluated prospectively, from 2012 to 2015, the incidence, types and risk factor for hais, microbiological and resistance patterns.resultsthe incidence of hais was 6.3%. the most common types were urinary infections (70.5%) and surgical site infections (22.1%). univariate analysis showed an increased risk of hais among patients with american society of anesthesiologists physical status classification system iii iv (odds ratio [or ], 1.39 ; p<0.001), immunosuppression (or, 1.80 ; p=0.013), previous urinary infection (or, 4.46 ; p<0,001), and urinary catheter before admission (or, 1.74 ; p<0.001). the surgical procedures with the highest incidence of hais were radical cystectomy (54.2%) and renal surgery (8.7%). the most frequently isolated microorganisms were escherichia coli (25.1%), enterococcus spp. (17.5%), klebsiella spp. (13.5%) and pseudomonas aeruginosa (12.3%). enterococcus sp was the most common microorganism after radical cystectomy and in surgical site infections, e. coli showed resistance rates of 53.5% for fluoroquinolones, 9.3% for amikacin. the percentage of extended - spectrum betalactamase producing e. coli was 24.7%. klebsiella spp. showed resistance rates of 47.8% for fluoroquinolones, 7.1% for amikacin and 4.3% for carbapenems. enterococcus spp showed resistance rates of 1.7% for vancomycin and ; p. aeruginosa of 33.3% for carbapenems and 26.2% for amikacin.conclusionscomorbidities, previous urinary infections, and urinary catheter are risk factors for hais. the microorganisms most commonly isolated were e. coli, enterococcus and p. aeruginosa. prospective monitoring may decrease the incidence of infections. |
our understanding of the interleukin-1 family (il-1f) has recently expanded to encompass 11 members : il-1f1-il-1f11. these cytokines are also termed il-1, il-1, il-1 receptor antagonist (il-1ra), il-18, il-36ra, il-36, il-37, il-36, il-36, il-38, and il-33, respectively (table 1). these protein molecules play a prominent role in inflammation and immune responses, acting as the first line of defense against invasive pathogenic microorganisms and physical damage. il-38 is a novel member of the il-1f identified in 2001 by a unique high throughput cdna screening approach taking advantage of a set of oligonucleotide probes to hybridize successive arrays of human cdnas from various tissues [3, 4 ]. il-38 is the 10th member of il-1f and its receptor is termed il-1 receptor - related protein 2 (il-1rrp2, il-36r). il-38 is an il-36 antagonist and functions as a typical receptor antagonist similar to il-1ra and il-36ra. il-38 reduces inflammation by preventing the binding of agonist receptor ligands to il-36r, a specific receptor of il-38. the novel il-1-like gene, il-38, is located in the il-1 family cluster (except il-18 and il-33) on human chromosome 2q13 - 14.1 near the il-1ra gene (il-1rn) and il-36ra gene (il-36rn). the il-38 gene is located 49,479 bp upstream from il-1rn on the same dna strand. the primary translated product is an il-38 precursor, 152 amino acids in length and with 16.9 kd molecular mass. sequence analysis indicated that the il-38 protein shares 41% homology with il-1ra and 43% homology with il-36ra [4, 7 ] and lower homology (1430%) with il-1 and other il-1 family proteins. in mammalian chinese hamster ovary cells, recombinant il-38 protein was synthesized into two forms, a major form at 25 kd and a minor form at 17 kd. lin. suggested that the major form of il-38 might be a result of posttranslational protein modifications, such as phosphorylation. however, studies have shown that the il-38 protein lacks n - glycosylation and o - glycosylation consensus sites in chinese hamster ovary cells. as is typical of the il-1 family, including il-36ra, il-36, il-36, and il-36, il-38 lacks a signal peptide and caspase-1 consensus cleavage site [3, 7 ]. furthermore, the natural n terminus for il-38 is still unclear. using the multiple alignment sequence profile - based searching method (psi - blast), an automated sequence and structure searching procedure (high throughput modeling), and a fold recognition method (seqfold), three - dimensional structural models of il-38 the il-38 structural model displays a 12--stranded trefoil structure and shares similarity with the crystal structure of il-1ra and il-1 [3, 8 ]. in addition, il-38 was categorized in the il-36 subfamily according to the length of its precursor. thus, il-38 likely also belongs to this subfamily because it has the ability to bind to the common receptor il-36r [2, 9 ]. to date, no reports have described the components and properties of il-38. we adopted the protparam tool [14, 15 ] application to analyze the amino acid composition of il-38, which consists of 19 amino acids. a (alanine), e (glutamic acid), and l (leucine) were the most prevalent amino acids (9.2%) in il-38, followed by g (glycine, 7.9%), p (proline, 6.6%), and s (serine, 6.6%). only h (histidine) was not present in il-38 (figure 1). the molecular weight of il-38 is 16.9 kd and is consistent with a previous study, as predicted by protparam tool. il-38 has a half - life of 7 h, an isoelectric point (pi) of 4.94, and the molecular formula of c757h1164n198o226s9, as analyzed by sopma (self - optimized prediction method from alignment) [15, 16 ]. moreover, the second structure of il-38 was composed of a -turn, random coil, -sheet, and -helix. analysis further indicated that the random coil and -sheet were uniformly distributed in the protein chain. by multitissue first - strand cdna pcr analysis, il-38 mrna was measured in a range of tissues, including heart, placenta, fetal liver, skin, spleen, thymus, and tonsil. il-38 was expressed mostly in the skin and in proliferating b cells of the tonsil. however, in nonimmune tissues, such as human heart and placenta, il-38 was present at low levels, similar to other il-1f members. some il-1f members are constitutively produced, whereas others have inducible expression, being rapidly induced by bacteria or inflammatory mediators [7, 17, 18 ]. in 2001, it was speculated that il-38 acted as an il-1 receptor antagonist because of its amino acid homology to the naturally occurring il-1ra and the observation that il-38 could bind to the soluble il-1 receptor type i (il-1ri). il-1ri was once considered a receptor for il-38 [3, 5 ]. however, the binding affinity of recombinant il-38 is significantly lower than that of il-1ra and il-1. recently, researchers doubted whether il-1ri was a receptor for il-38. il-1rrp2 was regarded as an il-38-specific receptor and was also called il-36r. in a report by van de veerdonk, the combining capacity between il-38 and il-1ri, il-36r, il-18r, and il-1r accessory proteins (il-1rap, il-1racp) was compared, in the presence of increasing concentrations of il-38. il-38 bound to il-36r but did not bind to the other immobilized receptors. furthermore, il-38 binding to immobilized il-36r was comparable to il-36ra binding to the same receptor. it was observed that increasing the concentration of il-38 resulted in increased optical density, reaching a plateau at 16.7 g / ml, a higher value than that obtained for il-36ra. based on the binding studies, these data suggest il-38 could act by blocking the il-36r pathway. these cytokines combine with the cell - surface receptor il-1r and induce downstream signaling, including downstream nuclear transcripts such as nuclear factor-b (nf-b) and activator protein-1 (ap-1). furthermore, as a feedback and adjustment mechanism, these signaling molecules induced the expression of cyclooxygenase, nitric oxide synthase, and other inflammatory mediators to promote the development of inflammation [19, 20 ]. in line with the characteristics of il-38 and the homology of il-38 and il-36ra, it can be concluded that il-38 has a role in inflammatory disease by il-36ra pathway - related molecules (figure 2). the biological function of il-38 is to inhibit il-36 cytokine (il-36, il-36, and il-36) binding to il-36r, similar to il-36ra. according to its activity as a receptor antagonist il-38 might also be related to il-1r and il-18r signaling pathways, although there is no evidence regarding its role in these specific signaling pathways. because of its homology with other il-1f members, il-38 is thought to have the biological activity of il-1f members. il-1 cytokines are primarily proinflammatory cytokines as they stimulate the expression of genes associated with inflammation and immunological diseases. il-1 or il-1 binds to its primary receptor il-1ri, which recruits a second receptor subunit, il-1racp. formation of the receptor heterodimer induces biological responses typically involving the activation of nf-b and mitogen - activated protein kinase (mapk) pathways. il-1f6 (il-36), il-1f8 (il-36), and il-1f9 (il-36) also activate nf-b and mapks similar to il-1. therefore, most molecules involved in il-1f - induced signaling, such as cytokines, chemokines, adhesion molecules, and enzymes, are mediators of inflammatory diseases [22, 23 ]. il-1ra (il-1f3) is the receptor antagonist of il-1, a protein composed of two major subunits, il-1 and il-1. il-1ra is synthesized and released in response to the same stimuli that lead to il-1 production. il-1ra, a potent anti - inflammatory cytokine, competitively inhibits stimulation by inflammatory mediators by binding to il-1r1 and preventing the recruitment of il-1racp. il-1ra is associated with severe autoimmune and inflammatory diseases such as periodontitis, vaginitis, non - hodgkin 's lymphoma, gastric cancer [28, 29 ], osteoarthritis, precancerous lesions, and inflammatory bowel diseases. deficiency of the il-1-receptor antagonist (dira), caused by mutations in il1rn, can lead to an autosomal recessive autoinflammatory disease. dira allows unopposed actions of il-1, resulting in life - threatening excessive systemic il-1-mediated inflammation with skin and bone involvement. a study by korthagen. aimed to elucidate the influence of polymorphisms in il1rn on idiopathic pulmonary fibrosis (ipf) susceptibility and mrna expression. polymorphisms of il1rn manifested as a variable number tandem repeat (vntr), which affected il-1ra mrna expression, suggest that lower levels of il-1ra predispose to developing ipf. il1rn may be a future novel therapeutic target with high specificity, low toxicity, and side effects for the treatment of specific diseases. it is important to enhance our understanding of il1rn functions, such as its interaction with other genes and the influence of environmental factors on its production, to develop treatments with reduced side effects. in addition, il1ra - deficient (il-1ra/) mice, good animal models for experimental studies, spontaneously develop several inflammatory diseases, resembling arthritis, aortitis, intervertebral disc degeneration, and psoriasis in humans due to unopposed excess il-1 signaling. the current knowledge also suggested that il-1ra, an endogenous inhibitor of il-1, related to alcoholic steatohepatitis, liver damage, lung damage, fat mass, and aqueous - deficient dry eye in autoimmune diseases. il-36ra (il-1f5) shares 44% homology with il-1ra and is an antagonist of il-36, il-36, and il-36. il-36ra has a -stranded trefoil structure, similar to other family members of il-1, with a conserved hydrophobic core. the mechanism of il-36ra antagonism is analogous to il-1ra by forming a functional signaling complex. il-36ra protein starting at val-2 is fully active and inhibits il-36, il-36, and il-36. the a - x - asp motif is conserved in all il-1f members at the n - terminal, where val-2 of il-36ra lies in this 9-amino acid conserved sequence. all four il-36 cytokines lack a conventional signal sequence and function as extracellular cytokines although it is unclear how they are secreted. in addition, il-36ra antagonist activity requires removal of the n - terminal methionine present in the primary translation product. more extensive n - terminal amino truncation of il-36, il-36, and il-36 can dramatically increase their specific activities. il-36ra plays a key role in innate and adaptive immunity by stimulating helper t - cell responses and it is associated with many inflammatory diseases. recessive homozygous mutations in il36rn are the major cause for the development of generalized pustular psoriasis [4649 ]. mutations in il36rn, including premature termination codon mutations, frameshift mutations, and substitutions of amino acid lead to incorrect folding of the il-36ra protein. these mutations inhibit the activity of il-36ra, thus failing to antagonize il-36 signaling pathways and inducing inflammatory skin disease due to the high levels of il-36, il-36, and il-36. excessive il-36 levels in the skin of mice lead to symptoms to human psoriasis. therefore, treatment with a combination of il-36ra and il-36r might improve psoriasis by inhibiting il-36 stimulation and might be an ideal treatment strategy for inflammation of human skin. il-36ra functions as an anti - inflammatory cytokine in the brain and enhances the hippocampal expression of il-4. this is a consequence of its interaction with the orphan receptor, single ig il-1r - related molecule (sigirr)/tir8. collectively, in vitro il-4 mrna and protein expression in glia induced by the interaction of il-36ra and sigirr / tir8 play a critical role in its anti - inflammatory properties. il-36 cytokines also have a significant association in the pathogenesis of rheumatoid arthritis [9, 51, 52 ], inflammatory lung diseases [53, 54 ], obesity, bile duct occlusion disorder, and chronic glomerulonephritis. data strongly suggest that il-36ra might be a useful treatment for il-36-related diseases. in recent years, scholars identified a novel cd4 t - cell subtype, which was different from t - helper 1 (th1) and th2 cells. these cells were named th17 cells due to the expression of il-17 and these discoveries have improved our understanding of inflammatory processes. th17 cells are different from natural t - cell precursors, and the mature cells secrete a variety of cytokines such as il-17 and il-22 [57, 58 ]. th17 plays an important role in a variety of autoimmune diseases and has an independent regulatory mechanism for their differentiation and development. th17 is associated with the pathogenesis of systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, psoriasis, inflammatory bowel disease, and autoimmune thyroid diseases. previous studies demonstrated the functions of il-38 and th17 cells by blocking the il-1r, il-18r, and il-36r pathways. these data suggested that the influence of il-38 on th17 cells was similar to blocking il-1r and il-36r pathways, which suppressed il-17 and il-22 secretion. consistent with binding data and the suppression of il-17 and il-22, we suggest that il-38 has similar biological effects on th17 cells. il-38 gene polymorphisms are associated with psoriatic arthritis (psa), ankylosing spondylitis (as) [5961 ], and cardiovascular disease, suggesting that il-38 is strongly correlated with these inflammatory diseases. the frequencies of th17 cells are significantly increased in the peripheral blood of patients with psa and as [8, 6368 ]. in addition, the number of th17 cells and serum il-17 levels were strongly related to systemic disease activity both at the onset and during disease progression of psa and as. il-38 reduced the expression of c. albicans - induced il-17 and il-22 from peripheral blood mononuclear cells (pbmcs) by reducing the stimulation of proinflammatory cytokines in the tissues. a recent study reported that low concentrations of il-38 were more effective than higher concentrations in inhibiting il-17 and il-22 production because higher concentrations modestly increased il-22. similar to il-36ra, blocking il-38 suppressed c. albicans - induced th17 cytokine production [5, 69 ]. both il-38 and il-36ra inhibited the production of il-17 and il-22 by specifically binding to the cell surface - specific protein receptor il-36r. however, neither il-38 nor il-36r functions as a classic receptor antagonist. in pbmcs, the dose - response suppression of il-38 and il-36ra by il-36-derived il-8 was not similar to that of il-1ra. il-38 decreased the production of proinflammatory cytokines similar to il-36ra [5, 70 ]. in contrast, il-38 and il-36ra have parallel effects on the production of lipopolysaccharide - induced il-6 from dendritic cells (dcs), inducing a twofold increase. il-6 has two adverse effects on immune cells : il-6 is proinflammatory but also suppresses inflammation in tissues injured by burns or other damages. il-1ra and il-38 have a comparable dose - effect regarding their antagonist activities and function as classic receptor antagonists ; the higher the concentration of il-22, the stronger its inhibition. compared with il-1ra and il-38, il-36ra does not behave as a typical receptor antagonist. il-38 and il-36ra function as antagonists at high concentrations, but at low concentrations, they inhibit the binding of coreceptors. thus, il-38 and il-36ra are defined as partial receptor antagonists, although they mimic the effects of il-1ra on the production of inflammatory cytokines. il-1 and most related family members are primarily proinflammatory cytokines that induce the expression of genes associated with inflammatory diseases. only il-37 acts as an anti - inflammatory cytokine. the binding of il-1ra and il-36ra to their receptor reduces inflammation by blocking the binding of receptor ligands. the production of fungal - induced il-17, il-22, and il-36-derived il-8 was decreased by il-38, which may play an important anti - inflammatory role in inflammatory diseases. many articles have demonstrated that il-1ra and il-36ra are associated with arthritis and psoriasis, respectively. in addition il-36 cytokine has significant in vivo effects on dcs and t cells in human immune responses via its role in the differentiation of inflammatory th1 cells [7072 ]. in conclusion, the current knowledge supports the concept that il-38 may be closely associated with il-36-mediated inflammatory diseases. thus far, the il-1 receptor antagonist anakinra, the soluble decoy receptor rilonacept, and the neutralizing monoclonal anti - il-1 antibody canakinumab have been approved as il-1-targeting agents for the treatment of specific diseases. another study demonstrated the beneficial use of a monoclonal antibody directed against the il-1 receptor and a neutralizing anti - il-1 antibody in clinical trials. the il-38-related signaling pathway is poorly understood and requires further study. the increasing knowledge of the mechanisms that regulate chronic inflammatory conditions such as rheumatoid arthritis may provide a potential strategy for the development of anti - inflammatory treatments for autoimmune diseases and establish a theoretical basis for clinical trials and drug development. | interleukin- (il-) 38 is a recently discovered cytokine and is the tenth member of the il-1 cytokine family. il-38 shares structural features with il-1 receptor antagonist (il-1ra) and il-36ra. il-36r is the specific receptor of il-38, a partial receptor antagonist of il-36. il-38 inhibits the production of t - cell cytokines il-17 and il-22. il-38 also inhibits the production of il-8 induced by il-36, thus inhibiting inflammatory responses. il-38-related cytokines, including il-1ra and il-36ra, are involved in the regulation of inflammation and immune responses. the study of il-38 and il-38-related cytokines might provide new insights for developing anti - inflammatory treatments in the near future. |
brain - derived neurotrophic factor (bdnf) is a neurotrophic factor that appears throughout the mammalian central nervous system1. it is frequently found in the hippocampus of the brain (hippocampal formation), as well as in the cerebral cortex and amygdalus. bdnf is reported to affect learning ability and memory, as it operates in the areas that regulate neuronal growth, and repair and neuroplasticity. it is also a feature of the brain during brain formation before birth and its influence continues through adolescence2,3,4. bdnf is reported to be an important material for the growth and development of nerve cells in different areas both within and outside of the brain, increasing resistance to damage and improving the survival of neurons1. it is a key factor in growth and differentiation during neural development, and is often used as an index to measure the impact of exercise on the rehabilitation of damaged nerves. it is further reported that both exercise and learning induce growth of the motor cortex, increase in hippocampal bdnf, and increase in the plasticity of the brain, which in turn induces development of the cell body and axon of the associated neurons5,6,7. bdnf expression in the hippocampus has been reported to improve the production and survival of brain cells8, and bdnf expression is reported to be stimulated by exercise6, 9, 10. it has also been reported that exercise has a positive impact on bdnf expression in the animal brain11. for young people, increases in bdnf induced by aerobic exercises would enhance neuronal growth and development in the hippocampus, which would in turn enhance cognitive ability, learning ability and memory, greatly improving educational performance. therefore exercise is considered to be essential for the development of the adolescent brain. insulin - like growth factor 1 (igf-1), one of the factors involved in bdnf expression, is known to mediate regulation of genes involved in bdnf - related neurogenesis12, and it is involved in the growth and differentiation of neuron units of the brain13. in association with exercise, igf-1 increases in both the brain and peripheral nerves after exercise and neurotransmitters are also increased in the blood vessels14. on the other hand, psychological stress has negative effects on nerve cells and heightened stress reduces bdnf mrna expression6. a fast - paced lifestyle, coupled with education policy and environmental changes in south korea are thought to have increased the psychological stress of the korean youth population. corticotrophin - releasing factor (crf) is secreted by the hypothalamus, which in turn induces the release of adrenocorticotrophic hormone (acth) from the anterior pituitary. cortisol is released not only in response to psychological stress, but also as a product of the energy generation required for exercises. with exercise, cortisol is released through the hypothalamic - pituitary - adrenal axis into the circulation of the human body. cortisol, which is one of the major corticosteroids, plays a role in the conversion of glycogen in muscles and the liver into blood glucose, or fats into fatty acids for the purpose of metabolic energy production. also, cortisol responds sensitively to self - defensive stimuli such as psychological oppression, anxiety, or fear, similar to the response to energy generation for the stimulation of movement. the relationship between stress and bdnf mrna (messenger ribonucleic acid) reveals that increased stress will lower the level of bdnf expression6 and significantly disturbs the creation of the nerve cells in the hippocampus15. most previous studies of bdnf have used experimental animals and many of their results correlate with a particular disease. in those experiments, animals were forced to perform aerobic exercises, with stress induced for the initiation and maintenance of the exercising condition. when these animals are given a stimulating stressor, such as electrical shocks or swimming as exercise, the stress elicits strong instincts to escape and initiates changes in the peripheral circulation. it also activates circulation in the central brain, thus affecting the whole body. in addition to the need to use a stressor to initiate exercise conditions in animals, the results use of these animal models have obvious limitations regarding their application to the human body. in order to investigate the effect of cortisol on the expression of bdnf and igf-1 through aerobic exercise, such experiments must ultimately be carried out using voluntary exercises performed by human subjects, negating the need for stress induced exercise states which can confound results. previous studies have not yet sufficiently proved the relationship of cortisol expression and nerve cell growth in response to aerobic exercise, as well as the involvement of bdnf and igf-1. the purpose of this study was therefore, to identify effect of aerobic exercise on the expression of cortisol, bdnf and igf-1 related to nerve cell growth. twenty healthy junior high school students, who did not regularly exercise and were not previously diagnosed as having abnormal glucose metabolism volunteered to participate in the study. this study was approved and reviewed by the institutional review board of the human care and use committee of the exercise physiology research institute of dankook university, and written consent forms were obtained from all of the subjects parents. bdnf, igf-1, and cortisol levels of all the subjects were measured at the start of the study. after baseline measurement, the subjects were randomly assigned an exercise group (n = 10) or a control group (n = 10). the characteristics of the subjects are shown in table 1table 1.the characteristics of the subjects (msd)variables groupage (yrs)height (cm)body weight (kg)%fateg (n=10)15161.2 2.556.3 6.819.8 4.6cg (n=10)15162.6 3.455.7 7.718.9 3.8eg : exercise group, cg : control group. eg : exercise group, cg : control group the exercise group participated in 8 weeks of supervised aerobic exercise training. the exercise group trained for 3 days a week for 8 weeks, while the control group was asked to continue their normal sedentary activities. maximal oxygen uptake (vo2max) of all the exercise group participants was measured in order to prescribe aerobic exercise intensity for the purpose of the intervention. the exercise group subjects were instructed to maintain a typical diet and a particular activity pattern throughout the study, while the subjects in the control group were asked to continue their daily normal and sedentary activities. pre - assessment blood samples were obtained 2 days before, and 2 days after the end of the exercise intervention for the assessment of bdnf, igf-1, and cortisol (table 2table 2.baseline values of bdnf, igf-1, and cortisol in eg and cgvariableseg (n=10)cg (n=10)m sdm sdbdnf (pg / ml)23,321.7 6,247.024,071.3 6,139.2igf-1 (ng / ml)397.1 131.1420.0 172.3cortisol (g / dl)13.6 4.114.5 4.6eg : exercise group, cg : control group. there were no significant differences (independent t - test) maximal oxygen uptake (vo2max) was measured using a computerized metabolic (quinton, usa) and a modified bruce protocol at dankook university fitness center, korea. graded exercise assessment was stopped once subjects attained an rer 1.10 or if subjects indicated that they wished to stop the test17. whole blood was centrifuged at 3,000 rpm for 15 minutes and to separate it into serum and plasma. bdnf concentration was measured using an elisa kit (sandwich enzyme - linked immunosorbant assay). igf-1 and cortisol were measured using a -counter (cobra-5010 quantum, packard, usa). elisa kits (promega, usa), igf - i - ria - ct kits (toshiba yba-200 frneo, japan) and coat - a - count cortisol kits (siemens, usa) were used to determine the concentrations of bdnf (pg / ml), igf-1 (ng / ml) and cortisol (ng / ml), respectively. the exercise program of this study consisted of an 8-week, 3 days a week aerobic exercise regimen at dankook university fitness center in gyeonggi - do, korea. pre - test vo2max, maximal heart rate (hrmax), and resting heart rates (hrrest) were measured by ecg supporting a computerized metabolic (quinton, usa). exercise was performed on treadmills and the exercise intensity was set between 40% and 60%vo2r based on the scale recommended by acsm17. in order to standardize the work performed, exercise time was individually prescribed so that each subject burned 200 kcal in an exercise session. during the testing and intervention periods, all subjects were asked not to undertake any exercise other than the ones designed for the experiments, with cooperation from their schools. all the results of this study are presented as mean standard deviation. the independent t - test was performed to examine differences in baseline characteristics of the subjects between the 2 groups. the paired t - test was performed to examine the changes between pre- and post - intervention in each group. statistical significance was chosen as p < 0.05, and all analyses were performed using spss version 18.0 (spss, chicago, il, usa). the changes in bdnf, igf-1 and cortisol after exercising 3 days per week for 8 weeks are shown in table 3table 3.the results of the paired t - test for bdnf, igf-1, and a cortisolvariablesgrouppre - testpost - test(m sd)(m sd)bdnf (pg / ml)eg23,321.7 6,247.027,566.0 5,647.6cg24,071.3 6,139.224,921.2 7,821.5igf-1 (ng / ml)eg397.1 131.1596.6 104.1cg420.0 172.3432.3 165.2cortisol (g / dl)eg13.6 4.111.2 2.3cg14.5 4.615.3 4.3values are meanssd. : significantly different within group by the paired t - test, : p < 0.05, : p < 0.001. : significantly different within group by the paired t - test, : p < 0.05, : p < 0.001 after performing 8 weeks of aerobic exercise, the change in bdnf of the exercise group was significant (p < 0.01), increasing from 23,321.7 6,247.0 the change in igf-1 was also significant (p < 0.05), increasing from 420.0 172.3 ng / ml to 432.3 165.2 ng / ml. the change in cortisol, however, was not significant, declining from 13.6 4.1 g / dl to 11.2 2.3 g / dl. the control group did not show any significant differences in bdnf, igr-1 or cortisol between pre- and post - intervention. the differences in bdnf, igf-1 and cortisol after exercise are shown in table 4table 4.the significance of differences (independent t - test) between eg and cg after 8 weeks of exercisevariableseg (n=10)cg (n=10)msdmsdbdnf (pg / ml)27,566.0 5,647.624,921.2 7,821.5igf-1 (ng / ml)596.6 104.1432.2 165.2cortisol (g / dl)11.2 2.315.3 4.3values are meanssd. : significantly different by the independent t - test, : p < 0.05, : p < 0.01. : significantly different by the independent t - test, : p < 0.05, : p < 0.01 research has been actively conducted on changes in the secretion of bdnf as it relates to brain function, so it is well known that bdnf is secreted in peripheral veins and skeletal muscles as well as in various brain regions. recently more research has been conducted to measure the reaction of human serum bdnf levels and targeted cognitive function of adult and elderly human subjects following long - term aerobic exercise and short sessions of aerobic exercise18,19,20,21,22,23. uysal. reported that when the rats performed regular aerobic exercises the density of hippocampal neurons in rats increased24., seven adult male subjects performed 12 weeks of endurance aerobic exercise and as a result bdnf serum significantly increased25. examined the effects of both short and long - term aerobic exercise on adolescents, whose brains are thought to still be undergoing significant development21. they showed enhanced bdnf expression as a result of a single session of aerobic exercise, and a higher level of enhancement after the long - term aerobic exercise. it has also been reported that endurance exercise increases bdnf protein expression in the skeletal muscle of rodents26. reported increases in the amount of bdnf as a result of long term regular exercise both when measured, during the exercise and at rest compared to groups that did not exercise at all3. in this study, after 8 weeks of regular aerobic exercise, the exercise group showed significantly increased serum bdnf expression levels, and a significant difference compared to the control group. in the study of erickson., 120 elderly subjects performed 40 minutes of walking exercise (5060% intensity) for 7 weeks, and they demonstrated, significant increases in maximal oxygen uptake, serum bdnf, cognitive ability, and the size of the hippocampus20. also recent studies have reported the tendency of bdnf to increase at rest when endurance aerobic exercise was performed11, 20, 25, 27, 28. however, a study by ferris. reported that cycling for a duration of 30 minutes at an intensity of 55% vo2max (20% below the lactate threshold) did not affect serum bdnf concentrations29. we believe the, increases in serum bdnf levels at rest and after regular aerobic exercise are related to the intensity