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lipid nanoparticles (lnp) are the leading delivery systems for enabling the therapeutic potential of sirna for systemic applications. lnp sirna systems containing optimized ionizable cationic lipids can exhibit remarkable in vivo potencies at doses as low as 0.02 mg sirna / kg body weight for silencing liver (hepatocyte) target genes in rodents following intravenous (i.v.) injection. these systems are relatively nontoxic, leading to therapeutic indices in mice approaching 1000, indicating potential clinical utility. despite this progress, the structure of these lnp sirna systems is unclear. some models of lnp sirna suggest a bilayer vesicle structure of the lnp with sirna on the inside in an aqueous interior, however other observations suggest that such models may be incorrect. for example, recent cryo - transmission electron microscopy (cryo - tem) studies of lnp sirna systems formed by mixing an ethanol stream containing lipid with an aqueous stream containing sirna using a t - tube mixing system results in lnp that have electron - dense cores rather than the less dense aqueous cores observed for vesicular systems. in addition, formulation of lnp sirna systems using the t - tube mixer can result in sirna encapsulation efficiencies above 70%, an observation that is difficult to reconcile with bilayer vesicular structure. this is because encapsulation depends on the presence of cationic lipid and it would therefore be expected that the maximum encapsulation efficiency should be approximately 50% for bilayer systems, assuming that the cationic lipid is equally distributed on both sides of the bilayer. in this work, we characterize the structure of lnp sirna systems produced using a rapid microfluidic mixing technology by employing a variety of biophysical assays as well as in silico simulations. cryo - tem studies show that these lnp sirna systems exhibit an electron - dense core (in contrast to bilayer vesicle systems), and that limit size systems can be generated at high peg - lipid contents that are consistent with the ability of sirna to stimulate formation of inverted micelles in association with cationic lipid. fluorescent energy resonance transfer (fret), p nmr, and rnase digestion studies show that encapsulated sirna is associated with internalized cationic lipid, is effectively immobilized on the nmr time scale and is fully protected from external rnase. density gradient studies show that the density of lnp sirna systems can vary from being significantly less dense than bilayer vesicle systems to exhibiting increased densities at higher sirna contents. taken together, these experimental results suggest that sirna resides in inverted micelles within the overall lnp sirna structure. these results are consistent with molecular modeling studies that indicate these lnp sirna systems have a nanostructured core consisting of a periodic arrangement of aqueous compartments, some of which contain sirna duplexes. the lipids 1-palmitoyl-2-oleoyl - sn - glycero-3-phosphocholine (popc), 1,2-distearoyl - sn - glycero-3-phosphocholine (dspc), 1,2-dioleoyl - sn - glycero-3-phosphoserine (dops), 1,2-dioleoyl - sn - glycero-3-phosphoethanolamine - n-(7-nitro-2 - 1,3-benzoxadiazol-4-yl) (nbd - pe), and 1,2-dioleoyl - sn - glycero-3-phosphoethanolamine - n-(lissamine rhodamine b sulfonyl) (rh - pe) were obtained from avanti polar lipids (alabaster, al). 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (hepes), cholesterol, sephadex g-50, tetrasodium edta, trizma base, and xylene cyanol were obtained from sigma - aldrich (st. louis, mo). triton x-100 and 2-(n - morpholino)ethanesulfonic acid (mes) were obtained from bdh (westchester, pa). ammonium acetate, boric acid, sodium acetate, and sodium chloride were obtained from fisher scientific (fair lawn, nj). bovine pancreatic rnase a was purchased from applied biosystems / ambion (austin, tx). the phosphodiester sirna used in this study was obtained from invitrogen (carlsbad, ca), and phosphorothioate sirna were 25-mer blunt - end duplexes (sense -5 -gccuuaacuuuggugaucaaggaua-3) and were obtained from dharmacon (lafayette, co). the ionizable cationic lipid 2,2-dilinoleyl-4-(2-dimethylaminoethyl)--dioxolane (dlinkc2-dma) and n-[(methoxy polyethylene glycol 2000 carbamyl]-1,2-dimyristyloxlpropyl-3-amine (peg - c - dma) were obtained from alcana technologies inc., cholesterol e total cholesterol assay kit was obtained from wako diagnostics (richmond, va). quant - it ribogreen rna assay kit was obtained from molecular probes (eugene, or). bilayer popc / cholesterol (1:1 ; mol / mol) vesicles were prepared by hydration of a dried lipid film with pbs, and the dispersion was then freeze thawed five times using liquid nitrogen. unilamellar vesicles were then prepared by extruding the frozen and thawed lipid suspension 10 times through two stacked 80 nm pore size polycarbonate filters. lnp were prepared by mixing appropriate volumes of lipid stock solutions in ethanol buffer with an aqueous phase containing sirna duplexes employing a microfluidic micromixer as described elsewhere. for the encapsulation of sirna, the desired amount of sirna was dissolved in 25 mm sodium acetate, ph 4.0. equal volumes of the lipid in ethanol and the sirna in buffer were combined in the microfluidic micromixer using a dual - syringe pump (model s200, kd scientific, holliston, ma) to drive the solutions through the micromixer at a combined flow rate of 2 ml / min (1 ml / minute for each syringe). the mixed material was diluted into an equal volume of 25 mm sodium acetate buffer, ph 4.0, upon leaving the micromixer outlet, thus reducing the ethanol content to 25%. the lipid mixture was then dialyzed for 4 h against 1000 volumes of 50 mm mes/50 mm sodium citrate buffer (ph 6.7) followed by an overnight dialysis against 1000 volumes of 1 phosphate buffered saline, ph 7.4 (gibco, carlsbad, ca) using spectro / por dialysis membranes (molecular weight cutoff 12 00014 000 da, spectrum laboratories, rancho dominguez, ca). the mean diameter of the lnp after dialysis was 41.3 14.9 nm as determined by dynamic light scattering (number mode ; nicomp 370 submicrometer particle sizer, santa barbara, ca). lipid concentrations were determined by measuring total cholesterol using the cholesterol e enzymatic assay from wako chemicals usa (richmond, va). encapsulation efficiency was calculated by determining unencapsulated sirna content by measuring the fluorescence upon the addition of ribogreen (molecular probes, eugene, or) to the sirna - lnp (fi) and comparing this value to the total sirna content that is obtained upon lysis of the lnp by 1% triton x-100 (ft) : % encapsulation = (ft fi)/ft 100. limit size particles were prepared by mixing 20 mm dlinkc2-dma / peg - c - dma (90/10, mol %) dissolved in ethanol with five volumes of 25 mm sodium acetate, ph 4.0 containing sirna. mixing of the two streams are accomplished using the above - mentioned herring micromixer device with a flow - rate of 0.5 ml / min for the lipid / ethanol stream and 2.5 ml / min for the aqueous stream. ethanol was removed by first diluting the lipid mixture with sodium acetate buffer, ph 4.0 and then removing the ethanol - containing buffer with an amicon ultra, 10 000 mwco, regenerated cellulose concentrator (millipore, billerica, ma). cyro - tem samples were prepared by applying 3 l lnp at 1020 mg / ml total lipid to a standard electron microscopy grid with a perforated carbon film. excess liquid was removed from the grid by blotting and then the grid was plunge - frozen in liquid ethane to rapidly freeze the sample using a vitrobot system (fei, hillsboro, oregon). images were taken under cryogenic conditions (88 k) at a magnification of 50 000 with an amt hr ccd side mount camera. samples were loaded with a gatan 70 degree cryo - transfer holder in an fei g20 lab6 200 kv tem (fei, hillsboro, or) under low dose conditions with an under - focus of 46 m to enhance image contrast. experiments were performed at the university of british columbia bioimaging centre (vancouver, bc). particle diameters were measured from the micrographs with the aid of imagej (national institute of health, bethesda, md). factor vii sirna was encapsulated with lnp formulations containing 40% dlinkc2-dma, 11.5% dspc, 47.5% cholesterol, and 1% peg - c - dma (mol %). an amount of 1.0 g of sirna (entrapped in lnp) was incubated with 0.05 g of bovine pancreatic rnase a (ambion, austin, tx) in 50 l of 20 mm hepes (ph 7.0) at 37 c for 1 h. at the end of the incubation, a 10 l aliquot of the reaction mix was added to 30 l of fa dye (deionized formamide, tbe, pbs, xylene cyanol, bromophenol blue, triton x-100) to halt the rnase reaction. gel electrophoresis was performed using 20% native polyacrylamide gel, and nucleic acids were visualized by staining with sybr - safe (invitrogen, carlsbad, ca). proton - decoupled p nmr spectra were obtained using a bruker avii 400 spectrometer operating at 162 mhz. free induction decays (fid) corresponding to 10 scans were obtained with a 15 s, 55 degree pulse with a 1 s interpulse delay and a spectral width of 64 khz. an exponential multiplication corresponding to 50 hz line broadening was applied to the fid prior to fourier transformation. experiments were performed at the centre for the drug research and development, vancouver, bc. fusion between lnp sirna nanoparticles and anionic dops bilayer vesicles was assayed by employing a fluorescence resonance energy transfer lipid mixing assay. labeled dops vesicles containing nbd - pe and rh - pe (1 mol % each) were prepared by hydration of the lipid in a thin film with 20 mm hepes buffer at ph 7.0 followed by 10 extrusions through two stacked 100 nm pore size polycarbonate filters (nuclepore) using the extruder (northern lipids, vancouver, bc). lnp composed of 40% dlinkc2-dma, 11.5% dspc, 47.5% cholesterol, and 1% peg - c - dma were prepared with an sirna - to - total lipid ratio (d / l ratio, wt / wt) of 0, 0.06, and 0.24. a d / l ratio of 0.24 represents a charge ratio of negative (sirna phosphate) to positive (cationic lipid amine) of one. unlabeled lnp were added to a stirred cuvette containing nbd - pe / rh - pe labeled dops vesicles at a 2:1 lipid molar ratio (200 m lnp/100 m dops vesicles) in 2 ml of 10 mm ammonium acetate, 10 mm mes, 10 mm hepes, and 130 mm nacl equilibrated to ph 5.5. fluorescence of nbd - pe was monitored using 465 nm excitation and 535 nm emission using a perkin - elmer ls-55 fluorimeter with a 1 1 cm cuvette under continuous low speed stirring. lipid mixing was monitored for approximately 10 min, after which 20 l of 10% triton x-100 was added to disrupt all lipid vesicles, representing infinite probe dilution (0.1% triton x-100 vol / vol final). lipid mixing was expressed as a percentage of infinite probe dilution determined using the equation : % lipid mixing = (f f0)/(fmax f0) 100, where f is the fluorescence intensity measured by the assay, f0 is the initial fluorescence intensity of nbd - pe / rh - pe / dops vesicles, and fmax is the maximum fluorescence intensity at infinite probe dilution after the addition of triton x-100 (0.1% v / v final). solutions of 1%, 2.5%, 5%, 10% and 15% sucrose (wt / vol) were prepared in distilled water and used to make a 10 ml step gradient. gradients were prepared successively overlaying 2 ml of less concentrated sucrose on top of the more concentrated ones. five hundred l of sample was applied to the gradient and was centrifuged at 39000 rpm in a beckman sw41 swing bucket rotor using a beckman coulter optima le-80k ultracentrifuge (brea, ca) for 18 h. these conditions results in an average centrifugal force of approximately 190 000 g in the middle of the tube. lipid content from each fraction was determined using the cholesterol e enzymatic assay from wako chemicals usa (richmond, va) described above. the lnp containing nucleic acids was formed by first simulating the self - assembly of a smaller building block, then creating a large particle by spatial translations of the building block, coating the large particle with a polymer - grafted lipid, and resolvating the system. the building block was self - assembled starting from random configurations in a small system (figure 7a). the small system contained all components excluding the polymer lipid and was prepared at low, intermediate, and high hydration levels (12, 20, and 40 water molecules per lipid, respectively). self - assembly resulted in a periodic nanostructure with enclosed water compartments (figure 7b). we also simulated self - assembly of a medium - sized system with all components including the polymer - grafted lipid as a test case. it resulted in the structure similar to the small system at low hydration, and the low - hydration building block was selected for simulations of the large system. the solvent was removed, and the building block was multiplied 3 3 3 times to obtain a large nanoparticle, retaining a small spacing (< 1 nm) between the translated unit cells. the coating layer contained the polymer lipid in random conformations distributed within a thin slab (3 nm), and was placed in close proximity (1 nm) at all facets of the nanoparticle. this system was then resolvated (water placed inside and outside of lnp) in a large simulation box. the small systems contained 8 duplex 12 bp dna complexes, 576 dlinkc2-dmalipids, 144 dspc lipids, and 576 cholesterol molecules. the medium - sized system contained 64 dnas, 4608 dlinkc2-dmalipids, 1152 dspc, 4608 cholesterols, and 1152 peg - lipids. the large system contained 216 dnas, 15552 dlinkc2-dmalipids, 3888 dspc, 15 552 cholesterols, and 3888 peg - lipids. the total ratio in the medium and large systems was dlinkc2-dma / dspc / cholesterol / peg - lipid 4:1:4:1 (molar), and dna to lipid ratio 0.05 wt / wt, similar to experimental conditions. the small system at low, intermediate, and high hydration levels contained 4030, 6530, and 12 560 water particles, respectively ; the medium and large systems contained 400 000 and 900 000 water particles, respectively ; cl ions were added to all systems to neutralize the charge. the large system included 1 400 000 particles in total, had a box size of 54 54 54 nm, and was simulated for 10 s (actual simulation time is indicated). two independent runs of 5 s each were performed for the medium - sized system, and six copies of the small system (two for each level of hydration) were simulated for 1 s. the martini coarse - grained (cg) force field was employed. in this force field, typically one martini particle represents 4 heavy atoms. sirna duplexes were represented by 12 base pair double stranded dnas ; the rna molecules would have minor differences in the lennard jones interactions, and lower chain flexibility which would be important for longer strands. models for dna and dspc lipid were downloaded from the martini web site (http://md.chem.rug.nl/cgmartini/index.php/downloads). the headgroup of dlinkc2-dmalipid comprised charged (qd type) and apolar (c4) particles, and the linker was constituted by a ring of sc4 and 2 sp1 particles. available models for sphingomyelin and peg polymer were combined to produce the peg - grafted ceramide lipid (containing a chain of 7 beads). while longer polymer chains are used in experimental studies to control the nanoparticle size, here a short chain length was incorporated to avoid unnecessary and computationally expensive increase of the simulation box volume (to accommodate longer chains). short polymers combined with a moderate water volume surrounding the nanoparticle were sufficient to keep it disconnected from its periodic image in our simulations. however, a somewhat larger molar concentration of the short peg - lipid (as compared to longer polymers in the experimental setups) was required to cover the whole nanoparticle surface. jones potential was shifted to zero between 0.9 and 1.2 nm, the coulomb potential was shifted to zero between 0 and 1.2 nm, with a relative dielectric constant of 15. the time step was 10 fs with the neighbor list updates every 10 steps. the system was coupled to an isotropic pressure of 1 bar using the berendsen barostat with a time constant of 4 ps. lipids, water, and nucleic acids were coupled separately to a temperature of 310 k using the velocity rescaling thermostat with a time constant of 1 ps. as noted above, high sirna encapsulation levels for sirna in lnp systems are inconsistent with bilayer structure, where a maximum encapsulation efficiency of 50% would be expected. here we characterized the encapsulation efficiencies of lnp sirna systems over a range of sirna / cationic lipid charge ratios using the microfluidic mixing process as described in the experimental section and employing the lipid mixture dlinkc2-dma / dspc / chol / peg - lipid (40/11.5/47.5/1 ; mol / mol). the resulting lnp sirna systems exhibited diameters of approximately 50 nm (number mode) as measured by dynamic light scattering. essentially complete (95% as indicated using the ribo - green assay) encapsulation was achieved over a wide range of sirna / cationic charge ratios, including charge ratios as high as 1.25 (see figure 1, supporting information). these observations suggest that the microfluidic mixing technique for formulating sirna allows somewhat more efficient encapsulation than the t - tube mixing technique where trapping efficiencies of 70% have been noted. in the next set of experiments, the cryo - tem characteristics of the lnp sirna systems produced by microfluidic mixing were investigated for the dlinkc2-dma / dspc / chol / peg - lipid (40/11.5/47.5/1 ; mol / mol) lipid composition. as shown in figure 1a and b, lnp sirna systems prepared at sirna / total lipid ratios of 0.06 and 0.24, corresponding to sirna - to - cationic charge ratios of 0.25 and 1, exhibited an electron dense core similar to the electron dense lnp sirna structure contrasts with the less dense interior of a vesicle system with an aqueous interior generated from popc / cholesterol (1:1 ; mol / mol) (figure 1c) and is visually similar to the electron dense interior exhibited by cryo - tem of a colloidal fat emulsion as reported by kuntsche. lnp containing dlinkc2-dma exhibit electron dense cores both in the presence and absence of encapsulated sirna as indicated by cryo - tem. lnp were prepared by microfluidic mixing employing a herringbone mixer as indicated in the experimental section. popc / cholesterol (50/50 ; mol / mol) bilayer vesicles were prepared by extrusion through polycarbonate filters with 80 nm pore size. (a) cryo - tem micrograph obtained from lnp sirna with lipid composition dlinkc2-dma / dspc / chol / peg - lipid (40/11.5/47.5/1 ; mol / mol) and sirna at a sirna / lipid ratio of 0.06, wt / wt, corresponding to an sirna / cationic lipid charge ratio of 0.25. (b) lnp with the same lipid composition as for (a) but prepared at an sirna / lipid ratio of 0.24 wt / wt, corresponding to an sirna / cationic lipid charge ratio of 1. (c) cryo - tem micrograph of popc / cholesterol (1:1) vesicles. (d) lnp with the same lipid composition as for (a) and (b) but prepared in the absence of sirna. the lnp sirna formulation employed in figure 1a contains sirna at a 0.06 sirna / lipid (w / w) ratio which corresponds to an sirna - to - cationic lipid charge ratio of 0.25. as a result, when the lnp sirna is formulated at ph 4.0, approximately 75% of the cationic lipid is not complexed to sirna in the lnp. it is therefore of interest that the lnp sirna particles observed in figure 1a exhibit an electron dense interior with no evidence of an internal aqueous core. this suggests that the cationic lipid may contribute to the electron dense interior even when not complexed to sirna. in order to determine whether this is the case, lnp systems with the same lipid composition but no sirna were formulated employing the microfluidic process and characterized by cryo - tem. as shown in figure 1d, an electron dense core was observed in the absence of sirna, indicating that cationic lipids such as dlinkc2-dma, possibly in combination with dspc and cholesterol, contribute to electron dense structures in the lnp interior. the structures that could give rise to the electron dense cores detected by cryo - tem are of interest. in the absence of sirna it may be proposed that the cationic lipid, in association with a counterion, adopts an inverted structures such as inverted micelles, consistent with the propensity of these lipids for highly curved inverted these structures are inverted in the sense that the polar headgroups are oriented toward interior aqueous cores with diameters as small as 3 nm. the actual equilibrium radius of an inverted micelle could be larger as dictated by the intrinsic or spontaneous radius of curvature of the constituent lipids. in turn, assuming a bilayer thickness of 4 nm this would suggest that lnp systems composed of pure cationic lipid should exhibit limit sizes with diameters in the range of 11 nm or larger, which is essentially a bilayer surrounding an aqueous interior with diameter as small as 3 nm. alternatively, in the presence of sirna, it is logical to suppose that the limit size particle consists of a distorted inverted micelle of cationic lipid surrounding the sirna oligonucleotide. in turn, this would suggest a limit size system as small as 14 nm diameter, assuming that the sirna contained in this inverted micelle is surrounded by an inner monolayer of cationic lipid within an outer monolayer of surface lipid and that the dimensions of the sirna are 2.6 nm in diameter and 5.8 nm in length. here we explored whether limit size particles compatible with such structures could be generated using peg - lipid as the surface lipid. in this regard, a vesicle containing an internal aqueous core of 3 nm diameter has an outside - to - inside surface area ratio of 6.8 (assuming a bilayer thickness of 4 nm), indicating that the outer monolayer requires the presence of lipids that provide an interfacial area approximately 7 times larger than the inner monolayer area. assuming that the interfacial area for a lipid such as dlinkc2-dma is similar to that of dioleoylphosphatidylcholine (0.7 nm), it is straightforward to show that approximately 10 mol % peg2000-lipid (surface area per molecule 36 nm) would be required to coat inverted micelles composed of dlinkc2dma with an aqueous core 3 nm in diameter. we therefore examined the limit size lnp that could be achieved for a dlinkc2-dma / peg - c - dma system (90:10, mol : mol) produced by the microfluidic mixing process. the size determined from cryo - tem micrographs (figure 2a) was 14.7 6.9 nm, consistent