of the exercise, and a threshold intensity of 60% vo2max seems necessary to elicit a response in bdnf levels. more research should be done to further elucidate the relationship between exercise intensity and bdnf expression in response to exercise. studied the effects of both short- and long - term exercise21, examining changes in serum bdnf levels and cognitive performance. they included the use of a face - name matching task test and a colour - word test for cognitive performance evaluation (memory). only the group which conducted 5 weeks of regular aerobic exercise showed a significant improvement in the face - name matching task test. previous studies have failed to identify a relationship between the level of expression of bdnf and improvement in brain function in humans. therefore, future studies relating the expression of bdnf and measures of brain function should also consider the effects of fifferent levels of exercise intensity on functional improvements as well as bdnf. igf-1 (insulin - like growth factor 1) is reported to be a carrier located in the upper part of a signaling pathway which induces the expression of bdnf. igf-1 has been reported to be a factor contributing to neural cell growth, differentiation and survival, and it shows increases in expression in the brain and periphery in response to various forms of exercise12. previous studies of the relationship between exercise and igf-1 have reported that growth hormone increased due to regular aerobic exercise through the promotion of igf-1 synthesis in cells. it was reported that igf-1 increased through an interaction with a variety of cells in the liver30. in this study, 8 weeks of regular aerobic exercise resulted in a significant increase in serum igf-1 expression levels post - intervention, and the exercise group also showed significant differences from the group that did not exercise regularly. in general, skeletal muscle growth and atrophy is regulated by insulin - like growth factor-131. lee and rhyu reported that igf-1 induced changes in the nervous system through aerobic exercise, which is a necessary condition for increased bdnf expression. they also showed that igf-1 was a carrier located in the upper part of a signaling pathway which regulates the expression of bdnf32. also, larnon, avitzur and klinger reported that physical activity acted in combination with growth hormones and intermediate mediators to elicit changes in bdnf expression. they further reported that when children or young people are at rest, physical activity - associated changes in growth hormone induce increases in the level of igf-1 through interactions with liver cells, which are important for protein synthesis, bone growth, and cell proliferation33. cortisol is a stress hormone secreted from the adrenal cortex of the kidneys, and it is expressed in response to external stimuli which induce psychological stress, and plays a role in increasing blood pressure and glucose levels, enabling the body to produce maximum energy as a part of the stress response mechanism. a study of the relationship between exercise and cortisol showed that short - term medium to low intensity strength exercises did not change plasma cortisol levels or may have even slightly reduced these levels, whereas high - intensity exercises increased plasma cortisol levels in the body34. in the present study, the group which performed regular aerobic exercise for 8 weeks showed decreased blood plasma cortisol levels, but the difference was not statistically significant. we believe this shows that while stress - induced exercise affects the expression of cortisol, as described in the murine studies of zheng., the negative relationship between bdnf and cortisol is likely the result of changes in cortisol due to stress rather than the exercise performed16. this study was conducted on adolescent subjects, whose brains were still undergoing development, with the purpose of identifying the effect of an 8-week aerobic exercise protocol on the expression of bdnf, igf-1 and cortisol. the results of this study suggest that chronic aerobic exercise has a positive effect, enhancing resting serum bdnf and igf-1 expression in adolescents whose brains are still developing. | [purpose ] this study was conducted on adolescent subjects whose brains are still developing with the purpose of identifying the effect of 8 weeks duration of aerobic exercises on the expression of bdnf, igf-1 and cortisol, to identify effect of aerobic exercise on the expression of cortisol, bdnf and igf-1 related to nerve cell growth. [subjects and methods ] the subjects were 20 junior - high school students with no history of physical illness. the students were divided into an exercise group and a control group. the exercise group performed 3 treadmill exercise times per week for 8 weeks. the exercise time for the consumption of 200 kcal was calculated and the exercises were performed by each individual for 8 weeks. [results ] the exercise group showed statistically significant in increases serum bdnf and igf-1 after 8 weeks, but cortisol showed no significant change. there were statistically significant differences between the groups in serum bdnf and igf-1 after 8 weeks, but the difference in cortisol levels was not significant. [conclusion ] we found that long - term regular aerobic exercises has a positive effect on the enhancement of serum bdnf levels at rest and igf-1 of adolescents who are still undergoing through brain developments. |
000150, vidhyasom, thailand), doxycycline hyclate (batch no. 20071121, huashu pharmaceutical corporation., n - methyl-2-pyrrolidone (nmp) (fluka, new jersey, usa) was used as received. sabouraud dextrose agar (sda), sabouraud dextrose broth (sdb), tryptic soy agar (tsa), and tryptic soy broth (tsb) (difco, usa) were used as media for antimicrobial test. the 20% w / w lutrol f127 aqueous solutions were prepared by the cold method as described previously. briefly, lutrol f127 was slowly added to cold water containing different amounts of nmp (4). each of dispersions was left in a refrigerator for 24 h at 4 until the solution was clear. to manipulate the various formulation properties, zno and dh the systems containing lutrol f127 (20% w / w), dh (5% w / w), and different amount of zno were also prepared. the thermal properties of prepared systems were analyzed by a differential scanning calorimetry (pyris sapphrie dsc, standard 115v, perkin elmer instruments, japan). samples (5 - 7 mg) were accurately weighed into aluminum pans and sealed. the heating rates were 5 and 10/min under nitrogen purge at the temperature of 30 - 300. normally, an abrupt change in the storage modulus or viscosity reflects the sol - gel transition. therefore, the textural analysis of 150 ml prepared systems was done using a ta.xt2i texture analyzer (charpa techcenter, godalming, stable micro systems ltd., uk) equipped with a 5 kg load cell and texture expert software. the sol - gel transition temperature of lutrol f127 gels containing various ingredients was determined from oscillating measurements at 1 hz and heated in a temperature controllable water bath from 4 to 45 at a heating rate of 1/min. the sol - gel transition temperature was calculated by time cure test obtained by plotting the g as a function of temperature. the gelling temperature was defined as the point where the g was halfway between g for the solution and g for the gel as mentioned previously. the ease with which gel systems containing samples could be expelled after filling into syringes and through their fitted needles was measured using a texture analyzer in compression mode. a sample was filled into a 1 ml syringe and was held up with a clamp at the upper probe of the texture analyzer. a constant force of 0.1 n was applied to the base and the distance required to expel the contents for a barrel length of 20 mm was measured. all of the experiments were triplicately done (n=3) at temperatures of 4 and 20. force displacement profiles were performed, which the force at a distance of 10 mm was selected to indicate the syringeability as the force to expel the sample during drug administration. antimicrobial activities of prepared dh and zno - dh gels were determined using agar cup diffusion method. the standard microbes used in this study were s. aureus (atcc 6538p), e. coli (atcc 10536), and c. albicans (atcc 17110). the actively growing broth culture of microbes was prepared and the turbidity was adjusted to obtain 10 cells / ml approximately. then the swab was spread onto the agar plate in three directions to ensure that the plate area was completely covered and the spread culture was dried at ambient condition. the prepared gels were filled into the cylinder cup and incubated at 37 for 24 h. the antimicrobial activities were measured as the diameter (cm) of clear zone for growth inhibition. one gram of gel was put in a dialysis tube (mw cutoff, 6000 - 8000). the dialysis tube was then placed in a glass bottle containing 100 ml phosphate buffer ph 7.2, maintained at 37, and stirred at 100 rpm. release medium of each sample (10 ml) was collected periodically and replaced with fresh dissolution medium. the concentration of dh was determined using a uv / vis spectrophotometer (perkin - elmer, germany) at 349 nm (n=3). the analysis of drug release from these developed systems was done following a previous report. samples after dissolution test were freeze dried using the freeze dryer (triad labconco, kansan city, mo, usa) for 48 h until the samples were dried to avoid the collapse of porous structures. the dried sample was placed on a double adhesive carbon that adhered to an aluminum foil which was stuck on metal stub before being sputter - coated with gold using the sputter coater (cressington 108, cressington scientific instruments, watford, england) prior to testing. micrographs were taken with sem (maxim 200 camscan, cambridge, england) at an accelerating voltage of 15 kv. the morphology of samples was observed for the porosity of pore structure, surface structure, and drug crystalline. the samples containing zno were also prepared as with the above mentioned method before observing with sem by both routine secondary electron imaging (sei) and backscattered electron imaging (bei) modes. the 20% w / w lutrol f127 aqueous solutions were prepared by the cold method as described previously. briefly, lutrol f127 was slowly added to cold water containing different amounts of nmp (4). each of dispersions was left in a refrigerator for 24 h at 4 until the solution was clear. to manipulate the various formulation properties, zno and dh the systems containing lutrol f127 (20% w / w), dh (5% w / w), and different amount of zno were also prepared. the thermal properties of prepared systems were analyzed by a differential scanning calorimetry (pyris sapphrie dsc, standard 115v, perkin elmer instruments, japan). samples (5 - 7 mg) were accurately weighed into aluminum pans and sealed. the heating rates were 5 and 10/min under nitrogen purge at the temperature of 30 - 300. normally, an abrupt change in the storage modulus or viscosity reflects the sol - gel transition. therefore, the textural analysis of 150 ml prepared systems was done using a ta.xt2i texture analyzer (charpa techcenter, godalming, stable micro systems ltd., uk) equipped with a 5 kg load cell and texture expert software. the sol - gel transition temperature of lutrol f127 gels containing various ingredients was determined from oscillating measurements at 1 hz and heated in a temperature controllable water bath from 4 to 45 at a heating rate of 1/min. the sol - gel transition temperature was calculated by time cure test obtained by plotting the g as a function of temperature. the gelling temperature was defined as the point where the g was halfway between g for the solution and g for the gel as mentioned previously. the ease with which gel systems containing samples could be expelled after filling into syringes and through their fitted needles was measured using a texture analyzer in compression mode. a sample was filled into a 1 ml syringe and was held up with a clamp at the upper probe of the texture analyzer. a constant force of 0.1 n was applied to the base and the distance required to expel the contents for a barrel length of 20 mm was measured. all of the experiments were triplicately done (n=3) at temperatures of 4 and 20. force displacement profiles were performed, which the force at a distance of 10 mm was selected to indicate the syringeability as the force to expel the sample during drug administration. antimicrobial activities of prepared dh and zno - dh gels were determined using agar cup diffusion method. the standard microbes used in this study were s. aureus (atcc 6538p), e. coli (atcc 10536), and c. albicans (atcc 17110). the actively growing broth culture of microbes was prepared and the turbidity was adjusted to obtain 10 cells / ml approximately. then the swab was spread onto the agar plate in three directions to ensure that the plate area was completely covered and the spread culture was dried at ambient condition. the prepared gels were filled into the cylinder cup and incubated at 37 for 24 h. the antimicrobial activities were measured as the diameter (cm) of clear zone for growth inhibition. one gram of gel was put in a dialysis tube (mw cutoff, 6000 - 8000). the dialysis tube was then placed in a glass bottle containing 100 ml phosphate buffer ph 7.2, maintained at 37, and stirred at 100 rpm. release medium of each sample (10 ml) was collected periodically and replaced with fresh dissolution medium. the concentration of dh was determined using a uv / vis spectrophotometer (perkin - elmer, germany) at 349 nm (n=3). the analysis of drug release from these developed systems was done following a previous report. samples after dissolution test were freeze dried using the freeze dryer (triad labconco, kansan city, mo, usa) for 48 h until the samples were dried to avoid the collapse of porous structures. the dried sample was placed on a double adhesive carbon that adhered to an aluminum foil which was stuck on metal stub before being sputter - coated with gold using the sputter coater (cressington 108, cressington scientific instruments, watford, england) prior to testing. micrographs were taken with sem (maxim 200 camscan, cambridge, england) at an accelerating voltage of 15 kv. the morphology of samples was observed for the porosity of pore structure, surface structure, and drug crystalline. the samples containing zno were also prepared as with the above mentioned method before observing with sem by both routine secondary electron imaging (sei) and backscattered electron imaging (bei) modes. the critical micellization temperature (cmt) was taken as the onset of the endothermic peak. there were endothermic peaks appearing for the systems containing 60 and 80% w / w nmp (fig. the cmt and gelation temperature of lutrol f68 and lutrol f127 were found to interconnect and influenced by cosolutes, such as electrolytes and hydrophobic substances. both cmt and gelation temperature increased upon diluting the system with water, and decreased with an increasing ph of the system due to the solubility reduction of active ingredients. the ratio between two block copolymers presented in the system had an impact on cmt and the gelation temperature, resulting in a decrease of onset temperature of both processes with an increased lutrol f127 dsc thermograms of systems containing 20% w / w lutrol f 127 and different amount of nmp with heating rate of 10/min (a) and 5/min (b). from top to bottom thermograms represents 0, 10, 20, 30, 50, 60 and 80% nmp in both figures. practically, the texture analysis could provide the informative data on the dynamic properties, such as the g and the viscous modulus (g). the sol - gel transition temperature for the lutrol f127 systems is shown in fig. 2. the g was notably low for a solution and increased drastically with temperature as a result of gel formation as previously reported. when the sol - gel transition occurred g became independent of further increase in temperature. the sol - gel transition temperature of the lutrol f127 system containing 5% w / w dh and 20% w / w nmp was about 28 whereas it was decreased with increasing zno amount. the results indicated the change of sol - gel transition which was similar to those where a sol - gel transition was determined by the visual method. storage modulus (g) of different systems : lutrolr f127 20% ---- ; lutrolr f127 20% + 5%dh + 20%nmp ---- ; lutrolr f127 20% + 5%dh + 20%nmp + 1%zno ---- and lutrolr f127 20% + 5%dh + 20%nmp + 5%zno ----- syringeability of various lutrol f127 systems was examined to determine the effects of the incorporated excipient and temperature on the force required to expel the product. the syringeability was tested at the storage temperature (4) and expelled temperature (20). the syringeability of system 1 was 7.110.14 n at 4 and 13.681.49 n at 20, which was a two fold increase in resistance to syringing over this temperature range. when the 5% w / w dh was added into lutrol f127 system (system 2), the syringeability was increased to 8.650.48 n at 4 and 17.311.77 n at 20 that were slightly higher than those of system 1. the syringeability of the 20% w / w lutrol f127 system containing 5% w / w dh and 20% w / w nmp (system 3) were 17.150.54 n and 21.211.37 n at 4 and 20, respectively. n at 4 and 25.792.30 n at 20. the syringeability correlated well with the rheology results indicating that the added zno increased shear stress of the dh - lutrol f127 systems. therefore, the addition of dh, zno, and nmp affected the syringeability of systems. these results corresponded to the experiment previously determining the rheology of poloxamer for ophthalmic use. syringeability of various systems antimicrobial activities of 20% w / w lutrol f127 gels containing dh are shown in fig. 3. the inhibition zone diameter against c. albicans of the gel base containing 20% w / w nmp was 1.80.1 cm, whereas the gel base with or without 20% w / w nmp did not exhibit the inhibition zone against s. aureus and e. coli, indicating that the addition of 20% w / w nmp could promote the antifungal activity against c. albicans. the addition of nmp into the gel system increased the inhibition zone against c. albicans ; therefore, nmp enhanced the antifungal activity of dh. the solubility parameter of nmp is similar to that of ethanol and dmso ; therefore, it could solubilize the lipid in fungal cell membrane and enhance the drug penetration. inhibition zone diameter of 20% w / w lutrol f 127 gel containing different concentrations of dh. inhibition zone diameter of 20% w / w lutrol f 127 gel containing different concentrations of dh obtained from the agar diffusion method against s. aureus ; e. coli and c. albicans (solid symbols represent the prepared gel without nmp, and open symbols represent the prepared gels with 20% w / w nmp) (n = 3). effect of zno on the antimicrobial activity of dh gels is shown in fig. inhibition zone diameters against s. aureus and e. coli of dh gels with or without nmp were not different whereas 20% w / w nmp could enhance the antifungal activity against c. albicans. the inhibition zone of the prepared gel containing 0.5% w / w zno were lower than that without zno and indicated that the diffusion of dh from gel was decreased with zno. for s. aureus, the inhibition zone of dh - lutrol f127 gel decreased as amount of zno was increased, whereas the inhibition zone of the prepared gel with 20% nmp did not depend on the zno amount. for e. coli and c. albicans, the inhibition zone of the dh - lutrol f127 gel with or without 20% w / w nmp decreased when the amount of zno were increased. these results showed that the amount of zno affected the diffusion of drug from gels. this species could attach either to the tricarbonyl methane or the phenol diketone area of this drug molecule. therefore, the drug release depended on the dissociation of this complex which retarded the drug diffusion into the agar of antimicrobial test and also prolonged drug release in dissolution study as presented in our previous work. the prolongation of drug release with zno and better antimicrobial activity were the properties of systems for sustaining dh. inhibition zone of 5% w / w dh gels containing different zno concentrations with and without nmp. inhibition zone of 5% w / w dh gels containing different zno concentrations with and without nmp against s. aureus e. coli and c. albicans (open symbols show the prepared gel with nmp. surface and cross section morphology of the prepared systems after dissolution test were characterized using sem. digital images acquired in a sem could be obtained by sei mode or bei mode. the sei mode is generally used for imaging the surfaces of structures. in the bei mode, the obtained image contrast depends on the atomic number of the structure under the electron beam. the higher the average atomic weight the brighter the region will appear in the image. freeze - drying technique was utilized for preparing samples to maintain the porous structure without any collapse during water sublimation. the sem images of lutrol f127 dried gel containing 5% w / w dh and different amounts of zno at different magnifications are shown in figs. the porous surface was evident for 20% w / w lutrol f127 system (fig. the enlarged images of the surface of the wall were found at magnification 100 and 350 which the porous structure was more evident. the structures of 20% w / w lutrol f127 systems were notably different from those of systems comprising dh. the structure of the dh - lutrol f127 system contained the interconnected pores, whereas the wall surface was smooth (fig. the pores were decreased with an increased zno contents and the porous structure was more compact and smaller. this was explained with entanglement between zno and lutrol f127 being enhanced by increasing zno content. for samples of 0.5, 1.0, 2.0, 5.0, and 10.0% w / w zno content, the sem micrographs were further magnified to clarify their structure as presented in figs. the addition of aerosil could transform the structure of poloxamer system from the cubic into the hexagonal phase. therefore, this study suggested that increasing the zno amount could decrease the size of the interconnected pores and increase the compactness of the wall surface. topography from bei mode of sem analysis revealed the distribution of zno particles in freeze - dried structure. the denser zno particles were evident in the system containing higher amount of this compound. sem micrographs of the dried gel systems ; 20% w / w lutrol f127 system (a) and 5% w / w dh - lutrol f127 systems (b) containing with 0.5% w / w zno (c) and 1.0% w / w zno (d) with different magnifications (20x (sei), 100x (sei) and 350x (sei)). the white bar presented 1 mm (left), 200 m (center) and 60 m (right). sem micrographs of 20% w / w lutrol f127 systems containing 5% w / w dh and different amount of zno. lutrol f127 systems containing 5% w / w dh and different amount of zno with different magnifications (20x (bei), 100x (bei) and 100x (sei)) ; (a) 2% w / w zno (b) 5% w / w zno and (c) 10% w / w zno. the white bar presented 1 mm (left) and 200 m (center and right). the sem photographs of cross - sectional morphology of lutrol f127 dried gel containing 5% w / w dh, 20% w / w nmp and different amount of zno after release test for 216 h in phosphate buffer ph 7.2 at different magnifications are shown in fig. the structure of lutrol f127 dried gel seemed fragile and the pore surface was reduced. lutrol f127 has a molecular weight of 12 600, which could not diffuse passing the dialysis tube (mw cutoff : 6000 - 8000). therefore lutrol f127 systems containing 20% w / w nmp, 5% w / w dh and different amount of zno after release test in phosphate buffer ph 7.2. lutrol f127 systems containing 20% w / w nmp, 5% w / w dh and different amount of zno after release test for 216 hours in phosphate buffer ph 7.2 with different magnifications (20x (sei), 100x (sei) and 350x (sei)) (a) 0.5% w / w zno (b) 1% w / w zno (c) 2% w / w zno and (d) 5% w / w zno. the white bar presented 1 mm (left), 200 m (center) and 60 m (right). the physical properties of the gel systems depended on the ingredients added into the systems. the sol - gel transition temperature of the lutrol f127 system containing 5% w / w dh and 20% w / w nmp was about 28 and decreased as the zno amount was increased. the syringeability of dh - lutrol f127 system was increased as increasing the amount of zinc oxide. however, their syringeability decreased when the temperature was decreased. the inhibition zones against s. aureus, e. coli, and c. albicans were smaller since zno delayed the diffusion and prolonged the dh release. the morphology of freeze - dried lutrol f127 system showed the interconnected pores with small porosity on their surfaces, whereas the addition of dh changed the structure of lutrol f127 that was the interconnected pores and the surfaces was smooth. the sizes of interconnected pores decreased with increasing zno content and the structure was more compact and smaller. however, the structure of the lutrol f127 system comprising zno after studying dh release was the interconnected pore. | to characterize the thermal behavior and texture analysis of doxycycline hyclate thermosensitive gels developed for periodontitis treatment containing zinc oxide prepared by using poloxamer (lutrol f127) as polymeric material and n - methyl pyrrolidone was used as cosolvent. the thermosensitive gel comprising doxycycline hyclate, lutrol f127, and n - methyl pyrrolidone were characterized for the thermal behavior and texture analysis. the topography of the system after the dissolution test was characterized with scanning electron microscope. differential scanning calorimetric thermogram exhibited the endothermic peaks in the systems containing high amount of n - methyl pyrrolidone in solvent. the sol - gel transition temperature of the systems decreased as the zinc oxide amount was increased. the addition of doxycycline hyclate, zinc oxide, and n - methyl pyrrolidone affected the syringeability of systems. the addition of zinc oxide into the doxycycline hyclate - lutrol f127 systems decreased the diameter of inhibition zone against staphylococcus aureus, escherichia coli, and candida albicans since zinc oxide decreased the diffusion and prolonged release of doxycycline hyclate. from scanning electron microscope analysis, the porous surface of 20% w / w lutrol f127 system was notably different from that of gel comprising doxycycline hyclate which had interconnected pores and smooth surfaces. the number of pores was decreased with increasing zinc oxide and the porous structure was smaller and more compact. therefore, the addition of zinc oxide could increase the syringeability of doxycycline hyclate - lutrol f127 system with the temperature dependence. zinc oxide decreased inhibition zone against test microbes because of prolongation of doxycycline hyclate release and reduced size of continuous cells. furthermore, zinc oxide also increased the compactness of wall surfaces of lutrol f127. |
hiv - infected individuals are at increased risk of developing a wide variety of ophthalmologic opportunistic infections with advanced degrees of immunosuppression. retinitis or acute retinal necrosis (arn) is commonly caused by various viruses, such as cmv, hsv and vzv, in patients with aids,,,. other non - viral etiologies of retinitis or arn also include toxoplasmosis, lymphoma and syphilis,,. although syphilis can involve any anatomical structures of the eye, anterior uveitis is the most common presentation in immunocompetent hosts. importantly, however, posterior segment involvement has been more commonly described in patients with aids. for example, seronegative secondary syphilis has been reported in hiv - infected patients,,,. reported a case of secondary syphilis in an hiv - infected man with kaposi sarcoma. serology testing of syphilis was repeatedly nonreactive which necessitated biopsy of a skin lesion for diagnosis. non - treponemal tests may also be negative in hiv - infected patients with ocular syphilis. syphilitic retinitis generally responds well to intravenous penicillin leading to favorable visual outcome, thus a high clinical suspicion and recognition of syphilitic retinitis in hiv - infected individuals followed by prompt initiation of treatment are crucial for clinicians even in the absence of objective evidence of syphilis. a 39-year - old african american female with a history of aids was in her usual state of health until 5 days prior to presentation when she developed sudden onset of loss of vision. she was diagnosed with hiv infection 2 months prior to presentation and she was not yet on highly active antiretroviral therapy (haart). two days later, she also developed decreased vision with blurriness in the right eye. she denied eye pain, conjunctival redness, and flashes of light or floaters in her visual fields. her past medical history was also significant for gonorrhea and syphilis diagnosed 13 years before. the temperature was 98.2 f, blood pressure 111/65 mm hg, pulse 95 beats per minute, respirations 16 breaths per minute and oxygen saturation 95% on room air. a 0.5 cm ulcerative lesion with irregular edges was noted in the genital area posterior to the anus. the ophthalmologic examination revealed visual acuity of hand motion in right eye and light perception in the left. the pupil size was 2.5 mm in the right eye and 4.5 mm in the left. fundoscopy showed vitritis and extensive white - yellow retinal lesions, compatible with necrotizing retinitis. laboratory studies revealed a white blood cell count of 4800 cells / mm, hemoglobin of 12.3 g / dl, and platelets of 157,000 cells / mm. the cd4 count was 29 cells/l and the hiv viral load was 3,100,000 copies / ml. based on the fundoscopic examination demonstrating necrotizing retinitis, a presumptive diagnosis of cmv retinitis was made and the patient was started on intravenous ganciclovir. on the third day after admission, based on the patient s past medical history of syphilis and positive treponema antibody (tp - pa) on admission, the patient was started on intravenous penicillin for suspected syphilitic retinitis, though serum rpr was negative. subsequently, the patient underwent lumbar puncture ; csf was colorless with wbc 8 cells / mm, protein 69 mg / dl and glucose 34 mg / dl. the patient demonstrated dramatic improvement of her vision and fundoscopic examination of both eyes. at the time of completion of 10 days of penicillin therapy, her visual acuity was 20/30 in the right eye and 20/50 in the left. in this report, we described a case of necrotizing retinitis due to syphilis in a patient with aids. retinal involvement due to syphilis has been described in individuals with advanced hiv infection,,,. our case posed a diagnostic challenge since the fundoscopic findings were suspicious for viral retinitis, particularly cmv. although the patient has a remote history of presumably treated syphilis, rpr was non - reactive at the time of admission. in fact, the patient was initially treated with ganciclovir for a presumptive diagnosis of cmv retinitis. after various viral pcr assays obtained from the anterior chamber aspiration returned negative, the decision was made to initiate penicillin which resulted in dramatic clinical improvement. in hiv - infected individuals with syphilis, more severe clinical manifestations, lack of response to penicillin therapy and inappropriate antibody responses, have been described in the literature. non - treponemal tests may be negative in hiv - infected patients with ocular syphilis. our patient demonstrated inadequate serology responses to rpr due to her immunosuppressed status with advanced stages of hiv - infection. conversely, polyclonal gammopathy and possible coexistent anticardiolipin antibodies in the presence of hiv may make non - treponemal tests be falsely positive. in addition, a failure to decrease serologic titers of syphilis in the absence of immune reconstitution with antiretroviral therapy has been reported. considering the uncertainty regarding syphilis serology testing in hiv - infected individuals, clinical judgment with a low threshold for the ocular manifestations of syphilis are diverse since it can involve any anatomical structures of the eye. in a study of 22 cases of ocular syphilis in hiv - negative individuals, although anterior uveitis is common in immunocompetent individuals, posterior segment involvement has been described more commonly in hiv - infected individuals with advanced stages of immunosuppression. in a retrospective study of ocular syphilis in hiv - infected patients, tran. investigated ocular manifestations due to syphilis for 20 eyes in 12 hiv - infected individuals ; necrotizing retinitis was noted in 7 eyes (35%), posterior chorioretinitis in 6 eyes (30%) and optic nerve involvement in 5 eyes (25%). in addition, syphilitic retinitis is more commonly seen in hiv - infected individuals with more advanced stages of immunosuppression. in a study of 101 hiv - infected patients, posterior uveitis was significantly more common in those with cd4 count < 200 cells / mm (p = 0.002). ocular syphilis may be complicated by central nervous system involvement, thus investigation for neurosyphilis should be considered especially for patients with aids. in a study of neurosyphilis during the aids epidemic in san francisco, another study reported a high proportion of neurosyphilis in hiv - infected patients with syphilitic uveitis ; 7 of 9 patients (77.8%) demonstrated csf abnormalities. even with no evidence of neurosyphilis, syphilitic retinitis should be treated with the same regimen for neurosyphilis ; a 1014 day course of intravenous penicillin is recommended. although ophthalmologic manifestations of syphilis are diverse, posterior segment involvement is common in hiv - infected individuals with more advanced stages of immunosuppression. lack of appropriate clinical responses to ganciclovir for presumed viral retinitis, negative pcr analysis for viral retinitis, remote history of syphilis led us to initiate penicillin therapy empirically with excellent visual outcome. recognition of syphilitic retinitis in patients with aids is paramount for clinicians and a threshold to initiate penicillin should be lower even in the absence of objective evidence of syphilis. | the ocular manifestations of syphilis are varied. ocular syphilis can occur during any stage of infection and involve virtually any part of the eye. in immunocompetent individuals, the most common etiologies include syphilitic uveitis. although the clinical presentation of ocular syphilis in hiv - infected patients is also widespread, posterior segment involvement has been more commonly described particularly in patients with aids. the diagnosis of syphilitic retinitis is challenging since its clinical presentation mimics retinitis caused by other viral etiologies. in addition, hiv - infected individuals with syphilis are more likely to develop aberrant serologic responses. recognition of syphilitic retinitis and prompt initiation of penicillin therapy is of critical importance since syphilitic retinitis generally responds well to treatment and loss of vision is reversible. in this report, we describe a 39-year - old female with advanced stages of aids who developed necrotizing retinitis due to syphilis. prompt initiation of intravenous penicillin led to excellent visual outcome for this patient despite significantly decreased visual acuity on presentation. |