with an ability of the cationic lipid to form inverted micellar structures with interior aqueous diameters in the range of 8 nm. on the other hand, the limit size of lnp sirna systems formulated at an sirna - to - cationic lipid charge ratio of one resulted in limit size systems of 22.7 6.1 nm in diameter (figure 2b), consistent with the presence of inverted micelles with interior diameters of approximately 15 nm consisting of cationic lipid complexed to internalized sirna. lnp exhibit limit sizes consistent with inverted micelle structure in presence and absence of sirna. limit size lnp were prepared by microfluidic mixing as indicated in the experimental section. (a) cryo - tem micrograph obtained from lnp with lipid composition dlinkc2dma / peg - lipid (90/10 ; mol / mol) in the absence of sirna. (b) lnp with the same composition as for (a) but prepared with sirna at an sirna - to - cationic lipid charge ratio of 1. (c) size distribution of lnps in figure 2a and (d) size distribution of lnps in (b). particle diameters are determined with the aid of image j (nih, bethesda, md), and average diameters are calculated from over 150 particles. if the sirna is complexed to cationic lipid and localized in an inverted micelle inside the lnp, it would be expected to be less mobile than if freely tumbling in the aqueous interior of a bilayer vesicle system. the mobility of the sirna can be probed using p nmr techniques. in particular, it would be expected that limited motional averaging would be possible for complexed sirna, leading to very broad solid state p nmr resonances due to the large chemical shift anisotropy of the phosphate phosphorus. if, on the other hand, the sirna is able to freely tumble in an aqueous environment, rapid motional averaging would be expected to lead to narrow, readily detectable p nmr spectra. in order to avoid conflicting p nmr signals arising from phosphorus in phospholipids and sirna, phosphorothoiate sirna, which gives a p nmr signal that is shifted downfield from the normal phosphate resonance, was used to ascertain the motional environment of the sirna. as shown in figure 3a, the p nmr signal from free phosphorothioate sirna is a doublet peak, shifted approximately 56 ppm downfield from the phosphate resonance. the doublet structure may be attributed to the presence of rp and sp isomers of the sirna strands. for lnp sirna systems with the lipid composition dlinkc2-dma / dspc / chol / peg - c - dma (40/11.5/47.5/1 mol %) and containing sirna (0.06 sirna / lipid ; wt / wt) no p nmr signal was observable for the encapsulated sirna (figure 3b), consistent with immobilization within the lnp core. detergent solubilization of the lnp particle using sodium dodecyl sulfate (1% vol / vol) resulted in recovery of the sirna signal (figure 3c). note that phosphothioate sirna, which gives rise to a p nmr resonance 56 ppm downfield of the phosphodiester peak, was used to avoid overlap with the p nmr signal arising from the dspc phosphorus. (b) p nmr spectrum of phosphothioate sirna encapsulated at a sirna / lipid ratio of 0.06 (w / w) in lnp containing dlinkc2-dma / dspc / chol / peg - lipid (40/11.5/47.5/1 ; mol / mol). (c) p nmr signal arising from the same sample as (b) after the addition of 1% sds to solubilize the particle. the spectra depicted were obtained from 15 000 transients as described in the experimental section. sirna sequestered in the lnp core should be fully protected from degradation by externally added rnase. lnp sirna systems with the lipid composition dlinkc2-dma / dspc / chol / peg - c - dma (40/11.5/47.5/1, mol %) were incubated with bovine pancreatic rnase a to determine the protection of encapsulated sirna. as shown in figure 4, gel electrophoresis indicates that free sirna is degraded, while the sirna encapsulated in the lnp particles formulated by the microfluidic method is completely protected. addition of the detergent triton x-100 dissolves the lnp, releases the sirna, and results in sirna degradation in the presence of rnase. sirna was either employed in the free form or encapsulated in lnp containing dlinkc2-dma / dspc / chol / peg - lipid (40/11.5/47.5/1 ; mol / mol) at an sirna / lipid ratio of 0.06 (w / w). the integrity of the sirna was challenged with 1 g / ml bovine pancreatic rnase a. 5% triton x-100 was added to solubilize the lnp. gel electrophoresis was performed on 20% native polyacrylamide gel and sirna visualized by staining with sybr - safe. as indicated above, the electron dense core of the lnp sirna systems may be suggested to consist of encapsulated sirna complexed to cationic lipid and the remaining lipid (cationic lipid, phospholipid, cholesterol and peg - lipid) is either present in the core in inverted micellar or related nanostructures, or is resident on the lnp exterior. it would then be expected that at high sirna contents corresponding to sirna - to - cationic lipid charge ratios of 1, where all the cationic lipid is complexed with internalized sirna, little or no cationic lipid would be present on the external monolayer. in order to test whether this is the case, a fluorescence resonance energy transfer (fret) assay for exterior cationic lipid was developed. this assay, which is essentially a membrane fusion assay, utilized negatively charged bilayer lipid vesicles composed of dioleoylphosphatidylserine (dops) that contained the fret pair, nbd - pe and rh - pe at 1 mol % each. lnp sirna systems consisting of dlinkc2-dma / dspc / chol / peg - lipid (40/11.5/47.5/1 mol %) were added to the dops vesicles and incubated at ph 5.5. the pka of dlinkc2-dma is 6.7 and thus more than 90% of the dlinkc2-dma on the outside of the lnp will be charged at ph 5.5, potentially promoting fusion with negatively charged dops lnp. as indicated elsewhere fusion is observed as an increase in the nbd - pe fluorescence at 535 nm as the nbd - pe and rh - pe probes become diluted following lipid mixing. as shown in figure 5, when the lnp systems contained no sirna, substantial fusion was observed, consistent with a considerable proportion of the dlinkc2-dmaresiding on the outer monolayer of the lnp system. when the lnp systems contained sirna at a sirna - to - total lipid ratio of 0.06 (w / w), which corresponds to an sirna - to - cationic charge ratio of 0.25, however, the extent of fusion was reduced (figure 5), and for lnp sirna systems prepared with an sirna - to - cationic lipid charge ratio of one little or no fusion was observed, indicating that little or no dlinkc2-dmawas present on the lnp sirna exterior. this supports the hypothesis that in lnp with high sirna content all the cationic lipid is complexed with sirna and sequestered in the lnp interior. the amount of external cationic lipid in lnp sirna systems was assayed as a function of the sirna phosphate - to - cationic lipid charge ratio using the fret lipid mixing assay described in the experimental section. three lnp systems dlinkc2-dma / dspc / chol / peg - lipid (40/11.5/47.5/1 ; mol / mol) were prepared at charge ratios of 0 (solid line), 0.25 (dotted line), and 1 (dash line). the lipid mixing assay was performed at ph 5.5 to ensure that essentially all external dlinkc2-dma was positively charged. the reaction was initiated by injecting the lnp (at t = 30 s) into a stirred cuvette containing the anionic dops / nbd - pe / rh - pe (98:1:1 molar ratio) vesicles. a remaining question concerns why the maximal dequenching for the unloaded sample plateaus at approximately 25%. it is logical to suppose that the dops vesicles fuse with positively charged lnp to the point that all available cationic lipid is complexed with dops, after which there is no electrostatic attraction driving lnp - dops vesicle fusion and therefore no further dilution of the fret pairs. the above experiments were performed using 200 mol lnp (80 mol cationic lipid) and 100 mol dops vesicles, indicating that when all possible fusion has occurred, approximately 20% of the dops vesicles would remain intact and the fret pairs in the remaining 80% of the dops this may be insufficient to produce maximum dequenching. in order to test this hypothesis, the concentration of fret pairs in the dops vesicles was lowered to maximize dequenching effects. as shown in supporting information figure 2, the maximal dequenching then increased to approximately 80% on fusion with unloaded lnp, consistent with hypothesis. if the lnp sirna systems exhibit a lipid core, they would be expected to exhibit a different density as compared to vesicles with an aqueous core. in the absence of sirna where the interior consists of inverted micelles of cationic lipid the density should be less than vesicular systems, and the density should increase as more sirna is encapsulated. as shown in figure 6, when empty lnps without sirna and lnps containing sirna at 0.06 sirna - to - total lipid ratio are centrifuged on a 115% sucrose step gradient as described under in the experimental section, the lnps remained on the top of the gradient (figure 6). in a parallel experiment, popc / cholesterol (1:1 ; mol / mol) bilayer vesicles were centrifuged on an identical gradient and the vesicles distributed as a broad peak centered at around fraction 7 of the gradient. increasing sirna content in the lnp results in an increase in density, as lnps containing sirna at a 0.24 sirna - to - total lipid ratio (corresponding to a 1:1 sirna / cationic lipid charge ratio) were much denser, exhibiting a peak centered at fraction 15 of the column. in an additional experiment (data not shown) empty lnps and lnp containing sirna at 0.06 sirna - to - lipid ratio were introduced first, at the bottom of the centrifuge tube, and a 110% sucrose step gradient layered on top. after centrifugation, the lnp were found to have redistributed to the top of the gradient. these results provide strong evidence that lnp sirna systems contain a lipid core with a density dependent on the amount of sirna encapsulated. the density of lnp sirna systems is consistent with a hydrophobic lipid core as indicated by density gradient ultracentrifugation.. fractions (500 l) were successively removed from the top of gradient following centrifugation at 190 000 g for 18 h and were assayed for cholesterol in popc / cholesterol bilayer vesicles (open circles), empty lnp system (filled squares), lnp sirna systems at a sirna / cationic lipid charge ratios of 0.25 and 1 (filled triangles and filled diamonds, respectively). the first step in computer modeling was to simulate the self - assembly of a putative unit cell for a lipid nanoparticle. to this end, a mixture of dlinkc2-dma, distearoylphosphatidylcholine (dspc), cholesterol, and nucleic acids was placed in a small box in a random configuration (figure 7a). the self - assembly was performed in several independent simulations at different hydration levels to explore possible structures formed in the mixture. we also simulated self - assembly of a medium - sized system containing a polymer - grafted lipid in addition to other components. comparing resulting structures, we found that a small system at low hydration (figure 7b) was similar to the core of the self - assembled medium sized system and selected it as a building block for a larger lnp. the large lnp was constructed by multiplying the building block, coating the resulting structure with a peg - lipid layer, and resolvating the system ; see the experimental section. small spacing between the unit cells and between the polymer layer and the nanoparticle was allowed to adjust the water contents upon equilibration of the lnp. self - assembly from a random configuration (a) into a building block (b) for a lipid nanoparticle (lnp). a mixture of dlinkc2-dma, dspc and cholesterol (576 dlinkc2-dmalipids, 144 dspc lipids and 576 cholesterol molecules ; 44/11/44 ; mol / mol) is placed in a small simulation box at a low hydration level ; see text. dlinkc2-dmais shown in yellow, cholesterol in pink, dspc in gray, lipid polar moiety in cyan, and nucleic acids (12 bp duplex dna) in red ; water not shown for clarity. during the equilibration (1 s), the water compartments closed and the peg - grafted lipids adsorbed on its surface with polymer chains oriented toward the solution. on the simulation time, the lnp gradually transformed into a smooth rounded capsule with an averaged diameter of ca. the outer layer of the lnp is constituted by a roughly homogeneous coating of peg - lipids. inside the lnp, irregular water filled compartments of diameters ranging from ca. 3 to 9 nm are separated by bilayer membranes with dnas bound to the membrane surface (see figure 8b d). the structure of the lnp core resembles an inverted hexagonal phase (hii) distorted at a high hydration level. 6 waters / lipid which corresponds to 0.10 l/mol lipid, in good agreement with experimental data (data not shown). given the significant fraction of lipids on the lnp surface (1/4), we expect this number to be slightly higher for a larger size lnp. a lipid nanoparticle (lnp) contains irregular water - filled cavities separated by bilayer membranes, with nucleic acids bound to membrane surfaces : side (a), cross - section (b, c), and zoom - in (d) views. cationic lipid dlinkc2-dmais shown in yellow, cholesterol in pink, dspc in gray, lipid polar moiety in cyan, peg - lipid in violet, and nucleic acids (duplex dna) in red ; water not shown for clarity. the lipid composition was dlinkc2-dma / dspc / cholesterol / peg - lipid (4:1:4:1 ; mol / mol) and dna to lipid ratio 0.05 wt / wt. in the lnp core, the numbers of nearest neighbors in the first coordination shell are given in table 1. there are strong preferential interactions between the dna phosphates and positively charged groups of dlinkc2-dmalipids. increased numbers of dlinkc2-dma- dspc neighbors originate from favorable interactions of the positively charged group of the cationic lipid with the negatively charged phosphate groups of dspc. these interactions lead to formation of molecular pairs which manifest in the radial distribution function (rdf) as a pronounced second peak at a distance of 1 nm (data not shown). estimated numbers in the absence of preferential interactions the numbers are calculated for dna phosphates, positively charged moiety of dlinkc2-dma, the cholesterol polar group, and the dspc phosphate group (or choline group in the case of dna neighbors) by integrating the rdfs over the first maximum. the densities of headgroups are higher in the lnp core (figure 9a) than on the lnp surface (figure 9b). this is expected from a high negative curvature of the water - filled compartments inside the lnp, and the presence of polymer - grafted lipids on the surface. the distribution of dlinkc2-dmaaround dspc lipids is shifted towards the dna phosphates, and cholesterol is distributed somewhat away from dlinkc2-dma(figure 9a). due to a high cationic lipid to dna ratio these free (figure 9c) and bound (figure 9d) cationic lipid fractions have a noticeably different 3d neighborhood. a somewhat heterogeneous surrounding of dspc and cholesterol aligned along the linker of the free lipid becomes for the bound lipid displaced away and to the side by dna phosphates. spatial density distributions for selected molecular groups around dspc in the lnp core (a) and on the lnp surface (b), and dlinkc2-dmain the lnp core free (c) and bound to dna (d). the surfaces of constant number densities are plotted at the values corresponding approximately to the average density in the first coordination shell. in the molecular representations, dspc headgroup is shown as dark gray, glycerol - ester region as light gray, and hydrocarbon chains as transparent gray beads ; dlinkc2-dmaheadgroup is shown as yellow, linker as light yellow, and chains as transparent yellow beads. in the density distributions, dna phosphates are colored in semitransparent red, dlinkc2-dmaheadgroup in cyan and linker in yellow, cholesterol polar group in pink, dspc headgroup in dark gray, and glycerol - ester in light gray. the model for lnp sirna structure developed by molecular simulation (figure 8) is fully consistent with the experimental results presented here. the model shows that encapsulated sirna is located in internalized distorted inverted micelles complexed to cationic lipid, with remaining lipids organized around small internal aqueous compartments. such organization could account for the increased electron density as compared to bilayer vesicle systems as evidenced by cryo - tem studies. this organization is also consistent with the ability of particles composed of dlinkc2-dma and peg - lipid to adopt structures as small as 14 nm diameter in the absence of sirna and 23 nm diameter in the presence of sirna, as well as the lack of external cationic lipid in lnp sirna systems prepared at high sirna - to - cationic lipid charge ratios and the essentially complete protection of encapsulated sirna from external rnase. previously dspc has been included to provide increased stability to the preformed vesicles used in the preformed vesicle formulation method and has been thought to play a stabilizing role in the lnp structure formed. the results presented here suggest that dspc could also be forming ion pairs between the dspc phosphate group and cationic lipid headgroup, leaving the dspc choline function to associate with sirna phosphates. a final point is that, as suggested by the molecular modeling results, it is likely that cholesterol is distributed roughly homogeneously in the lnp core and surface, given its ability to partition into both lamellar and inverted lipid structures such as the hexagonal hii phase. a major advantage of the model presented in figure 8 is that it can explain how sirna encapsulation efficiencies approaching 100% can be achieved during the formulation process. specifically, during the microfluidic synthesis process, the following steps are achieved : first, rapid mixing between the aqueous phase containing sirna and the ethanol phase containing cationic lipid, second the association of cationic lipid with sirna to form hydrophobic nucleating structures, and third, as the polarity of the medium increases further, coating of the nucleating structures by remaining lipids as they reach their solubility limits in the ethanol / water system, forming the final lnp sirna structure. in this picture, it is clear how essentially all of the sirna could be incorporated into lnp systems at nonsaturating sirna loading, leading to complete encapsulation. there are, however, other structures that could be compatible with high encapsulation efficiencies. in previous work, we have encapsulated antisense oligonucleotides into lnp systems containing ionizable cationic lipids with encapsulation efficiencies of up to 70% using the pfv method. the lnp formed are small multilamellar vesicles where the oligonucleotide is apparently located at the lamellar interfaces. these systems contained higher levels of the bilayer forming lipid dspc and lower levels of cationic lipid than employed here. in order to show that the systems we observe here are part of a continuum of structures that are sensitive to the proportions (and types) of lipid components, we examined the cryo - tem morphology of an lnp sirna system containing a larger proportion of dspc and a lower proportion of cationic lipid (dlinkc2-dma / dspc / chol / peg - lipid in the proportions 20/31.5/47.5/1 ; mol / mol, as opposed to 40/11.5/47.5/1 ; mol / mol). this system also exhibited high encapsulation efficiencies above 90% but also showed evidence of multilamellar structure, as shown in supporting information figure 3. it would therefore appear that the internal structure of lnp sirna systems changes from multilamellar to inverted micellar as the proportion of bilayer - forming species is reduced, as may be expected based on polymorphic phase preferences of component lipids. it is likely that the results presented here for lnp sirna systems produced by microfluidic mixing will also extend to lnp sirna systems composed of the same or similar lipids but prepared by other in - line mixing protocols such as the t - tube mixer, as long as the mixing rates are sufficiently fast. in this regard, it may be noted that lnp sirna systems containing ionizable cationic lipids prepared by t - tube mixing show the distinctive electron - dense core by cryo - tem. in summary, the results presented in this work demonstrate a new type of lipid nanoparticle with a structured core formed by the rapid mixing of cationic lipid - containing ethanol solutions with aqueous solutions of sirna oligonucleotides. such structures are consistent with the electron dense core observed by cryo - tem and the efficient loading characteristics associated with these particles. this work also provides, for the first time, an understanding of the mechanism of formation of lnp sirna formulations formed by mixing cationic lipids in ethanol with sirna in aqueous solution. it is anticipated that this understanding will lead to the design of more sophisticated lnp structures. | lipid nanoparticles (lnp) containing ionizable cationic lipids are the leading systems for enabling therapeutic applications of sirna ; however, the structure of these systems has not been defined. here we examine the structure of lnp sirna systems containing dlinkc2-dma(an ionizable cationic lipid), phospholipid, cholesterol and a polyethylene glycol (peg) lipid formed using a rapid microfluidic mixing process. techniques employed include cryo - transmission electron microscopy, 31p nmr, membrane fusion assays, density measurements, and molecular modeling. the experimental results indicate that these lnp sirna systems have an interior lipid core containing sirna duplexes complexed to cationic lipid and that the interior core also contains phospholipid and cholesterol. consistent with experimental observations, molecular modeling calculations indicate that the interior of lnp sirna systems exhibits a periodic structure of aqueous compartments, where some compartments contain sirna. it is concluded that lnp sirna systems formulated by rapid mixing of an ethanol solution of lipid with an aqueous medium containing sirna exhibit a nanostructured core. the results give insight into the mechanism whereby lnp sirna systems are formed, providing an understanding of the high encapsulation efficiencies that can be achieved and information on methods of constructing more sophisticated lnp systems. |
many proteomics experiments are performed on the peptide level (namely, bottom - up proteomics). for this purpose, the proteome is enzymatically digested, and the peptide and peptide fragment masses are measured by tandem mass spectrometry after online separation by reverse - phase liquid chromatography (lc ms / ms). for the analysis of lc ms / ms data, peptides are statistically scored by search engines such as mascot, sequest, omssa, maxquant, x!tandem, ms - gf+, ms amanda, myrimatch, or comet. all of these tools compare input peptide ms / ms spectra to theoretical spectra generated by the in silico digest of protein databases. the peptides can, dependent on the settings of the search engine, contain missed cleavage sites and variable or fixed amino acid modifications. all of the in silico generated spectra constitute the so - called search space (i.e., spectra of all peptides and variants thereof that are considered a potential match to the input spectrum). the search engine settings and database used are crucial for the quality of the results obtained because discrepancies between biological sample content and peptides present in the search space can lead to several undesirable effects. for the statistical evaluation of the results, in most cases, false discovery rates (fdr) are calculated. fdr is defined as the ratio of false positives to the total number of assignments (false positives + true positives) and is often estimated with the help of decoy databases (i.e., randomized or reversed databases). usually, most of the proteins not contained in the target database are sample contaminants from different species, either introduced by accident or as a reagent during sample preparation. the former can be reduced by careful sample handling, and among the best known examples is human keratin. the latter type of common contaminants in proteomics experiments includes proteases, which are added to the sample to digest proteins to the more readily analyzed peptides. the most widely used protease is trypsin due to its specificity, efficiency, availability, and relatively low cost. trypsin cleaves the amino acid sequences c - terminal to lysine and arginine residues (with the exception of the occurrence of n - terminal proline) and gives rise to peptides of suitable length and charge for lc however, trypsin is also subject to autolysis because it contains 11 lysine and 4 arginine residues. because trypsin is used in high quantities to promote efficient digestion (trypsin to sample protein ratio : 1:201:100), trypsin - derived peptides are highly abundant and result in high - quality spectra during ms / ms. in the case where the trypsin sequence is not present in the database it is therefore widely accepted in the community to concatenate a contaminant list that includes trypsin with sample specific databases for the reasons given above. the groups of ron beavis (http://www.thegpm.org/crap/) and matthias mann (http://www.maxquant.org/downloads.htm) make their list of contaminants publicly available. although the database should cover all possible components of a sample, care should be taken not to choose a too - large database (e.g., the entire uniprot trembl database) because large databases not only increase the search time but also, more importantly, increase the number of random peptide hits. for the prevention of the autolysis of trypsin and the associated loss of activity, the protease is often modified by acetylation (new england biolabs) or reductive methylation (promega, sigma - aldrich) of lysines, the latter being the most common approach. both modifications yield a trypsin that is more resistant to autolysis while retaining its catalytic function. here, we show that the most widely employed trypsin preparations are chemically modified and that including native trypsin in the contaminant database is not sufficient to prevent false - positive identifications of trypsin - derived spectra. instead, it is necessary to add modified trypsin to the list of contaminants. we present a new database search strategy with a minimal increase in search space and the concomitant loss of statistical significance. finally, this approach can be easily implemented into existing workflows for many widely used search engines. the list of common repository of adventitious proteins (crap) was downloaded from ftp://ftp.thegpm.org/fasta/crap (version : january 30, 2015). the protein identifiers were then uploaded to uniprot retrieve / id mapping tool to generate a fasta file in uniprotkb format (february 2, 2015). databases from search strategies a e were reversed using the compomics dbtoolkit 4.2.3. swiss - prot_human or swiss - prot_yeast was used as the database ; carbamidomethylation of cysteines was set as a fixed modification. oxidation of methionine and protein n - terminal acetylation were set as variable modifications. swiss - prot_human or swiss - prot_yeast with concatenated crap was used as the database. oxidation of methionine and protein n - terminal acetylation were set as variable modifications. swiss - prot_human or swiss - prot_yeast with concatenated crap was used as the database. oxidation of methionine and protein n - terminal acetylation were set as variable modifications. for data set 2, acetylation of lysine was allowed as an additional variable modification ; for all other data sets, dimethylation of lysine was allowed as an additional variable modification. swiss - prot_human or swiss - prot_yeast with concatenated crap was used as the database. oxidation of methionine and protein n - terminal acetylation were set as variable modifications. swiss - prot_human or swiss - prot_yeast with concatenated crap was used as the database. furthermore, modified trypsin was added to the database (where all lysines (k) were replaced by dimethylated lysines (j) to detect dimethylated lysines in trypsin). oxidation of methionine, protein n - terminal acetylation, loss of one methylation of dimethylated lysines (j) (to detect monomethylated lysines in trypsin), and loss of dimethylation of dimethylated lysines (j) (to detect unmethylated lysines in trypsin) were set as variable modifications. data set 1 : saccharomyces cerevisiae ; pxd002726 data set 2 : phosphorylation sites of numb (human cell line) with mouse recombinant protein numb ; pxd000997 data set 3 : human breast cancer study ; pxd000246 data set 4 : identified spectra from 209 public human data sets from the pride repository (see supporting table s4) data set 5 : human brain 611 reference map ; prd000151 database search was performed using mascot ver. 2.4.1 or ms amanda standalone_windows_1.0.0.3948 customized to contain dimethylated lysine j = 156.125710 (kindly provided by viktoria dorfer). data were searched against human and s. cerevisiae swiss - prot database as specified in the database section. for fdr estimation, a target peptides mass tolerance was set to 10 ppm (or 100 ppm for data set 5) and fragment mass tolerance to 0.8 da. carbamidomethylation of cysteine was set as a fixed modification, and oxidation of methionine was set as a variable modification. to detect peptides containing peptide c - terminal artificial amino acid j, we changed the cleavage specificity for trypsin to j, k, and r, not after p. furthermore, to detect mixed (i.e., peptides including methylated and unmodified lysine) and monomethylated peptides, we added the loss of monomethylation (14.0153759 da) and loss of dimethylation (28.0307517 da) of the artificial amino acid (j) as variable modifications. data set 4 was searched using x!tandem (version : piledriver (2015.04.01.1)). because x!tandem restricts the amino acids that may be redefined, o was used to represent dimethylated lysine. the mass of o was set to 156.12571 using the protein, modified residue mass file carbamidomethylation of cysteine was set as a fixed modification, and oxidation of methionine, n - terminal acetylation and a loss of 28.0307517 da on o representing loss of dimethylation and a loss of 14.0153759 da on parent - mass error was set to 2 da, and fragment mass error was set to 0.4 da. a total of two missed cleavages were allowed, the specificity of trypsin was adapted as described before, and refinement mode was disabled. fdr for separate target decoy searches (data set 3) was calculated for mascot ion scores as fdr being equal to the number of decoy hits divided by the number of target hits, with score increments of 0.01 until fdr was smaller than 1%. log(2) score distribution for separate decoy and target database searches of data set 3 using different search strategies. left panel : decoy database searches ; right panel : target database searches. a : search strategy a, swiss - prot_human ; b : search strategy b, swiss - prot_human plus list of common contaminants ; c : search strategy c, swiss - prot_human plus common contaminants and additional variable modification dimethyl (k) ; d : search strategy d, swiss - prot_human plus common contaminants and additional variable modifications methyl (k) and dimethyl (k). e : search strategy e, swiss - prot_human plus common contaminants and methylated lysines of trypsin considered as artificial amino acids. marked in black are peptides that pass the fdr 1% cutoff (mascot ion score of 20.50 for searches a, b, and e, 20.94 for search c, and 22.21 for search d). gel bands were cut out and destained using 50% acetonitrile in 100 mm ammonium bicarbonate. after dehydration with 100% acetonitrile, gel pieces were dried and then reduced with 10 mm dithiothreitol for 30 min at 56 c and subsequently alkylated with 55 mm iodoacetamide for 20 min at 37 c in the dark. gel pieces were dehydrated and dried again and then incubated with 0.19 g of sequencing - grade modified trypsin (promega, v5111) overnight. peptides were extracted in four sequential steps : 15 l of 25 mm ammonium bicarbonate, 150 l of acetonitrile, 40 l of 5% formic acid, and 150 l of acetonitrile. the dried peptide extracts were resuspended in 5% acetonitrile and 0.1% trifluoroacetic acid, and peptides were separated on an acclaim pepmap rslc c18, 2 m, 100, 75 m i.d. 50 cm column employing a dionex ultimate 3000 rslc coupled to a ltq - orbitrap velos (thermo fisher scientific). data - dependent top 10 cid ms and ms / ms data were acquired in the orbitrap analyzer at a resolution of 60 000 and in the ion trap employing normal scan rate settings, respectively. the mass spectrometry proteomics data was deposited to the proteomexchange consortium (http://proteomecentral.proteomexchange.org) via the pride partner repository with the data set identifier pxd002726 and 10.6019/pxd002726. despite the above - mentioned chemical modifications, peptides originating from trypsin are among the most commonly high - scoring identified peptides in bottom up searches. this can be seen when looking at the pride cluster resource, where many of the largest clusters containing several thousand spectra represent peptides from trypsin (e.g., these unmodified peptides are the fraction of peptides that were not modified during reductive dimethylation, which could be due to the native state of trypsin during the modification. assuming that the majority of accessible lysines are methylated but still many cleavage events occur, it is reasonable to expect peptides containing methylated lysines upstream of the unmodified lysine at the cleavage site. we therefore reanalyzed several of our own data sets as well as publicly deposited data sets to assess the presence of methylated peptides and the impact of different search strategies. the first data set we examined was a set of in gel digests from s. cerevisiae prepared in our laboratory (data set 1, pxd002726), which was analyzed with different search strategies (table 1). initially, we searched only against the swiss - prot_yeast database without accounting for potential contaminants (search strategy a). second, we included the list of common contaminants (search strategy b). and third, to find dimethylated trypsin, we allowed dimethylation of lysine as a variable modification (search strategy c). search c identified porcine trypsin with a higher score than search b because more peptides could be identified. we also identified numerous peptides with dimethylated lysines at their c - termini, challenging the assumption of complete inhibition of tryptic activity at the c - terminal of dimethylated lysines (figure 1). moreover, we performed a search with the additional variable modification monomethylation of lysine (search strategy d) to check for incomplete conversion during chemical stabilization. indeed, peptides harboring monomethylated lysines were identified, being a fraction of roughly 30% of total methylated lysines (supporting table s1). when analyzing a trypsin self - digest, we could identify the same peptides, confirming our assumption that the methylated peptides indeed originated from trypsin (supporting table s2). using a similar approach, we also found acetylated peptides from trypsin in a human epithelial cell data set (data set 2, pxd000997) (supporting table s3). the yeast data set (data set 1) was searched against swiss - prot_yeast (search strategy a) or swiss - prot_yeast plus common repository of adventitious proteins (search strategy b) to identify yeast and known contaminant proteins from our laboratory. carbamidomethyl (c) as a fixed modification and oxidation (m) as a variable modification were set for all searches. dimethylation (k) was set as a variable modification in the search strategy c, and both monomethylation (k) and dimethylation (k) were set as variable modifications in search strategy d. shown are the highest scoring proteins for each search strategy. methylated peptides of trypsin (highlighted in bold) can indeed be found in samples digested with modified trypsin when the appropriate search settings are used (searches c and d ; see figure 1 and supporting table s1), making it the top - scoring protein hit in this in - gel digest. blue : sequence coverage of peptides identified in data set 1 by search c. peptides are listed in supporting table s1.. other colors : methylated lysine. in searches c and d (by allowing variable modifications of lysine), we identified both methylated lysines and unmodified lysines in trypsin - derived peptides. although the strategy led to an increased score of porcine trypsin (table 1), this approach has a severe drawback. the most severe is the impact on result statistics because more random matches will occur when all possible permutations are tested. enlargement of the database leads to a rise of false - positive hits, increasing both the number of false positives but also false negatives when applying the same fdr threshold. however, ignoring the modified peptides can have the detrimental effect of leading to the identification of false positives. to circumvent this, we devised a method that considers the chemically modified tryptic autolysis peptides without unnecessarily inflating search space. we envisaged introducing dimethylated lysine as an artificial amino acid into the database to be able to limit its occurrence to the actually modified protein in the database. mascot allows the easy introduction of artificial amino acids via the unimod.xml file and the two capital letters j and o for user - defined amino acids. we therefore introduced the artificial amino acid j, having a total monoisotopic mass of 156.125710 da (j = k + dimethyl (+ c2h4, + 28.0313)). for a copy of the porcine trypsin entry, we then replaced all lysines (k) with dimethylated lysines (j) (fictional accession number 012345, try_art) and added it to the database including the contaminants. in addition, the cleavage specificity of trypsin was set to the c - terminal of k, r, and j (except when followed by p). using the database including artificial trypsin and the search settings explained above, we successfully identified peptides of trypsin containing dimethylated and unmodified lysines in data set 1. to identify mixed peptides, including both dimethylated and unmodified lysines within one peptide, we chose a rather counterintuitive approach : we included the loss of dimethylation of dimethylated lysines as a variable modification. this approach limits the increase of search space to the smallest degree necessary (i.e., only peptides that contain j are multiplied while still covering all possible combinations of dimethylated and unmethylated lysines). the same approach can also be used to identify monomethylated lysines by allowing the loss of one methyl group for j (because j minus methyl is equal to k plus methyl). in contrast to the addition of two global variable modifications, we could not observe any impact on search specificity with this strategy (search strategy e), as is detailed in the next section. we next assessed the influence of the different search parameters on peptide spectrum matches (psms), peptide scores, and protein scores using five raw files from a human breast - cancer data set (data set 3, pxd000246). search strategy a identified a lower number of peptides because it was unable to assign spectra to contaminants. search strategy b, representing the community standard to employ a contaminant database without considering modifications of trypsin, yielded a higher number of total psms as well as more psms after applying a 1% fdr cutoff. to visualize the impact of the increased search space in search strategies c and d, we depict histograms of mascot ion scores of a representative raw file (site1_cell_line_erneg_her2neg_pool1_1d_rep1.raw) from data set 3 in figure 2. it is clearly visible that both the decoy and the target populations contain more hits when employing search strategies c and d. less obviously, the mean of the log(2) decoy scores is shifted to slightly higher values (1.47 (a, b, and e) versus 1.64 (c)). these effects are further amplified when adding another variable modification (search strategy d ; figure 2 ; mean 1.95). because of the bimodal distribution of the target populations even after log(2) transformations, the kruskal wallis test was employed for statistical testing and showed highly significant changes (p < 10) in both score distributions for search strategies c and d. calculating the 1% fdr score threshold for the target decoy pairs, search strategies a, b, and e required an ion score of 20.50 to pass the cutoff, while this threshold was increased to 20.94 in search strategy c and 22.21 in search strategy d. although search strategies c and d lead to the highest number of total psms, the number of psms passing the 1% fdr threshold decreases significantly (see figure 2, supporting figure s1). naturally, this also reduces the average protein score (average protein scores were 262.0, 262.3, 256,6, 252,5 and 262.7 for searches a, b, c, d, and e, respectively, with only the scores from search c and d being significantly lower than the average scores from all other searches) (figure s1). in summary, the use of global variable modifications leads to more total psms but at the expense of specificity, as can be seen after applying 1% fdr. the use of the artificial amino acid based search strategy e did not yield a statistically significant improvement over search strategy b in either total psms or psms after applying 1% fdr. however, individual spectra were incorrectly assigned to unrelated proteins by search strategy b rather than modified trypsin, as revealed by search strategy e. this is depicted in figure 3, showing which fractions of false positives of search strategy a can be explained by the common contaminant approach (i.e., search strategy b (23%) and the artificial amino acid search strategy e (additional 7%, total 30%)). to rule out search - engine - specific effects, we processed the same data set with a customized version of ms amanda, a scoring algorithm especially suited for data sets with high - resolution ms / ms spectra. employing search strategy b however, with both search engines, search strategy e resulted in much higher probabilities for the dimethylated trypsin derived peptide to be the correct identification, as shown for one example spectrum in figure 4. fractions of false positives explained by different search strategies (data set 3). search strategy b reveals that 23% of all false positives from search strategy a are caused by common - contaminant - derived peptides (including unmodified trypsin). search strategy e identifies an additional 7% in this data set (overall range 066% and mean 6.6% in all human pride data sets (supporting table s4)), which are exclusively caused by methylated trypsin peptides. individual spectrum misassigned by two separate search engines despite including contaminants in the database (data set 3). indicates that the accession number for dimethylated trypsin can be set at discretion, avoiding occupied accession numbers. depicted in figure 4 is an exemplary case, which may easily lead to identifications passing the 1% fdr threshold and subsequently increases the chance of wrong protein inference using search strategy b. we want to emphasize that search b was done using a database of appropriate size (swiss - prot_human) and included common contaminants. this widely used approach results in statistically valid but still incorrect assignments, which were revealed to actually represent dimethylated trypsin derived peptides with a very high probability, employing search strategy e. to assess the prevalence of these incorrectly assigned spectra, we reprocessed the identified spectra from 209 public human data sets (data set 4) stored in the pride database (see supporting table s4). the extracted identified spectra were identified using x!tandem and the new artificial amino acid based search strategy (search strategy e). we were able to identify methylated trypsin peptides in 38 out of these 209 experiments. for the 171 experiments in which we could not identify methylated trypsin, it is unclear whether unmodified trypsin was used. it has to be noted that most publications only specify the trypsin vendor, and most vendors offer different types of trypsin preparations. the number of spectra identified as dimethylated trypsin peptides ranged from 1 to 600 per project (see supporting table s4). we want to point out that these spectra constitute a substantial amount of all false - positive peptides in community gold standard search strategy b. at an fdr of 1%, the spectra reassigned by search strategy e to methylated peptides originating from trypsin correspond to up to 66% of all false positive hits the distribution of unmodified to monomethylated to dimethylated trypsin derived peptides was on average 25:35:40%. it is also important to highlight that we only reanalyzed the submitted, identified spectra because only these may have led to incorrect identifications. because of the way data is submitted to pride, it is often not possible to know whether all