arsenic is a naturally occurring element that exists ubiquitously in the environment in both organic and inorganic forms combined with other elements such as oxygen, chlorine, and sulfur, as well as carbon and hydrogen (cullen and reimer, 1989 ; who, 2000 ; iarc, 2004). it is classified chemically as a metalloid, having properties intermediate between a metal and a nonmetal ; however, it is frequently referred to as a metal (iarc, 1980). elemental arsenic is the 20 most common in the earth s crust, and is emitted to the environment as a result of volcanic and industrial activities. mining, smelting of non - ferrous metals and burning of fossil fuels are the major anthropogenic sources of arsenic contamination of air, water, and soil. arsenic and arsenic compounds are used in pharmaceuticals, wood preservatives, agricultural chemicals (e.g. pesticides, insecticides, and herbicides), and applications in the mining, metallurgical, glass - making, and semiconductor industries (mandal, 2002). especially, the historical usage of arsenic - containing pesticides and wood preservatives led to contamination of the agricultural land and groundwater (who, 2001). human exposure to arsenic has become a major global health problem because the worldwide contaminations and adverse health effects. in most populations, the main source of arsenic exposure is the drinking water (nrc, 1999, 2001 ; smith., 2002 ; the current maximum contamination levels of arsenic were lowered from 50 g / l to 10 g / l by usepa and who (iarc, 2004). arsenic in drinking water is mainly inorganic arsenic and more harmful than in food such as grains and vegetables (akter. moreover, inorganic arsenic is more toxic than organic arsenic and can exist in two major oxidation states : a trivalent form, arsenite (as), and a pentavalent form, arsenate (as). biologically, arsenite is 2~10-fold more active and toxic than arsenate (kosentt, 1994). of the inorganic arsenic compounds, arsenic trioxide (as2o3), sodium arsenite (naaso2) and arsenic trichloride (ascl3) are the most common trivalent compounds, and arsenic pentoxide (as2o5), arsenic acid (ash3o4) and arsenates [e.g. sodium arsenate (na2haso4), lead arsenate (pbhaso4) and calcium arsenate (as2ca3o8) ] are most common pentavalent compounds. the toxic effects of arsenic that are of most concern to humans are those that occur from chronic low - level exposure. epidemiological studies have indicated that chronic exposure to arsenic is associated with increased risks of various cancers, including those of the skin, lung, bladder, kidney, and liver, as well as numerous other non - cancer illnesses including vascular and cardiovascular disease, diabetes, reproductive and developmental problems, and neurologic and cognitive problems (abernathy. conversely, arsenic has been considered as an effective chemotherapeutic agent in the treatment of certain human cancers (douer & tallman, 2005). among arsenic compounds, arsenic trioxide (as2o3) has successfully been employed in the treatment of acute promyelocytic leukemia (apl) (soignet., 1998). it has also been shown that as2o3 efficiently induces apoptosis in malignant apl cells in vitro (hu., 1999). these observations has been confirmed that as2o3 can induce apoptosis in other leukemia cells and solid tumor cells, including gastric cancer cells, neuroblastoma cells, and prostate and ovarian carcinomas (zhang el al., 1999 ; uslu., 2000). however, arsenic is commonly regarded as a potent carcinogen that can induce the formation of various types of solid tumors (smith., 1992 ; liu & waalkes, 2008). our previously study demonstrated that as2o3 effectively provokes cytotoxicity and apoptotic cell death in mouse testicular sertoli cells in vitro (kim., 2011). these investigations indicates that the male reproductive toxicity of arsenic may be directly associated with damage of testicular cells. in addition, various experimental models have been developed to understand how arsenic exposure causes these diverse disease outcomes. however, the reproductive and developmental toxicity of arsenic is poorly understood, and specific relationships between experimental and human exposures are not established. several epidemiologic studies have reported that arsenic exposure in utero increased spontaneous abortion and stillbirth and decreased birth weight (ihrig., 1998 ; ahmad., 2001 the reproductive and developmental toxicity of arsenic have primarily been documented through murine studies, suggesting arsenic as a risk to the developing fetus. in animals, some experimental studies have reported that arsenic intoxication is associated with spermatotoxicity (waalkes., 2003 ; pant., 2004), inhibition of testicular steroidogenesis and reduction of the weight of the testes and accessory sex organs (sarker., 2003). arsenic exposure also has been associated with inhibition of ovarian steroidogenesis and gonadotrophins secretion (chattopadhyay., 1999), elevation of adrenocortical steroidogenesis and plasma corticosterone level (ghosh., 1999), as well as with severe metabolic disorders such as diabetes in humans (tseng., 2002). however, the actual molecular events resulting in reproductive and developmental toxicity from exposure of arsenic remain unclear. the purpose of this paper is to review and summarize the major scientific developments on the topic of reproductive and developmental toxicity associated with arsenic exposure, integrating evidence from epidemiological and experimental studies. arsenic is a major global health concern throughout the world widely based on its carcinogenic potential after occupational or environmental exposure (iarc, 1987 ; nrc, 1999). arsenic is commonly present in air, water, and soil and could find their routes into the human body through inhalation, ingestion and skin absorption. the primary route of arsenic exposure for the most population is via the ingestion of contaminated food or water. inhalation or skin absorption of arsenic from contaminated sources is a minor exposure route for the general population (atsdr 2007). most ingested and inhaled arsenic is well absorbed through the gastrointestinal tract and lung into the blood stream. arsenic enters into the human body through these routes, and is distributed in a large number of organs including the lung, liver, spleen, kidney, intestine, skin, and vascular and lymphatic systems, as well as reproductive and nervous systems (hunter., 1942 ; atsdr, 2007). after absorption through lung and the gastrointestinal tract, 95 to 99% of the arsenic is located in erythrocytes, bound to the globin of hemoglobin and is then transported to the other parts of the body. about 70% of the arsenic most arsenic absorbed into the body is converted by the liver to less toxic methylated form that is efficiently excreted in the urine (who, 1981 ; atsdr, 1989). arsenic is a multi - site carcinogen in humans, causing tumors in a variety of tissues including lung, skin, liver, kidney, and bladder, as well as uterus and prostate (nrc, 1999 ; waalkes., 2000, 2003). acute arsenic exposure may cause gastrointestinal tract disorders (goebl., 1990), whereas chronic exposure may exert degenerative, inflammatory, and neoplastic changes of the respiratory, hematopoietic, cardiovascular, and nervous systems (naiger & osweiler, 1989). although epidemiological data have firmly established arsenic to be a human carcinogen, animal studies are less well defined. most laboratory animals appear to be substantially less susceptible to arsenic than humans (naqvi. it has been reported that chronic oral exposure to inorganic arsenic (0.05 - 0.1 mg / kg / day) causes neurological and hematological toxicity in humans but not in monkeys, dogs, and rats exposed to arsenite or arsenate at doses of 0.72 to 2.8 mg / kg / day (byron., 1967). exposure of humans to inorganic arsenic results in the urinary excretion of inorganic arsenic and two major methylated metabolites, methyl arsenic and dimethyl arsenic (crecelius, 1977 ; smith., 1977 ; yamauchi & yamamura, 1979). in many organisms, including humans, inorganic arsenic is reduced in the blood from pentavalent arsenate (as) to trivalent arsenite (as), then taken up by hepatocytes and oxidatively methylated to monomethyl arsenic acid (mma) and then to dimethyl arsenic acid (dma) (fig. 1) (aposhian, 1997 ; healy., 1998 ; pott., 2001 ; thomas., the reduction is facilitated by reductases and the reduced form of glutathione (gsh) along with possibly other thiols as electron donors. oxidative methylation is carried out by methyltransferases, and s - adenosylme - thionine (sam) serves as the main methyl donor (drobna., 2005). arsenate (as) is reduced by as reductase with glutathione (gsh) to yield arsenite (as) and glutathione disulfide (gssg). as is converted to pentavalent monomethylarsonic acid (mma), a reaction catalyzed by arsenic-3-methyltransferase (as3mt), with sadenosylmethione (sam) serving as the methyl donor ; in the process, sam is hydrolyzed to sadenosyl homocysteine (sah). after the reduction of mma to mma, a second methylation step result in the synthesis of dimethylarsinic acid (dma), and then it is reduced to dma. inorganic arsenic in water is abundant in the form of arsenate ; it is negatively charged at physiological ph and slowly taken up by cells (cohen., 2006). arsenate is rapidly converted to arsenite in vivo (vahter and envall, 1983 ; vahter & marafente, 1985) which is taken up by cells much more quickly than arsenate (tseng, 2009). methylation of arsenicals facilitates their excretion from the cell and therefore was long considered a detoxification process, but recent evidence indicates that monomethylated and dimethylated arsenicals have many cyto- and genotoxic effects including increased oxidative stress (kligerman & tennant, 2007), chromosomal aberrations, and oxidative dna damage (schwerdtle. methylated trivalent metabolites are highly reactive and are more potent inhibitors of gsh reductase and thioredoxin reductase compared with arsenite or pentavalent metabolites (styblo., 1997 ; lin., 1999). thioredoxin reductases catalyzes the nadph - dependent reduction of the disulfide bond in oxidized thioredoxin, which is a critical antioxidant enzyme controlling the cellular redox balance (rossman, 2003). in experimental animals, prenatal exposure to inorganic arsenic has resulted in arsenic accumulation in fetuses, including fetal brain (rodriguez., 2002 ; miyazaki., 2005), and detection of monomethyl and dimethyl arsenicals in fetuses (hall., 2007). in humans, dimethylarsinic acid (dma) accounts for nearly 90% of all detected arsenic in the blood plasma of both the newborns and their mothers (concha., 1998). this form of arsenic accounts for 60 to 70% of the total detected arsenic in urine of the general population, whereas urine from pregnant women contained more than 90% arsenic as dma. this suggested that arsenic methylation may be increased during pregnancy and that dma is the major form of arsenic transferred to the fetus. the elevated evidence now suggests that arsenic exposure in utero also poses health risks to the developing fetus (vahter, 2009). various epidemiological studies have found significant associations between prenatal arsenic exposure and adverse infant outcomes, such as infant mortality, low birth weight, and birth defects (ahmad., 2001 ; milton., 2005 these health problems were most evident in individuals exposed to high - level arsenic (iarc, 2004). during pregnancy, high - level exposure to arsenic in drinking water causes pregnancy complications, including fetal loss and premature delivery (chakraborti., 2003), and low - level exposure to arsenic affects uterus and placental growth results in progeny birth weight (hopenhayn., 2003 ; rahman., 2009). conclusions from these epidemiological studies are further supported by results from animal models (ferm and carpenter, 1968 ; hood and bishop, 1972). several cross - sectional studies have been reported that woman exposure of chronic arsenic in drinking water increases adverse pregnancy outcomes including premature delivery, spontaneous abortion, stillbirth and neonatal death in bangladesh (milton., 2005), northeastern taiwan (yang., 2003), and chile (hopenhayn - rich., 2000). despite the strong association between arsenic exposure and a range of child health concerns, various animal studies have shown that arsenic can produce developmental toxicity, including malformation, growth retardation and fetal death (tabocova., 1996 ; furthermore, developmental toxicity critically depends on dose, route and gestation day following the arsenic exposure (golub., 1998). fetal malformations were only reported when pregnant rats and mice were intravenous (i.v.) and intraperitoneal (i.p.) injected with inorganic arsenic at early gestation (stump., 1999 ; desesso, 2001). maternal inhalation or oral ingestion of inorganic arsenic affected fetal development and behavior, but did not cause malformations (holson., 1999 ; stump. it is then transported to the liver and may undergo first - pass metabolism prior to being delivered to the uterus. intraperitoneal injections, on the other hand, allow arsenic to be taken up by blood vessels directly and may bypass first - pass metabolism (stump., 1999 ; desesso, 2001). in pregnant rats, the maternal blood and embryonic arsenic concentration from i.p. injected mother was significantly higher than those from the orally exposed mother (hood. inhalation is the least effective means of increasing maternal or embryonic arsenic concentrations, compared to i.p. and i.v. intraperitoneal injection of 45 mg / kg sodium arsenate in swiss mice during gestation day (gd) 8 induced fetal renal and skeletal malformations without affecting maternal weights (fascineli., 2002). however, oral exposure to arsenic did not cause neural tube defects, even at maternally toxic dose levels. oral gavage with high - dose arsenic trioxide (10 mg / kg / day) in female crl : cd(sd)br rats during gd 14 to 19 decreased fetal weights, but did not induce changes in mating index, fertility index, implantation, or fetal malformation (holson. nevertheless, dimethylarsinic acid (dma), an arsenic metabolite, given to pregnant mice and rats by oral gavage caused developmental toxicity (rogers. cd-1 mice were orally gavaged with dma at 200~600 mg / kg / day during gd 7 to 16. these mice showed lower maternal weight gain and fetal weight at 200 mg / kg / day, and a higher incidence of cleft palate at 400 mg / kg / day. similarly, during gd 7 to 16, cd rats were orally gavaged with dma at 7.5 to 60 mg / kg / day (rogers., 1981). maternal weight gain and fetal weight were decreased at > 40 mg / kg / day. fetal mortality was increased at 50 or 60 mg / kg / day, but no fetal gross malformations were seen in these rats. in a later study, dma was given to pregnant sprague - dawley (sd) rats at 40 mg / kg / day by oral gavage during gd 6 to 15. this treatment did not induce maternal weight reduction or maternal lethality, but decreased fetal weight (chernoff., furthermore, postnatal developmental changes were observed when arsenic was given in the drinking water to pregnant and lactating rats and continually to the newborns (rodriguez. ingestion of drinking water with sodium arsenite (36.7 mg / l) in sd rats during gd 15 to postnatal day (pnd) 16 induced developmental indices including delayed pinna detachement on pnd 12 and early eye opening on pnd 14 in more litters, compared to the untreated controls (rodriguez., pinna detachment occurs between pnd 3 and 4, and eye opening is observed on pnd 16. these data indicate that arsenic induces an asynchrony of the maturational processes during postnatal development. overall, it is assumed that prenatal exposure to arsenic may cause adverse pregnancy outcomes and children s health problem, however, the exact mechanism underlying arsenic - induced developmental toxicity and a direct relationship with reproductive toxicity remains to be elucidated. in males, arsenic may induce gonad dysfunction through declined testosterone synthesis, apoptosis and necrosis (davila - esqueda. however, the male reproductive dysfunction of arsenic exposure on human health is not well established. recently, few epidemiologic studies have shown that arsenic exposure significantly causes infertility and low sperm quality, as well as erectile dysfunction in men (nie., 2006 ; hsieh., 2008 ; meeker., 2010). several experimental studies have been demonstrated that significant accumulation of arsenic in testes and accessory sex organs, such as epididymis, seminal vesicle, and prostate gland (danielsson., 1984 ; pant., 2001). male mice exposed to sodium arsenite at 20 or 40 mg / l for 5 weeks in drinking water showed decreased epididymal sperm counts and testicular weight (chang., 2007). similarly, in male rats, daily exposure to sodium arsenite at 5 mg / l for 4 weeks in drinking water also resulted in decreased testicular weights, accessory sex organ weights, and epididymal sperm counts, as well as extensive degeneration of a wide variety of germ cells at stage vii of the spematogenic cycle (jana., 2006). furthermore, arsenic - exposed mice exhibited dose - dependent gradual reductions in seminiferous tubular diameter and various gametogenic cell populations, such as resting spermatocyte, pachytene spermatocyte, and elongated spermatid (sanghamitra., in addition, arsenic exposure also reduced plasma levels of testosterone and gonadotropin in male mice and rats (sarkar.. 2003 ; chinoy., 2004 ; injections at 4, 5, or 6 mg / kg / day for 26 days. at 5 and 6 mg / kg / day, relative testicular weight, accessory sex organ weights and epididymal sperm counts were decreased. moreover, sodium arsenite - treated rats were accompanied by decreases of plasma concentrations of luteinizing hormone (lh), folliclestimulating hormone (fsh), and testosterone after arsenic exposure. these suggested that arsenic may target on the brain and/or pituitary as well as directly on the germ cells. they might exert a direct inhibitory action on the testis, or they might affect the hypothalamic - pituitary axis causing changes in plasma concentrations of lh and fsh. reduction of plasma lh might impair leydig cell function and result in a consequent reduction in testosterone production. testosterone is a prerequisite for normal spermatogenesis, while fsh is also required for normal testicular function and spermatogenesis (fig. 2) (jana., 2006). on the other hand, a possible mechanism for decreased sperm motility might be associated with a direct binding of arsenic to sperm (uckun., 2002). nuclear chromatins of sperm have large amounts of thiol - rich protamines, and the sperm flagellum is rich in thiols. another possible cause of the reduction in serum lh, fsh, and testosterone levels could be high serum corticosterone levels. 1988 ; hardy. 2005), and has been reported in sodium arsenite - treated rats (biswas., 1994). luteinizing hormone (lh) acts directly upon the leydig cells to stimulate testosterone (t) production. t and folliclestimulating hormone (fsh) act upon the sertoli cells within seminiferous epithelium to support development and maturation of germ cells. in addition to spermatogenesis, in mice, cholesterol metabolism and testosterone production were affected by arsenic exposure (chinoy., 2004). male mice orally exposed to arsenic trioxide (as2o3) at 0.5 mg / kg for 30 days showed increased cholesterol levels and decreased expression of 3-hydroxysteroid dehydrogenase (hsd) and 17-hsd, two important regulatory enzyme of steroidogenesis. in the testis, 3-hsd mainly converts pregnenolone to progesterone, and 17-hsd converts androstenedione into testosterone. in the seminiferous tubules in the testis, cholesterol in the membrane of developing cells these data suggest that low plasma level of testosterone after arsenic exposure may be due to low enzymatic conversion by the decreases of 3-hsd and 17-hsd, rather than a lack of the synthetic precursor cholesterol. arsenic exposure may induce cell death or apoptosis in the testicular germ cells or somatic sertoli cells in vitro (celino., 2009 ; kim., 2011). in japanese eel, low - dose (0.1~1 m) of arsenic inhibits spermatogenesis via steroidogenesis suppression, while high - dose (100 m) of arsenic induces oxidative stress - mediated germ cell apoptosis (celino., 2009). we previously showed that arsenic trioxide (as2o3) at 10 m efficiently induces reactive oxygen species (ros)-related cytotoxicity and apoptotic cell death in mouse tm4 sertoli cells (kim. the testicular sertoli cells interact directly with developing germ cells throughout the process of spermatogenesis. these cells have an indispensable able in the development and movement of germ cells (russell, 1993 ; griswold, 1998). thus, spermatogenic dysfunction of arsenic toxicity may be associated with direct damages of testicular component cells (fig. 3). arsenic may inhibit spermatogenesis and sperm maturation following certain key molecular mechanisms including reductions of testosterone and gonadotrophins lh and fsh, disruptions of steroidogenic enzymes 3-hsd and 17-hsd, negative regulations of lh and fsh by increased corticosterone, decreases of sperm motility and viability following the direct binding of arsenic (as) to sperm, and direct damages or apoptosis of testicular component germ cells or sertoli cells. lh, luteinizing hormone ; fsh, follicle - stimulating hormones ; 3-hsd, 3-hydroxysteroid dehydrogenase ; 17-hsd, 3-hydroxysteroid dehydrogenase. these toxic effects are influenced by the forms, sources, and routes, as well as doses and periods of arsenic exposure. in vivo studies demonstrated that inorganic arsenic, such as sodium arsenite, arsenic trioxide, and dimethyl arsinic acid, an arsenic metabolite, causes reproductive and developmental toxicity. injection routes causes fetal malformation ; in contrast, oral and inhalational exposure to inorganic arsenic affects fetal development, including growth retardation and fetal death. in drinking water, oral exposure to inorganic arsenic causes dysfunctions of spermatogenesis, reductions of testosterone and gonadotrophins, and disruptions of steroidogenesis. however, the reproductive and developmental toxicity of arsenic is poorly understood, and the molecular mechanism of arsenic - induced reproductive toxicity remains unclear. therefore, we further investigated some of the possible mechanisms that are affected by arsenic causing reproductive toxicity. the crucial mechanisms of arsenic - induced reproductive toxicity may be associated with hormonal regulation and function, binding to sperm, and regulation of steroidogenesis, as well as direct effects of testicular component cells. | arsenic is a toxic metalloid that exists ubiquitously in the environment, and affects global health problems due to its carcinogenicity. in most populations, the main source of arsenic exposure is the drinking water. in drinking water, chronic exposure to arsenic is associated with increased risks of various cancers including those of skin, lung, bladder, and liver, as well as numerous other non - cancer diseases including gastrointestinal and cardiovascular diseases, diabetes, and neurologic and cognitive problems. recent emerging evidences suggest that arsenic exposure affects the reproductive and developmental toxicity. prenatal exposure to inorganic arsenic causes adverse pregnancy outcomes and children s health problems. some epidemiological studies have reported that arsenic exposure induces premature delivery, spontaneous abortion, and stillbirth. in animal studies, inorganic arsenic also causes fetal malformation, growth retardation, and fetal death. these toxic effects depend on dose, route and gestation periods of arsenic exposure. in males, inorganic arsenic causes reproductive dysfunctions including reductions of the testis weights, accessory sex organs weights, and epididymal sperm counts. in addition, inorganic arsenic exposure also induces alterations of spermatogenesis, reductions of testosterone and gonadotrophins, and disruptions of steroidogenesis. however, the reproductive and developmental problems following arsenic exposure are poorly understood, and the molecular mechanism of arsenic - induced reproductive toxicity remains unclear. thus, we further investigated several possible mechanisms underlying arsenic - induced reproductive toxicity. |
it affects nearly 100 million people in tropical and subtropical areas of europe, africa, latin america, and asia. most cases are chronic and asymptomatic, but severe infections such as hyperinfection syndrome (his) and disseminated strongyloidiasis (ds) occur when cellular immunity is compromised. in his (1.52.5 % of cases), manifestations are confined to the respiratory and gastrointestinal (gi) systems. ds, infection with multiple organ involvement other than the respiratory and gi systems such as the brain or the skin, may have a mortality of up to 87 %. bacterial meningitis caused by enteric flora is a potentially fatal complication of ds / his. localized strongyloidiasis, as well as ds / his, can predispose individuals to bacterial meningitis with enteric organisms such as klebsiella pneumoniae (k. pneumoniae), escherichia coli (e. coli), and enterococcus spp. in the absence of evidence for strongyloidiasis outside the gi tract [2, 3 ]. human t - lymphotropic virus-1 (htlv-1) is a retrovirus that infects 1020 million people worldwide. the virus is highly prevalent in japan, sub - saharan africa, the caribbean, and south america. southern japan is the area with the highest htlv-1 prevalence, with over 10 % of the general population being infected. htlv-1 can be transmitted from mother to child, through sexual contact, and through contaminated blood products. most cases are asymptomatic, but adult t cell leukemia / lymphoma (atll) and htlv-1-associated myelopathy / tropical spastic paresis may occur. co - infection with s. stercoralis and htlv-1 predisposes individuals to ds / his, possibly because htlv-1 interferes with th2 cell responses to s. stercoralis, including eosinophil activation and production of interleukins and ige. glucocorticoids, malignancies, human immunodeficiency virus (hiv), organ or marrow transplantation, hypogammaglobulinemia, leprosy, tuberculosis, and malnutrition also increase the risk. this report delineates the clinical features of bacterial meningitis associated with strongyloidiasis seen at a single center in subtropical japan, in an area endemic for both strongyloidiasis and htlv-1. cases with both bacterial meningitis and strongyloidiasis were extracted from a review of the discharge diagnoses for all hospitalizations at okinawa chubu hospital between january 1990 and may 2010. the prevalence of strongyloidiasis and htlv-1 in this area is reportedly 6.3 and 14 %, respectively. about 39,000 patients visit the emergency department and nearly 14,000 patients are hospitalized per year. after review, we included cases that met the following inclusion criteria : (1) typical signs and symptoms of meningitis (headache, temperature > 38 c, nuchal rigidity, and/or altered consciousness) ; (2) cerebrospinal fluid (csf) findings consistent with bacterial meningitis, including : neutrophil (pmn) predominance, low glucose, high protein, high opening pressures, organisms on gram stain, or recovery of microorganisms from csf or blood ; (3) proof of coexistent strongyloidiasis (larvae or eggs from any specimens). in addition to the inclusion criteria, we collected the following data : gender, age, outcome, symptoms, csf findings, s. stercoralis results, hiv testing results, medical / social history, glucocorticoid use, colonoscopy results, and use of antibiotics and antihelminthics. we performed a mann whitney u - test for csf statistical analysis and a one - sided fisher s exact test for outcome statistical analysis, using stata software (version 12.1 ; statacorp, college station, tx, usa). the median age at diagnosis was 58 years and 11/21 (52.4 %) patients were male. table 1 shows the gender, age, culture results for csf and blood, results of csf gram staining, serological results for htlv-1, specimens from which s. stercoralis was detected, past history, initial antibiotics and antihelminthics (antibiotics were subsequently chosen according to culture results), and outcome of the patients. six episodes of acute meningitis without proven concurrent strongyloidiasis were included because each of these patients had previous or subsequent episodes of proven strongyloidiasis associated with meningitis. five of 21 patients (23.8 %) had more than one episode of meningitis. of these five patients, four were treated with thiabendazole, and one was not treated during the first episode because he was not diagnosed with strongyloidiasis. among the eight cases treated with ivermectin, none developed recurrent episodes of either strongyloidiasis or meningitis. table 1clinical features of all episodes abpc ampicillin, atll adult t cell leukemia / lymphoma, ca carcinoma, ctrx ceftriaxone, ctx cefotaxime, dm diabetes mellitus, e. coli escherichia coli, gnr gram - negative rods, gpc gram - positive cocci, k. pneumoniae klebsiella pneumoniae, l. lactic lactococcus lactis, pmn polymorphic nuclear neutrophils, ra rheumatoid arthritis, s. bovis streptococcus