identifications were submitted or only the ones passing the fdr threshold applied by the authors. nevertheless, methylated trypsin peptides passing 1% fdr of the cluster search do occur in a considerable number of public data sets and thus lead to incorrect identifications. in total, 3960 modified trypsin peptides were identified from wrongly assigned spectra (listed in table s4). one interesting example was found in data set 5 from human brain tissue (prd000151). when we first reanalyzed this sample set with search strategy b, we were unable to reproduce the original assignment of a spectrum to human trypsin isoform 2. comparing the search settings, we found that the original publication used a higher parent - mass tolerance, namely 100 ppm for orbitrap data. with the same parent - mass tolerance, we were also able to identify the peptide from human trypsin isoform 2, with a parent mass of approximately 25 mmu. search e yielded a peptide from dimethylated porcine trypsin and from human trypsin isoform 2 with almost identical scores for both mascot and ms amanda searches (see figure 5). comparing the sequences of the two peptides, we found that the human isoform contains an arginine instead of the lysine at the same position in the porcine trypsin. interestingly, arginine and dimethylated lysine have a mass difference of 24.6 mmu, almost perfectly fitting the mass difference of expected to measured mass for the human isoform 2 peptide. because of the expected instrument accuracy and the biological origin of the sample (brain), we are inclined to propose the dimethylated peptide (with mass difference 1 ppm) as the correct match. a precursor mass of 25 mmu as for the initial match might exclude a peptide from the list of potential candidates in search strategies in which stringent precursor mass filtering is applied before peptide matching. however, when using wide parent - mass windows for the initial search, as suggested recently by bonzon - kulichenko. for database - dependent scoring functions (e.g., mascot expect value), other matches can actually outscore and outrank the correct match (as shown in figure 5). it therefore has to be ensured to include all potential matches irrespective of score and rank until applying post - filtering according to precursor mass when using this approach. peptide originating from dimethylated trypsin assigned to human trypsin isoform 2 (data set 5). even though the score for dimethylated trypsin is lower in the mascot search, the mass difference and the biological origin of the sample implicate that the dimethylated trypsin is the correct assignment. analysis with ms amanda showed identical scores and probabilities for both dimethylated porcine trypsin and human trypsin isoform 2. methylation of trypsin is employed to protect against autolysis and concomitant enzymatic activity loss. as expected, our results show that the enzyme is not completely modified because the modification of the natively folded protease can only take place at lysine residues accessible to the reagent. more interestingly, we also show that methylation does not completely protect against autolysis. in many of the analyzed samples, we identified peptides of trypsin that were actually methylated directly at the cleavage site. the use of appropriate databases in proteomics is crucial for identifying correct peptides and avoiding false - positive peptide hits that might result in inferring wrong proteins. even if common contaminants are included in the database, methylated trypsin derived peptides are not considered in the search space, and the presence of these modified peptides leads to false - positive peptide identifications. unfortunately, the most intuitive solution, to consider methylated lysines in the search (i.e., adding variable modifications), has been reported to result in reduced specificity of a search. while this approach is able to efficiently reduce the number of unassigned spectra, it also increases the peptide score threshold at a constant fdr because of the considerably larger search space, as demonstrated in this study. peptides filtered out due to the now more stringent score cutoff also can not add to protein scores, which results in a lower average protein score (supporting figure s1). the same effect can be observed whenever considering variable modifications, which might occur naturally or during sample preparation (e.g., phosphorylations, carbamidomethylation artifacts, and nontryptic cleavages). therefore, one has to carefully balance the beneficial effect of additionally assigned spectra to the detrimental effects of increased search space on a sample specific basis. this is depicted in figure 2, where either two or three global variable modifications plus one variable protein n - terminal modification are used. additional variable modifications lead to a further combinatorial expansion of search space, reducing search specificity and resulting in a net loss of statistically valid peptide hits. the same is true to an even bigger extent when allowing for semi- or nonspecific enzymatic cleavages (supporting figure s1). thus, we suggest including the modification of the employed digestion enzymes into the contaminant database as an artificial amino acid. this approach does not have significant drawbacks regarding the search space, as demonstrated in this study, and can be used alongside other sample - specific search parameters without a loss of search specificity. several search engines such as mascot, x!tandem, and comet (see box 1) allow for the easy introduction of non - natural amino acid, but for some, all amino acid letters and masses are hard - coded. examples for the latter are the (discontinued) omssa engine, andromeda (maxquant), sequest, and ms amanda. sequest, for example, does in our hands not accept additional amino acids defined in the unimod.xml, and maxquant in its current version has no option to add non - natural amino acids. in our opinion, it would be useful to either include a special letter for the omnipresent case of dimethylated lysine from trypsin in each search engine or to make the amino acid code customizable by the user. adaption of the employed contaminant databases to include an additional trypsin copy in which lysines are replaced by the letter of choice is usually trivial. however, care has to be taken that the letter is not used as an ambiguity symbol or for special amino acids in the employed database (e.g., j is used for i / l in ncbinr). in general, it might be worth considering increasing the namespace for amino acids to be more flexible when searching for nonstandard amino acids. at the moment, one is usually limited to the 26 letters of the latin alphabet, 20 of them being already used for the major amino acids, with the employed search engines. in mascot, four of the remaining six letters are hard - coded, which leaves only two letters for the users to add amino acids such as selenocysteine, pyrrolysine, or, in our case, dimethyl - lysine. mascot : nonstandard amino acids can be defined via the unimod.xml file ; letters j and o are available for modification ; http://www.matrixscience.com/blog/non-standard-amino-acid-residues.html x!tandem : the protein, modified residue mass file can be used to change masses associated with individual letters except the ambiguity letters b, j, x, z, leaving letters o and u available to define nonstandard amino acids ; http://thegpm.org/tandem/api/pmrmf.html comet : user amino acids can be added by using the static modifications parameter with available letters b, j, u, x, z, (e.g., add_b_user_amino_acid = 156.125710) ; http://comet-s.sourceforge.net/parameters/parameters_201501/ an alternative approach could rely on the ability of more recent versions of search engines to provide the functionality to use multiple databases in one search. if the search engine would allow the setting of different variable modifications for the individual source databases, one could then include a database containing only trypsin and set mono- and dimethylation as variable modifications for just this one database, resulting in the same search space as with our artificial amino acid method. another feasible approach would be to combine the in silico generated search space with measured spectral libraries from contaminants. finally, the likely ideal solution would be to store known post - translational modifications directly in the sequence database file, as suggested for the hupo psi extended fasta format (peff). this would not only remove false positives caused by a well - defined subset of peptides but also directly allow the assigning of all peptides with known post - translational modifications even without the use of variable modifications. unfortunately, this file format is not yet supported by any of the search engines used in this study. here, we show that the current state - of - the - art standard proteomic data analysis has severe drawbacks and suggest a new strategy that improves the accuracy of proteomics experiments. autolysis - protected trypsin (a standard tool for proteomics) leads to false - positive peptide identifications. when reanalyzing all publically available human proteomic data sets (pride database), we found hundreds of statistically significant wrong assignments in more than 18% of the data sets because of methylated peptides originating from trypsin, corresponding to up to 66% of all false positive hits. furthermore, we present a simple yet efficient search strategy accounting for this problem that can be readily implemented in many widely used search engines. we have demonstrated that our adapted search strategy employing an artificial amino acid for modified trypsin yields more accurate results than the community gold standard employing contaminant databases solely. this is accomplished by limiting the inflation of search space to a minimum while efficiently avoiding false - positive peptide identifications. | chemically modified trypsin is a standard reagent in proteomics experiments but is usually not considered in database searches. modification of trypsin is supposed to protect the protease against autolysis and the resulting loss of activity. here, we show that modified trypsin is still subject to self - digestion, and, as a result, modified trypsin - derived peptides are present in standard digests. we depict that these peptides commonly lead to false - positive assignments even if native trypsin is considered in the database. moreover, we present an easily implementable method to include modified trypsin in the database search with a minimal increase in search time and search space while efficiently avoiding these false - positive hits. |
ankylosing spondylitis (as), an inflammatory arthritis of the spine, is the prototype hla - b27-associated seronegative spondylarthritis.1 although several studies have examined the pathogenesis of as, the disease is not yet completely understood. paraoxonase (pon), an enzyme produced by the liver, is associated with high - density lipoprotein particles and is known to have an antioxidant function.24 in humans, the pon gene family has three members (pon1, pon2, and pon3) that are located on chromosome 7q21.322.1.5 the physiological roles and natural substrates of these enzymes are not well known.6 pon1 has three known enzymatic molecules, including pon, arylesterase (are), and dyazoxonase. pon1 hydrolyzes organophosphates such as paraoxon, hydrolyzes aromatic esters such as phenylacetate, and also decreases the accumulation of lipid peroxidation products.7 are, a thiol enzyme, is reactivated by 2-mercaptoethanol and cysteine but not by reduced glutathione. are acts on phenyl acetate to release phenol, which can produce a stable indophenol dye with 4-aminoantipyrine and potassium ferricyanide.8 human serum pon1 and are are both esterase enzymes that have lipophilic antioxidant characteristics.9 these enzymes play a role in decreasing oxidative stress. pon1 in particular is an important endogenous free radical scavenging system in the human body.10 serum pon1 acts in conjunction with are to function as a single enzyme.6,11 recently, a number of studies investigated serum pon and are activities in response to inflammatory arthritic conditions such as rheumatoid arthritis (ra), psoriasis, and systemic lupus erythematosus (sle) with conflicting results.2,1217 to the best of our knowledge, serum pon and are activities have not previously been assessed in patients with as. therefore, the aim of the present study was to assess whether pon and are levels may be related to disease activity and functional capacity in as patients. thirty - two patients with as and 25 sex- and age - matched healthy controls were enrolled in the study. the diagnosis of as was made according to the modified new york criteria.18 patients with as consumed non - steroidal anti - inflammatory drugs, but no tnf- inhibitory drugs, during this study. none of the participants in the present study was obese, nor were any of them taking lipid lowering agents or antioxidant drugs. we used the bath ankylosing spondylitis disease activity index (basdai) and the bath ankylosing spondylitis functional index (basfi) questionnaires for clinical assessment of the patients with as. these two self - limited questionnaires are the gold standard for measuring disease activity and functional capacity in as patients. the basdai consists of a 1 through 10 scale (one indicating no problem and 10 indicating the worst problem) that is used to answer six questions pertaining to the five major symptoms of as (fatigue, spinal pain, joint pain, and enthesitis, as well as morning stiffness duration and severity).19 the basfi also has a 1 through 10 scale (1 indicating easy and 10 indicating impossible) and consists of ten questions, eight of which relate to the functional capacity of patients and two of which relate to the patient s ability to cope with everyday life.20 this study was performed in accordance with the ethical standards set by the declaration of helsinki and was approved by the local ethics committee. all participants were analyzed in terms of complete blood count and erythrocyte sedimentation rate (esr), as well as c - reactive protein (crp), serum pon, and are levels. the serum was then separated from blood cells by centrifugation and immediately stored at 80c until analysis. a nephelometric method was used for measuring crp levels (beckman array protein system, usa). assays were made without additional nacl (baseline activity) and with 1 mol / l nacl included in the assay buffer (salt - stimulated activity), by following the formation of p - nitrophenol (assessed by the absorbance at 405 nm) for 5 min. the assay buffer contained 0.125 mol / l tris - hcl (ph 8.5), 1.25 mmol / l cacl2, and, for each set of assays, 6 mmol / l of a freshly prepared paraoxon (o, o - diethyl-0-p - nitrophenylphosphate ; sigma chemical co) substrate solution and 1 mol / l nacl (ph 8.5). the assay tube contained 750 l of tris buffer, 50 l of serum (diluted 1:2 with water), and 200 l of 6 mmol / l paraoxon. the enzymatic reaction was started at 37c by the addition of the substrate solution. using a techcomp 8500 ii spectrophotometer, one unit of pon1 activity was defined as the enzyme quantity that disintegrated 1 mol of paraoxon substrate in 1 minute.21,22 the percent stimulation of pon1 was calculated as follows : [(pon1 activity with 1 mol / l nacl)-(basal activity)/basal pon1 activity ] x100.23 are activity was measured using phenylacetate as the substrate. the assay tube contained 750 l of 0.1 mol / l tris - hcl at ph 8.5, 1 mmol / l cacl2, 125 l of 12 mmol / l phenylacetate, and 125 l of serum (diluted 1:10 with water). all participants were analyzed in terms of complete blood count and erythrocyte sedimentation rate (esr), as well as c - reactive protein (crp), serum pon, and are levels. the serum was then separated from blood cells by centrifugation and immediately stored at 80c until analysis. a nephelometric method was used for measuring crp levels (beckman array protein system, usa). assays were made without additional nacl (baseline activity) and with 1 mol / l nacl included in the assay buffer (salt - stimulated activity), by following the formation of p - nitrophenol (assessed by the absorbance at 405 nm) for 5 min. the assay buffer contained 0.125 mol / l tris - hcl (ph 8.5), 1.25 mmol / l cacl2, and, for each set of assays, 6 mmol / l of a freshly prepared paraoxon (o, o - diethyl-0-p - nitrophenylphosphate ; sigma chemical co) substrate solution and 1 mol / l nacl (ph 8.5). the assay tube contained 750 l of tris buffer, 50 l of serum (diluted 1:2 with water), and 200 l of 6 mmol / l paraoxon. the enzymatic reaction was started at 37c by the addition of the substrate solution. using a techcomp 8500 ii spectrophotometer, one unit of pon1 activity was defined as the enzyme quantity that disintegrated 1 mol of paraoxon substrate in 1 minute.21,22 the percent stimulation of pon1 was calculated as follows : [(pon1 activity with 1 mol / l nacl)-(basal activity)/basal pon1 activity ] x100.23 are activity was measured using phenylacetate as the substrate. the assay tube contained 750 l of 0.1 mol / l tris - hcl at ph 8.5, 1 mmol / l cacl2, 125 l of 12 mmol / l phenylacetate, and 125 l of serum (diluted 1:10 with water). the as group was comprised of 32 patients ; nine were female and 23 male. the mean age of these patients was 33 years (range 2065), and the disease duration was 8.13 years (range 125). in the control group (n=25), seven healthy volunteers were female and the others male. there was no statistically significant difference between patients and controls with regard to demographic data (p>0.05) (table 1). when laboratory findings were compared between the as and control groups, the esr but not crp levels differed significantly between the as and control group (p0.05) (table 1). in addition to these findings, we found no correlations among basdai and basfi scores, serum pon, or are values in the as group in the present study. finally, there were no statistically significant correlations between serum pon and are levels or demographic variables (e.g., gender or disease duration). the inflammation primarily affects the axial skeleton, peripheral joints, and extra - articular structures. the pathophysiological basis of as has not yet been well identified, but immunological mechanisms have been implicated.24 various functions of neutrophils have been shown to be enhanced in as ; these stimulated neutrophil functions mediate oxidative stress, which may have an important role in the pathogenesis of as.2527 oxidative stress describes situations in which the production of damaging oxidants exceeds the organism s capacity to neutralize them.2628 a decisive role of oxidative stress in inflammatory diseases like as has not yet been fully elucidated. however, reactive oxygen species produced by activated neutrophils during inflammatory reactions play important functions in the pathogenesis of many inflammatory diseases.24,29 inflammatory reactions trigger oxidative stress that leads to increased oxidant levels, which in turn decrease the level of antioxidants. the antioxidant defense system is responsible for protecting cells against the potentially harmful effect of oxidizing agents 30,31 in addition to these findings, a limited number of studies investigated the association between the pathogenesis of as and oxidative stress. for example, ozgocmen.32 reported that oxidative stress and lipid peroxidation were accelerated in untreated patients with active as but found no significant correlation between oxidant / antioxidant levels and disease activity. in another study, karakoc and coworkers24 indicated that increased oxidant and decreased antioxidant capacity might be related to the pathogenesis of as, but again no significant correlation between antioxidant / oxidant parameters and disease activity was found. we found no statistically significant difference in antioxidant enzyme levels in as patients upon comparison to healthy controls and also detected no significant correlation between antioxidant parameters and disease activity. however, various other reports in the medical literature have indicated alterations in serum pon and are activities for patients with different rheumatic diseases, such as ra, psoriasis, sle, and behcet s disease.2,1217 in many of these studies, pon and are activities were reported to be significantly lower in patients with rheumatic diseases than in healthy controls.2,1216 in another study, kiss and coworkers 16 indicated that are activity in patients was not different from that of controls, despite reduced pon activity in patients with sle. in contrast, toker.17 reported that serum pon1 and are activities were significantly higher in patients with psoriasis than in controls. thus, the range of findings remains complicated and controversial. to the best of our knowledge, the present study is the first to assess serum pon and are activities in patients with as. we found no significant difference in serum pon and are activities in patients with as when compared to those of healthy controls. in addition, there was no significant correlation among antioxidant parameters, disease activity, or the patient s functional ability. serum pon and are activities may be within normal ranges and may not be associated with disease activity and functional capacity in patients with as. further research is needed to provide a deeper understanding of this disease. | objectivesthe aim of this study was to investigate the activities of serum paraoxonase and arylesterase in patients with ankylosing spondylitis with respect to those of healthy controls, to assess whether these enzyme levels are related to disease activity and functional capacity.methodsthe study included 32 patients with ankylosing spondylitis whose diagnoses were made according to the modified new york criteria as well as 25 healthy controls matched for age and sex. the bath ankylosing spondylitis disease activity index and the bath ankylosing spondylitis functional index were applied to the ankylosing spondylitis patients. as laboratory parameters, the erythrocyte sedimentation rate and serum c - reactive protein level were measured in patients and control subjects. paraoxonase and arylesterase enzyme activities were measured using appropriate methods.resultsno statistically significant differences (p>0.05) were found between the ankylosing spondylitis patients and controls in terms of serum paraoxonase or arylesterase levels. furthermore, there was no correlation between clinical and laboratory parameters in patients with ankylosing spondylitis.conclusionserum paraoxonase and arylesterase levels in ankylosing spondylitis patients may not differ from those of healthy controls, and there is no significant correlation between antioxidant parameters and the bath ankylosing spondylitis disease activity index or bath ankylosing spondylitis functional index scores in ankylosing spondylitis patients. further research is needed to provide deeper understanding of this disease. |