bovis, s. sanguinis streptococcus sanguinis, vcm vancomycin indicates the year when diagnosis was made oral glucocorticoid was prescribed clinical features of all episodes abpc ampicillin, atll adult t cell leukemia / lymphoma, ca carcinoma, ctrx ceftriaxone, ctx cefotaxime, dm diabetes mellitus, e. coli escherichia coli, gnr gram - negative rods, gpc gram - positive cocci, k. pneumoniae klebsiella pneumoniae, l. lactic lactococcus lactis, pmn polymorphic nuclear neutrophils, ra rheumatoid arthritis, s. bovis streptococcus bovis, s. sanguinis streptococcus sanguinis, vcm vancomycin indicates the year when diagnosis was made oral glucocorticoid was prescribed table 2 summarizes the culture results for csf and blood. k. pneumoniae was the most frequent pathogen, followed by streptococcus bovis (s. bovis) and e. coli. no organism was isolated from csf or blood cultures in 14/33 (42.4 %) episodes. among culture - negative cases, gram - positive cocci (gpc) were detected in csf and/or blood cultures in 9/33 (27.3 %) episodes. table 2summary of culture resultsorganismcsf culture (%) blood culture (%) k. pneumoniae 5 (15.2)7 (21.2) s. bovis 5 (15.2)5 (15.2) e. coli 2 (6.1)3 (9.1) l. lactis 1 (3.0)1 (3.0) enterococcus spp.1 (3.0)0 s. sanguinis 1 (3.0)0negative18 (54.5)18 (54.5)one case of mixed infection with k. pneumoniae and s. bovis is included in the blood culture results k. pneumoniae klebsiella pneumoniae, s. bovis streptococcus bovis, e. coli escherichia coli, l. lactis lactococcus lactis, s. sanguinis streptococcus sanguinis summary of culture results one case of mixed infection with k. pneumoniae and s. bovis is included in the blood culture results k. pneumoniae klebsiella pneumoniae, s. bovis streptococcus bovis, e. coli escherichia coli, l. lactis lactococcus lactis, s. sanguinis streptococcus sanguinis serology for htlv-1 was positive in 16/21 (76.2 %) patients, including two cases of acute atll and four cases with smoldering atll. among five cases without htlv-1, two had previous neurosurgeries, two were diagnosed with malnutrition, and one received glucocorticoid for uveitis. three patients had negative hiv test results (patients 6, 13, and 17), but no other patients were examined for hiv due to the low prevalence in japan. twenty - seven of 33 cases (81.8 %) improved without neurological complications and 6/33 episodes (18.2 %) ended in fatalities. only patient 21 received three doses of dexamethasone, 0.15 mg / kg every 8 h, which was discontinued after strongyloidiasis was diagnosed. all patients with gpc meningitis recovered without neurological complications, except for patient 20, who developed acute atll and died. there was no significant difference in outcome between culture - positive cases and culture - negative cases. fatal cases comprised 4/19 (21.0 %) in culture - positive cases and 2/14 (14.2 %) in culture - negative cases (p - value 0.490). among 33 episodes of meningitis, fever was seen in 31 (93.9 %), headache in 30 (90.9 %), nausea and vomiting in 27 (81.8 %), rigors in 17 (51.5 %), and altered mental status in 15 (45.5 %). s. stercoralis was detected in the stool in 26/33 (78.8 %) episodes, in gastric juice in 10/33 (30.3 %) episodes, in sputum in 5/33 (12.1 %) episodes, and in ascites in 1/33 (3.0 %) episodes. s. stercoralis was detected only in the gi tract specimens in 23/33 (69.7 %) cases. only two patients underwent colonoscopy ; patient 13 had diverticulosis and patient 21 had benign adenoma, both of whom had s. bovis meningitis. nineteen of 21 (90.5 %) patients, including one patient with previously resected colon cancer, did not undergo colonoscopy. medians and interquartile ranges of csf cell count, pmn percentage, sugar, csf / blood sugar ratio, and protein were 1,440/mm (5402,500), 84 % (7196), 48 mg / dl (2564), 39 % (1253), and 117 mg / dl (84237), respectively. there were no significant differences between culture - positive and culture - negative cases. we present a series of 21 patients with neutrophil - dominant meningitis associated with strongyloidiasis, including 14 cases with negative csf and blood cultures. s. bovis was the etiology for six patients, all of whom were positive for htlv-1. s. bovis is a gpc in the gut flora of 1016 % of healthy individuals. it may cause bacteremia, endocarditis, and meningitis in patients with gi lesions or colon cancer, and in neonates [7, 8 ]. s. bovis has been reported as a rare cause of bacterial meningitis associated with strongyloidiasis. only four cases of bacterial meningitis due to s. bovis have been reported with strongyloidiasis ; three had defective cellular immunity (two with hiv and one due to steroids) and one was reportedly immunocompetent [6, 911 ]. we detected six cases in a single center over 20 years, which suggests that the incidence of s. bovis meningitis in patients with strongyloidiasis may be higher than previously suspected. this interaction could impact the management of meningitis in geographic areas where htlv-1 and strongyloidiasis are endemic. the practice guidelines for the management of bacterial meningitis published by the infectious diseases society of america (idsa) recommend dexamethasone for adults with suspected pneumococcal meningitis, with dosage continued if the csf gram stain shows gram - positive diplococci. this approach risks exacerbating strongyloidiasis in cases of meningitis due to gpc associated with strongyloidiasis, which can not be reliably distinguished from pneumococci on gram stain. glucocorticoid use is the strongest known risk factor for disseminated strongyloidiasis, due to the acquired defect in cell - mediated immunity. glucocorticoids may act as ecdysteroids that accelerate the conversion of larvae from the rhabditiform state to the invasive filariform state [1, 13 ]. one case of ds was reported after only a single dose of dexamethasone prior to stereotactic radiosurgery. distinguishing pneumococcal meningitis from meningitis due to other gpc on clinical grounds or gram stain alone is problematic. a modified approach to corticosteroid use in gpc meningitis would, therefore, be advisable in areas where strongyloidiasis is endemic. patients with non - pneumococcal meningitis do not benefit from steroids, which are given to mitigate the harmful csf inflammatory response. patients with underlying immunologic diseases are less likely to mount self - damaging inflammatory responses. the good clinical outcomes seen in our cases, which were managed without glucocorticoids, may be attributable to these factors. a comprehensive microbiology approach using expanded techniques such as rapid latex agglutination testing may be helpful for the early identification of patients that can safely receive glucocorticoids, if this testing is locally available. of our cases, 42.4 % had negative cultures of both blood and csf despite clinical meningitis and csf pleocytosis, which were clinically indistinguishable from culture - positive cases. the postulated mechanism by which strongyloidiasis causes bacteremia and meningitis is that parasitic ulceration / perforations of the intestinal mucosa create a portal of entry for enteric bacteria to reach the bloodstream. mantovani. suggested that the bacteria are co - transported with the migrating parasites. this hypothesis assumes that the parasite invades the meninges without any attending bacteria, and that s. stercoralis alone induces the intense inflammatory changes that mimic the csf findings of bacterial meningitis. given that the sensitivity of csf or blood cultures in patients with bacterial meningitis is reportedly > 70 % [1719 ], our frequent observation of culture - negative suppurative meningitis lends support to this hypothesis. among 14 patients treated with thiabendazole for s. stercoralis, four (28.6 %) developed recurrence after treatment ; no patient treated with ivermectin relapsed. these facts suggest that ivermectin may eradicate s. stercoralis more effectively than thiabendazole, in which case ivermectin should be considered for patients with underlying immunodeficiency or associated meningitis. the fact that s. stercoralis was detected only in gi tract specimens in 69.7 % of cases supports the previously suggested theory that localized strongyloidiasis can cause enteric meningitis. limitations of this retrospective report of a single - center experience include the low percentage of cases in which colonoscopy excluded an additional portal of entry, such as structural lesions / cancers, and the infrequency of hiv testing. however, during the long - term follow - up of our geographically restricted cases, neither colon cancer nor hiv was diagnosed in any patient. another limitation is a lack of information regarding the s. bovis speciation (streptococcus gallolyticus or streptococcus infantarius). further observation of the association between these three infectious entities in additional institutions and other geographical areas will help establish the generalizability and importance of these observations. in areas where strongyloidiasis and human t - lymphotropic virus-1 (htlv-1) are endemic, clinical meningitis is surprisingly common and frequently recurrent. culture - negative meningitis and streptococcus bovis meningitis may represent a larger proportion of cases than the meningitis seen in non - tropical areas. clinicians should be aware that these cases may represent a special scenario in which the conventional use of glucocorticoids can pose a previously unrecognized hazard. further study is needed in order to clarify the mechanisms by which these infections interact, and to define the prevalence and significance of accompanying structural colonic lesions. | meningitis caused by enteric flora is a known complication of strongyloidiasis, and human t - lymphotropic virus-1 (htlv-1) predisposes individuals to severe strongyloidiasis. we reviewed the clinical features of bacterial meningitis associated with strongyloidiasis seen at a single center in subtropical japan, in an area endemic for both strongyloidiasis and htlv-1. we found 33 episodes in 21 patients between 1990 and 2010. the results were remarkable for the high incidence of meningitis due to gram - positive cocci (27.3 %), especially streptococcus bovis, and culture - negative cases (42.4 %). given the high incidence of gram - positive meningitis, a modified approach to corticosteroid use would be advisable in areas where strongyloidiasis is endemic, due to the potentially adverse consequences of glucocorticoid therapy. |
the plasma samples included in this study were collected from patients suspected of having lung cancer due to clinical symptoms. patients were examined from may 2011 until january 2014 at the department of pulmonary medicine, aarhus university hospital, denmark. one hundred and nine patients with nsclc adenocarcinoma stage iiia iv were consecutively collected. a control group was made of 110 patients who were diagnosed to be cancer free based on computed axial tomography and, if relevant, negative biopsies. are frequently observed amongst patients with lung cancer, acute or chronic infection or inflammation of any kind were not considered reason for exclusion. by doing so, a control group resembling the clinical situation in a diagnostic outpatient department could be made. microarray printing was performed on a spotbot extreme protein edition microarray printer with a 946mp4 pin (arrayit corporation, ca, usa). as a positive control 100 g / ml of biotinylated human igg antibodies were printed on epoxy - coated slides (75.6 mm25.0 mm ; schott nexterion, de), which were left to dry at room temperature overnight prior to further analysis. a total of 37 anti - human antibodies were used and listed in the following with the corresponding clone, if available : cd146 (p1h12), flotillin-1, hb - egf (4g10), her3 (2f9), her4 (h4.77.16), hsp90 (igf1), n - cadherin (8c11), p53 (pab240), tag72 (0.n.561), and tsg101 (5b7) from abcam (ma, usa) ; sftpd (vif11) from acris antibodies gmbh (de) ; egfr and egfrviii from antibodies-online.com (de) ; cd63 (mem-259) from biolegend (ca, usa) ; her2 (29d8) from cell signalling (ma, usa) ; cd9 and cd81 from lifespan biosciences (wa, usa) ; spa (6f10) from novus biologicals (co, usa) ; cd13 (498001), cd14 (50040), cd142 (323514), cd151 (210127), cd206, cea (487609), muc1 (604804), tnf ri, tnf rii and tspan8 (458811) from r&d systems (mn, usa) ; areg (s-13), epcam (0.n.277), mucin16 (x306), ny - eso-1 (e978) and plap (8b6) from santa cruz biotechnologies (tx, usa) ; cd171 from sigma - aldrich (mo, usa) ; c - met (016) and pd - l1 from sino biological inc. (china) ; cd163 (mac2 - 158) from trillium diagnostics, llc (me, usa). all antibodies were diluted with pbs with 5% glycerol and printed in triplicates at 75200 g / ml. (15) with modifications. in short, the microarray slides were initially blocked (50 mm ethanolamine, 100 mm tris, 0.1% sds, ph 9.0) prior to incubation with 10 l plasma sample diluted (1:10) in wash - buffer (0.05% tween20 in pbs). the incubation was performed in multi - well hybridization cassettes (arrayit corporation) at rt for 2 hours followed by overnight incubation at 4c. following a short wash, the slides were incubated with a cocktail of biotinylated detection antibodies (anti - human - cd9, -cd63 and -cd81, lifespan biosciences, wa, usa) diluted 1:1,500 in wash - buffer. after a wash, a subsequent 30-minute incubation step with cy5-labelled streptavidin (life technologies) diluted 1:1,500 in wash - buffer was carried out for detection. prior to scanning, the slides were washed in wash - buffer, then in ultrapure / deionized water and finally dried using a microarray high - speed centrifuge (arrayit corporation). scanning and spot detection was performed as previously described (15). graphs and statistics were carried out using graphpad prism (version 6.04, graphpad software, inc., san diego, ca, usa), excel (version 2013, microsoft, redmond, wa, usa) and roccet (version 03/17/2014, tmic, ab, canada). heat maps and hierarchical cluster analysis were produced using genesis (version 1.7.6, igb tu graz, graz, austria). random forests was used for classification and monte - carlo cross validation (mccv) using balanced subsampling was employed to compute roc curves and to calculate confidence intervals of their area under the curve (auc). in each mccv, two thirds (2/3) of the samples were used to evaluate the feature importance. the top important features were then used to build a classification model, which was validated on the 1/3 of the samples that were left out. five hundred iterations were used to calculate performance of the model. prior to analysis missing value estimation was performed by replacing missing values by min. a top 20 of ratios (based on p - values) was computed and included into the analysis prior to data transformation and normalization by log2. the plasma samples included in this study were collected from patients suspected of having lung cancer due to clinical symptoms. patients were examined from may 2011 until january 2014 at the department of pulmonary medicine, aarhus university hospital, denmark. one hundred and nine patients with nsclc adenocarcinoma stage iiia iv were consecutively collected. a control group was made of 110 patients who were diagnosed to be cancer free based on computed axial tomography and, if relevant, negative biopsies. are frequently observed amongst patients with lung cancer, acute or chronic infection or inflammation of any kind were not considered reason for exclusion. by doing so, a control group resembling the clinical situation in a diagnostic outpatient department could be made. microarray printing was performed on a spotbot extreme protein edition microarray printer with a 946mp4 pin (arrayit corporation, ca, usa). as a positive control 100 g / ml of biotinylated human igg antibodies were printed on epoxy - coated slides (75.6 mm25.0 mm ; schott nexterion, de), which were left to dry at room temperature overnight prior to further analysis. a total of 37 anti - human antibodies were used and listed in the following with the corresponding clone, if available : cd146 (p1h12), flotillin-1, hb - egf (4g10), her3 (2f9), her4 (h4.77.16), hsp90 (igf1), n - cadherin (8c11), p53 (pab240), tag72 (0.n.561), and tsg101 (5b7) from abcam (ma, usa) ; sftpd (vif11) from acris antibodies gmbh (de) ; egfr and egfrviii from antibodies-online.com (de) ; cd63 (mem-259) from biolegend (ca, usa) ; her2 (29d8) from cell signalling (ma, usa) ; cd9 and cd81 from lifespan biosciences (wa, usa) ; spa (6f10) from novus biologicals (co, usa) ; cd13 (498001), cd14 (50040), cd142 (323514), cd151 (210127), cd206, cea (487609), muc1 (604804), tnf ri, tnf rii and tspan8 (458811) from r&d systems (mn, usa) ; areg (s-13), epcam (0.n.277), mucin16 (x306), ny - eso-1 (e978) and plap (8b6) from santa cruz biotechnologies (tx, usa) ; cd171 from sigma - aldrich (mo, usa) ; c - met (016) and pd - l1 from sino biological inc. (china) ; cd163 (mac2 - 158) from trillium diagnostics, llc (me, usa). all antibodies were diluted with pbs with 5% glycerol and printed in triplicates at 75200 g / ml. the microarray slides were initially blocked (50 mm ethanolamine, 100 mm tris, 0.1% sds, ph 9.0) prior to incubation with 10 l plasma sample diluted (1:10) in wash - buffer (0.05% tween20 in pbs). the incubation was performed in multi - well hybridization cassettes (arrayit corporation) at rt for 2 hours followed by overnight incubation at 4c. following a short wash, the slides were incubated with a cocktail of biotinylated detection antibodies (anti - human - cd9, -cd63 and -cd81, lifespan biosciences, wa, usa) diluted 1:1,500 in wash - buffer. after a wash, a subsequent 30-minute incubation step with cy5-labelled streptavidin (life technologies) diluted 1:1,500 in wash - buffer was carried out for detection. prior to scanning, the slides were washed in wash - buffer, then in ultrapure / deionized water and finally dried using a microarray high - speed centrifuge (arrayit corporation). scanning and spot detection was performed as previously described (15). microarray printing was performed on a spotbot extreme protein edition microarray printer with a 946mp4 pin (arrayit corporation, ca, usa). as a positive control 100 g / ml of biotinylated human igg antibodies were printed on epoxy - coated slides (75.6 mm25.0 mm ; schott nexterion, de), which were left to dry at room temperature overnight prior to further analysis. a total of 37 anti - human antibodies were used and listed in the following with the corresponding clone, if available : cd146 (p1h12), flotillin-1, hb - egf (4g10), her3 (2f9), her4 (h4.77.16), hsp90 (igf1), n - cadherin (8c11), p53 (pab240), tag72 (0.n.561), and tsg101 (5b7) from abcam (ma, usa) ; sftpd (vif11) from acris antibodies gmbh (de) ; egfr and egfrviii from antibodies-online.com (de) ; cd63 (mem-259) from biolegend (ca, usa) ; her2 (29d8) from cell signalling (ma, usa) ; cd9 and cd81 from lifespan biosciences (wa, usa) ; spa (6f10) from novus biologicals (co, usa) ; cd13 (498001), cd14 (50040), cd142 (323514), cd151 (210127), cd206, cea (487609), muc1 (604804), tnf ri, tnf rii and tspan8 (458811) from r&d systems (mn, usa) ; areg (s-13), epcam (0.n.277), mucin16 (x306), ny - eso-1 (e978) and plap (8b6) from santa cruz biotechnologies (tx, usa) ; cd171 from sigma - aldrich (mo, usa) ; c - met (016) and pd - l1 from sino biological inc. (china) ; cd163 (mac2 - 158) from trillium diagnostics, llc (me, usa). all antibodies were diluted with pbs with 5% glycerol and printed in triplicates at 75200 g / ml. the ev array was prepared as described by jrgensen. (15) with modifications. in short, the microarray slides were initially blocked (50 mm ethanolamine, 100 mm tris, 0.1% sds, ph 9.0) prior to incubation with 10 l plasma sample diluted (1:10) in wash - buffer (0.05% tween20 in pbs). the incubation was performed in multi - well hybridization cassettes (arrayit corporation) at rt for 2 hours followed by overnight incubation at 4c. following a short wash, the slides were incubated with a cocktail of biotinylated detection antibodies (anti - human - cd9, -cd63 and -cd81, lifespan biosciences, wa, usa) diluted 1:1,500 in wash - buffer. after a wash, a subsequent 30-minute incubation step with cy5-labelled streptavidin (life technologies) diluted 1:1,500 in wash - buffer was carried out for detection. prior to scanning, the slides were washed in wash - buffer, then in ultrapure / deionized water and finally dried using a microarray high - speed centrifuge (arrayit corporation). scanning and spot detection was performed as previously described (15). graphs and statistics were carried out using graphpad prism (version 6.04, graphpad software, inc., san diego, ca, usa), excel (version 2013, microsoft, redmond, wa, usa) and roccet (version 03/17/2014, tmic, ab, canada). heat maps and hierarchical cluster analysis were produced using genesis (version 1.7.6, igb tu graz, graz, austria). random forests was used for classification and monte - carlo cross validation (mccv) using balanced subsampling was employed to compute roc curves and to calculate confidence intervals of their area under the curve (auc). in each mccv, two thirds (2/3) of the samples were used to evaluate the feature importance. the top important features were then used to build a classification model, which was validated on the 1/3 of the samples that were left out. five hundred iterations were used to calculate performance of the model. prior to analysis missing value estimation was performed by replacing missing values by min. a top 20 of ratios (based on p - values) was computed and included into the analysis prior to data transformation and normalization by log2. the ev array analysis was performed on 109 plasma samples from patients diagnosed with stage iiia iv nsclc adenocarcinoma and on 110 plasma samples from patients without a cancer diagnosis (table i). even though only 10 l unpurified plasma sample was loaded for each patient, sufficient signal for further analysis was obtained from all samples. selected baseline patient characteristics for the two study groups thirty - seven different antibodies were used for the analysis of plasma exosomes. in addition, antibodies targeting markers involved in nsclc pathology were optimised for the ev array analysis. for this purpose, members of the egf system and selected markers of inflammation (cd163, cd206) (16, 17) were selected (table ii). description of the markers selected for the ev array together with the outcome (p - value) of the non - parametric t - test comparing the log2 transformed data from the control group and the cancer group (supplementary fig. 1a and b) unless otherwise stated the marker level was higher in the control group. elevated in cancer patients. the univariate analysis of each marker using log2-transformed signal - values revealed generally a significantly higher marker - expression in control subjects compared to lung cancer subjects (table ii and supplementary fig. 1a and b). hierarchical cluster analysis for each of the groups depicted a high variety of expression among the individuals, which was also seen in a previously study of healthy donors (15). the hierarchical cluster analysis also showed co - variance among certain markers, for example cd9, cd81 and cd151 (marked with a box) and egfrviii, flotilin-1, her4, n - cadherin and cd163 (marked with a dashed box) (fig. the control group had a significantly higher (p<0.05) cd9, cd81 and cd151 expression when looking at log2-transformed data (fig. for the co - varying group consisting of egfrviii, flotilin-1, her4, n - cadherin and cd163, all markers were higher expressed in the control group, as well as when looking at log2-transformed data (fig. 2b). as the cancerous patients showed a tendency to have a lower amount of positive antigens over all (fig. 3a), a normalisation (calculation of the percentage) of the signals for each antigen to the total signal for each individual patient was performed (fig. 3a). taken together with the fact that the ev array signal detection relies on cd9-, cd63- and/or cd81-antibody binding, the relative expression of these markers was examined further (pie charts in fig. 3b), the cd9, cd63 and cd81 percentage was significantly higher (p<0.05) in the cancer group. two groups of markers show co - variance both in the control group and in the cancer group (marked with boxes). b) heat map illustration of all markers in the cancer group. ev array signal intensities for selected antigens. a) the ev array signal intensities for the exosomal markers cd9, cd63 and cd81 displayed in box plots. the co - variation of the signal intensities across the patient samples can be seen in fig. 1 and supplementary fig b) box plot of a group of antigens (flotilin-1, her4, egfrviii, n - cadherin and cd163) showing a high degree of co - variation (see fig. p<0.05 ; p<0.001 ; p<0.0001 ; ns = not significant. a) the signals for all analytes were summed for each individual patient and plotted ; controls indicated with green and cancer with red. for each individual patient the pie charts illustrate an example of the normalised data for a patient in each group with a total amount of signal of ~40. b) and c) box plot of the relative expression of markers from figure 2a and b in percentage (in relation to the total sum of exosomal signal). p < 0.05 ; p<0.01 ; p<0.0001 ; ns = not significant. normalised data for the co - varying group egfrviii, flotilin-1, her4, n - cadherin and cd163 was also investigated. for these markers none of the individual markers alone gained an auc indicating a diagnostic potential (fig. however, the diagnostic potential of a panel of markers was evaluated using multivariate analysis. for the multivariate analysis the univariate analysis of the normalised data for cd9, cd63 and cd81 (fig. therefore, these normalised values were accepted in the multivariate analysis together with the individual relations between the analysed markers. figure 4 shows the roc curves for the 3, 5, 10 and 30 best rated marker combinations. the 30-marker model has the largest auc of 0.83, ci : 0.770.90, a sensitivity of 0.75 and a specificity of 0.76. was observed when expanding the number of markers included beyond 30 (data not shown). the area under curve (auc) for top 3-, 5-, 10-, and 30-marker panels are given together with the 95% confidence interval. the importance of each marker or their individual relations for the model was assessed (fig. the most important marker was found to be cd81 followed by the relation between tag72 and the normalised values of cd63. the top 10 ranking of the importance did not change between the models including 3, 5, 10 or 30 markers. the mean average importance to the classification model using the 30-marker panel illustrated in fig. 4 for each of the analysed exosomal antigens, the normalised values (indicated by) and their internal relations (indicated by /). the top 10 ranking did not change between the models including 3-, 5-, 10- or 30-markers and the markers included in each model are visualised by the coloured lines. the ev array analysis was performed on 109 plasma samples from patients diagnosed with stage iiia iv nsclc adenocarcinoma and on 110 plasma samples from patients without a cancer diagnosis (table i). even though only 10 l unpurified plasma sample was loaded for each patient, sufficient signal for further analysis was obtained from all samples. selected baseline patient characteristics for the two study groups thirty - seven different antibodies were used for the analysis of plasma exosomes. in addition, antibodies targeting markers involved in nsclc pathology were optimised for the ev array analysis. for this purpose, members of the egf system and selected markers of inflammation (cd163, cd206) (16, 17) were selected (table ii). description of the markers selected for the ev array together with the outcome (p - value) of the non - parametric t - test comparing the log2 transformed data from the control group and the cancer group (supplementary fig. 1a and b) unless otherwise stated the marker level was higher in the control group. elevated in cancer patients. the univariate analysis of each marker using log2-transformed signal - values revealed generally a significantly higher marker - expression in control subjects compared to lung cancer subjects (table ii and supplementary fig. hierarchical cluster analysis for each of the groups depicted a high variety of expression among the individuals, which was also seen in a previously study of healthy donors (15). the hierarchical cluster analysis also showed co - variance among certain markers, for example cd9, cd81 and cd151 (marked with a box) and egfrviii, flotilin-1, her4, n - cadherin and cd163 (marked with a dashed box) (fig. the control group had a significantly higher (p<0.05) cd9, cd81 and cd151 expression when looking at log2-transformed data (fig. for the co - varying group consisting of egfrviii, flotilin-1, her4, n - cadherin and cd163, all markers were higher expressed in the control group, as well as when looking at log2-transformed data (fig. 2b). as the cancerous patients showed a tendency to have a lower amount of positive antigens over all (fig. 3a), a normalisation (calculation of the percentage) of the signals for each antigen to the total signal for each individual patient was performed (fig. 3a). taken together with the fact that the ev array signal detection relies on cd9-, cd63- and/or cd81-antibody binding, the relative expression of these markers was examined further (pie charts in fig. 3b), the cd9, cd63 and cd81 percentage was significantly higher (p<0.05) in the cancer group. two groups of markers show co - variance both in the control group and in the cancer group (marked with boxes). b) heat map illustration of all markers in the cancer group. ev array signal intensities for selected antigens. a) the ev array signal intensities for the exosomal markers cd9, cd63 and cd81 displayed in box plots. the co - variation of the signal intensities across the patient samples can be seen in fig. 1 and supplementary fig b) box plot of a group of antigens (flotilin-1, her4, egfrviii, n - cadherin and cd163) showing a high degree of co - variation (see fig. p<0.05 ; p<0.001 ; p<0.0001 ; ns = not significant. a) the signals for all analytes were summed for each individual patient and plotted ; controls indicated with green and cancer with red. for each individual patient the pie charts illustrate an example of the normalised data for a patient in each group with a total amount of signal of ~40. b) and c) box plot of the relative expression of markers from figure 2a and b in percentage (in relation to the total sum of exosomal signal). p < 0.05 ; p<0.01 ; p<0.0001 ; ns = not significant. normalised data for the co - varying group egfrviii, flotilin-1, her4, n - cadherin and cd163 was also investigated. for these markers none of the individual markers alone gained an auc indicating a diagnostic potential (fig. however, the diagnostic potential of a panel of markers was evaluated using multivariate analysis. for the multivariate analysis the univariate analysis of the normalised data for cd9, cd63 and cd81 (fig. therefore, these normalised values were accepted in the multivariate analysis together with the individual relations between the analysed markers. figure 4 shows the roc curves for the 3, 5, 10 and 30 best rated marker combinations. the 30-marker model has the largest auc of 0.83, ci : 0.770.90, a sensitivity of 0.75 and a specificity of 0.76. was observed when expanding the number of markers included beyond 30 (data not shown). the area under curve (auc) for top 3-, 5-, 10-, and 30-marker panels are given together with the 95% confidence interval. the importance of each marker or their individual relations for the model was assessed (fig. the most important marker was found to be cd81 followed by the relation between tag72 and the normalised values of cd63. the top 10 ranking of the importance did not change between the models including 3, 5, 10 or 30 markers. the mean average importance to the classification model using the 30-marker panel illustrated in fig. 4 for each of the analysed exosomal antigens, the normalised values (indicated by) and their internal relations (indicated by /). the top 10 ranking did not change between the models including 3-, 5-, 10- or 30-markers and the markers included in each model are visualised by the coloured lines. so far, only a limited number of studies have examined the diagnostic potential in relation to nsclc. until now, the studies have been limited to investigating exosomes captured by antibodies towards a single antigen, for example, epcam or cd9 (9, 13). in spite of these constraints, promising results were obtained through these studies and a subset of exosomal mirnas and the exosomal - bound cd91-protein was revealed as promising biomarkers. the present study examined plasma exosomes from 219 patients, initially suspected of having lung cancer, from a multi - marker aspect. investigation of 37 exosome capturing antibodies and the use of a cocktail of 3 different detecting antibodies against known exosome markers gave comprehensive information paving the way for multivariate analysis. our multivariate ev array analysis results in a 30-marker panel classifying 75% of the patients correctly. this result is superior to exosomal cd91-expression alone (61%) as recently reported by ueda. our data suggests that the ev array analysis could be a valuable complementary tool in diagnosing nsclc. the univariate t - test analysis revealed higher exosomal marker abundance on the control patient exosomes compared to the cancer patient exosomes. this is in concordance with the recent study of serum samples from nsclc patients and healthy blood donors performed by ueda.. the decreased overall signal could be due to less compact exosomes in lung cancer patients, for example, fewer proteins per exosome. presumably, this would result in fewer lung cancer exosomes being captured by the antibodies. previously, differences in the protein load of the exosomes of melanoma patients have also been shown by peinado. to have an influence on the survival rate (61). another possibility is that exosomes from nsclc patients express another subset of markers than the ones targeted in this analysis or that the exosomes carrying the cancerous markers are cleared by an activated immune system in the cancerous patients (62). additional markers could be explored to optimise the ev array and other detection antibodies could be evaluated for further investigation of this phenomenon. cd9 and cd81 remain well - described exosomal markers and may be expected to co - vary. interestingly, cd63, another well - known marker, does not follow this pattern, which has also been described previously (15, 63). the difference in expression of cd63 in relation to cd9 and cd81 highlights the advantage of detecting the signal with a cocktail of cd9, cd63 and cd81 antibodies in order to achieve sufficient information. egfrviii together with flotilin-1, her4, n - cadherin and cd163 constituted an additional co - varying group (fig. nevertheless, egfr truncation is not normally observed in nsclc and neither in normal tissue (64, 65). an obvious first thought to explain the level of egfrviii signal in both control and cancer patients could point to an unspecific antibody binding to normal egfr. however, since the patterns of these 2 markers (egfr and egfrviii) vary from each other this explanation does not seem to be the case. this phenomenon should be explored further in order to assess the biological bearing of this truncated receptor. as seen from the ranking of the 30 markers, their contribution differs in importance to the model. increasing the average importance with new and more efficient markers furthermore, as no increase in diagnostic potential was seen when including more markers, it is essential to substitute weak markers with better candidate markers in order to increase the average importance and thereby provide an even more valuable tool. in conclusion, the ev array constitutes a broad spectra tool offering exhaustive information on exosomal content with a minimal of sample requirement, which can be optimized and adjusted to fit individual sets of samples. the authors have not received any funding or benefits from industry or elsewhere to conduct this study. | backgroundlung cancer is one of the leading causes of cancer - related death. at the time of diagnosis, more than half of the patients will have disseminated disease and, yet, diagnosing can be challenging. new methods are desired to improve the diagnostic work - up. exosomes are cell - derived vesicles displaying various proteins on their membrane surfaces. in addition, they are readily available in blood samples where they constitute potential biomarkers of human diseases, such as cancer. here, we examine the potential of distinguishing non - small cell lung carcinoma (nsclc) patients from control subjects based on the differential display of exosomal protein markers.methodsplasma was isolated from 109 nsclc patients with advanced stage (iiia iv) disease and 110 matched control subjects initially suspected of having cancer, but diagnosed to be cancer free. the extracellular vesicle array (ev array) was used to phenotype exosomes directly from the plasma samples. the array contained 37 antibodies targeting lung cancer - related proteins and was used to capture exosomes, which were visualised with a cocktail of biotin - conjugated cd9, cd63 and cd81 antibodies.resultsthe ev array analysis was capable of detecting and phenotyping exosomes in all samples from only 10 l of unpurified plasma. multivariate analysis using the random forests method produced a combined 30-marker model separating the two patient groups with an area under the curve of 0.83, ci : 0.770.90. the 30-marker model has a sensitivity of 0.75 and a specificity of 0.76, and it classifies patients with 75.3% accuracy.conclusionthe ev array technique is a simple, minimal - invasive tool with potential to identify lung cancer patients. |
spinal cord injury (sci) is an insult to the spinal cord that results in lost or impaired function causing reduced mobility or feeling below the level of injury. common causes of sci are trauma, disease, and degeneration. up to 90% of cases are because of trauma. the reported annual incidence of sci ranges between 10.4 and 83 per million inhabitants worldwide and between 15 and 55 per million in north america. the incidence in taiwan is much higher, at 150 per million according to a nationwide health insurance database. with improvements in the technology used for the clinical care, treatment, and follow - up of individuals with sci, the life expectancy of these patients has been significantly increased and approached that of the general population. consequently, most patients with sci are likely to encounter aging - related problems such as cancer. the relationship between cancer risk and sci has been explored for decades, but most research has focused on genitourinary (gu) tract cancers. reports, however, have also noted an increased risk of bladder cancer in patients with sci. we previously found that patients with sci had a lower risk of prostate cancer compared with people without sci, but that the risk of bladder cancer did not differ between people with and without sci. scant information is available regarding the risk of non - gu cancers in patients with sci. studies investigating colonoscopic lesions in patients with sci have reported inconsistent results. in a study of veterans disabled by myelopathy, frisble found a 2- to 6-fold increase in the age - adjusted incidence of colorectal carcinoma in males with myelopathy compared with the general male population. spinal cord injury changes body composition, metabolism, and hormonal regulation over time and can cause chronic complications, several of which have been suggested to be associated with some cancer types ; these complications include osteoporosis, metabolic syndrome, and venous thromboembolism (vte). to our knowledge, no large - scale population - based study has evaluated how sci may affect the development of non - gu cancers. this study aimed to determine whether non - gu cancer risk was associated with sci in taiwan. a retrospective cohort design was adopted to analyze the database of the national health insurance program. we hypothesized that the risk of certain non - gu cancers may be increased among patients with sci. the data used in this study were obtained from the national health insurance research database (nhird) and covered the period from january 1, 2000 to december 31, 2011. the nhird, released by the national health research institutes (nhri) for research purposes, is a nationwide population - based claims database of the taiwan nhi program, which was implemented on march 1, 1995 and covers more than 23 million people, representing approximately 99% of the taiwan population. the nhird contains all reimbursement claims data, including inpatient and outpatient medical claims, hospital information, and patient demographic characteristics. before releasing the database, the national health research institute scrambles all personal information and provides an anonymous number for each insured person for linking with their claims data. the current study was approved by the institutional review board of china medical university and hospital (cmuh104-rec2115). the diagnoses in the nhird are based on the international classification of diseases, ninth revision, clinical modification (icd-9-cm). we identified adult patients newly diagnosed with sci (icd-9-cm codes 806 and 952 ; n = 45,692) between 2000 and 2010. patients with a history of cancer (icd-9-cm 140208) (n = 1037), an age of 5 years, those with sci, however, had a significantly higher risk of esophageal cancer compared with those without sci. cox model with hazard ratios and 95% confidence intervals of cancer types associated with spinal cord injury, stratified by follow - up duration our study revealed no significant difference in overall non - gu cancer risk between the sci and control groups. individual cancer risk analysis revealed that patients with sci exhibited a significantly higher risk of esophageal, liver, and hematologic malignancies. by contrast, a significantly lower risk of colorectal cancer was found in the sci cohort. analyses stratified by sex, age, and follow - up duration demonstrated various correlations between sci and non - gu cancer risk., a significantly lower risk of colorectal cancer was found in the sci group compared with the control group. this result differs from that of frisbie, who reviewed the records of 1023 veterans disabled by myelopathy and found that the age - adjusted incidence rates in the sixth to ninth decades of age were 2 to 6 times larger than the highest reported rates for the general male population (p < 0.05). morris conducted a case - control study in australia and found that the patients with sci had the same risk of malignancy that the general population did. in a study of colonoscopic lesions in patients with sci, han found no difference in cancer detection between an sci and control group ; inflammatory bowel disease, which is a risk factor for cancer, was more common in the sci group, but not significantly so (p = 0.06). the conflicting results between our study and previous reports may be partially because of less frequent colonoscopy screenings in the sci group than in the control group. because of the inconvenience and relative difficulty of the bowel preparation process necessary for the invasive procedure, patients with sci receive significantly fewer colonoscopy screenings than does the general population, according to a self - report survey. furthermore, considering several complications of sci suggested as being related to cancer risk, we expected that a possible association may exist between non - gu cancer and sci. osteoporosis is a common consequence of sci and occurs in almost every patient with sci. although the mechanism of this relationship remains unclear, a commonly suggested reason relates to the mechanical unloading of the paralyzed limbs and vascular dysfunction below the level of injury. studies examining whether osteoporosis increases cancer risk have yielded inconsistent results. in a cohort of 23,935 people with osteoporosis, mcglynn found a significantly lower risk of colorectal cancer among the female patients compared with the general female population, particularly in the 70 years age group. if the cancer risk of patients with osteoporosis can partially reflect that of patients with sci, then their results are consistent with ours. our data showed that both male and female patients with sci had a significantly lower risk of colorectal cancer compared with their counterparts, and the difference was greater for women. older patients with sci (50 year) had a significantly lower colorectal cancer risk, but this difference was not present among younger patients with sci. in addition, observational studies and meta - analyses have associated osteoporosis drugs with unchanged or decreased colorectal cancer risk. regarding other non - gu cancers, mcglynn reported that female patients with osteoporosis had a significantly higher risk of liver cancer and hematologic malignancy compared with female patients without osteoporosis. the women with sci in the current study also exhibited a significantly higher risk of liver cancer and hematologic malignancy than women without sci. other research has found inconclusive results for the risk of different non - gu cancers among patients with osteoporosis. changed body composition and inflammatory activity among patients with sci may contribute to the higher incidence of metabolic syndrome (obesity, elevated blood pressure, elevated fasting plasma glucose, high serum triglycerides, and low high - density lipoprotein levels). prospective cohort studies have shown associations between metabolic syndrome, and increased risk of breast cancer, colorectal cancer, esophageal adenocarcinoma, and hepatocellular carcinoma.although we matched and adjusted the risk factors of diabetes, hypertension, and hyperlipidemia in this study, these are considered comorbidities rather than complications in the analyses. our study revealed a significantly higher risk of esophageal and liver cancer among patients with sci and also concluded that metabolic syndrome after sci may play a role in this association. venous thromboembolism, including deep venous thrombosis and pulmonary embolism, is a well - documented complication during the hospital course of patients with acute sci. at least 3 major reasons for this association have been identified : stasis, hypercoagulability, and intimal injury. several large epidemiological studies have reported an increased risk of cancer after diagnosis of idiopathic vte. the vte factor may partially account for the higher incidence rate of some cancer types among the patients with sci in our study. factors other than sci - related complications may mediate the possible association between non - gu cancer and sci. substantial evidence suggests that chronic inflammation can play a pivotal role in the initiation, development, and progression of cancers. impaired immune response may also contribute to the development and progression of some types of cancer. the main strength of this study is its use of a nationwide population - based database, which ensures the high generalizability of the findings. the findings may be helpful to our government in considering future policy plan in the field of cancer surveillance. for example, we assumed that the sci group received less frequent colonoscopy screenings than did the control group, and it can mislead to an underestimated risk of colorectal cancer in sci group. by contrast, more hematologic malignancies were detected in the patients with sci, likely because these patients received more blood tests during their more frequent hospital stays. second, neither patient lifestyle and behavior information (such as tobacco use and alcohol consumption) nor tumor characteristics (such as histology, grade, and stage) were included in the nhird ; thus, these factors which may affect the results in either direction, could not be adjusted for in this study. third, patients with sci are highly prevalent with metabolism disarrangement, malnutrition, and inflammation ; however, the nhird lacks data of liver function, renal function, inflammation markers, and nutrition markers, so we can not adjust these factors to eliminate the possible confounding. fourth, the evidence derived from a cohort study is generally of a lower methodological quality than that obtained from a randomized trial because a cohort study design is subject to several biases related to adjusting for confounding effects. in a retrospective study, lack of baseline data, residual confounding, selection bias, and death competing factor are the major drawbacks. despite the intrinsic limitations of the nhird administrative data, the information that we obtained on the diagnosis of sci and cancer was highly reliable. in summary, our study suggested that sci may be related to the risk of specific types of cancer, either increased or decreased risks, and it is not fully consistent with previous reports. the mechanism of this relationship remains undetermined ; however, indirect links mediated by some complications of sci and cancer may account for it. the intricate interaction between cancer and different complications may complicate this association between sci and non - gu cancers. | abstractlittle information is available regarding the risk of nongenitourinary (gu) cancers in patients with spinal cord injury (sci). the authors conducted a nationwide population - based study to investigate whether a higher risk of non - gu cancer is seen among patients with sci.data retrieved from the national health insurance research database of taiwan were used in this study. a total of 41,900 patients diagnosed with sci between 2000 and 2011 were identified from the national health insurance research database and comprised the sci cohort. each of these patients was randomly frequency matched with 4 people from the general population (without sci) according to age, sex, comorbidities, and index year. cox proportional hazards regression analysis was used to calculate adjusted hazard ratios and 95% confidence intervals and determine how sci affected non - gu cancer risk.no significant difference in overall non - gu cancer risk was observed between the sci and control groups. the patients with sci exhibited a significantly higher risk of developing esophageal, liver, and hematologic malignancies compared with those without sci. by contrast, the sci cohort had a significantly lower risk of colorectal cancer compared with the non - sci cohort (adjusted hazard ratio = 0.80, 95% confidence interval = 0.690.93). additional stratified analyses by sex, age, and follow - up duration revealed various correlations between sci and non - gu cancer risk.the patients with sci exhibited higher risk of esophageal, liver, and hematologic malignancies but a lower risk of colorectal cancer compared with those without sci. the diverse patterns of cancer risk among the patients with sci may be related to the complications of chronic sci. |
diabetic nephropathy (dn) is a major cause of end - stage renal disease (esrd) and high mortality in diabetic patients. for example, high blood pressure, high glomerular filtration rate, glycemic control, and race were reported to be associated with dn development. genetic susceptibility may also be an important determinant of the incidence and severity of dn. interleukin-10 (il-10) is an immunoregulatory cytokine produced by th2 cells, regulatory t cells, and monocytes / macrophages. il-10 is an anti - inflammatory cytokine that can inhibit the synthesis of cytokines such as il-6, il-1, il-1, and tnf- in activated macrophage and ifn by t cells. myliwska. found elevated concentration of circulating il-10 in diabetes mellitus (dm) patients with dn, compared to dm patients without dn. wong. suggested that plasma concentrations of il-10 exhibited significant positive correlation with urine albumin / creatinine ratio in dn patients. in addition, changes in il-10 levels correlated with the extent of renal damage in dn. taken together, these reports indicate that il-10 might contribute to the pathogenesis of dn. several previous studies have studied the association between il10 - 1082a / g polymorphism and dn risk [713 ]. however, the results were inconclusive and contradictory. for example, babel. suggested that il10 - 1082a / g polymorphism may increase susceptibility to dn.. concluded that the il-10 (1082g / a) gene polymorphism was not associated with the development of dn in turkish patients with type 2 diabetes. therefore, we performed a meta - analysis to investigate the association between il10 - 1082a / g polymorphism and dn risk. all relevant studies were searched by using the pubmed and embase (the last retrieval date was oct 10, 2014 using the search terms : diabetic nephropathy and interleukin-10 or il10). all searched studies were retrieved and only published studies with full - text articles were included. when there were multiple publications with duplicate samples, only the newest study was used in this research. our study was approved by the ethics committee of the first affiliated hospital of henan university of science and technology. the inclusion criteria were : (1) the research was a case - control study or a cohort study ; (2) the study investigated the association between il10 - 1082a / g polymorphism and dn risk ; (3) the il10 - 1082a / g genotypes of individual groups were provided. the exclusion criteria were : (1) no usable data reported ; (2) animal studies ; (3) reviews or abstracts ; (4) duplicates. two authors extracted the data independently, including first author, year, ethnicity, age, sex, diabetes type, and sample size. statistical analysis was conducted using stata software 11.0 (statacorp, college station, tx, usa). hardy - weinberg equilibrium (hwe) test in healthy control group was conducted using the test. odds ratio (or) with a 95% confidence interval (ci) are presented for dichotomous data, and the significance level was 0.05. q - statistic and i - statistic were used to measure statistical heterogeneity and the significance level was 0.10. a fixed - effects model was selected when there was no significant heterogeneity ; otherwise, a random - effects model was used. publication bias was tested using begg s test and funnel plot (significant level was 0.05). all relevant studies were searched by using the pubmed and embase (the last retrieval date was oct 10, 2014 using the search terms : diabetic nephropathy and interleukin-10 or il10). all searched studies were retrieved and only published studies with full - text articles were included. when there were multiple publications with duplicate samples, only the newest study was used in this research. our study was approved by the ethics committee of the first affiliated hospital of henan university of science and technology. the inclusion criteria were : (1) the research was a case - control study or a cohort study ; (2) the study investigated the association between il10 - 1082a / g polymorphism and dn risk ; (3) the il10 - 1082a / g genotypes of individual groups were provided. the exclusion criteria were : (1) no usable data reported ; (2) animal studies ; (3) reviews or abstracts ; (4) duplicates. two authors extracted the data independently, including first author, year, ethnicity, age, sex, diabetes type, and sample size. statistical analysis was conducted using stata software 11.0 (statacorp, college station, tx, usa). hardy - weinberg equilibrium (hwe) test in healthy control group was conducted using the test. odds ratio (or) with a 95% confidence interval (ci) are presented for dichotomous data, and the significance level was 0.05. q - statistic and i - statistic were used to measure statistical heterogeneity and the significance level was 0.10. a fixed - effects model was selected when there was no significant heterogeneity ; otherwise, a random - effects model was used. publication bias was tested using begg s test and funnel plot (significant level was 0.05). two studies were conducted in asian populations and the rest of the studies were performed in caucasian populations. thus, a total of 9 case - control studies with 4165 subjects were included in this meta - analysis [713 ]. previous studies showed that the il10 - 1082aa genotype was associated with increased il-10 production. therefore, we investigated the association between il10 - 1082a / g polymorphism and dn risk in the recessive models (aa vs. ga+gg). we found that il10 - 1082a / g polymorphism significantly associated with an increased dn risk (or=1.21 ; 95% ci 1.071.37 ; p=0.002 ; figure 1). in the subgroup analysis by race, both caucasians and asians with il10 - 1082a / g polymorphism showed increased dn risk (or=1.25 ; 95% ci 1.031.52 ; p=0.03 and or=1.25 ; 95% ci 1.041.49 ; p=0.02), respectively. when we deleted the study without dm type, the result was not altered (or=1.21 ; 95% ci 1.021.44 ; p=0.03). in the subgroup analysis by sample size, studies with large sample size and studies with small sample size both showed increased dn risk (or=1.16 ; 95% ci 1.021.31 ; p=0.02 and or=1.50 ; 95% ci 1.141.98 ; p=0.004), respectively. cumulative meta - analysis was conducted by sorting the studies by publication time (figure 2). to determine the stability of the result we found that no single study impacted the pooled or, indicating that the results of our research are statistically robust (figure 3). egger s test and begg s funnel plot were conducted to assess the publication bias. two studies were conducted in asian populations and the rest of the studies were performed in caucasian populations. thus, a total of 9 case - control studies with 4165 subjects were included in this meta - analysis [713 ]. previous studies showed that the il10 - 1082aa genotype was associated with increased il-10 production. therefore, we investigated the association between il10 - 1082a / g polymorphism and dn risk in the recessive models (aa vs. ga+gg). we found that il10 - 1082a / g polymorphism significantly associated with an increased dn risk (or=1.21 ; 95% ci 1.071.37 ; p=0.002 ; figure 1). in the subgroup analysis by race, both caucasians and asians with il10 - 1082a / g polymorphism showed increased dn risk (or=1.25 ; 95% ci 1.031.52 ; p=0.03 and or=1.25 ; 95% ci 1.041.49 ; p=0.02), respectively. when we deleted the study without dm type, the result was not altered (or=1.21 ; 95% ci 1.021.44 ; p=0.03). in the subgroup analysis by sample size, studies with large sample size and studies with small sample size both showed increased dn risk (or=1.16 ; 95% ci 1.021.31 ; p=0.02 and or=1.50 ; 95% ci 1.141.98 ; p=0.004), respectively. cumulative meta - analysis was conducted by sorting the studies by publication time (figure 2). to determine the stability of the result we found that no single study impacted the pooled or, indicating that the results of our research are statistically robust (figure 3). egger s test and begg s funnel plot were conducted to assess the publication bias. several meta - analyses have been performed to assess the association between il10 - 1082a / g polymorphism and dm risk [1517 ]. however, no meta - analysis study evaluated the association between il10 - 1082a / g polymorphism and dn risk. thus, this is the first meta - analysis of the association between il10 - 1082a / g polymorphism and dn risk. the present meta - analysis of 9 case - control studies evaluated the association between il10 - 1082a / g polymorphism and dn risk. we found that il10 - 1082a / g polymorphism was a risk factor for dn. this result indicates that il10 - 1082aa genotype might increase dn risk more than the ag or gg genotypes. in the race subgroup analysis, both caucasians and asians with il10 - 1082a / g polymorphism showed increased dn risk. this result suggests that il10 - 1082a / g polymorphism played the same role in the pathological mechanism of dn in different races, but there were only 2 studies in asian populations. we also found that il10 - 1082a / g polymorphism could increase dn in the t2 dm patients. since no study with t1 dm was included, more studies with these populations are needed. studies with large sample size and studies with small sample size showed increased dn risk. this result suggests that sample size did not change the result of this meta - analysis. considering all of these results, we confirmed that il10 - 1082a / g polymorphism might contribute to the development of dn. a large body of evidence suggests that the pathogenesis of dm is related to the presence of a chronic low - grade inflammatory state and the activation of the innate immune system. additionally, inflammatory cytokines were reported to be involved in the development of microvascular diabetic complications, including dn. il-10 was a candidate gene in the pathophysiologic mechanism of auto - immune / inflammatory disease, because it could regulate both cellular and humoral immunity. a previous study suggested that il-10 was elevated in the sera of t2 dm patients with dn. first, the studies included in our meta - analysis were small ; therefore, our meta - analysis had low statistical power to assess the association of the il10 - 1082a / g polymorphism with the risk of dn. second, the number of the included studies was moderate ; therefore, the results of this meta - analysis might be influenced by factors such as publication bias, but no evidence of publication bias was found in our study. third, our study did not address gene - gene and gene - environment interactions. last but not least, this meta - analysis lacked sufficient reliability to confirm or refute the results of subgroup analysis in a definitive manner. our meta - analysis study confirmed that il10 - 1082a / g polymorphism might contribute to the susceptibility for dn. | backgroundstudies have assessed the association between interleukin-10 (il-10) -1082a / g polymorphism and diabetic nephropathy (dn) risk, but the results were inconclusive and contradictory. therefore, we performed a meta - analysis to investigate the association between il10 - 1082a / g polymorphism and dn risk.material/methodsall relevant studies were searched by using pubmed and embase. data were extracted by 2 authors independently. odds ratios (ors) and corresponding 95% confidence intervals (cis) were calculated.resultsnine case - control studies with 4165 subjects were included in this meta - analysis. we found that il10 - 1082a / g polymorphism was significantly associated with an increased dn risk (or=1.21 ; 95% ci 1.071.37 ; p=0.002). in the subgroup analysis by race, both caucasians and asians with il10 - 1082a / g polymorphism showed increased dn risk (or=1.25 ; 95% ci 1.031.52 ; p=0.03 and or=1.25 ; 95% ci 1.041.49 ; p=0.02), respectively. when we deleted the study without diabetes type, the result was not altered (or=1.21 ; 95% ci 1.021.44 ; p=0.03). in the subgroup analysis by sample size, both studies with large sample size and studies with small sample size showed increased dn risk (or=1.16 ; 95% ci 1.021.31 ; p=0.02 and or=1.50 ; 95% ci 1.141.98 ; p=0.004), respectively.conclusionsthis meta - analysis confirmed that il10 - 1082a / g polymorphism might contribute to the susceptibility for dn. |