for the surgical treatment of stage ib1 cervical cancer, open radical hysterectomy with pelvic lymph node dissection has been the gold standard for over 100 years and has undergone little modification since it was first described by wertheim and later by meigs. techniques for laparoscopic radical hysterectomy and lymph node dissection were developed in the early 1990s [3, 4 ] and considerable experience has been gained since with over 1,000 cases reported in literature. after initial experience with robotic systems that are no longer available, the introduction of the da vinci surgical robotic system (intuitive surgery, mountain view, ca, usa) and subsequent fda approval for gynaecological use in 2005, has made the laparoscopic approach to complex radical gynaecologic operations more feasible. the robotic system has an advantage over the traditional laparoscopic approach regarding improved articulation of instruments, stereoscopic vision, tremor reduction and motion downscaling. these features suggest that a shorter learning curve may be achieved compared to conventional laparoscopy. robot - assisted laparoscopic surgery is used in gynaecology for benign hysterectomy, myomectomy, tubal reanastomoses, radical surgery, lymph node dissections and sacrocolpopexias [9, 10 ]. robot - assisted laparoscopic radical hysterectomy (ralrh) for cervical cancer has been recently introduced. a total of 313 cases in 11 reports have now been published [1120 ]. these early reports describe operation time, blood loss, hospital stay, lymph node count and complications, but the aspect of the learning curve is not well described. one report describes the aspect of the learning curve more in detail. in the present study, we compare 14 open radical hysterectomies (rh) with 14 ralrhs including sentinel node detection. we analysed patient - related aspects (blood loss, operating time, radicality of surgery (lymph node count), hospital stay and follow - up) and surgeon - related aspects (operating time) of the learning curve of the surgical team in the transition from open to robot - assisted laparoscopic radical hysterectomy. a total of 14 cases underwent an open rh between july 2004 and july 2006, and subsequently 14 patients were operated with the use of the da vinci robot between august 2006 and january 2008 at the vu university medical centre in the netherlands. all the operations were performed by the same surgical team. in one case from the robot group,, we introduced the laparoscopic pelvic lymph node dissection (lplnd) with sentinel node (sn) detection for the surgical treatment of figo ia2iia cervical cancers in 2000. when the sn contains a metastasis, the procedure is abandoned, and the patient subsequently receives chemo - radiotherapy. the aim of this approach is to reduce the number of patients undergoing radical hysterectomy followed by chemo - radiation as this leads to substantially more morbidity than either treatment alone, without obvious better survival [23, 24 ]. our surgical team first obtained experience with the da vinci robot system in a training facility ; consequently, experience was broadened on pelvic lymphadenectomy and sentinel node procedures. also, simple hysterectomies were performed with the system while continuing to perform radical hysterectomies by laparotomy. we subsequently started to perform robotic radical hysterectomies, using da vinci robotic system with four robotic arms (intuitive surgery, mountain view, ca, usa) (fig. 1) before and after this date, the last open procedures and the first robotic procedures by the same team were compared with respect to number of lymph nodes retrieved (as a marker for radicality of surgery), theatre time, blood loss, hospital stay and complications. 1four arm da vinci robot (intuitive surgery, mountain view, ca, usa) four arm da vinci robot (intuitive surgery, mountain view, ca, usa) technically, the robot - assisted procedure starts with the injection of tc99-nanocol the day prior to surgery and a scintigram is made to detect and localise the sns. on the day of surgery, immediately prior to the procedure 2-ml patent blue (patent blue v, 2.5% solution, laboratoire guerbet, roissy, france) is injected in the cervix around the tumour. a mccartney tube (4.5 cm) is inserted in the vagina and fixed to the cervix, by means of a suture through the cervix, which is attached to the end of the tube. the abdomen is insufflated through a verres needle just above the umbilicus where an 11-mm camera port is introduced. two 8-mm robotic troicarts are introduced at the level of and 10 cm laterally to the supra - umbilical incision. a 5th 11-mm port is introduced in the left upper quadrant for the assistant. the patient is then placed in extreme trendelenburg position ; and at this point, the robot system is attached. the procedure then continues with opening of the retroperitoneum lateral to the external iliac artery and all spaces paravesical and pararectal as well as the obturator fossa are opened in search for the sn, both visually and by use of a laparoscopic radiosensitive probe (europrobe, strassbourg, france) (fig. 2). if the sn contains no metastasis, the radical hysterectomy is started by dissection of the ovarian vessels and uterine artery by bipolar coagulation and selective coagulation and cutting. the specimen can now be removed vaginally with the mccartney tube, and the vaginal vault was sutured. all radical hysterectomies were performed at the rutledge type iii level both for the open and robotic procedure. statistical analysis was done using the statistical software package spss 15.0 (spss inc, chicago, il). the outcome for robot - assisted and open groups were compared using the chi - square test and fisher extract test for categorical variables and two sample student t test for continuous variables. 2sentinal node detection after injection of patent blue and with a laparoscopic radiosensitive probe sentinal node detection after injection of patent blue and with a laparoscopic radiosensitive probe the median age in the robot group is 43 years compared to 46 years in the open group. the distribution of histology was similar in both groups, squamous cell carcinoma 's predominated. in the open group high grade histology predominated whereas in the robot group most cases were graded as moderately differentiated (table 1). table 1patient characteristics, histology and stagerobot groupopen groupn1314agemedian (range)43 (3178)46 (3268)histologysquamous (%) 10 (77)11 (79)adeno (%) 2 (15)3 (21)endometrioid (%) 1(8)0 (0)grade100296348stage (figo)ib11112other(one endometrial cancer stage iib, one stage ib2 after neo - adjuvant chemo)(one stage ib2) patient characteristics, histology and stage the median number of lymph nodes removed was slightly higher in the robot group 29 versus 26 in the open group (not statistically significant) (table 2). blood loss was calculated by the difference in the total amount of suctioned and irrigated fluids. median blood loss in the open group was 2,000 ml (range, 1,0004,600 ml). for the robot group, median blood loss was 300 ml (range, 501,000 ml). in both groups, blood loss for sn+lplnd and radical hysterectomy was combined. hospital stay for the radical hysterectomy in the open group was at a median of 9 days (range, 716 days). in the robot group, the median hospital stay was 4 days (range, 314 days). one temporary ureteric obstruction occurred post - operatively, and the hydronefrosis was decompressed by percutaneous drainage. one complication occurred : an accessory ureter was cut requiring percutaneous drainage and re - anastomosis in a second procedure. theatre times are represented in real theatre time spent and are calculated from the start of anaesthetic preparations. theatre time ends when the patient leaves the operating table. for the open group, the median theatre time for the radical hysterectomy was 3 h:45 min (range, 2:505:30). for the robot group, this was 7 h:23 min (range, 4:2410:10) and includes the sn+lplnd in 10 of 13 cases. in the remaining three cases, the sn+lplnd was performed in a separate surgical session, and the console time of the sn+lplnd was added to the total theatre time of the radical hysterectomy. the theatre times are decreasing dramatically over time in the robot group, whereas theatre time in the open group has obviously remained stable (fig. 3). table 2comparison of the theatre time, lymph nodes removed, blood loss, hospital stay, complications and recurrences for radical hysterectomy patientsrobot group (n = 13)open group (n = 14)p valuetheatre time (min)434 (264610)225 (170330)<0.001 median (range)ln removed (n)29 (1976)26 (1041)0.064 median (range)blood loss (ml)300 (501,000)2,000 (1,0004,600)<0.001 median (range)hospital stay (days)4 (214)9 (716)<0.001 median (range)complications (n)13 numberfollow - up (months)26 (1732)42 (3154)<0.001 median (range)recurrences (n)21 number comparison of the theatre time, lymph nodes removed, blood loss, hospital stay, complications and recurrences for radical hysterectomy patients in a median follow - up of 42 months (range, 3154), for the open group, one pelvic sidewall recurrence occurred 7 months after the radical hysterectomy. the robot group has a mean follow - up of 26 months (range, 1732) in which two recurrences occurred. one pelvic sidewall recurrence and a port metastasis occurred 12 months after primary surgery. this was treated with excision of the port metastasis and chemo - radiation on the pelvis. the second recurrence occurred 17 months after primary surgery and was located in the rectum, a posterior exenteration was performed. all but one patient received single modality treatment. in one patient from the open group, post - operative radiotherapy was required due to a narrow vaginal margin. in this study, we could confirm the findings of previous authors describing the ralrh for the treatment of cervical cancer [11, 1318, 20 ]. they also conclude that this technique is feasible and could be of benefit to the patient regarding reduction of blood loss and reduction of hospital stay. we found a significant reduction of blood loss, and hospitalisation was reduced by 5 days in the robotic group indicating significant quicker recovery compared to open surgery. one aspect of quality of surgery is the number of pelvic lymph nodes removed. in this study, lymph node counts were higher in the robotic group, although this was not statistically significant. most other authors could not find a statistically significant difference in lymph node counts compared with open surgery, but there seems to be a trend towards a higher lymph node retrieval in patients operated with robot assistance [14, 15, 17 ]. an explanation could be that the studies were too small to show a statistically significant difference. recent larger reports concerning the ralrh confirm this trend and found a statistically significant higher lymph node count in the robotic group [11, 12 ]. it is likely that this may be attributed to instrument articulation, 3d view and motion downscaling, allowing a very selective dissection in robot - assisted surgery. in our study, the complication rate was lower in the robot - assisted group ; this is consistent with the data described by other authors [11, 12, 14, 15 ]. there where twice the number of recurrences in the robotic group, and the follow - up was shorter in the robotic group (table. 2). however, numbers are too small to draw statistical conclusions for recurrences and complication rates. in addition, we showed that theatre time may rapidly be reduced to the level of an open radical hysterectomy. a learning curve of 14 cases reduced the team 's total theatre time by 48%. our learning curve is comparable with the only other published detailed learning curve for the ralrh described by fanning.. we plotted their learning curve of the first 14 robotic cases together with our learning curve (fig. their reduction in theatre time is 35% after 14 cases and 59% after 20 cases. reported their skin - to - skin time for the ralrh (with or without sentinel node) and found also a rapid decrease of surgery time. the learning curve for the robot hysterectomy with pelvic and para - aortic lymphadenectomy for endometrial cancer is described in detail by seamon. they conclude the learning curve for this procedure seems to be approximately 20 cases ; however, efficiency continues to improve after the first 20 cases. 3theatre time for the radical hysterectomy (open procedure and robot procedure) also plotted the learning curve from fanning. theatre time for the radical hysterectomy (open procedure and robot procedure) also plotted the learning curve from fanning. our team gradually introduced the use of the robot and had experience in laparoscopic oncological surgery and open radical surgery for many years. when a more rapid transition to robotic surgery is chosen, the learning curve may be longer. in our study, we purposely measured real theatre time as it is our experience that a significant amount of time may be lost in preparing and positioning of the patient as well as positioning and introducing the robotic system. this is also stated by seamon. who looked in detail at theatre time, skin - to - skin time and console times for the robotic hysterectomy with lymphadenectomy for endometrial cancer. measuring pure console time, as is done in many other studies on robotic surgery, does not reflect the effort of the whole team 's learning curve and may therefore not represent a true comparison with open surgery. in our study this is mainly due to the sn technique where the anatomical spaces are all opened before starting the lymph node in search of the sn. a dedicated surgical and anaesthesiological team tremendously increases efficiency and reduces the time spent in this phase of the procedure. anaesthesiological aspects of robotic surgery should be well appreciated before embarking on robotic surgery [29, 30 ]. interestingly, anaesthesiological complications did not occur despite prolonged surgical times with the patient in extreme trendelenburg position. there is still a lack of literature about the learning curve for robot - assisted gynaecological procedures. in urology, the aspect of the learning curve for radical prostatectomies is well described, and compared to conventional laparoscopy, the learning curve for robotic surgery is significantly shorter [31, 32 ]. recently, the learning curve of the robot - assisted sacrocolpopexy was described, a reduction of operative time of 25% after ten cases was found, again suggesting a relatively short learning curve. lenihan. performed 113 robot - assisted procedures (mainly hysterectomies, but also myomectomy, sacrocolpopexy and oophorectomy). with the use of a dedicated team, they were able to set up the robot for surgery in 45 min after 20 cases and in 35 min within 50 cases. robot console times and total operative times were consolidated after approximately 50 cases at about 50 min for console time and 90 min for total operative time. forty cases of benign gynaecologic procedures by a single surgeon are described by pitter. after 20 cases, a statistical improvement in operative time is reached. both studies do not differentiate between the procedures performed. in conclusion our data suggest a relatively short learning curve with quick improvement of patient and surgeon - related parameters. | we analysed the introduction of the robot - assisted laparoscopic radical hysterectomy in patients with early - stage cervical cancer with respect to patient benefits and surgeon - related aspects of a surgical learning curve. a retrospective review of the first 14 robot - assisted laparoscopic radical hysterectomies and the last 14 open radical hysterectomies in a similar clinical setting with the same surgical team was conducted. patients were candidates for a laparoscopic sentinel node procedure, pelvic lymph node dissection and open radical hysterectomy (rh) before august 2006 and were candidates for a laparoscopic sentinel node procedure, pelvic lymph node dissection and robot - assisted laparoscopic radical hysterectomy (ralrh) after august 2006. overall, blood loss in the open cases was significantly more compared with the robot cases. median hospital stay after ralrh was 5 days less than after rh. the median theatre time in the learning period for the robot procedure was reduced from 9 h to less that 4 h and compared well to the 3 h and 45 min for an open procedure. three complications occurred in the open group and one in the robot group. ralrh is feasible and of benefit to the patient with early stage cervical cancer by a reduction of blood loss and reduced hospital stay. introduction of this new technique requires a learning curve of less than 15 cases that will reduce the operating time to a level comparable to open surgery. |
the inappropriate disposal of dyes in waste water constitutes an environmental problem and can cause damage to the ecosystem wastewater containing dyes cause water pollution by lowering light penetration and photosynthesis and toxicity from heavy metals associated with pigments. synthetic dyes are widely used in industries such as textiles, leather, paper, and plastics to color final products. reactive dyes are the most common dyes used due to their advantages, such as bright colors, excellent colorfastness, and ease of application but effluents are highly toxic to aquatic life. these dyes are mostly resistant to biodegradation and therefore are not removed by conventional treatment techniques. dyes are colored substances which colored a substrate as they retained in it by adsorption. these are soluble completely or partially soluble in a media from which they are applied in contrast with pigments. synthetic dyes have complex aromatic structures which provide them physiochemical, thermal, biological, and optical stability and used in photographic paper as indicator and as biological stains. commercial uses of dyes include the coloration of textile paper, leather, wood, inks, fuels, food items and metals. this dye may cause problems in, respiratory tract, gastrointestinal tract, irritation to skin, and redness of eyes. prasad. studied the biosorption of coomassie brilliant blue by acid treated coir pith in batch mode. effect of time, initial concentration of dye and initial adsorbent concentration on biosorption was investigated. the investigation shows that the acid - treated coir pith is better suited for the removal of dyes from the textile industry effluent. katarzyna and watala studied a new photometric method for determining the surface area of plants, which relies on the adsorption of coomassie brilliant blue g-250 on the plant surfaces. the method was applied to a variety of aquatic plants and found to provide a routine procedure to determine plant surface area. compared to activated carbon and/or carbon fiber adsorption, in this research, the characteristics and mechanism of adsorption were examined with the aim to develop an efficient dyestuff adsorption system using okara. the amount of the saturation dye adsorption, qm (mg - dye / g - okara) obtained from the langmuir adsorption isotherm after 24 h at ph 3 was 197. it was found that okara 's adsorption ability is approximately 66 times higher than the ability of activated carbon. rauf. studied three organic dyes namely, coomassie blue, malachite green and safranin orange by adsorption on sand at 298 k. characteristics of local sand sample used as an adsorbent in this work were initially found from the low - temperature adsorption of nitrogen on sand samples at 77 k. conditions for maximum adsorption of these dyes on sand sample were then optimized. the adsorption behavior of the dyes was also investigated in terms of added cations and anions and it was found that adsorption of coomassie blue and safranin orange decreased substantially in the presence of sulphate, thiosulphate, acetate, potassium, and nickel and zinc ions. wei. studied the interactions of coomassie brilliant blue g-250 (cbb) with bovine serum albumin (bsa) and 7-globulin at low ph that are investigated by a spectrophotometric method. it is considered that the binding of cbb to protein is because of the weak interactions (ionic, van dar waals, hydrogen bonding, and hydrophobic). factors which influence the sensitivity of the cbb protein assay are studied using this method. ionic strength and acidity are found to have significant effect on the binding of cbb to protein. the objective of the present study was to assess the ability of locally available bran for the removal of coomassie brilliant blue (cbb). the study was carried out with the aim to optimize conditions for maximum removal of this dye from aqueous solutions by using wheat bran. besides this, the adsorption data was fitted to various equations to obtain constants related to the equilibrium of the adsorption phenomena. pure coomassie brilliant blue r-250 was used for the preparation of standard coomassie brilliant blue r-250 stock solution. the main characteristics of comassie brilliant blue are and max = 580 nm, color index (c.i) = 42660, chemical formula = c45h44n3nao7s2, and m.w. = 825.97 g / mol and is insoluble in cold water and slightly soluble in hot water. wheat has a hard outer layer. when it is processed, this layer becomes a byproduct, and is called bran. in the case of processing wheat to make wheat flour, after three days wheat bran was put in oven at 60c for twenty four hours. dried wheat bran was thoroughly washed with distilled water to remove all dirt and was dried at 100c till constant weight. then the dried wheat bran was grinded and sieved with a 30-mesh sieve, to prepare homogenous particle size. this biosorbent was again washed with distilled water to remove adhearing dirt and foreign particles. the average particle size of adsorbents was measured and it was 500 m and bet surface area was measured by the brunauer, emmett and teller (bet) method using n2 gas adsorption (quanta - chrome inst., nova 2200e surface area analyzer) and it was calculated as 8.62 m / g. 100 mg / l comassie brilliant blue stock solution was prepared in distilled water and stored in brown reagent bottles in