accidents with a pneumatic nail gun, which has been used in the construction industry, have increased.2) however, reports on management of a patient with a nail gun injury to the cranium are rare. the authors report on a case of a patient treated with simple retraction after small skin and prophylactic antibiotic administration. a 56-year - old man had five nail gun - shots due to attempted suicide and was transferred to the emergency room. on his physical examination, his mental status was drowsy and there was no neurological deficit except for nails on his cranium. in his past history, there was no psychological background, however, he used to drink often and became depressed. on plain skull radiography, five nails were found (figure 1a). among the five nails, two penetrated glabella. two nails were located in the right parietal bone and another was located in the left parietal bone. due to the head of the nail, the direction was confined to the outer table of the skull. in addition, a subarachnoid hemorrhage was observed on brain computed tomography (ct)(figure 1b). all procedures were performed in the operating room under general anesthesia in order to treat the emergent condition as rapidly as possible. the nail head was located beneath the scalp and tightly attached to the skull, it was not visible from the outside. a small incision to advance a forceps and a rongeur was necessary, and, all nails were removed successfully (figure 1c). during the period of conservative management antibiotics including vancomycin, ceftriaxone, and metronidazole were administered for two weeks and carbamazepine was administered for a week. conventional angiography was performed, and there was no vascular invasion or newly formed fistula (figure 1d). head injury by pneumatic nail gun injury is rare, and there have been some case reports on management, treatment. according to lee and park,4) the speed of the launched nail and presence of the nail head are important determinants of the extent of nail gun related head injuries. if major artery or sinuses are not involved, extraction from the nail head could be the main treatment. ct is typically the first - line radiologic examination in the emergency room for head - injured patients. in general, ct shows excellent details in bone and average value in soft tissue. failure to recognize cerebral damage following penetrating brain injuries, especially when there is retained foreign body, can lead to serious life - threatening complications.1) in addition, because involvement of the large vessels is critical to determining the operative strategy, vascular integrity should be evaluated with ct angiography or ct enhanced image in the initial evaluation in the emergency room. in this case, nails were removed by a small scalp incision due to the fact that major artery or sinuses were not involved. if this is not the case, management would be direct ligation or vascular bypass surgery. despite many controversies regarding use of prophylactic antibiotics, the authors believe that broad spectrum antibiotics are appropriate for patients with penetrating brain injury since it is also recommended that antibiotics should be started as soon as possible.3) post - traumatic epilepsy is a major cause of disability and inadequate social integration. although there is no prospective study to indicate the efficacy of prophylactic anti - epileptic medication following a traumatic brain injury, it has been recommended that patients be covered for about the first week after injury with a medication such as phenytoin or carbamazepine.5) in the patient with head injury by pneumatic nail gun, the first evaluation should focus on the shape of the nail head and vascular integrity from simple x - ray and ct. if the nail head is confined to the depth of skull penetration, cautious extraction of the nail head can be performed without postoperative complication and early start of prophylactic antibiotics can be the next treatment. | a 56-year - old man had five nail gun - shots on his skull due to attempted suicide and was transferred to the emergency room. because the nail head played a role as a brake, the launched nail made a hole in the skull but did not entirely pass through it. if major artery or sinuses are not involved, cautious retrieval after a small scalp incision can be performed and prophylactic antibiotics be administered for treatment. |
apvd implies partial or total failure of the pulmonary veins to reach the left atrium. instead, pulmonary venous drainage is anomalously connected to systemic vein / s, typically to the superior or inferior vena cava (svc or ivc) or directly to the right atrium (ra). apvd accounts for up to 2% of all congenital heart disease and is usually associated with left to right shunting at atrial level through some form of atrial septal defect (asd). the haemodynamic effects of apvd are also those of left to right shunting at atrial level, thereby exacerbating right heart volume overload. the most common form of partial avpd is connection to the svc or svc - ra junction in association with a superior sinus venosus defect. scimitar syndrome is rarer, with the right pulmonary vein / s draining to the inferior vena cava. this abnormal form of drainage is visible on plain chest radiography, and was first described in 1836.1 the apvd is seen as a curvilinear density along the right heart border, similar in shape to the classic turkish curved sword. scimitar syndrome was coined by neill in 1960 to describe the cxr appearance.2 scimitar syndrome must be differentiated from the pseudoscimitar syndrome in which an abnormal descending vein pursues an aberrant course in the right lung but drains normally into the left atrium, and from kartagener 's syndrome. scimitar syndrome is also known as halasz 's syndrome, mirror - image lung syndrome, hypogenetic lung syndrome, epibronchial right pulmonary artery syndrome and vena cava bronchovascular syndrome. it occurs more commonly in females and is occasionally familial.2 the left lung is very rarely involved and the reason for this is unknown.3 the true incidence of this condition is unknown since the syndrome may remain undetected in asymptomatic patients who do not undertake a cxr. scimitar syndrome overlaps with pulmonary sequestration and the term venolobar syndrome has been coined to include these associated pulmonary and vascular malformations.4 the condition is associated with:4 partial agenesis or hypoplasia of the right lung with bronchial isomerismdiverticulum or hypoplasia of the right bronchial system.hypoplasia or agenesis of the right pulmonary artery. this may cause mediastinal shift to the right side and the scimitar vein may be difficult to appreciate or even completely obscured.abnormal systemic blood supply to at least part of the right lung, most frequently the posterior basal segment of the lower lobe, usually arising from the infradiaphragmatic descending aorta.dextrocardia due to right lung hypoplasia with mediastinal shift.accessory diaphragm or eventration or partial absence of the diaphragm.phrenic cysthorseshoe lungoesophageal and gastric lungabsence of the pericardiumother congenital cardiac malformations (25% of cases) including asd, ventricular septal defect, coarctation of the aorta, tetralogy of fallot, pulmonary stenosis, absent inferior vena cava with azygos continuation to superior vena cava and persistent left superior vena cava.5 moreover, the apvd may also be partially obstructed, further contributing to pulmonary hypertension.6 partial agenesis or hypoplasia of the right lung with bronchial isomerism diverticulum or hypoplasia of the right bronchial system. this may cause mediastinal shift to the right side and the scimitar vein may be difficult to appreciate or even completely obscured. abnormal systemic blood supply to at least part of the right lung, most frequently the posterior basal segment of the lower lobe, usually arising from the infradiaphragmatic descending aorta. dextrocardia due to right lung hypoplasia with mediastinal shift. accessory diaphragm or eventration or partial absence of the diaphragm. oesophageal and gastric lung absence of the pericardium other congenital cardiac malformations (25% of cases) including asd, ventricular septal defect, coarctation of the aorta, tetralogy of fallot, pulmonary stenosis, absent inferior vena cava with azygos continuation to superior vena cava and persistent left superior vena cava.5 moreover, the apvd may also be partially obstructed, further contributing to pulmonary hypertension.6 the detection of any of these anomalies should lead to a search for other components. the clinical spectrum of scimitar syndrome ranges from severely ill infants to asymptomatic adults, and cases may present in one of three ways:78 in the neonatal period with respiratory and/or cardiac failure. this is most commonly caused by pulmonary hypertension due to cardiac and/or right lung anomalies. treatment is surgical and outcome is dependant on the nature and severity of the anomalies.9 heart failure may also be caused because of a large arterial supply from the abdominal aorta to a sequestered lobe. in these situations, cardiac catheterisation may be used to embolise the aberrant pulmonary blood supply. this not only relieves heart failure but also makes the surgical field more bloodless. at any stage in life due to recurrent chest infections, usually affecting the right lower lobe that often has an abnormal arterial blood supply and venous drainage. lobectomy or even right pneumonectomy may be required to deal with bronchiectasis and prevent further chest infections. affected individuals may also present with haemoptysis due to pulmonary hypertension.10 at any stage in life as an incidental finding e.g. due to the detection of a murmur or due to the evident cxr abnormalities.11 useful investigations include echocardiography, angiography and computerised chest tomography.12 magnetic resonance imaging also delineates abnormal vessels. apvd with significant left to right shunting is corrected surgically, in this case, by baffling anomalous pulmonary venous return to the left atrium through the inferior vena cava or by reimplantation of the scimitar vein to the left atrium. we present a case of scimitar syndrome that presented in the late teens with a murmur. the patient (female) was well and asymptomatic, with no cardiac or respiratory problems. echocardiography showed a dilated right heart and left to right shunting across a moderate asd. transoesophageal echocardiography confirmed the defect and showed left pulmonary veins draining to left atrium, but failed to demonstrate the right pulmonary veins. cardiac catheterisation easily demonstrated both left pulmonary veins but failed to show the right pulmonary veins (figures 1 and 2). left upper pulmonary vein to left atrium (hand injection) left lower pulmonary vein to left atrium (hand injection) right pulmonary artery angiogram showed classical scimitar syndrome, with right pulmonary veins joining to a descending (scimitar) vein that drained to the inferior vena cava (figures 35). right pulmonary artery angiogram - arterial and venous phases superimposed in retrospect, the scimitar vein was also visible on direct fluoroscopy (figure 6). fluoroscopy only - scimitar vein clearly seen right pulmonary artery angiogram saturations confirmed left to right shunting with step - up at the level of the inferior vena cava (figure 8). saturations showing step - up at the level of the inferior vena cava plain cxr (figure 9) and lateral cxr (figure 10) also amply demonstrated the scimitar vein along with no other pulmonary pathology. cxr - scimitar vein seen as a curvilinear density along the right heart border (arrow) lateral cxr - scimitar vein seen as a curvilinear density (arrows) chest computerised tomography was performed to exclude the possibility of pulmonary sequestration. sequestration was not present, but the descending vein could be clearly seen within the right lung (figure 11), and inferiorly coursing medially towards the inferior vena cava (figure 12). chest ct slice showing descending vein in right lung field (arrrow) chest ct slice showing descending vein in right lung field coursing towards the inferior vena cava (arrow) surgical repair was successfully and uneventfully carried out by baffling the orifice of the scimitar vein at its entry into the inferior vena cava to the asd. the patient was placed on bypass with bicaval drainage and aortic return and cooled to 18 degrees celcius. due to the close proximity of the hepatic vein confluence and the inferior vena cava, the latter could not be snared in order to isolate the venous return to the right atrium. right atriotomy showing atrial septal defect (asd) and margins of enlarged asd (dotted lines) right atriotomy with more inferior exposure showing scimitar vein opening freshly harvested autologous pericardial patch right atriotomy showing pericardial patch bafflling pulmonary venous return from scimitar vein opening (a) to atrial septal defect (b) therefore into the left atrium the patient had an uneventful postoperative course. the predischarge echocardiogram showed residual dilatation of the pulmonary veins and right ventricle and unobstructed baffle flow (figures 1720). ra = right atrium, rv = right ventricle, aov = aortic valve, la = left atrium apical four chamber view showing baffle on its way to asd. b = baffle, ra = right atrium, tv = tricuspid valve, rv = right ventricle, cs = coronary sinus parasternal short axis view showing baffle just below the level where it drains into the left atrium. b = baffle, ra = right atrium, rv = right ventricle, la = left atrium, lvot = left ventricular outflow tract parasternal short axis view showing baffle draining into the left atrium (arrow). b = baffle, ra = right atrium, rv = right ventricle, la = left atrium, lvot = left ventricular outflow tract | scimitar syndrome is a form of partial anomalous pulmonary venous drainage that is dramatically visible on plain chest radiography (cxr). in these individuals the entire venous drainage from the right lung enters a single anomalous large vein that descends to the inferior vena cava. this descending vein is visible on cxr as a curvilinear density along the right heart border and resembles the curved turkish sword that gives the condition its name. scimitar syndrome forms part of the large spectrum of associated conditions known as venolobar syndrome. these include right lung hypoplasia or sequestered segments of right lung, congenital heart disease and various others. we report the case of a young woman who presented incidentally, with a murmur, at 16 years of age. full investigation including angiography showed a large atrial septal defect with right heart dilation and scimitar syndrome. she underwent surgical correction with uneventful and complete correction by baffling of the scimitar vein from its entry into the inferior vena to the left atrium through the enlarged atrial septal defect. |
eukaryotic topoisomerase i is a monomeric enzyme that catalyzes the relaxation of supercoiled dna during important processes including dna replication, transcription, recombination and chromosome condensation (13). human topoisomerase i (htop1) is composed of 765 amino acids, and proteolytic experiments have shown that the enzyme is composed of four different domains : n - terminal domain (residues 1214), core domain (residues 215635), linker domain (residues 636712), and c - terminal domain (residues 713765) (4). the 3d structure of the enzyme without the n - terminal domain (topo70), complexed with a linear double - helix dna substrate, has permitted a further classification of the core domain in subdomain i (residues 215232 and 320433), ii (residues 233319) and iii (residues 434635), and has shown that topoisomerase i is a bi - lobed protein that clamps completely around duplex dna through protein one lobe is constituted by the c - terminal catalytic domain and core subdomain iii, the other one (called cap) by core subdomains i and ii. the protein forms a substantially closed clamp between loops, designated lip1 and lip2, belonging to core subdomains i and iii, respectively. the two lips interact directly through a non - covalent interaction between the carboxylic lateral group of glu497 and the side - chain amino group of lys369 (5). the catalytic cycle of the enzyme is composed of five steps : dna association, cleavage, strand rotation, ligation and dissociation (6). a complete understanding of the dna recognition event and the conformational changes that follow dna binding would require determination of the 3d structure of the enzyme in the free and dna - bound states. large domain movements are required for the protein to bind the supercoiled dna substrate, likely through the flexible hinge that has been hypothesized to be located between the cap and subdomain iii (helices 8 and 9) (5). indeed a molecular dynamics (md) simulation study suggested the occurrence of a movement of 1014 between the lips, during the dna rotation step (7). two articles have demonstrated that introduction of two cysteines on the two lips, by site - directed mutagenesis, traps htop1 in the closed conformation, through a disulfide bridge upon dna addition, confirming the existence of ample conformational changes (8,9). until now, the structure of the protein has been solved only in the closed form, when it is fully embracing the dna, and no information is available concerning the preferred conformation in the absence of the substrate. a paper describing the open state of the topoisomerase ib from deinococcus radiodurans has recently appeared (10). the bacterial enzyme is much smaller than the human one, being constituted by a c - terminal domain of about 250 residues, comparable to the core subdomain iii and c - terminal domain of the human enzyme, and by an n - terminal domain of about 90 residues that has no counterpart in the human enzyme. the two domains are splayed apart in an open conformation in which the surface of the catalytic domain containing the active site is exposed for dna binding. in the case of the human enzyme, the internal flexibility of the protein in the absence of dna, and the consequent conformational heterogeneity, makes its crystallization and analysis through x - ray diffraction very difficult. md simulation is a powerful technique that is widely used to probe the flexibility of proteins, dna, and protein dna complexes, and also has been used to examine the properties of dna - topoisomerase i complexes (1114). starting from the htop1 closed crystallographic structure, the flexibility of the different domains have been monitored, but it has not been possible to monitor the so - called open state conformation, because of the strict non - covalent interactions occurring between the protein domains and dna (11). md can also be applied to sample large conformational changes after introducing a perturbation or a force that imposes the system to sample events occurring on a timescale not accessible by the current computational resources. this approach has been used to study the conformational changes of several proteins (15,16), and to propose a model for the relaxation of positive and negative dna supercoils in the htop1dna complex (7). in this work, we have carried out a simulation of the human topoisomerase enzyme, starting from the closed configuration coordinates of the topo70-dna non - covalent complex after elimination of the 22-bp dna duplex oligonucleotide coordinates. a repulsion force between the two lips has been introduced for a short time to induce the destabilization of the local minimum, and then an unperturbed simulation was carried out for 10 ns. analysis indicates the occurrence of a large reorientation of the different domains that however, fully maintain their secondary and tertiary structures. beside such a large conformational change, which induces movements as large as 8090 between the domains, the active site residues remain preassembled, and the open structure of the protein displays a vast positive region with the active site located nearly at its center, perfectly suited to interact with the negatively charged dna substrate. the initial configuration of htop1 was modeled obtaining the starting position for residues 215633 and 641765 from the crystal structure 1a36 (6), and those for residues 203214 from the crystal structure 1ej9 (17). the seven residues constituting the loop region that connects the linker to the core domain (residues 634640, which are lacking in the 1a36 pdb structure because of thermal fluctuation) were added to the system by molecular modeling, as already described (14), using the sybyl program (st louis, mo). the same procedure has been followed to reconstruct the partially missing side chains of the residues not fully detected in the x - ray diffraction structure. the spatial environment of each new residue was checked for close contact or overlap with neighboring residues, and stereochemical regularization of the structures was obtained by the powell minimization method implemented in the sybyl program. the system was modeled with the amber95 all - atom force field (18), and placed in a rectangular box (111 152 133 the dimension of the box was sufficiently large to accommodate the protein also in the open state, without any interactions with its periodic images. the system was, in fact, simulated in periodic boundary conditions, using a cutoff radius of 9 for the non - bonded interactions, and updating the neighbor pair list every 10 steps. the shake algorithm (22) was used to constrain all bond lengths involving hydrogen atoms. optimization and relaxation of solvent and ions were initially performed, keeping the solute atoms constrained to their initial position with decreasing force constants of 500, 25, 15, and 5 kcal/(mol). the system has been simulated at a constant temperature of 300 k using berendsen 's method (23) and at a constant pressure of 1 bar with a 2-fs time step. simulation a refers to the protein in the closed state without the dna double strand and in the presence of 21 cl counterions to neutralize the system (a total of 219 655 atoms, with 9415 protein atoms and 70 073 water molecules) ; in simulation b, the ionic strength has been increased introducing 21 atoms of na and 21 more cl atoms (a total of 219 487 atoms, with 9415 protein atoms and 70 003 water molecules) ; in c, a positive charge value has been given to the lateral chain of the glu 497 residue to introduce a repulsion with the close lys369 in the opposite lip ; in d, the repulsion between the two lips has been increased, introducing a positive charge value also to the main - chain carbonyl group of glu497 and to the lateral chain carbonyl group of asn366 ; in the final part of the simulation (simulation e), the charge perturbation was eliminated and an unperturbed simulation has been carried out for 10 ns. table 1.conditions and lengths of the different simulations carried out for htop1simulationabcdecharge perturbationnoneglu497glu497 and asn 366nonetime (ps)350050020020010 000main - chain perturbation.side-chain perturbation. conditions and lengths of the different simulations carried out for htop1 main - chain perturbation. the atomic rmsf have been computed by using the following definition : where the averages have been computed over the equilibrated md trajectory of simulation a, i.e. from 500 to 3500 ps. the per - residue root mean square deviations (rmsds) between two aligned structures x and y have been computed by using the following definition : where the distances between all atoms in a certain residue are averaged. the average per - residue rmsd for simulation e has been calculated by least - square fitting a group of main chain atoms to their initial position, calculating the per - residue rmsd with the previous equation, and averaging the resulting value on the entire 10 ns simulation time. all the analyses have been carried out using the gromacs md package version 3.3.1 (24). figures 2, 4 and 5, and the movies of md dynamics, shown as supplementary data at nar online, were created using the vmd visualization package (25). in the first 3500 ps of simulation in the absence of dna, defined here as simulation a, the protein is maintaining a conformation very close to the crystallographic state, being the largest rmsd calculated on the main chain atoms of 3.5. this value is very close to that found for the protein simulated in complex with the dna substrate for an identical simulation time length (13). this result indicates that the closed configuration of the protein is very stable, independently of the presence of dna, and that the intra - domain and inter - domain protein interactions are sufficient to maintain the closed conformation. also, the overall flexibility of the protein with or without dna is quite similar, as can be observed in figure 1, where the per - residue rmsf in the absence of dna, evaluated for the last 3000 ps of simulation a, is compared to the rmsf evaluated on the same time window for the simulation previously carried out on the protein dna complex (13). the maxima and minima of the rmsf are identical in the two simulations, being very similar to the protein regions characterized by the largest flexibility. in fact, in the absence of dna, the linker and the loop connecting -strands 12 and 13 centered on gly520 are the regions characterized by the largest fluctuations, as already observed for the enzyme in the closed conformation in the presence of dna (12). differences are observed in the absolute values of the rmsf of definite regions, such as the nose - cone helices (helices 5 and 6 in core subdomains ii and i, respectively) and the small loop at the n - terminal of the linker domain (residues pro636-met645), which show larger rmsfs in the simulation without dna than those observed in the simulation of topo70 in complex with dna (figure 1). residues 634640 are known to be highly flexible, and are not detected in several topoisomerase dna complex crystals because of their thermal fluctuations, while they are seen in the structure of the topo70dna topotecan ternary complex (26). this region seems to display a scale of disorder, being more ordered in the ternary topo70dna topotecan complex, less ordered in the binary topo70dna complex and even more disordered in the absence of dna. on the contrary, the maximum fluctuations, located in the loop of the linker domain between helices 18 and 19, are less ample in the simulation without the dna. figure 1.average per - residue rmsf represented as a function of the residue number of htop1 in the absence of dna for the initial unperturbed simulation a (full line) in comparison with the ones previously calculated for the protein in complex with dna (dashed line). figure 2.four snapshots of the final unperturbed simulation e taken at different times, showing the opening of the protein. core subdomains i, ii and iii are rendered in yellow, blue and red, respectively. linker and c - terminal domains are rendered in green and cyan, respectively. the hinge residue glu445 is indicated by an arrow in panel d. average per - residue rmsf represented as a function of the residue number of htop1 in the absence of dna for the initial unperturbed simulation a (full line) in comparison with the ones previously calculated for the protein in complex with dna (dashed line). four snapshots of the final unperturbed simulation e taken at different times, showing the opening of the protein. core subdomains i, ii and iii are rendered in yellow, blue and red, respectively. linker and c - terminal domains are rendered in green and cyan, respectively. the hinge residue glu445 is indicated by an arrow in panel d. per - residue - rmsds (data not shown) give higher values in the simulation in the absence of dna, for the linker domain and the nose - cone helices, which move away from the space previously occupied by dna. both these regions have been shown to form several direct and water - mediated contacts with the dna phosphate groups in the strands downstream of the scissile base (5,11,13). it is worth noting, therefore, that the greater changes of dynamics and structure, in the absence of dna but in the closed conformation, are located in the two regions involved in the controlling of the rotation of the scissile strand during the relaxation step. the x - ray diffraction study describes a single salt bridge direct contact between lys369 and glu497 (5). in the simulation in the absence of dna, a new interaction is observed from 900 and 2500 ps between the lateral chain of lys369 and the carbonylic main - chain group of glu497, with a distance of 3. after this time, the distance between these two groups increases to 5 and the lateral chain of asn366 and the asp500 main chain start to interact, their distance being 3 (see figure 7, in supplementary data). it is interesting to note that this new inter - lip interaction is also observed in the last part (from 1 to 3.5 ns) of the simulation of the protein in the presence of dna (13). a perturbation of the inter - lip interactions found at the end of simulation a, occurring between charged or polar groups (glu497coo ; glu497co ; asn366od1asp500nh), has been attempted by increasing the ionic strength of the system. in details, 21 na and cl atoms have been added to the system, which has been simulated for 500 ps (simulation b). however, since the energetic stability of the inter - lip interactions is not sufficiently perturbed by the increase of ionic strength (see figure 7 in supplementary data), a not physical perturbation (i.e. change of electrostatic charges) has been introduced in the system, in order to create a destabilization within a simulation time compatible with the currently computational resources. it is interesting to note that the introduction of a positive charge replacing the negative one at the carboxylic group of glu497 is not enough to push the two lips far away from each other. in fact, the introduction of the positive charge on the coo group of glu497 leads to an increase of the glu497lys369 distance because of electrostatic repulsion (black line in figure 7 in supplementary data, simulation c). however, lys369 changes its molecular partner and forms a strong interaction with the glu497 main - chain carbonyl group, as evidenced by the decrease of the relative distance of the glu497 main - chain and lys369 lateral chain, respectively (red line in figure 7 in supplementary data, simulation c). the same occurs for the interaction between the lateral chain of asn366 and the main - chain of asp500 (green line in figure 7 in supplementary data). therefore, the destabilization at the level of the salt bridge between glu497 and lys369 lateral chains, due to the introduction of the positive charge on the glu497 lateral chain, is compensated for by the strengthening of other lip1lip2 interactions that allow the protein to maintain a stable closed configuration. only after the introduction of two additional positive charges, one on the carbonyl group of glu497 and the other one on the lateral chain of asn366, the two lips start to separate each other, as can be observed by monitoring the above - mentioned distances in simulation d (figure 7 in supplementary data). the system has been allowed to evolve with this additional perturbation for 200 ps. after this time, the positive charges have been eliminated, bringing the modified residues charges back to their initial values. the minimum distance between the lips, in the first configuration of the following unperturbed simulation (simulation e), is only 6.5, and the overall rmsd of this structure is 3.5 from the initial x - ray configuration (deviation of the main - chain atoms fitted on the main - chain atoms themselves). the initial configuration and a dynamic visualization of the md dynamics is available as supplementary data at nar online. the first important observation is that the protein domains change their relative position, reaching an open conformation able to bind and trap the dna substrate, but the secondary and tertiary structures of the single domains are very well conserved during the entire simulation period. this is an important observation, since it implies that the different domains act as independent and internal coherent units that maintain their overall structure, although strongly changing their relative orientation. core subdomains i and ii (colored yellow and blue, respectively) show coordinated motion, separated from that of core subdomain iii (colored red). this last subdomain, on the other hand, remains strongly interacting with the c - terminal domain (colored cyan) throughout the simulation. a quantitative analysis of the relative protein domain motion is represented in figure 3, where the average per - residue rmsd has been calculated, overlapping the protein on the core subdomains i and ii (black line), the core subdomain iii (red line), the linker (green line), or the c - terminal domain (blue line). the analysis indicates a very strong and coherent motion of the core subdomains i ii with respect to the other protein domains, with maximum rmsd (about 100) when the protein is fitted on the linker domain. the destabilization of the lip interactions permits a rotation of the core subdomains i ii nose - cone helices, initially orientated parallel to the linker domain (figure 2a), of nearly 90 after 2.5 ns (figure 2b) and of 180 after 5 ns (figure 2c). this motion permits the opening of the two halves that embrace the dna, formed by core subdomains i ii on one side and core subdomain iii and c - terminal domain on the other side. ii continues during the last 5 ns of the simulation time, although with a lower extent. the region that permits these relative domain motions is the n - terminal portion of helix 8 (residues 434444, figures 2d and 4), and can be identified also by the intersection of the black and red lines in figure 3. the c - terminal portion of helix 8, on the contrary, remains strongly interacting with helix 16 located in the c - terminal domain, during the whole opening dynamics (figure 4). these two regions remain in close contact because of the presence of a cluster of hydrophobic residues. on the opposite site of this portion of helix 16 is located arg590, a residue belonging to the catalytic pentad (27) that, due to these hydrophobic interactions, is maintained in an orientation similar to the one found in the crystallographic structures. actually, all five active site residues maintain, for the whole simulation time, an orientation very similar to the one found in the closed configuration detected through x - ray diffraction (5). the similar orientation can be appreciated in figure 5a where the active site region of the last frame of simulation e (gray) is overimposed on the x - ray structure (red). as a matter of fact, the largest deviation of the active site backbone atoms from the 1a36 crystallographic structure is only 1.7. the recent 3d structure of deinococcus radiodurans (dra) topoisomerase ib, the only one crystallized in an open conformation in the absence of dna, has indicated that the five active site residues are preassembled, being in a position nearly identical to those found in the dna - bound human htop1 (10). in line, the orientation of the active site residues of the human enzyme in our simulation is also very similar to that of the bacterial enzyme, as shown in figure 5b, where the last frame of simulation e (gray) is overimposed to the structure of dra (blue). figure 3.average per - residue rmsd of htop1 for the final unperturbed simulation e represented as a function of the residue number. the average per - residue rmsd have been calculated overlapping the protein on the initial position of the core subdomains i and ii (black line), the core subdomain iii (red line), the linker (green line) or the c - terminal domain (blue line). figure 4.snapshot of the final configuration of simulation e, showing only the core subdomain iii, the c - terminal, and the linker domain regions of the protein. helices 8, 9 and 16 and the catalytic arg590 are highlighted in the figure to evidentiate the hydrophobic interactions between the c - terminal portion of helix 8 and helix 16. figure 5.