dark place to prevent oxidation of the dye solution. various concentrations of comassie brilliant blue solutions ranging from 1 to 10 ppm were prepared from the comassie brilliant blue stock solution in 100 ml measuring flasks.. with the help of double beam spectrophotometer absorbance of comassie brilliant blue was measured at wavelength of 580 nm in a 1.0 cm matched quartz curettes against a reagent blank (without soln.) on through mixing. at this wavelength the values obtained from this experiment were given in table 1 and graph shown in figure 1. the adsorption of comassie brilliant blue on wheat bran was studied by a batch technique. the general method used for these studies 0.1 g of the dried sieved wheat bran was equilibrated with 10 cm of the comassie brilliant blue solution of the known concentration in a stopper pyrex glass flasks at fixed temperature in a shaker for a known period of time. the flasks containing the weighed amounts of wheat bran and comassie brilliant blue solution were separately kept in the before mixing for a sufficient period of time to attain the desired experimental temperature. after equilibration the suspension was centrifuged in a stopper tube for 10 minutes at 3500 rpm. the concentration of comassie brilliant blue in a known volume of the supernatant was determined by spectrophotometrically. adsorption of comassie brilliant blue on wheat bran was determined in terms of equilibrium concentration (ce), percentage removal p%, adsorption capacity qe determined as, the equilibrium concentration (ce) is determined. the amount adsorbed per unit weight of the wheat bran, x / m, was calculated spectrophotometrically from the initial (known comassie brilliant blue concentrations) and final absorbance of the solutions. the adsorption of c.b.b on wheat was studied as a function of shaking time at 35c. a sample of 50 cm of c.b.b (10 ppm) solution was taken in nine titration flasks, labeled them from 19, and shaken with 0.1 g of wheat bran in each flask for different intervals of time ranging from 5 min to 60 min in a shaker. after 15 minutes all the solutions were filtered and absorbance was determined photometrically at wavelength 560 nm. from it, calculate the equilibrium concentration (ce) of c.b.b, percentage removal, and equilibrium time was determined by plotting a graph between different time intervals and percentage removal. figure 1 shows the variation of % age adsorption as a function of shaking time. the result shows that the adsorption of c.b.b on wheat bran is symmetrically time dependant which increases with the shaking time rapidly and gets to an equilibrium stage after 30 minutes which dose not change latter on. the values of equilibrium concentration (ce) and % age adsorption were shown in figure 1. as adsorption and desorption occur side by side so after an appropriate time equilibrium takes place. composition of wheat bran also plays a major role in this regard. at 30 minute, the maximum adsorption is observed due to the attractive forces developed between cbb and wb. after complete adsorption on outer surface, the dye enters to the inner surface via pores. the adsorption of c.b.b on wheat bran was studied as a function of different ph. 110, while keeping all other parameters, that are, c.b.b concentration (10 ppm), shaking time (30 min). after equilibration, the suspension was centrifuged for 30 minutes at 3500 rpm. after 15 minutes all the solutions were filtered and absorbance was determined photometrically at wavelength 560 nm. from it, calculate the amount of equilibrium concentration (ce) of c.b.b, percentage removal, and determined the adsorption of c.b.b on wheat bran at different ph by plotting a graph between different ph and percentage removal. figure 2 shows the variation of equilibrium concentration (ce) and percentage removal as a function of different ph. the result shows that the adsorption of c.b.b on wheat bran increases with increasing ph and attains to maximum at ph 2.0. both ce and % age adsorption also increase with increasing ph till ph 2.0 and then decreased sharply. the values of equilibrium concentration (ce) and % age adsorption were shown in figure 2.. it may be due to the increased positive charges on the surface of adsorbent that attracts the functional groups of a dye carrying negative charge. the adsorption of c.b.b on wheat bran was studied as a function of different amount of adsorbent, that is, 0.11.0 g, while keeping all other parameters that is, c.b.b concentration (10 ppm), shaking time (30 min), and ph (2.0), of the suspension constant. calculate the equilibrium concentration (ce) of c.b.b and % age adsorption and determine the adsorption of c.b.b on wheat bran at different amount of adsorbent by plotting a graph between different amount of adsorbent and % age adsorption. figure 3 shows the variation of % age adsorption as a function of different amount of adsorbent. the results show that the adsorption of c.b.b on wheat bran increases spontaneously with increasing adsorbent concentration due to availability of greater active sites. on the basis of these results, 0.1 g/50 cm of c.b.b solution was selected for further studies. the values of equilibrium concentration (ce) and % age adsorption were shown in figure 3. the decrease in % age removal of dye with increased amount of adsorbent is due to the fact that adsorption sites remain unsaturated during adsorption reaction. the adsorption of c.b.b on wheat bran was studied as a function of its different concentrations, that is, 530 ppm, while keeping all other parameters that is, shaking time (30 min) and ph (2.0), sorbent concentration (0.1 g) constant. calculate the equilibrium concentration (ce) of c.b.b and % age adsorption and determined the adsorption of c.b.b on wheat bran at its different concentrations by plotting a graph between different concentration of sorbate and % age adsorption. figure 4 shows the variation of % age adsorption as a function of different sorbate concentrations. the results show that the adsorption of c.b.b on wheat bran increases spontaneously with increasing sorbate concentration till 30 ppm and then become constant onward. % age adsorption increases continuously with increasing c.b.b concentration. on the basis of these results 0.2 g/1000 cm of c.b.b solution the values of equilibrium concentration (ce) and % age adsorption were shown in figure 4. the adsorption of c.b.b on wheat bran was studied as a function of different temperature, that are, 30c, 40c, 50c, 60c, 70c, and 80c, while keeping all other parameters that are, c.b.b concentration (10 ppm), shaking time (30 min), ph (2.0), and sorbent concentration (0.1 g), constant. from it calculate the equilibrium concentration (ce) of c.b.b and % age adsorption and determined the effect of temperature on adsorption of c.b.b on wheat bran by plotting a graph between different temperature and % age adsorption. figure 5 shows the variation of % age adsorption as a function of different temperature. the results show that the adsorption of c.b.b on wheat bran was increased spontaneously with increasing temperature. the values of equilibrium concentration (ce) and % age adsorption were shown in figure 5. increase in adsorption at elevated temperature is due to the enlargement of pore size from where dye adsorbed. the adsorption of c.b.b on wheat bran was studied as a function of different c.b.b concentration that is, 530 ppm, while keeping all other parameters that are, shaking time (30 min), ph (2.0), and sorbent concentration (0.1 g) constant. 50 cm of c.b.b (5 ppm to 30 ppm) solution was shaken with 0.1 g of wheat bran for 30 min in a shaker. after 15 minutes, the absorbance was determined photometrically at wavelength 560 nm. then calculated the adsorbed amount of c.b.b and equilibrium concentration (ce) and determine the adsorption isotherms c.b.b on wheat bran by plotting a graph between extent of adsorption (logq) and equilibrium concentration log ce (moldm) the adsorption isotherms were used to investigate the relationship between the concentration of sorbed species and sorption capacity of sorbing species. langmuir adsorption isothermlinear form of langmuir adsorption isotherms is shown as follows : (1)1qe=1qmax + 1bqmax1ce qe : sorption capacity, ce : equilibrium concentration, qmax : maximum possible amount of dye that can be adsorbed per unit dry weight of sorbent, b : empirical constant, indicating the affinity of sorbent towards the sorbate. linear form of langmuir adsorption isotherms is shown as follows : (1)1qe=1qmax + 1bqmax1ce qe : sorption capacity, ce : equilibrium concentration, qmax : maximum possible amount of dye that can be adsorbed per unit dry weight of sorbent, b : empirical constant, indicating the affinity of sorbent towards the sorbate. a curve 1/qe versus 1/ce has been plotted to investigate the fitting of langmuir model for the equilibrium data of wb - dye sorption a straight line was obtained. the correlation coefficient r was found to be 0.9732 indicating that the data was fitted well the langmuir model (figure 6). from the langmuir plot, qmax = 6.410 mg / g and b = 3.502 l / mg. the characteristics of the langmuir isotherm can be expressed as another constant separation factor or equilibrium parameter given by rl. (2)rl=11+klco, where kl is langmuir 's equilibrium constant which is related to the affinity of binding sites and kl = qmax b. co is the initial dye concentration. according to mckay. is found to be 0.0042636 and again the adsorption is found to be favorable. freundlich absorption isothermlinear form of freundlich adsorption isotherms is shown as [1618 ]. (3)logqe = logkf+1nlogce, where qe is the equilibrium dye concentration sorbed in sorbent (mg g), ce is the dye equilibrium concentration in solution (mg kf is freundlich constant related to the sorption capacity, and 1/n is an empirical parameter related to sorption intensity, which varies with heterogeneity of the material. the value of n is between 01 indicating favorable sorption of cbb (figure 7). (3)logqe = logkf+1nlogce, where qe is the equilibrium dye concentration sorbed in sorbent (mg g), ce is the dye equilibrium concentration in solution (mg kf is freundlich constant related to the sorption capacity, and 1/n is an empirical parameter related to sorption intensity, which varies with heterogeneity of the material. the value of n is between 01 indicating favorable sorption of cbb (figure 7). this equation is used to determine sorption mean energy which enabled us to estimate whether adsorption is carried out by ion exchange mechanism, physisorption or, chemisorptions. (4)lnqe = lnqm2,=rtln(1 + 1ce), where qm is a theoretical saturation capacity, is a constant related with sorption energy, molj, is a polonyi potential and is equal to rtln(1 + 1/ce). mean sorption energy e is determined by (5)es=12. is calculated from slope of a linear graph lnqe versus shown in figure 8. the value of sorption mean energy is 0.26 kjmol. it is positive and less than 8 kjmol. the effect of a change in temperature on the sorption system was studied to determine the thermodynamic parameter and to investigate the nature of the process. thermodynamic parameters for adsorption of c.b.b on wheat bran such as heat of adsorption (enthalpy change, h), entropy change, s, free energy of specific adsorption and g were calculated from the binding constant kc obtained from langmuir plot by using the following equation. (6)go=hotso. below is the equation of straight line : (7)go=rtlnkd, kd = qece, lnkd=ho2.303rt+so2.303r. for activation energy, following equation is used (8)lnk = lnaea2.303rt.ea can be calculated from slope of a graph lnk versus 1/t as shown in figure 10. where r is the gas constant, kc is the equilibrium constant, t is the temperature (k), co is the equilibrium concentration of c.b.b on adsorbent (mg / l), and ce is the equilibrium concentration of c.b.b in the solution (mg / l). the values of thermodynamic parameters h and s for the c.b.b. on wheat bran calculated from above equations the negative values of g indicates the spontaneous nature of adsorption. as the temperature increases, the g values increase, indicating more driving force and hence resulting greater adsorption capacity at higher temperatures. the lagergrens kinetics equation has been most widely used for the adsorption of an adsorbate from an aqueous solution. the model is based on the assumption that rate is directly proportional to the number of free sites according to lagergren equation : (9)log(qeqt)=logqekt2.303. linear plot of log (qe qt) versus t was plotted to evaluate this kinetic model and to determine rate constant k. the values of r and k are 0.469 and 0.002073, respectively, shown in figure 11. it depicts that lagergren model is not fitted in sorption of cbb on wb. the pseudo - second - order model is based on the biosorption followed by second - order mechanism nearby the rate of sorption is proportional to the square of number of unoccupied sites and can be represented as (10)tq=1k2qe2+tqt. the parameters qe and k2 are calculated from slope and intercept of a plot t / qe versus time shown in figure 12. the value of r is 0.8396 that shows pseudo - second - order model is best fitted. the result of this study indicates that wb is an effective sorbent for the uptake of toxic dye from aqueous solution. the optimum parameters for equilibrium study are time of contact 30 min, ph 2, and dose of w.b 2 gl under optimum conditions. the sorption of cbb dye by wb followed a monolayer sorption model langmuir isotherm rather than multilayer model. | the sorption studies of coomassie brilliant blue (cbb) from aqueous solution have been carried out on wheat bran (wb). coomassie brilliant blue on wheat bran was used to study the adsorption behavior under various parameters such as ph, dosage amount, and contact time. it was observed that under optimized conditions up to 95.70% dye could be removed from solution onto wb. langmuir and freundlich adsorption isotherms were used to elaborate the results. freundlich model was found to be fitted well and favored multilayer adsorption. the freundlich constants n and kf were determined as 0.53 and 2.5 104. thermodynamic parameters such as g, h, and s studied were taking into account, showed spontaneous and favorable reaction for coomassie brilliant blue on wheat bran. the maximum adsorption capacity qm was found to be 6.410 mg / g. the investigations show that non treated wb is a low - cost adsorbent for the removal of dyes from textile industry effluents. |
we report the presentation and management of a single case of human toxicity of sodium chlorite. a 65-year - old man presented to hospital after accidentally ingesting a small amount of a sodium chlorite solution. his principal manifestations were mild methemoglobinemia, severe oxidative hemolysis, disseminated intravascular coagulation, and anuric acute kidney injury. he was managed with intermittent hemodialysis, followed by continuous venovenous hemofiltration for management of acute kidney injury and in an effort to remove free plasma chlorite. concurrently, he underwent two red cell exchanges, as well as a plasma exchange, to reduce the burden of red cells affected by chlorite. these interventions resulted in the cessation of hemolysis with stabilization of serum hemoglobin and platelets. this is only the second case of sodium chlorite intoxication reported in the medical literature and the first to report the use of renal replacement therapy in combination with red cell exchange in its management. sodium chlorite is a powerful oxidizing agent used to generate chlorine dioxide, which has several applications, including bleaching of pulp, paper, and textiles. when ingested, oxidizing agents can lead to methemoglobinemia, and intravascular hemolysis may ensue. we report the only known case of accidental sodium chlorite ingestion leading to mild methemoglobinemia, severe hemolysis, and acute kidney injury (aki) successfully treated with hemodialysis, followed by hemofiltration and concurrent red cell and plasma exchange. a 65-year - old man presented to his local hospital with nausea, vomiting, diarrhea, and dark urine after ingesting a small amount of a 28 % sodium chlorite solution. he had diluted the sodium chlorite in a cup with an unmeasured amount of water and was using this solution to clean his fruit. he then accidentally drank a mouthful of the solution after confusing this cup with another that contained only water. upon ingestion, he immediately self - induced vomiting, and only after 4 h when he noticed dusky finger tips and lips, did he present to the hospital. he was transferred to our institution and, upon arrival, was hemodynamically stable, mildly confused, and anuric. g / l, white blood cell count 22.1 10/l, normal platelet count, creatinine 144 mol / l, sodium 141 mmol / l, potassium 5.5 mmol / l, bicarbonate 19 mmol / l, chloride 114 mmol / l, calculated anion gap 14, ionized calcium 1.13 mmol / l, phosphate 1.37 mmol / l, and haptoglobin of < 0.08 bilirubin and lactate dehydrogenase were unable to be processed by the laboratory, but the serum was described as being brownish in color. urine was brown, and examination showed 3 hemoglobin, with few red blood cells and many hemegranular casts. initial arterial blood gas showed ph 7.35, paco2 35 mmhg, pao2 256 mmhg, methemoglobin (methb) 6.7 %, and lactate 1.8 mmol / l. he was admitted to the intensive care unit and treated with an 8-h session of hemodialysis to manage hyperkalemia and to attempt removal of sodium chlorite. we used a high - flux dialysis filter (toray ts-1.6sl polysulfone filter) and prescribed a blood flow of 400 ml / min with a dialysate flow of 500 ml / min to maximize clearance. the dialysate contained electrolytes in the following concentrations : bicarbonate 35 mmol / l, sodium 140 mmol / l, potassium 2 mmol / l, magnesium 0.75 mmol / l, calcium 1.5 mmol / l, and glucose 8 mmol / l. he was not treated with methylene blue or ascorbic acid but was started on a high - dose n - acetylcysteine infusion. we used the 21-h intravenous n - acetylcysteine regimen recommended in acetaminophen toxicity (150 mg / kg over 1 h, followed by 50 mg / kg over 4 h then 100 mg / kg over 16 h). while on dialysis, his methb levels decreased to normal, but upon its completion, he developed an increasing lactate (8.2 mmol / l), a drop in hemoglobin to 87 g / l, and hemodynamic instability requiring intravenous fluid resuscitation and vasopressor support with norepinephrine (10 mcg / min). a serum glucose-6-phosphate dehydrogenase (g6pd) screen revealed deficiency, and plasma - free hemoglobin mg / l. a peripheral blood film showed blister and bite cells consistent with oxidative hemolysis, with no evidence of microangiopathic hemolysis. it also showed marked thrombocytopenia due to peripheral consumption with platelets of 28 10/l. in addition to thrombocytopenia, elevated pt inr (1.4) and d - dimer (15.43 mg / l) were consistent with the diagnosis of disseminated intravascular coagulation (dic). high fio2 was delivered via a non - rebreather mask, and continuous venovenous hemofiltration (cvvh) with a total effluent of 50 ml / kg was initiated. the filter used was gambro st100 an69 membrane, and replacement fluid was gambro prismasol with electrolyte concentrations as follows : bicarbonate 32 mmol / l, sodium 140 mmol / l, potassium 3 mmol / l, chloride 108 mmol / l, magnesium 0.5 mmol / l, and lactate 3.0 mmol / l. concurrently, red cell exchange was initiated via a separate 12 french dual lumen central venous catheter. upon completion of the 2-h long red cell exchange (total of 3.3 l exchanged), his symptoms improved, and the lactate and plasma - free hemoglobin decreased to 4.8 this was accompanied by worsening nausea and back pain, as well as an increase in lactate to 9.5 mmol / l and a recurrence of hemodynamic instability. he underwent a second session of red cell exchange (2 h with total exchange of 2 l), which was followed by plasma exchange (3.5 h with 5.2 l exchanged). with this exchange 1serum hemoglobin, lactate, and plasma - free hemoglobin (pfh) over time serum hemoglobin, lactate, and plasma - free hemoglobin (pfh) over time he subsequently remained stable with no further drops in hemoglobin or rises in lactate. repeat screens for g6pd deficiency at 4 days and 12 months were negative. he was continued on continuous renal replacement for 96 h in total at which time he was transitioned to intermittent hemodialysis as he remained anuric ; 17 days after admission to hospital, he was no longer dialysis - dependent and was discharged home. over the following 2 months, its primary commercial application is that of a bleaching agent in the pulp and paper and textile industries. outside of its commercial uses, it has numerous unfounded claims as a natural health cure for a multitude of health issues. although there is not an abundance of data regarding the physiologic effects of sodium chlorite, several animal and human studies have documented its ability to cause methemoglobinemia, hemolysis, and glutathione depletion. more physiologic data exist for the related compound, sodium chlorate [3, 4 ]. a study by singelmann. examined the effect of incubating human red cells with varying concentrations of chlorate, showing that these red cells develop glutathione depletion and methemoglobinemia. this drug - induced g6pd deficiency, in addition to glutathione depletion and membrane rigidity, leads to oxidative hemolysis and explains the lack of efficacy of methylene blue in these patients [3, 4 ]. importantly, steffen. noted that the presence of methemoglobin was necessary for inhibition of g6pd and cross - linking of membrane proteins. if, however, hypochlorite was used instead of chlorate ; oxidation of hemoglobin and oxidative hemolysis occurred independent of methemoglobin. this feature may explain the severity of hemolysis in our case, despite the presence of only mild methemoglobinemia. only one previous case of acute sodium chlorite toxicity has been reported in the literature. lin. described a case of intentional ingestion of sodium chlorite leading to severe methemoglobinemia, hemolysis, dic, and renal failure. the patient presented with a methb level of 59 %, which did not respond to administration of multiple doses of methylene blue. our patient presented with similar gastric upset, likely as a result of chlorite - induced irritation of the gastric mucosa. unlike the previous case,, he developed inhibition of g6pd and went on to suffer life - threatening oxidative hemolysis. this suggests that methemoglobinemia is not a prerequisite for chlorite - induced enzyme inhibition and membrane protein cross - linking. due to the low levels of methb, acute g6pd deficiency, and previous lack of efficacy reported for chlorate and chlorite ingestions, we chose not to administer methylene blue or ascorbic acid. we did, however, employ high - dose n - acetylcysteine in an attempt to replenish depleted glutathione stores. although we had no data to suggest its use, we felt that there was a theoretical benefit with minimal risk of adverse consequences. it is unclear what factors contributed to the discrepancy between methb levels and hemolysis in the two cases. it may be that a smaller ingested amount of chlorite resulted in less methb, although this leaves unexplained the similar degree of hemolysis in the two cases. it is plausible, as explained above, that oxidative hemolysis with chlorite may occur independently of methemoglobinemia. in the absence of severe methemoglobinemia, we believe that the intermittent lactic acidosis resulted from a combination of decreased oxygen - carrying capacity and reduced tissue perfusion. the patient had hemolyzed more than half of his red cells, thereby reducing oxygen - carrying capacity of more than 50 %. in addition, massive hemolysis can lead to hypotension due to peripheral vasodilation, thereby leading to reduced tissue perfusion and lactic acidosis. this explains the simultaneous occurrence of hemolysis, hypotension, and increasing serum lactate levels. lin. reported a diagnosis of acute interstitial nephritis in a patient with sodium chlorite toxicity. findings on renal biopsy included acute tubulointerstitial changes, lymphocytic infiltration, and severe interstitial edema. they also described moderate tubule damage with hemorrhage and necrosis along with focally collapsed loops. however, several of these findings, such as necrosis, are not usual findings of interstitial nephritis. in addition, the extremely rapid progression to anuria is not consistent with interstitial nephritis. several studies of chlorate, a chemically similar compound to chlorite, suggest that renal toxicity is due to a combination of methemoglobinuria, direct proximal tubular toxicity, and possibly lesions similar to those seen with the hemolytic uremic syndrome [4, 6 ]. in the absence of severe methemoglobinemia, it is likely that the patient s aki is accounted for by a combination of tubular toxicity from hemoglobin and renal ischemia due to poor oxygen delivery. the renal medulla has a very low oxygen tension in normal conditions and is, therefore, extremely sensitive to reduced systemic oxygen delivery. cases of renal failure with other forms of oxidative hemolysis support this theory. with a molecular weight of 67.45 daltons, unbound chlorite should be effectively removed by hemodialysis with a standard or high - flux dialysis membrane. our patient s hemoglobin and hemodynamics remained stable while receiving ihd, which we theorize may be related to removal of free chlorite. it is important to note that the amount of free versus bound chlorite in serum is unknown, and that hemodialysis was undertaken both to manage aki and in an attempt to remove any free chlorite. it is difficult to know whether or not extracorporeal removal of chlorite played a significant role in improving the patient s outcome, and it remains possible that it was of little benefit. in trying to remove small toxins, such as chlorite, high efficiency clearance achieved with intermittent the volume of distribution of chlorite is unknown ; therefore, a prolonged session of intermittent dialysis, similar to that used in a lithium overdose, is prudent. our patient developed worsening hemolysis after completion of a session of intermittent hemodialysis, which we suspected was due to a rebound increase in serum chlorite levels secondary to redistribution from the extravascular space. due to intermittent hemodynamic instability and in an attempt to avoid rebound increases in serum chlorite levels, continuous renal replacement therapy was initiated. we prescribed a relatively high dose of cvvh (50 ml / kg) to maximize clearance of chlorite. pure hemofiltration was chosen over dialysis in an effort to enhance clearance of free hemoglobin and limit renal tubular damage. it is, however, unlikely that removal of free hemoglobin altered the course of aki as the patient was already anuric. although not previously used in chlorite intoxication, red cell exchange has been used in numerous chlorate ingestions, with variable success [6, 8 ]. we undertook red cell exchange to reduce the burden of red cells affected by chlorite. it is unclear if his subsequent deterioration was due to residual abnormal red cells, serum chlorite affecting transfused red cells, or progressive dic from the persistent presence of hemolytic waste products in the plasma. a second red cell exchange was therefore performed together with a plasma exchange, resulting in cessation of hemolysis and continued physiologic stability. in conclusion, it is important to consider sodium chlorite ingestion when encountered by a patient with hemolysis, with or without methemoglobinemia. although no clinical evidence exists for its use, high - dose n - acetylcysteine may be considered for its theoretical benefit along with minimal risk. this is the first reported case of sodium chlorite toxicity managed with hemodialysis, hemofiltration, and concurrent red cell and plasma exchange. without larger case series or prospective studies, it is difficult to suggest this as standard of care ; but with physiologic reasoning, our successful patient outcome, and minimal risk, it should be strongly considered in the critically ill patient with related exposure. this article is distributed under the terms of the creative commons attribution license which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. | introductionsodium chlorite is a powerful oxidizing agent with multiple commercial applications. we report the presentation and management of a single case of human toxicity of sodium chlorite.case reporta 65-year - old man presented to hospital after accidentally ingesting a small amount of a sodium chlorite solution. his principal manifestations were mild methemoglobinemia, severe oxidative hemolysis, disseminated intravascular coagulation, and anuric acute kidney injury. he was managed with intermittent hemodialysis, followed by continuous venovenous hemofiltration for management of acute kidney injury and in an effort to remove free plasma chlorite. concurrently, he underwent two red cell exchanges, as well as a plasma exchange, to reduce the burden of red cells affected by chlorite. these interventions resulted in the cessation of hemolysis with stabilization of serum hemoglobin and platelets. the patient survived and subsequently recovered normal renal function.discussionthis is only the second case of sodium chlorite intoxication reported in the medical literature and the first to report the use of renal replacement therapy in combination with red cell exchange in its management. |
adenomatoid tumors, although rare, are the most common tumors of the paratesticular tissues, accounting for approximately 30% of all paratesticular tumors. in men, they are located in the epididymis, testicular tunicae, and rarely, the spermatic cord with most arising in or adjacent to the lower or upper pole of the epididymis. although benign in nature but because they are difficult to differentiate from testicular malignancy these men are often subjected to radical orchiectomy. recently, some authors have tried to define the imaging characteristics of these tumors but since these tumors are rare and experience is limited imaging is still not considered as diagnostic. we report a case that was diagnosed on the basis of clinical suspicion, biochemical investigations, fine - needle aspiration cytology findings and radiological tests and thereby high orchiectomy could be avoided. a 52-year - old male presented with complaints of a painless nodule in right scrotal sac for last 2-years that was gradually but very slowly increasing in size. there was no history suggestive of urinary infection, genitor - urinary tuberculosis or epididymo - orchitis. examination revealed a pea - sized firm, well - marginated, smooth swelling present at the lower pole of testis but that could not be separated with epididymis. scrotal ultrasonography revealed a solid mass at the lower pole of right testis that was relatively hyperechoic as compared to the testicular parenchyma [figure 1 ]. scrotal mri revealed a well - defined lenticular lesion at lower part of right testis. the lesion displayed signals isointense to slightly hypointense on both t1- and t2-weighted images with adjacent hypointense band. fine - needle aspiration from the mass revealed absence of malignant cells. in view of long clinical history, negative tumor markers, findings typical of adenomatoid tumor on sonography and mri, as reported previously, and absence of malignant cells on cytology local excision was performed under local anesthesia through inguinal incision. at exploration scrotal ultrasonography shows a solid mass at the lower pole of righttestis that was relatively hyperechoic as compared to the testicular parenchyma scrotal mri shows a well - defined lenticular lesion at lower part of right testis. the lesion displays signals isointense to slightly hypointense on both t1 and t2-weighted images with adjacent hypointense band most of these tumors have been present for several years and uniformly behave in a benign fashion. cellular atypia and local invasion have been observed occasionally. nonetheless, because of the long history of these tumors and benign nature surgical excision is adequate. a practical problem faced by clinicians is to differentiate this condition preoperatively from the much commoner testicular malignancy so as to avoid high inguinal orchiectomy especially in younger men. because of the adenomatoid tumor does not produce any characteristic pattern on ultrasound that would allow it to be distinguished from malignant testicular tumors because they can be hypo, hyper or even isoechoic as regards the adjacent parenchyma. it provides additional morphological evidence to allow precise localization of the origin of the mass, may also show contrast - enhancement features that enable further confidence of a benign diagnosis, and may allow conservative management. however, in our case we were able to diagnose this condition after thorough evaluation and thereby could conserve the testis. | paratesticular tumors are rare tumors that are difficult to diagnose preoperatively and therefore, many patients are subjected to inguinal orchiectomy. however, radical orchiectomy can be avoided as the diagnosis of paratesticular tumor can be made on the basis of clinical suspicion, findings of tumor markers and radiological tests. |
proton pump inhibitors (ppis) potently inhibit gastric acid secretion, and are widely used for prevention and treatment of various acid - related diseases including peptic ulcers and gastro - esophageal reflux diseases. although their acid inhibitor potency is far stronger than that of histamine h2 receptor antagonists (h2ras), ppis are reported to have some weak points in comparison with those drugs. a disadvantage of the acid inhibitory effect of ppis is the strong influence of cyp2c19, a hepatic drug metabolizing enzyme that degrades ppis. in patients with high activity of the cyp2c19 enzyme (extensive metabolizers), the effect of ppi administration is not adequately strong, because of enzymatic degradation. on the other hand, in patients with a low cyp2c19 enzyme activity (poor metabolizers), another disadvantage is slow onset of the acid inhibitory effect after ppi administration. to improve these weak points, new types of ppis rabeprazole is a new type of ppi that is not strongly influenced by cyp2c19 enzyme activity, because it is not mainly degraded by cyp2c19. in addition, this drug is reported to inhibit acid secretion more quickly than first generation ppis including omeprazole and lansoprazole. another agent is esomeprazole, an s - isomer of omeprazole that is a mixture of s- and r - isomers. esomeprazole has also been reported to not be effectively degraded by cyp2c19 and its effect is not strongly influenced by its enzyme activity. the acid suppressing effects of rabeprazole (20 mg) and esomeprazole (40 mg) have been investigated, with those of the latter reported to be equal or superior to the former. the standard doses of rabeprazole and esomeprazole in japan are 10 and 20 mg, respectively, per day. those ppis at those doses have not been directly compared in regard to quickness of acid inhibition and acid inhibitory potency in cases with different cyp2c19 enzyme activities. it is considered that intra - gastric ph monitoring soon after acute single administration of a ppi is an ideal experimental design to investigate its quick acid inhibitory effects. in the present study, a single standard dose of rabeprazole or esomeprazole was administered to normal volunteers in a multicenter double - blind randomized prospective study with a cross - over design and their effects on intra - gastric ph were compared. fifty - seven healthy volunteers were enrolled at 9 university hospitals ; shimane university, hokkaido university hospital, gunma university hospital, nippon medical school, yokohama city university hospital, hamamatsu university school of medicine, osaka city university, osaka medical college, and saga medical school. their mean age was in the 20s and none of the subjects was infected by helicobacter pylori (h. pylori), which was determined by testing for the presence of the h. pylori antibody in serum and urine samples. the cyp2c19 genotype was tested by a polymerase chain reaction - restriction fragment length polymorphism (pcr - rflp) assay, as previously reported. twenty - nine of the 57 enrolled volunteers were randomly enrolled in the pre - meal ppi administration protocol. the subjects were investigated by ph monitoring twice, once with 10 mg of rabeprazole and once with 20 mg of esomeprazole. the ppis were delivered in identical opaque gelatin capsules and discrimination between them was impossible during the study period. the identical opaque gelatin capsules containing 10 mg rabeprazole or 20 mg esomeprazole were prepared by an author pharmacist (kn) and packaged in marked bags, and then delivered to each participating hospital. the key code of the drugs was kept by kn and opened firstly after fixing the final ph data. the 2 ph monitoring examinations were separated by at least a 1-week interval. an intra - gastric ph monitoring sensor catheter (zenetics medical, salt lake city, ut) was introduced into the gastric body and monitoring was started at 17:00, as previously reported. a single oral dose of the ppi was administered at 15 min before supper, at 18:45. the subjects started eating their supper (carbohydrates 112.8 g, protein 16.3 g, fat 27.3 g, calories 762 kcal) at 19:00 and were asked to finish within 30 min. the subjects were then requested to lie on their bed from 23:00 to 7:00 next morning. breakfast (carbohydrates 34 g, protein 5.8 g, fat 2.8 g, calories 85 kcal) and lunch (carbohydrates 74.4 g, protein 17.1 g, fat 11.4 g, calories 531 kcal) were also consumed within 30 min, starting at 8:00 and 12:00, respectively. all conditions were the same as above, except that rabeprazole or esomeprazole was administered orally 30 min after the end of supper at 20:00. in the postprandial administration protocol, the supper (carbohydrate 52.8 g, protein 8.8 g, fat 9.2 g, calorie 330 kcal) and breakfast (carbohydrate 94 g, protein 13.3 g, fat 20.9 g, calorie 617 kcal) were also slightly different from those used in the pre - meal administration protocol. median ph for each monitored hour and the percentage of time at which intra - gastric ph was below 4.0 were calculated for the total 24-h period, as well as the daytime (7:0023:00) and nighttime (23:007:00) periods. statistical analysis was performed using a wilcoxon signed rank test when results of a friedman test showed significant differences. the chronological data shown in fig. 1, 3, 4 and 6 were analyzed by linear mixed models. the sample size of the study was calculated based on the previous studies comparing 40 mg esomeprazole and 20 mg rabeprazole on their first administration day. calculated the number of necessary subjects as 18 based on parametric assumption and found the statistically significant results in their study. therefore, in this study, 27 healthy subjects were enrolled in two different protocols (administration before or after a meal). fifty - seven healthy volunteers were enrolled at 9 university hospitals ; shimane university, hokkaido university hospital, gunma university hospital, nippon medical school, yokohama city university hospital, hamamatsu university school of medicine, osaka city university, osaka medical college, and saga medical school. their mean age was in the 20s and none of the subjects was infected by helicobacter pylori (h. pylori), which was determined by testing for the presence of the h. pylori antibody in serum and urine samples. the cyp2c19 genotype was tested by a polymerase chain reaction - restriction fragment length polymorphism (pcr - rflp) assay, as previously reported. twenty - nine of the 57 enrolled volunteers were randomly enrolled in the pre - meal ppi administration protocol. the subjects were investigated by ph monitoring twice, once with 10 mg of rabeprazole and once with 20 mg of esomeprazole. the ppis were delivered in identical opaque gelatin capsules and discrimination between them was impossible during the study period. the identical opaque gelatin capsules containing 10 mg rabeprazole or 20 mg esomeprazole were prepared by an author pharmacist (kn) and packaged in marked bags, and then delivered to each participating hospital. the key code of the drugs was kept by kn and opened firstly after fixing the final ph data. the 2 ph monitoring examinations were separated by at least a 1-week interval. an intra - gastric ph monitoring sensor catheter (zenetics medical, salt lake city, ut) was introduced into the gastric body and monitoring was started at 17:00, as previously reported. a single oral dose of the ppi was administered at 15 min before supper, at 18:45. the subjects started eating their supper (carbohydrates 112.8 g, protein 16.3 g, fat 27.3 g, calories 762 kcal) at 19:00 and were asked to finish within 30 min. the subjects were then requested to lie on their bed from 23:00 to 7:00 next morning. breakfast (carbohydrates 34 g, protein 5.8 g, fat 2.8 g, calories 85 kcal) and lunch (carbohydrates 74.4 g, protein 17.1 g, fat 11.4 g, calories 531 kcal) were also consumed within 30 min, starting at 8:00 and 12:00, respectively. all conditions were the same as above, except that rabeprazole or esomeprazole was administered orally 30 min after the end of supper at 20:00. in the postprandial administration protocol, the supper (carbohydrate 52.8 g, protein 8.8 g, fat 9.2 g, calorie 330 kcal) and breakfast (carbohydrate 94 g, protein 13.3 g, fat 20.9 g, calorie 617 kcal) were also slightly different from those used in the pre - meal administration protocol. median ph for each monitored hour and the percentage of time at which intra - gastric ph was below 4.0 were calculated for the total 24-h period, as well as the daytime (7:0023:00) and nighttime (23:007:00) periods. statistical analysis was performed using a wilcoxon signed rank test when results of a friedman test showed significant differences. the chronological data shown in fig. 1, 3, 4 and 6 were analyzed by linear mixed models. the sample size of the study was calculated based on the previous studies comparing 40 mg esomeprazole and 20 mg rabeprazole on their first administration day. calculated the number of necessary subjects as 18 based on parametric assumption and found the statistically significant results in their study. therefore, in this study, 27 healthy subjects were enrolled in two different protocols (administration before or after a meal). of the 57 enrolled subjects, 2 in the preprandial administration group and 1 in the postprandial group were not analyzed because of intolerance to the second ph monitoring examination. there were no significant differences in regard to gender, age, height, body weight, bmi, and cyp2c19 genotypes between the administration protocol groups (table 1). when administered before the meal, the median intra - gastric ph after esomeprazole administration tended to be higher than after rabeprazole administration (fig. 1), whereas intra - gastric ph in the 2:003:00 time period was significantly higher after esomeprazole administration. we also calculated percent time of intra - gastric ph > 4.0 over the 24-h period, as well as during the daytime and nighttime periods. again, esomeprazole tended to show a stronger acid inhibitory effect, though differences with rabeprazole were not significant (fig. 2). when the data was separately calculated for different cyp2c19 genotypes, esomeprazole raised intra - gastric ph more effectively in rapid metabolizers at 4 time points in 24-h observation period (fig. 3), while there was no apparent difference between intra - gastric ph between rabeprazole and esomeprazole in the intermediate and poor metabolizers, except at a single time point in poor metabolizers. when the ppis were administered after meals, there were no apparent differences in median intra - gastric ph at any time point after either administration (fig. 4). furthermore, after calculating percent time of intra - gastric ph > 4.0, there were no differences found during the daytime and nighttime periods (fig. 5). median intra - gastric ph was also calculated based on cyp2c19 genotype, and compared between rabeprazole and esomeprazole, with no significant difference found, except at a single time point in poor metabolizers (fig. the present results show that the acid inhibitory effects of 10 mg of rabeprazole and 20 mg of esomeprazole after single oral doses were similarly potent, especially when administered after meals. four kinds of ppis, omeprazole, lansoprazole, rabeprazole, and esomeprazole, are available for clinical practice in japan, which can be divided into 2 groups based on their degradability by the hepatic drug metabolizing enzyme cyp2c19. omeprazole and lansoprazole are easily degraded by cyp2c19, while rabeprazole and esomeprazole are not. asian individuals are known to have heterogeneous cyp2c19 enzyme activity, as 30% are extensive metabolizers with high enzyme activity, 20% are poor