(a) superimposition of the active site region of the last frame of simulation e (gray color) with the human enzyme x - ray structure (red color). (b) superimposition of the active site region of the last frame of simulation e (gray color) with the deinococcus radiodurans enzyme x - ray structure (blue color). average per - residue rmsd of htop1 for the final unperturbed simulation e represented as a function of the residue number. the average per - residue rmsd have been calculated overlapping the protein on the initial position of the core subdomains i and ii (black line), the core subdomain iii (red line), the linker (green line) or the c - terminal domain (blue line). snapshot of the final configuration of simulation e, showing only the core subdomain iii, the c - terminal, and the linker domain regions of the protein. helices 8, 9 and 16 and the catalytic arg590 are highlighted in the figure to evidentiate the hydrophobic interactions between the c - terminal portion of helix 8 and helix 16. (a) superimposition of the active site region of the last frame of simulation e (gray color) with the human enzyme x - ray structure (red color). (b) superimposition of the active site region of the last frame of simulation e (gray color) with the deinococcus radiodurans enzyme x - ray structure (blue color). in our simulation, despite the huge inter - domain protein motion that spans up to 100, the active site residues fully maintain their relative orientation, indicating that, in the human enzyme also, the active site is preassembled and in the appropriate position to cleave the dna substrate, at variance with what has been proposed for the vaccinia virus enzyme, where only the dna binding would trigger the active site assembly (28). the presence of a preassembled active site suggests that, in the open conformation, the enzyme is ready to interact with dna, and the only conformational change that the protein must undergo is the one needed to embrace the substrate. in line, electrostatic calculation of the open protein conformation shows that the protein displays a vast positive region (blue in figure 6) with the active site resides located nearly at its center, perfectly suited to interact with the negatively charged supercoiled dna substrate. figure 6.a snapshot of the open conformation of the final unperturbed simulation e represented with the same domain colors as figure 2 (left panel), together with its electrostatic potential mapped onto its molecular surface (right panel). red color represents negative and blue positive potential (5 to + 5 kbt). electrostatic potentials were calculated using the program grasp (29). a snapshot of the open conformation of the final unperturbed simulation e represented with the same domain colors as figure 2 (left panel), together with its electrostatic potential mapped onto its molecular surface (right panel). red color represents negative and blue positive potential (5 to + 5 kbt). in the first 3500 ps of simulation in the absence of dna, defined here as simulation a, the protein is maintaining a conformation very close to the crystallographic state, being the largest rmsd calculated on the main chain atoms of 3.5. this value is very close to that found for the protein simulated in complex with the dna substrate for an identical simulation time length (13). this result indicates that the closed configuration of the protein is very stable, independently of the presence of dna, and that the intra - domain and inter - domain protein interactions are sufficient to maintain the closed conformation. also, the overall flexibility of the protein with or without dna is quite similar, as can be observed in figure 1, where the per - residue rmsf in the absence of dna, evaluated for the last 3000 ps of simulation a, is compared to the rmsf evaluated on the same time window for the simulation previously carried out on the protein dna complex (13). the maxima and minima of the rmsf are identical in the two simulations, being very similar to the protein regions characterized by the largest flexibility. in fact, in the absence of dna, the linker and the loop connecting -strands 12 and 13 centered on gly520 are the regions characterized by the largest fluctuations, as already observed for the enzyme in the closed conformation in the presence of dna (12). differences are observed in the absolute values of the rmsf of definite regions, such as the nose - cone helices (helices 5 and 6 in core subdomains ii and i, respectively) and the small loop at the n - terminal of the linker domain (residues pro636-met645), which show larger rmsfs in the simulation without dna than those observed in the simulation of topo70 in complex with dna (figure 1). residues 634640 are known to be highly flexible, and are not detected in several topoisomerase dna complex crystals because of their thermal fluctuations, while they are seen in the structure of the topo70dna topotecan ternary complex (26). this region seems to display a scale of disorder, being more ordered in the ternary topo70dna topotecan complex, less ordered in the binary topo70dna complex and even more disordered in the absence of dna. on the contrary, the maximum fluctuations, located in the loop of the linker domain between helices 18 and 19, are less ample in the simulation without the dna. figure 1.average per - residue rmsf represented as a function of the residue number of htop1 in the absence of dna for the initial unperturbed simulation a (full line) in comparison with the ones previously calculated for the protein in complex with dna (dashed line). figure 2.four snapshots of the final unperturbed simulation e taken at different times, showing the opening of the protein. core subdomains i, ii and iii are rendered in yellow, blue and red, respectively. linker and c - terminal domains are rendered in green and cyan, respectively. the hinge residue glu445 is indicated by an arrow in panel d. average per - residue rmsf represented as a function of the residue number of htop1 in the absence of dna for the initial unperturbed simulation a (full line) in comparison with the ones previously calculated for the protein in complex with dna (dashed line). four snapshots of the final unperturbed simulation e taken at different times, showing the opening of the protein. core subdomains i, ii and iii are rendered in yellow, blue and red, respectively. linker and c - terminal domains are rendered in green and cyan, respectively. the hinge residue glu445 is indicated by an arrow in panel d. per - residue - rmsds (data not shown) give higher values in the simulation in the absence of dna, for the linker domain and the nose - cone helices, which move away from the space previously occupied by dna. both these regions have been shown to form several direct and water - mediated contacts with the dna phosphate groups in the strands downstream of the scissile base (5,11,13). it is worth noting, therefore, that the greater changes of dynamics and structure, in the absence of dna but in the closed conformation, are located in the two regions involved in the controlling of the rotation of the scissile strand during the relaxation step. the x - ray diffraction study describes a single salt bridge direct contact between lys369 and glu497 (5). in the simulation in the absence of dna, a new interaction is observed from 900 and 2500 ps between the lateral chain of lys369 and the carbonylic main - chain group of glu497, with a distance of 3. after this time, the distance between these two groups increases to 5 and the lateral chain of asn366 and the asp500 main chain start to interact, their distance being 3 (see figure 7, in supplementary data). it is interesting to note that this new inter - lip interaction is also observed in the last part (from 1 to 3.5 ns) of the simulation of the protein in the presence of dna (13). a perturbation of the inter - lip interactions found at the end of simulation a, occurring between charged or polar groups (glu497coo ; glu497co ; asn366od1asp500nh), has been attempted by increasing the ionic strength of the system. in details, 21 na and cl atoms have been added to the system, which has been simulated for 500 ps (simulation b). however, since the energetic stability of the inter - lip interactions is not sufficiently perturbed by the increase of ionic strength (see figure 7 in supplementary data), a not physical perturbation (i.e. change of electrostatic charges) has been introduced in the system, in order to create a destabilization within a simulation time compatible with the currently computational resources. it is interesting to note that the introduction of a positive charge replacing the negative one at the carboxylic group of glu497 is not enough to push the two lips far away from each other. in fact, the introduction of the positive charge on the coo group of glu497 leads to an increase of the glu497lys369 distance because of electrostatic repulsion (black line in figure 7 in supplementary data, simulation c). however, lys369 changes its molecular partner and forms a strong interaction with the glu497 main - chain carbonyl group, as evidenced by the decrease of the relative distance of the glu497 main - chain and lys369 lateral chain, respectively (red line in figure 7 in supplementary data, simulation c). the same occurs for the interaction between the lateral chain of asn366 and the main - chain of asp500 (green line in figure 7 in supplementary data). therefore, the destabilization at the level of the salt bridge between glu497 and lys369 lateral chains, due to the introduction of the positive charge on the glu497 lateral chain, is compensated for by the strengthening of other lip1lip2 interactions that allow the protein to maintain a stable closed configuration. only after the introduction of two additional positive charges, one on the carbonyl group of glu497 and the other one on the lateral chain of asn366, the two lips start to separate each other, as can be observed by monitoring the above - mentioned distances in simulation d (figure 7 in supplementary data). the system has been allowed to evolve with this additional perturbation for 200 ps. after this time, the positive charges have been eliminated, bringing the modified residues charges back to their initial values. the minimum distance between the lips, in the first configuration of the following unperturbed simulation (simulation e), is only 6.5, and the overall rmsd of this structure is 3.5 from the initial x - ray configuration (deviation of the main - chain atoms fitted on the main - chain atoms themselves). the initial configuration and three significant snapshots taken from simulation e are shown in figure 2. a dynamic visualization of the md dynamics is available as supplementary data at nar online. the first important observation is that the protein domains change their relative position, reaching an open conformation able to bind and trap the dna substrate, but the secondary and tertiary structures of the single domains are very well conserved during the entire simulation period. this is an important observation, since it implies that the different domains act as independent and internal coherent units that maintain their overall structure, although strongly changing their relative orientation. core subdomains i and ii (colored yellow and blue, respectively) show coordinated motion, separated from that of core subdomain iii (colored red). this last subdomain, on the other hand, remains strongly interacting with the c - terminal domain (colored cyan) throughout the simulation. a quantitative analysis of the relative protein domain motion is represented in figure 3, where the average per - residue rmsd has been calculated, overlapping the protein on the core subdomains i and ii (black line), the core subdomain iii (red line), the linker (green line), or the c - terminal domain (blue line). the analysis indicates a very strong and coherent motion of the core subdomains i ii with respect to the other protein domains, with maximum rmsd (about 100) when the protein is fitted on the linker domain. the destabilization of the lip interactions permits a rotation of the core subdomains i ii nose - cone helices, initially orientated parallel to the linker domain (figure 2a), of nearly 90 after 2.5 ns (figure 2b) and of 180 after 5 ns (figure 2c). this motion permits the opening of the two halves that embrace the dna, formed by core subdomains i ii on one side and core subdomain iii and c - terminal domain on the other side. ii continues during the last 5 ns of the simulation time, although with a lower extent. the region that permits these relative domain motions is the n - terminal portion of helix 8 (residues 434444, figures 2d and 4), and can be identified also by the intersection of the black and red lines in figure 3. the c - terminal portion of helix 8, on the contrary, remains strongly interacting with helix 16 located in the c - terminal domain, during the whole opening dynamics (figure 4). these two regions remain in close contact because of the presence of a cluster of hydrophobic residues. on the opposite site of this portion of helix 16 is located arg590, a residue belonging to the catalytic pentad (27) that, due to these hydrophobic interactions, is maintained in an orientation similar to the one found in the crystallographic structures. actually, all five active site residues maintain, for the whole simulation time, an orientation very similar to the one found in the closed configuration detected through x - ray diffraction (5). the similar orientation can be appreciated in figure 5a where the active site region of the last frame of simulation e (gray) is overimposed on the x - ray structure (red). as a matter of fact, the largest deviation of the active site backbone atoms from the 1a36 crystallographic structure is only 1.7. the recent 3d structure of deinococcus radiodurans (dra) topoisomerase ib, the only one crystallized in an open conformation in the absence of dna, has indicated that the five active site residues are preassembled, being in a position nearly identical to those found in the dna - bound human htop1 (10). in line, the orientation of the active site residues of the human enzyme in our simulation is also very similar to that of the bacterial enzyme, as shown in figure 5b, where the last frame of simulation e (gray) is overimposed to the structure of dra (blue). figure 3.average per - residue rmsd of htop1 for the final unperturbed simulation e represented as a function of the residue number. the average per - residue rmsd have been calculated overlapping the protein on the initial position of the core subdomains i and ii (black line), the core subdomain iii (red line), the linker (green line) or the c - terminal domain (blue line). figure 4.snapshot of the final configuration of simulation e, showing only the core subdomain iii, the c - terminal, and the linker domain regions of the protein. helices 8, 9 and 16 and the catalytic arg590 are highlighted in the figure to evidentiate the hydrophobic interactions between the c - terminal portion of helix 8 and helix 16. figure 5.(a) superimposition of the active site region of the last frame of simulation e (gray color) with the human enzyme x - ray structure (red color). (b) superimposition of the active site region of the last frame of simulation e (gray color) with the deinococcus radiodurans enzyme x - ray structure (blue color). average per - residue rmsd of htop1 for the final unperturbed simulation e represented as a function of the residue number. the average per - residue rmsd have been calculated overlapping the protein on the initial position of the core subdomains i and ii (black line), the core subdomain iii (red line), the linker (green line) or the c - terminal domain (blue line). snapshot of the final configuration of simulation e, showing only the core subdomain iii, the c - terminal, and the linker domain regions of the protein. helices 8, 9 and 16 and the catalytic arg590 are highlighted in the figure to evidentiate the hydrophobic interactions between the c - terminal portion of helix 8 and helix 16. (a) superimposition of the active site region of the last frame of simulation e (gray color) with the human enzyme x - ray structure (red color). (b) superimposition of the active site region of the last frame of simulation e (gray color) with the deinococcus radiodurans enzyme x - ray structure (blue color). in our simulation, despite the huge inter - domain protein motion that spans up to 100, the active site residues fully maintain their relative orientation, indicating that, in the human enzyme also, the active site is preassembled and in the appropriate position to cleave the dna substrate, at variance with what has been proposed for the vaccinia virus enzyme, where only the dna binding would trigger the active site assembly (28). the presence of a preassembled active site suggests that, in the open conformation, the enzyme is ready to interact with dna, and the only conformational change that the protein must undergo is the one needed to embrace the substrate. in line, electrostatic calculation of the open protein conformation shows that the protein displays a vast positive region (blue in figure 6) with the active site resides located nearly at its center, perfectly suited to interact with the negatively charged supercoiled dna substrate. figure 6.a snapshot of the open conformation of the final unperturbed simulation e represented with the same domain colors as figure 2 (left panel), together with its electrostatic potential mapped onto its molecular surface (right panel). red color represents negative and blue positive potential (5 to + 5 kbt). a snapshot of the open conformation of the final unperturbed simulation e represented with the same domain colors as figure 2 (left panel), together with its electrostatic potential mapped onto its molecular surface (right panel). red color represents negative and blue positive potential (5 to + 5 kbt). | the open state of human topoisomerase i has been probed by molecular dynamics simulation, starting from the coordinates of the closed structure of the protein complexed with dna, after elimination of the 22-bp dna duplex oligonucleotide. a repulsion force between the two lips of the protein has been introduced for a short time to induce destabilization of the local minimum, after which an unperturbed simulation has been carried out for 10 ns. the simulation shows that the protein undergoes a large conformational change due to rearrangements in the orientation of the protein domains, which however move as a coherent unit, fully maintaining their secondary and tertiary structures. despite movements between the domains as large as 8090, the catalytic pentad remains preassembled, the largest deviation of the active site backbone atoms from the starting crystallographic structure being only 1.7. electrostatic calculation of the open protein structure shows that the protein displays a vast positive region with the active site residues located nearly at its center, in a conformation perfectly suited to interact with the negatively charged supercoiled dna substrate. |
a drug delivery system (dds) is defined as a formulation or a device that enables the introduction of a therapeutic substance in the body and improves its efficacy and safety by controlling the rate, time, and place of release of drugs in the body. the process includes the administration of the therapeutic product, the release of the active ingredient by the therapeutic product, and subsequent transport of the active ingredients across the biological membranes to the site of action. oral route for the administration of therapeutic agents is the oldest and most convenient route because of low cost of therapy and ease of administration which leads to higher level of patient compliance. it has a wide acceptance up to 5060% of total dosage forms and has been explored for systemic delivery of drugs via pharmaceutical products of different dosage form. approximately 50% of the drug products available in the market are administered orally [13 ]. conventional dosage forms are rapidly absorbed, with the ascending and descending portions of the concentrations versus time curve reflecting primarily the rate of absorption and elimination, respectively. because of the rapid rate of absorption from conventional drug delivery systems, drugs are usually administered more than once daily, with the frequency being dependent on biological half - life (t(1/2)) and duration of pharmacological effect [46 ]. an appropriately designed controlled - release drug delivery system (crdds) can improve the therapeutic efficacy and safety of a drug by precise temporal and spatial placement in the body, thereby reducing both the size and number of doses required. the main objective of controlled / sustained release drug delivery is to make sure of safety and to improve effectiveness of drugs as well as patient compliance. moreover, this controlled drug delivery system fails to achieve the stated advantages due to lack of releasing the initial bolus dose dumping and failure to achieve site specific drug delivery. formulation of layers from different types of polymers allows manipulation over more than one rate - controlling polymer, thus enabling different kinds of drug delivery of one or more active ingredients, that is, where the drug may be released with a bolus and then at a controlled rate or by targeted drug delivery in the gi tract using ph dependent polymers [7, 8 ]. diabetes mellitus is a chronic metabolic disorder characterized by a high blood glucose concentration, hyperglycemia (fasting plasma glucose > 7.0 mmol / l, or plasma glucose > 11.1 mmol / l two hours after a meal) caused by insulin deficiency, often combined with insulin resistance, where continuous therapy is required. apart from diet and exercise, which are fundamental in the treatment of diabetes mellitus (dm), single oral antidiabetic drug sometimes fails to achieve and maintain glycemic control. consequently, a combination therapy using agents with complementary mechanism of action has become a cornerstone of type 2 diabetes mellitus (t2 dm) management. metformin hcl, the only available biguanide, remains the first line drug therapy for obese patients with t2 dm. daily dose of metformin hcl is 0.53 gm. in monotherapy under certain conditions, daily dose of pioglitazone hcl is 1530 mg. pioglitazone sensitizes peripheral tissues to insulin and hence may cause hypoglycemia when insulin is used concomitantly, though hypoglycemia can also occur with monotherapy. solubility of pioglitazone decreases with increase in ph and metformin is freely soluble drug, pioglitazone is formulated as immediate release layer, and metformin is formulated as controlled release layer. the release retardant guar gum which was used in the present study is one of the most promising dietary fibers, which is a gel - forming galactomannan obtained by grinding the endosperm portion of cyamopsis tetragonolobus l., a leguminous plant. all natural fiber diet works with body to achieve a feeling of fullness and to reduce hunger. reddy 's laboratories, hyderabad and msn formulations, hyderabad, respectively, hpmc k4 m and crospovidone were procured from nihal traders, hyderabad, guar gum is the grounded endosperm of cyamopsis tetragonolobus, and all the remaining chemicals are of analytical grade. immediate release layer of pioglitazone hcl was prepared by direct compression method (figure 4). pioglitazone hcl, crospovidone, avicel ph-102, and lactose were accurately weighed and passed through sieve number 40. talc and magnesium stearate were added after passing through sieve number 40 and mixed properly. controlled release layer of metformin hcl containing hpmc k4 m was prepared by direct compression method. metformin hcl, hpmc k4 m, pvp k30, and dicalcium phosphate were passed through sieve number 40. talc and magnesium stearate were added after passing through sieve number 40 and mixed properly. metformin hcl granules containing guar gum were prepared by wet granulation technique by adding pvp k 30 dissolved in distilled water as a granulating fluid. required quantities of metformin hcl, guar gum, and dicalcium phosphate were passed through sieve number 40 and were mixed thoroughly and a sufficient volume of granulating fluid was added slowly. the obtained granules were dried at 50c in hot air oven till a constant weight was obtained (until dry). talc and magnesium stearate were added after passing through sieve number 40 and mixed properly. optimization process is done for immediate release layer in order to select the composition to form a multilayered tablet. the first layer consists of immediate release and the second layer consists of controlled release. the first layer was placed in the die cavity which consists of immediate release layer and punched with low compression force and then the second layer was placed in the die cavity which consists of controlled release layer and allowed for punching and finally barrier layer containing 35 mg of ethyl cellulose was placed in the die cavity and compressed with maximum compression force in order to obtain multilayered tablets by using 12 mm punches of saimach smd 16 station tablet compression machine with an average hardness of 68 kg / cm. the optimized immediate release tablet f3 is formulated into multilayer tablet containing metformin hcl as controlled release layer with two different polymers. the granules of all formulations were evaluated for powder flow properties independently for both immediate and controlled release layers. bulk and tapped densities were determined by tapped density apparatus from which compressibility index and hausner 's ratio values were calculated. drug content of granules was determined spectrophotometrically and the granules prepared from wet granulation method (f9f13) were subjected to moisture content and loss on drying by using hot air oven. randomly 10 tablets selected were used for determination of thickness that was expressed in mean sd and unit is mm. the individual weight of 20 tablets was taken after selecting them randomly for weight variation. then their average weight and their mean and standard deviationwere calculatedand compared with the standards. the weight of the tablet being made is measured to ensure that it contains predetermined amount of drug. hardness is termed as the tablet crushing strength and it is the force required to break a tablet diametrically. hardness of tablets was measured by selecting 5 tablets randomly and the hardness of each tablet was measured with monsanto hardness tester. the preweighed tablets were exposed to repeated shocks in roche friabilator in which they are initially weighed (wo) and kept in a tumbling and rotating apparatus drum and were subjected to fall from 6 inches height. after completion of 100 rotations, the tablets were reweighed (w) and the percent loss in weight or friability (f) was calculated by the formula given below : (1)%friability = initial weightfinal weightinitial weight100. the disintegration time was determined at 37 0.5c using disintegration test apparatus in 0.1 n hcl. twenty tablets were powdered, and dose equivalent weight of powder blend was accurately weighed and transferred into a 100 ml volumetric flask. initially the solution in the volumetric flask was filtered, diluted suitably, and analyzed spectrophotometrically. the drug content should be within the range between 90 and 110% of standard amount : (2)%drug content = drug contentlabel claim100. in vitro dissolution studies was conducted using usp dissolution apparatus - i at 37 0.5c temperature and at 50 rpm and the volume of dissolution media is 900 ml. 0.1 n hydrochloric acid was used as dissolution medium for the first two hours and 6.8 ph phosphate buffer for the remaining time. samples of 5 ml were withdrawn at predetermined time intervals and replaced with 5 ml of fresh dissolution medium. the collected samples were suitably diluted with dissolution fluid, wherever necessary, and were analyzed for the pioglitazone hcl for the first two hours at 269 nm and for metformin hcl for the remaining time at 233 nm by using a double beam uv spectrophotometer. the rate and mechanism of metformin release from the prepared multilayer tablets were analyzed by fitting the data in zero order as cumulative amount of drug release versus time : (3)c = k0 t, where c is the amount of drug released at time t and k0 is the release rate constant, first order as log cumulative percent of drug remaining versus time : (4)log c = log c0kt2.303, where c0 is the initial concentration of drug and k is the first - order rate constant, higuchi model as cumulative percentage of drug released versus square root of time : (5)q = kt1/2, where q is the amount of drug released at time t and k is the diffusion rate constant, korsmeyer - peppas model as log time versus log cumulative percent drug release : (6)log(mtm)=logk+n log t, where mt is the amount of drug released at time t, m is the amount of drug release after infinite time, k is release rate constant, and n is the diffusion exponent indicative of the mechanism of drug release. the extent of swelling was measured in terms of % weight gain by the tablet. initially one tablet from each formulation was kept in a petridish containing 6.8 ph phosphate buffers. the tablet was removed, lightly blotted with tissue paper to remove excess buffer, and reweighed for every 1 h ; the weights of the tablet were noted. percentage weight gain by the tablet was calculated by the following formula : (7)s. i.={(wtw0)w0}100, where s.i. is the swelling index, wt the weight of tablet at time t (h), and w0 the weight of tablet at zero time. the tablets were packed in suitable packaging and the well - sealed tablets were kept in the humidity chamber and their stability studies were conducted as per ich and who guidelines to assess the drug content samples which were collected at the end of the studies and were evaluated for drug release and its content. the conditions prescribed by ich guidelines for accelerated studies are 40c 2c/75% rh 5% rh for 3 months. in order to investigate the possible interactions among pioglitazone hcl, metformin hcl, and different polymers / diluents, ftir studies were carried out (figures 1, 2, and 3). as the identical peaks were observed in all the cases, all the formulations showed good flow properties with angle of repose values between 25 and 35 and hausner 's ratio ranged between 1.10 and 1.19, whereas carr 's index values ranged between 11 and 15, and all these values are shown in tables 4 and 5. the tablets were visually observed and free from defects such as lamination, chipping, and capping. hardness of the tablets was in the range of 2.97.3 kg / cm, thickness of the tablets is within 2.655.20 mm, and the friability ranges between 0.17 and 0.81 percent. the drug release from f1 composed of 10% crospovidone is 98.14 0.3%, f2 composed of 15% crospovidone is 99.60 0.1%, f3 composed of 20% crospovidone is 99.89 0.4%, and f4 composed of 25% crospovidone is 99.86 0.2%. the concentration of avicel ph 102 was kept at constant in all the formulations, that is, 15%. the drug release at specific time intervals was plotted in a graph and from the drug release profile formulation f3 composed of 20% crospovidone showed good drug release profile (99.97 0.5%) with least disintegration time. hence, f3 was selected as the optimized formulation for immediate release layer for incorporation with metformin controlled release layer. the drug release from crospovidone may be due to its capillary activity with the dissolution media. the optimized f3 was formulated into bilayer tablets containing metformin controlled release layer along with it (table 2). the drug release from f11 was better and a barrier layer was incorporated to it in f13 to release the drug in a controlled manner. the drug release of pioglitazone hcl from f13 was found to be 99.94 0.7% at 2 hrs of dissolution in 0.1 n hcl. the drug release of metformin hcl from f13 was found to be 98.81 0.5% at 10 hrs of dissolution in 6.8 ph phosphate buffer. the release of drug not only depends upon the nature of matrix but also depends upon the drug polymer ratio. due to the differences in the rheological properties of the polymers guar gum at low concentrations (f9) can retard the drug release very well compared to hpmc k4 m (f5) that can retard the drug release at a slight higher concentration compared to guar gum, whereas the drug retarding capacity is similar at higher concentrations for hpmc (f7 and f8) and guar gum (f11 and f12). as hpmc k4 m is a semisynthetic polymer, by direct compression, high binder concentrations are required to maintain the tablet integrity due to the poor compressibility problems of metformin hcl compared to less concentrations of binder in wet granulation formulated with guar gum that acts as a good binding agent and maintains integrity of tablets up to 8 hrs of dissolution in 6.8 ph phosphate buffer. the filler dicalcium phosphate employed in the formulations (f5f13) is insoluble and nonswelling and also slows the drug release from the matrix [26, 27 ]. hpmc forms low density hydrocolloid system and a hydrogel layer would be formed which acts as a gel boundary for the delivery system that retards the drug release. the tablets swelled radially and axially during the in vitro swelling studies of hpmc and the dissolution of drug can be diffusion controlled by the formation of gel that forms a viscous layer acting as a protective barrier to both the influx of water and the efflux of the drug in solution from the polymer matrix as water uptake increases with increase in concentration of polymer, whereas the penetration of water into tablets formulated with guar gum (f9f13) was rather slow. the guar gum formulations showed an initial burst of drug release from the matrix - embedded system, diffusion coupled with erosion might be the mechanism of the drug release at low concentrations, and the slow drug release was observed with increase in the concentration of the polymer ; this might be due to the formation of thick gel layer with increasing viscosity around the tablets and this gel further inhibits the entry of release medium into the matrix and control release of the drug. the reason behind this is due to the slow erosion of the gelled layer, backbone mechanism for the drug release from the tablets containing higher amounts of guar gum [28, 29 ]. the data suggested that the release kinetics of the drug follow zero order drug release, as the values of regression coefficient obtained for zero order drug release profiles (r = 0.8524 to 0.9932) are higher as compared to first - order drug release profiles (r = 0.00980.4866). the in vitro release profiles of drug from all these formulations could be best expressed by higuchi 's equation as the plots showed the highest linearity (r = 0.9422 to 0.9966). this indicates that the drug release from the tablets was diffusion method. to confirm the exact mechanism of drug release further the data was fitted in korsmeyer - peppas equation and the results showed that the drug release follows diffusion mediated fickian model from the n value (n < 0.45) (table 8). the swelling index was found to be increased with the polymer concentration. the swelling index was found to be increased with polymer concentration. there was a good correlation between the swelling index and in vitro drug release (figure 7). the tablets were evaluated at initial, 1st month, 2nd month, and 3rd month for in vitro drug release. the in vitro release profile was the same as that of the initial one and the results were shown in figure 8, indicating that the formulation was quite suitable. the in vitro drug release profile of optimized formulation (f13) was compared with that of the marketed formulation (pio - fix mf 15). the % drug release of pioglitazone from marketed formulation was found to be 56.57 0.3% at 5 min and 99.97 0.5% at 2 hrs, whereas the drug release from the optimized formulation was found to be 60.57 0.8% at 5 min and 99.94 0.7% at 2 hrs, indicating that the drug release from the optimized formulation of immediate release layer of pioglitazone hcl showed better drug release compared to the innovator. the % drug release of metformin hcl from marketed formulation was found to be 97.5 0.8% and from f13 was 98.81 0.5% at 10 hrs. hence, the drug release from optimized formulation was better when compared to marketed formulation. pioglitazone hcl and metformin hcl along with the polymers and other excipients which are selected were compatible and were confirmed from the ftir studies. a barrier layer is incorporated in f13 to release the drug in a controlled manner, such that the drug is to be eliminated before the administration of next dosage form, and in night times the metabolic processes are slow and the plasma drug concentration levels decline slowly due to the reduced elimination rate. the drug release rate from immediate layer was found to be dependent on the concentration of superdisintegrants employed whereas the controlled release layer was found to be dependent on the type of and polymer concentration employed in the tablet formulation. this may be due to the variations in the viscosity, swelling index, and type of release mechanism involved. the formulation f13 formulated with crospovidone-20%, avicel ph 102 - 15% in immediate release layer, guar gum-22.5%, and pvp k30 - 2% in controlled release layer along with the barrier layer showed the required drug release profile. | in the treatment of type 2 diabetes mellitus a continuous therapy is required which is a more complex one. as in these patients there may be a defect in both insulin secretion and insulin action exists. hence, the treatment depends on the pathophysiology and the disease state. in the present study, multilayered tablets of pioglitazone hydrochloride 15 mg and metformin hydrochloride 500 mg were prepared in an attempt for combination therapy for the treatment of type 2 diabetes mellitus. pioglitazone hcl was formulated as immediate release layer to show immediate action by direct compression method using combination of superdisintegrants, namely, crospovidone and avicel ph 102. crospovidone at 20% concentration showed good drug release profile at 2 hrs. metformin hcl was formulated as controlled release layer to prolong the drug action by incorporating hydrophilic polymers such as hpmc k4 m by direct compression method and guar gum by wet granulation method in order to sustain the drug release from the tablets and maintain its integrity so as to provide a suitable formulation. the multilayered tablets were prepared after carrying out the optimization of immediate release layer and were evaluated for various precompression and postcompression parameters. formulation f13 showed 99.97% of pioglitazone release at 2 hrs in 0.1 n hcl and metformin showed 98.81% drug release at 10 hrs of dissolution in 6.8 ph phosphate buffer. the developed formulation is equivalent to innovator product in view of in vitro drug release profile. the results of all these evaluation tests are within the standards. the procedure followed for the formulation of these tablets was found to be reproducible and all the formulations were stable after accelerated stability studies. hence, multilayered tablets of pioglitazone hcl and metformin hcl can be a better alternative way to conventional dosage forms. |
for our analysis of rab gtpases, we exclusively used a gal4 targeting cassette (figs. 1, 2a). here we provide four additional cassettes : (1) a simple 3xp3 rfp/ kan cassette for making molecularly defined deletions and (2) hemagglutinin (ha), (3) flag and (4) green fluorescent protein (gfp) cassettes for tagging gene products (fig these cassettes, along with the gal4 cassette, all have the 3xp3 rfp / kan sequences flanked by loxp sites, which allows for positive selection in both bacteria and in flies and has proved to be invaluable for a number of different reasons (see below). moreover, this cassette can be removed in vivo using available cre transgenic lines (bloomington stock center : bl#1501). the activity of cre leaves behind a 34 bp scar that should be taken into account if one attempts to tag gene products at their n - termini. the gfp, ha and flag tagging cassettes share common 5 and 3 sequences, allowing one set of locus specific primers to be used across the entire cassette collection (fig. (b) schematic showing the specific sequence flanking each of the specific tagging cassettes. details on recombineering methods have been presented elsewhere. briefly, our protocol follows four basic steps for vector building (fig. 1) : (1) pcr amplify and pcr - soe 500 bp left arm (la) and right arm (ra) homology arms that flank both ends of the genomic region of interest together and clone them into the frt / i - sce i modified p[acman]-ko 1.0 vector. (2) use gap repair within recombination competent bacteria (dy380) to clone the entire region of interest into p[acman]-ko 1.0. (3) amplify the targeting cassette using oligos containing 100 bp of homology to either side of the gene being targeted. (4) transform dy380 cells carrying the p[acman]-ko - region of interest - plasmid with the pcr amplified targeting cassette. we simplified the first recombineering step by incorporating gateway cloning sequence into our modified p[acman]-ko vector. when designing primers, 25 bp of attb sequence is included on the 5 end of the forward la primer and the reverse ra primer. including these attb sequences on either end of the pcr - soe product allows one to clone this dna into p[acman]-ko using a bp reaction (invitrogen). incomplete digestion of the p[acman]-ko- pcr - soe product plasmid used for gap repair in the first recombineering reaction (fig. 1 ; therefore we use high - fidelity enzymes and allow the digestion of the p[acman]-ko- pcr - soe product to continue for at least three hours. while the number is variable from construct to construct, we typically obtain < 50 amp / tet resistant dy380 colonies after the first recombineering reaction. a large number of amp / tet resistant colonies after the gap repair reaction usually indicates the presence of the original unrecombineered p[acman]-ko pcr - soe product plasmid. when creating a targeting cassette specific for a particular gene, it is imperative to amplify from a completely linearized and purified cassette template. attempting to pcr directly off the plasmid carrying a particular cassette kanamycin resistance conveyed by a contaminating plasmid results in a high number of false positive dy380 colonies after the second recombineering reaction (fig. 1 ; step 4). because of this observation, we often run dna from colonies obtained after step 4 on an agarose gel to check for the presence of a low molecular weight high copy plasmid before transforming the final vector into epi300 cells. after sequence verification, correctly recombineered plasmids are transformed into epi300 cells, amplified using copy control solution (epicenter) and maxi- prepped for injection as outlined in chan. (2011). importantly, we found that freshly prepared dna works best for transformation and therefore always performed the maxi - prep not more than one day before injection. moreover, the large molecular weight dna should be stored at 4c and never frozen. despite these steps we still find that transformation efficiencies tend to be low for p[acman ] vectors over 50 kb (table 1). because of this low efficiency, we maximize the number of progeny obtained of every g0 fly. single g0 males were crossed to 35 virgin females and single g0 females were crossed to 3 males and all these vials are tossed up to three times. the dna corresponding to rab5, rab40, rabx1, rab2-atg and rab18 gal4 vectors was not diluted prior to transformation into epi300 cells, resulting in drosophila transformants that did not carry the gal4 targeting cassette. diluting the dna of remaining rabs prior to introducing the plasmid into epi300 cells alleviated this problem so that all the subsequent drosophila transformants carried the gal4 cassette. mobilization of the targeting cassette from the predefined insertion site in vivo is performed using existing transgenic sources of flpase and i - sce i (bloomington stock # s : 6934 and 6935). this mobilization results in the targeting of the knock - in / out cassette to the endogenous locus. p[acman]-ko transformants are identified based on the presence of the white+ gene contained within the backbone of the p[acman]-ko 1.0 vector. through the course of our experiments we sometimes isolated transformed flies that expressed the white+ marker but did not carry the 3xp3-rfp gene contained within the gal4 targeting cassette (table 1). furthermore, we isolated white+, rfp+ and white+, rfp- flies from the same g0 parent. these observations suggested that the dna used for transformation contained a mixture of p[acman]-ko plasmids : some that contained the gal4 3xp3-rfp cassette and others that did not. we considered the possibility that the p[acman]-ko vector was kept at a low copy number in dy380 cells but not at single copy. therefore it remained possible that the gal4 cassette inserted in some fraction of the p[acman ] plasmids within a particular cell but not all of them. if dna isolated after the final recombineering reaction is indeed a mixture, simply diluting the dna before transforming epi300 cells should greatly reduce the probability that any resulting bacterial colony would contain both types of plasmids. subsequent experiments showed that diluting dna to the point where one obtains 150 epi300 amp / kan resistant colonies on a 35 mm petri dish after electroporation virtually eliminates false positive (white+, rfp-) drosophila transformants. therefore inclusion of the positive rfp+ marker within the targeting and tagging cassettes provides insurance that drosophila transformants carry correctly recombineered vectors. the key step in a homologous recombination experiment in drosophila (and the main difference to mouse) is the mobilization of the targeting cassette from a defined site where it is already integrated in the genome. hence, instead of injection into embryonic stem (es) cells, the targeting cassette is mobilized by enzymatic excision in germ cells. consequently, selection screening for a correct targeting event, i.e., insertion of the targeting cassette into the correct endogenous locus, has to occur in flies instead of cells. this process requires three steps : first, the donor dna (targeting cassette) and transgenes encoding the enzymes needed to mobilize the cassette (hs - flp and hs - i - sce i) are crossed into the same genetic background. second, preliminary candidates are screened for mobilization of the targeting cassette away from its original site. this step is greatly facilitated if both the targeting cassette and the site from which it is mobilized are independently marked. indeed, this feature constitutes a major difference between our technique and other recent efforts, as outlined in detail below. the third step comprises mapping new insertions of the targeting cassette to the correct chromosome, followed by molecular and/or genetic verification. it is important to note that most insertions are not the desired clean homologous recombination event. several modifications have been introduced to optimize this process, which we would like to compare with our recently developed method. major improvements to ends - out homologous recombination have recently been introduced by huang. first, a dominant and heat shock dependent cell lethal construct (hs - hid) present on both the y and balancer chromosomes kill off all undesired progeny. second, a dominant cell lethal construct (uas - reaper) is fused behind the 3 homology arm of the targeting cassette. in a correct targeting event, this dna fragment is deleted ; however, in a faulty targeting event its presence leads to the death of the undesired offspring when driven by a neuronal or ubiquitous gal4 driver. the latter improvement (uas - reaper) has so far not been tested in conjunction with our technique, and a combined method might substantially improve targeting efficiency. a further major difference between our approach and the method by huang. use white+ as a positive selection marker of the targeting cassette but do not mark the site from which it is mobilized, leading to a difficulty in distinguishing failed mobilization or mobilization and re - insertion based on white+ alone. in theory, offspring in which the targeting cassette fails to mobilize should be killed by the uas - reaper dominant negative selection. hence, all viable and white+ progeny should represent cases where the targeting cassette was mobilized and re - integrated without the uas - reaper sequence. since random re - insertion is predicted to result in a higher rate of insertion on the correct chromosome, the majority of these false positive in contrast, our technique employs positive labeling of the targeting cassette with 3xp3-rfp and white+, yellow+ as independent markers for the site from which the targeting cassette is mobilized. selection for the targeting cassette (rfp+) and against the site from which it is mobilized (white- and yellow-) significantly reduces the number of preliminary candidates. report between 116 and 1700 preliminary candidates of which between 1 and 22 could be verified as correct targeting events. in contrast, our method leads to 20100 preliminary candidates of which 8090% are on the correct targeted chromosome. the accuracy of these targeting events depends on the homology region and was reported for one locus in chan. the recombineering methods outlined here and in our recent study provide a means to manipulate the drosophila genome with base - pair precision. recombineering represents a powerful and scalable method for determining gene function and for exploring how locus dependent and independent parameters contribute to the efficiency of in vivo homologous recombination in drosophila. | evaluating how an individual gene contributes to a particular biological process benefits greatly from a comprehensive understanding of all members of its gene family. such knowledge is ideally obtained using multicellular model organisms, which provide rapid and decisive platforms for determining gene function. we recently established a novel transgenesis platform in drosophila to systematically knock out all members of the rab small gtpase family of membrane regulators. this platform combines bac transgenesis / recombineering with ends - out homologous recombinations and gatewaytm technologies and provides a new rapid and scalable method that eases the manipulation of endogenous loci. this method not only allows for the generation of molecularly defined lesions, but also the precise replacement or tagging of genes in their endogenous loci. using this method, we found that up to half of all rab gtpases exhibit enriched expression at synapses in the nervous system. here we provide critical details about the underlying recombineering and transgenesis method, new cassettes for tagging endogenous loci and information on important parameters that will allow drosophila researchers to target members of other gene families. |
microbiological diagnosis is the main stay for the effective treatment of pulmonary tuberculosis (pt). for obtaining the correct sputum sample, patient education is imperative. however, even if the correct sample is expectorated, the bacillary population has to be at least 10000 per milliliter, to get the smear positive for acid fast bacilli (afb). moreover, it depends on the previous treatment, default behavior, and effective cough. as such, it opens a pandora 's box in the diagnosis, as there are no authentic radiological diagnosis criteria for pt. the computed tomography (ct) of the chest only increases the guessing probability for the clinician, but does not lead to a photo - finish diagnosis. however, a fiberscope can reach the pathological site of the bronchopulmonary segments and the lavage, brush smear, and post scopy sputum can reflect the correct collection of the specimen. hence fiberoptic bronchoscopy (fob) was done in cases having undiagnosed radiological panorama. all were sputum smear - negative for afb twice and exfoliative cytology was suggestive of inflammation. the highlight of this study was the usage of the trans oro pharyngeal spacer (tops), which was a multipurpose one. this avoided contamination with the nasal and oral flora, and if necessary, respiratory toilet or emergency intubation could be done through this. the objective of the study was to confirm the presence of afb in the pulmonary lesions in smear - negative pulmonary tuberculosis cases for earlier initiation of the treatment. this study has been conducted by the department of microbiology and department of respiratory medicine, ganni subba lakshmi (gsl) medical college, rajahmundry, from february 2007 to may 2010. our teaching hospital attached to the medical college has thousand beds and is a referral center for tb treatment in this area. in this study we considered patients with suspected pt, that is, cough of more than two weeks. patients who were not cooperative and children below 14 years were excluded from the study (getting sputum sample was difficult). first, smears were made with the sputum samples to detect afb as per the revised national tuberculosis control program (rntcp) guidelines. all smears positive for afb cases were referred to the clinician for anti - tuberculosis treatment (att) and smears negative for afb cases were preceded by fob, and were then included in the study. written informed consent was taken from all the individuals participating in the study, as also an endorsement from the ethical committee of the institute. algorithm of the study the fob was done by fujinon - bro yl2, japan, transorally, tops was used to avoid contamination. injection atropine 0.01mg / kg i m was given half an hour before the procedure. bronchodilator, salbutamol 1 ml + 1 ml normal saline was given via a nebulizer to all these subjects as they had bronchospasm earlier. the subjects were directed to rinse the oral cavity with 2 ml of lidocaine jelly before the procedure and lidocaine 2% was liberally used through the channel of fob, not exceeding 200 mg in total. bronchial lavage from the affected segment, brush smear, and post scopy sputum were also collected for smear examination to detect afb by ziehl neelsen (zn) staining. the main features of the study design were : the microbiologist picked up the correct portion of the bronchial lavage in the endoscopy room and the samples were examined immediately after zn staining.the total xylocaine used, on an average, was 3 ml from the vocal cords down to the bronchial tree.fob was done through tops, patented by the senior author and manufactured by the pran clinic and co., rajahmundry, 2004 model. this was a mechanical devise to deliver the aerosol medicine for the symptomatic treatment of bronchial asthma. it was 10 cm in length, with a 10 mm internal diameter [figure 2 ]. there was a universal adapter in front, which had a straight part and an arched section that bent twice with angles of 110 and 150. as per the mallampati classification, the oropharynx could be classified into classes i, ii, iii, and iv, depending upon the patency of the oropharyngeal lumen. the tops was much more useful [figure 3 ], as it lifted the uvula [figure 4 ], and fob or blind intubation could be done easily through tops. it also snugly fitted into the pharynx, the distal end being just above the epiglottis. the advantage of using this was avoidance of contamination with the oropharyngeal / nasal flora and easy passage of the scope. if there was bleeding, effective toileting could be done and intubation also could be achieved. the microbiologist picked up the correct portion of the bronchial lavage in the endoscopy room and the samples were examined immediately after zn staining. the total xylocaine used, on an average, was 3 ml from the vocal cords down to the bronchial tree. fob was done through tops, patented by the senior author and manufactured by the pran clinic and co., rajahmundry, 2004 model. this was a mechanical devise to deliver the aerosol medicine for the symptomatic treatment of bronchial asthma. it was 10 cm in length, with a 10 mm internal diameter [figure 2 ]. there was a universal adapter in front, which had a straight part and an arched section that bent twice with angles of 110 and 150. as per the mallampati classification, the oropharynx could be classified into classes i, ii, iii, and iv, depending upon the patency of the oropharyngeal lumen. the tops was much more useful [figure 3 ], as it lifted the uvula [figure 4 ], and fob or blind intubation could be done easily through tops. it also snugly fitted into the pharynx, the distal end being just above the epiglottis. the advantage of using this was avoidance of contamination with the oropharyngeal / nasal flora and easy passage of the scope. if there was bleeding, effective toileting could be done and intubation also could be achieved. tops in class iii oropharynx the highlight of the study is the use of the trans oro pharyngeal spacer (tops), which is an innovative product, by the senior author. it helps in easy passage of the tube, and bronchoscope bite is avoided. adding to this, there have been no complaints of discomfort from the patients while passing the scope. as tops out of 533 individuals in our study, there were 322 males and 211 females (mean age was 39.4 years). the radiological profile [table 1 ] revealed that 291 (54.5%) patients had unilateral minimal radiological extent and the remaining 242 (45.5%) had bilateral moderately advanced disease. in the bilateral disease, the the samples of lavage, brush smear, plus post scopy sputum were positive for afb, in 241 cases. only post scopy collection of sputum within 24 hours was positive for afb in 100 cases [table 2 ]. thus in 341(64%) subjects, the bacteriological diagnosis was clinched. in the afb - positive patients the quantitative analysis showed scanty (1 9 afb in 100 fields) results in 260 (76%) and 1 + (10 99 afb in 100 fields) bacillary load in the remaining 81 (24%) patients. this spectrum of cases, after establishing the cause and effect, were put on effective att. an editorial quoted that 40% of the cases with initial sputum afb - negative status continued to be sputum negative, while 60% of them had progressive disease later. previous reports in non - immunocompromised patients with sputum smear - negative tb indicated that bronchoscope specimens and post bronchoscope sputum are helpful in establishing the diagnosis. as per adelaithi m., diagnostic fob is often performed before the final results of the sputum analysis are available, to exclude other common infections, such as pneumocystis carinii pneumonia, to confirm pt. hence, the present study adumbrated a similar picture helping the clinician to treat the cases immediately not withstanding the radiological shadows. fiberoptic bronchoscopy through tops ensured ease of procedure and the secretions adhering inside the conduit were useful for microscopic examination. an immediate diagnosis of pt in smear - negative cases could be established with the bronchoscope, which was 45.3% in our study. similar results were reported by steve h. in a study. however, there was some difference in the afb yield of post scopy sputum. in our study 19% of the post scopy smears were positive for afb, whereas, it was 32% in the study by steve h. the usage of brush for smear is mandatory when doing bronchoscopy. we have improved over this recommendation by examining the bronchial lavage, brush smear, and post scopy sputum with a stand - by microbiologist. the specimens were examined and collated by both the pulmonologist and microbiologist, avoiding any false negative reports. one feature of this study was that the residual 191 (36%) cases (negative for afb by all means) were not administered treatment for pt and saved the grace. it was quite possible that they had treatment elsewhere inadvertently or non - tubercular granulomatous lung disease had to be entertained. the specimens collected by bronchoalveolar lavage, brush smear, and post scopy sputum, should be evaluated together for afb positivity in a smear negative for afb cases. they have to be examined immediately avoiding transit delay, contamination, and false reading. collection of post scopy sputum in the next 24 hours and examining post scopy sputum for afb is also an important aspect of this study. as per the revised national tuberculosis control program (rntcp) algorithm, in a symptomatic patient, if sputum smear is negative for acid fast bacilli (afb), we have to wait for 10 14 days, with broad spectrum antibiotics. after this time period also, if the sputum sample is negative for afb and if the chest x - ray is suggestive of tb, then the patient will be given anti - tb treatment by categorizing him / her as sputum negative pulmonary tb. in this manner, the two - week time period is wasted and without afb patient may have to start att. | introduction : the tuberculosis control program is based on a felt need oriented basis. the diagnosis is mainly microbiological. however, sputum smear - negative acid fast bacilli (afb) cases with suspected radiological findings can be problematic in diagnosis.objectives:to confirm the diagnosis of tuberculosis early, in smear - negative afb cases by using a fiberoptic bronchoscope.materials and methods : we embarked on fiberoptic bronchoscopy (fob) and spot scopy smear microscopy (ssm) for 533 suspected pulmonary tuberculosis (pt) cases (sputum smear negative and radiologically suggestive) from february 2007 to may 2010. fob was performed using a special device, a trans oro pharyngeal spacer (tops), as a conduit.results:the yield for positivity for afb was 341 (64%) out of 533 cases.conclusion and recommendation : the specimens collected by using the fiberoptic bronchoscope confirmed the disease in the smear - negative cases. hence, fob was recommended in smear - negative cases, to avoid delay in the treatment of tuberculosis. |
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