metabolizers with low enzyme activity, and the remaining 50% are intermediate metabolizers. therefore, different from western countries, the acid inhibitory effects of omeprazole and lansoprazole are known to be diverse among individuals. in cases with a high level of cyp2c19 enzyme activity, the acid inhibitory effects of these drugs are expected to be limited. to improve uncertainty, the more stable ppis rabeprazole and esomeprazole are increasingly used in clinical practice for japanese patients, with standard oral doses of 10 and 20 mg, respectively. rabeprazole is a newly developed racemic mixture compound reported to resist cyp2c19 degradation, while esomeprazole is an s - isomer of omeprazole and similarly resistant to cyp2c19. therefore, these ppis are considered to have a more consistent acid inhibitory effect irrespective of cyp2c19 enzyme activity. however, that of esomeprazole is considered to become submaximal when the drug is administered after a meal. there are 2 possible mechanisms regarding this weak point of esomeprazole to consider, decreased absorption and incomplete activation. the plasma concentration of esomeprazole was investigated and compared when administered during fasting and after meals. those results clarified that the plasma concentration of esomeprazole was higher when administered during fasting, though the precise mechanism related to that difference is not clear. all ppis need to be activated by the acidic environment in the secretory canaliculi of parietal cells. when administered after meals, an absorbed ppi will not be effectively activated because food - induced acid secretion and the highly acidic environment in the secretory canaliculi are nearly terminated when the plasma concentration of the drug reaches a peak level at 23 h after administration. mainly based on data obtained from esomeprazole trials in western countries, ppis are recommended to be administered 30 min before meals. on the other hand, the acid inhibitory effect of rabeprazole was shown to be not significantly influenced by timing of administration. in the present study, intra - gastric acidity after a single postprandial oral dose of rabeprazole (10 mg) or esomeprazole (20 mg) was similarly raised and remained nearly identical for 24 h. on the other hand, the acid inhibitory effect of the latter was slightly stronger than that of the former when each was administered before meals, though the difference was not statistically significant. these results confirm a previous report showing that esomeprazole had a stronger acid inhibitory effect when administered 30 min before meals. in the present study, direct comparisons of the acid inhibitory effects of the tested ppis between pre- and post - prandial administrations was difficult, since the foods taken during the monitoring periods were not identical. however, when we compared the pre- and post - prandial administrations, esomeprazole was stronger with pre - prandial administration, as previously reported, while rabeprazole was equally potent irrespective of the timing of administration. in japan, approximately 80% of physicians instruct their patients to take ppis after breakfast and approximately 10% after dinner. therefore, 90% of the patients take ppis after meals. in such an environment, the acid inhibitory effects of the present ppi administrations are considered to be nearly identical, though esomeprazole may show a statistically non - significant benefit when administered before meals. the first is lack of baseline intra - gastric ph data obtained without any drug administration. to more sensitively check the potency of any acid inhibitory effect, baseline ph data are necessary therefore, a comparison of the intra - gastric ph observed after rabeprazole and esomeprazole administrations is the only one possible in this study. therefore, we could not correlate the pharmacokinetic disposition of ppi with the intragastric ph. the influence of meal on the absorption of ppis and their acid inhibitory effects could not be made clear. another is the lack of ph data during chronic administration of the ppis, since these drugs are frequently used for chronic treatment. an additional study with chronic administrations of ppis as well as baseline data may be necessary in the future. in summary, we found that the intra - gastric ph values for 24 h after a single oral dose of rabeprazole (10 mg) or esomeprazole (20 mg) were nearly identical, especially when administered after meals. on the other hand the center for clinical research and the first department of medicine at hamamatsu university school of medicine have received grants from takeda pharmaceutical co., ltd., astrazeneca kk, eisai co., ltd., daiichi - sankyo co., ltd., and sugimoto m and furuta t have received lecture fees from takeda pharmaceutical co., ltd., astrazeneca kk, eisai co., ltd., daiichi - sankyo co., ltd. kusano m received lecture fee and research grant from eisai co., ltd., and lecture fee from astrazeneca kk and daiichi - sankyo co., ltd., takeda pharmaceutical co., ltd., and astrazeneca kk and received research funds from eisai co., ltd., takeda pharmaceutical co., ltd., otsuka pharmaceutical co., ltd., astrazeneca kk, astellas pharmaceutical co., ltd., and daiichi - sankyo co., ltd. higuchi h and fujimoto k received research grant and lecture fees from astrazeneca kk, eisai co., ltd., daiichi - sankyo co., ltd. naora k received research grants from astrazeneca kk, eisai co., ltd., and takeda pharmaceutical co., ltd. arakawa t received research grant from eisai co., ltd. and otsuka pharm co., and lecture fee from eisai co., ltd. kinoshita y received research grants and lecture fees from astrazeneca kk, eisai co., ltd. | comparisons between the acid inhibitory effects of rabeprazole and esomeprazole after single oral administration with standard doses have not been previously presented. we examined intra - gastric ph after oral administrations of these two proton pump inhibitors using 24-h ph monitoring. fifty - four normal volunteers not infected by helicobacter pylori were investigated. using a cross - over design, we administered 10 mg of rabeprazole or 20 mg of esomeprazole in 27 at 30 min after supper and in the remaining 27 subjects at 15 min before supper, and performed 24-h ph monitoring. intra - gastric ph data were nearly identical when the proton pump inhibitors were taken after meals. even if the data were compared in different cyp2c19 genotypes, rabeprazole and esomeprazole did not show the difference. in poor metabolizer, both of the drugs showed stronger acid inhibition. when taken before meals, intra - gastric ph after esomeprazole administration was slightly but not significantly higher than that observed after rabeprazole administration not only in daytime but also in nighttime period. in conclusion, rabeprazole and esomeprazole were similarly effective when administered after a meal. |
we recruited 17 current and 17 ex - smokers with copd to form our study sample. copd was diagnosed according to the gold criteria.1 participants were allowed to have any degree of bdr following inhalation of 200 g salbutamol, but no history of asthma as confirmed by two physicians independently. smoking intensity was defined both as pack - years, and number of cigarettes smoked on average per day when smoking, regardless of years of smoking history. all participants were in a stable condition at the time of the study and free from acute exacerbations of symptoms and/or from upper respiratory tract infections for at least one month preceding the study. participants did not have any active concurrent medical problems and/or a clinical history of allergic diseases (eg, rhinitis). atopic status was assessed by skin prick testing to a panel of seven common aeroallergens and a positive response was defined by a weal diameter of 3 mm. participants had not used corticosteroids in any form in the last three months or long acting bronchodilators in the past 4 weeks. pulmonary function tests were performed for all participants according to ats standards.15 bdr was assessed by remeasuring spirometry in all participants 20 minutes after the inhalation of 200 g of salbutamol from a metered dose inhaler. gas transfer factor was measured by the single breath carbon monoxide method,16 and lung volumes were measured in a body plethysmograph post bronchodilator. carboxyhemoglobin levels were measured during lung function testing and reviewed to confirm the nonsmoking status of the ex - smokers. sputum induction was performed with hypertonic saline inhalation, following well established methods.17,18 sputum plugs free of salivary contamination were selected and processed according to an adaptation of the method used by pin.17 bal and endobronchial biopsies were obtained and processed as described previously.19,20 briefly, participants were pre - medicated with nebulized (5 mg) and sedation was achieved with intravenous administration of midazolam (310 mg) and fentanyl (25100 g). lignocaine (4%) was applied topically to the nose, pharynx, and larynx, and 2% lignocaine was admistered below the cords. bronchoalveolar lavage was performed to yield three 60 ml aliquots from the right middle lobe using a standard protocol. total cell counts were determined on unfiltered balf using a neubauer hemocytometer and cytocentrifuge preparations were stained with toluidine blue and diff - quik (american scientific, mcgaw park, il, usa) to allow identification and determination of mast cells and balf absolute and differential cell counts, respectively. endobronchial biopsies were then taken from sub - segmental cariane of the right lower lobe of each patient, using alligator forceps (fb-15c ; olympus, toyko, japan). differential cell counts were performed on at least 500 cells per slide across the middle band of the cytocentrifuge spot,20,21 under 100x magnification using an oil - immersion lens. for mast cells, 2,5005,000 total cells (depending on cytospot density) were enumerated by presence of metachromasia. results were averaged and expressed as both a percentage and absolute count for each cell type.20 interleukin (il)-8 concentration in cell - free supernatants of sputum and bal was measured using a four - layer quantitative chemiluminescence sandwich enzyme linked immunosorbent assay (elisa), according to the manufacturer s instructions (r&d systems inc., a chain polymer technology system (envision plus, dakocytomation) was employed.22 monoclonal antibodies used were anti - neutrophil elastase (dako, glostrup, denmark), anti - cd68 (dako), anti - tryptase a (dako), anti- eg1, anti - eg2 (pharmacia diagnostics, uppsala, sweden), anti - cd4, and anti - cd8 (dako), as well as a negative isotype control. cells were quantified in the subepithelial lamina propria up to a depth of 150 m, excluding vessels, mucosal glands, and smooth muscle by light microscopy, by a single experienced observer, using an image analyzer (image pro plus 4.5, usa). two quality slides from one of the best tissue blocks from each participant were coded and scored blind. results were expressed as the number of positive cells per millimeter of epithelial basement membrane.19 statistical analysis was performed using spss (version 12.0.1 for windows, usa). for two group comparisons, t tests, and mann whitney and chi - square tests pearson s test and spearman s test were used for parametric and nonparametric data, respectively. data were expressed as mean standard deviation if parametric, and as median and interquartile range if nonparametric. we have previously demonstrated in endobronchial biopsies and bal studies that around 15 participants per group is an optimal number.23,24 preliminary repeatability data for copd airway samples were similar, indicating that the power versus participant number plot flattened markedly beyond this number of particpants with little extra gain in power with larger numbers. we recruited 17 current and 17 ex - smokers with copd to form our study sample. copd was diagnosed according to the gold criteria.1 participants were allowed to have any degree of bdr following inhalation of 200 g salbutamol, but no history of asthma as confirmed by two physicians independently. smoking intensity was defined both as pack - years, and number of cigarettes smoked on average per day when smoking, regardless of years of smoking history. all participants were in a stable condition at the time of the study and free from acute exacerbations of symptoms and/or from upper respiratory tract infections for at least one month preceding the study. participants did not have any active concurrent medical problems and/or a clinical history of allergic diseases (eg, rhinitis). atopic status was assessed by skin prick testing to a panel of seven common aeroallergens and a positive response was defined by a weal diameter of 3 mm. participants had not used corticosteroids in any form in the last three months or long acting bronchodilators in the past 4 weeks. pulmonary function tests were performed for all participants according to ats standards.15 bdr was assessed by remeasuring spirometry in all participants 20 minutes after the inhalation of 200 g of salbutamol from a metered dose inhaler. gas transfer factor was measured by the single breath carbon monoxide method,16 and lung volumes were measured in a body plethysmograph post bronchodilator. carboxyhemoglobin levels were measured during lung function testing and reviewed to confirm the nonsmoking status of the ex - smokers. sputum induction was performed with hypertonic saline inhalation, following well established methods.17,18 sputum plugs free of salivary contamination were selected and processed according to an adaptation of the method used by pin.17 bal and endobronchial biopsies were obtained and processed as described previously.19,20 briefly, participants were pre - medicated with nebulized (5 mg) and sedation was achieved with intravenous administration of midazolam (310 mg) and fentanyl (25100 g). lignocaine (4%) was applied topically to the nose, pharynx, and larynx, and 2% lignocaine was admistered below the cords. bronchoalveolar lavage was performed to yield three 60 ml aliquots from the right middle lobe using a standard protocol. total cell counts were determined on unfiltered balf using a neubauer hemocytometer and cytocentrifuge preparations were stained with toluidine blue and diff - quik (american scientific, mcgaw park, il, usa) to allow identification and determination of mast cells and balf absolute and differential cell counts, respectively. endobronchial biopsies were then taken from sub - segmental cariane of the right lower lobe of each patient, using alligator forceps (fb-15c ; olympus, toyko, japan). differential cell counts were performed on at least 500 cells per slide across the middle band of the cytocentrifuge spot,20,21 under 100x magnification using an oil - immersion lens. for mast cells, 2,5005,000 total cells (depending on cytospot density) were enumerated by presence of metachromasia. results were averaged and expressed as both a percentage and absolute count for each cell type.20 interleukin (il)-8 concentration in cell - free supernatants of sputum and bal was measured using a four - layer quantitative chemiluminescence sandwich enzyme linked immunosorbent assay (elisa), according to the manufacturer s instructions (r&d systems inc., minneapolis, usa). for staining of cell populations within endobronchial biopsies, a chain polymer technology system (envision plus, dakocytomation) was employed.22 monoclonal antibodies used were anti - neutrophil elastase (dako, glostrup, denmark), anti - cd68 (dako), anti - tryptase a (dako), anti- eg1, anti - eg2 (pharmacia diagnostics, uppsala, sweden), anti - cd4, and anti - cd8 (dako), as well as a negative isotype control. cells were quantified in the subepithelial lamina propria up to a depth of 150 m, excluding vessels, mucosal glands, and smooth muscle by light microscopy, by a single experienced observer, using an image analyzer (image pro plus 4.5, usa). two quality slides from one of the best tissue blocks from each participant were coded and scored blind. statistical analysis was performed using spss (version 12.0.1 for windows, usa). for two group comparisons, t tests, and mann whitney and chi - square tests pearson s test and spearman s test were used for parametric and nonparametric data, respectively. data were expressed as mean standard deviation if parametric, and as median and interquartile range if nonparametric. we have previously demonstrated in endobronchial biopsies and bal studies that around 15 participants per group is an optimal number.23,24 preliminary repeatability data for copd airway samples were similar, indicating that the power versus participant number plot flattened markedly beyond this number of particpants with little extra gain in power with larger numbers. there were no statistical differences in regard to age, sex, and atopic status between the two copd groups examined. although the ex - smokers had a higher mean pack year smoking history, the variability within groups was large and the difference was not significant. no significant differences were found in any of the lung function indices (table 2). the mean % bdr predicted was 7.4 and 7.2 in current and ex - smokers (ns). among current smokers there were six participants (35%) with bdr 10% (given as % of predicted) and in ex - smokers there were four (23.5% in the group ; not significantly different between groups according to the results of a chi - square test). in sputum, current smokers with copd had significantly increased absolute numbers of mast cells compared to ex - smokers, who in contrast demonstrated significantly increased absolute numbers and percentage differential counts of neutrophils (table 3). in bal, current smokers demonstrated a significant increase in absolute numbers and percentage differential counts of eosinophils compared to ex - smokers, who in turn exhibited overall decreased total cell counts, but with associated increases in lymphocyte, neutrophil, and epithelial differential cell percentages as well as a trend toward increased il-8 levels (p = 0.06) (table 4). in contrast, biopsy cellular profiles were very similar (table 5) between current and ex - smokers. there were few significant correlations between smoking intensities, airway inflammatory cells, and lung function, although there was a significant negative relationship between smoking intensity and lung function in current smokers as perhaps might be expected, ie, more smoking was associated with poorer lung function, but this relationship was not seen in the ex - smokers. in current smokers there was a weak relationship between smoking intensity and total cell counts, but this was in a negative direction and only in bal samples ie, more smoking was related to fewer total cells in bal. in ex - smokers, some relationships were seen in sputum, bal, and biopsy between smoking intensity and cell counts but again, these were mainly negative (for lymphocytes, mast cells and epithelial cells), although positive for macrophages ; there was little consistency between airway compartment types. in general, therefore, more total cells in sputum and bal tended to be associated across the board with better lung function. in airway biopsies, higher cell numbers were similarly associated with better lung function. for current smokers, the strongest such relationships were for neutrophils and cd4 lymphocytes, and for ex - smokers for cd4 lymphocytes and mast cells. cell profiles in sputum, bal, and airway biopsies were statistically unrelated to each other in either current or ex - smokers with copd. there were no statistical differences in regard to age, sex, and atopic status between the two copd groups examined. although the ex - smokers had a higher mean pack year smoking history, the variability within groups was large and the difference was not significant. no significant differences were found in any of the lung function indices (table 2). the mean % bdr predicted was 7.4 and 7.2 in current and ex - smokers (ns). among current smokers there were six participants (35%) with bdr 10% (given as % of predicted) and in ex - smokers there were four (23.5% in the group ; not significantly different between groups according to the results of a chi - square test). in sputum, current smokers with copd had significantly increased absolute numbers of mast cells compared to ex - smokers, who in contrast demonstrated significantly increased absolute numbers and percentage differential counts of neutrophils (table 3). in bal, current smokers demonstrated a significant increase in absolute numbers and percentage differential counts of eosinophils compared to ex - smokers, who in turn exhibited overall decreased total cell counts, but with associated increases in lymphocyte, neutrophil, and epithelial differential cell percentages as well as a trend toward increased il-8 levels (p = 0.06) (table 4). in contrast, biopsy cellular profiles were very similar (table 5) between current and ex - smokers. there were few significant correlations between smoking intensities, airway inflammatory cells, and lung function, although there was a significant negative relationship between smoking intensity and lung function in current smokers as perhaps might be expected, ie, more smoking was associated with poorer lung function, but this relationship was not seen in the ex - smokers. in current smokers there was a weak relationship between smoking intensity and total cell counts, but this was in a negative direction and only in bal samples ie, more smoking was related to fewer total cells in bal. in ex - smokers, some relationships were seen in sputum, bal, and biopsy between smoking intensity and cell counts but again, these were mainly negative (for lymphocytes, mast cells and epithelial cells), although positive for macrophages ; there was little consistency between airway compartment types. in general, therefore, more total cells in sputum and bal tended to be associated across the board with better lung function. in airway biopsies, higher cell numbers were similarly associated with better lung function. for current smokers, the strongest such relationships were for neutrophils and cd4 lymphocytes, and for ex - smokers for cd4 lymphocytes and mast cells. cell profiles in sputum, bal, and airway biopsies were statistically unrelated to each other in either current or ex - smokers with copd. our aim in this cross - sectional study was to investigate in detail, using different airway compartment sampling techniques, the differences between airway inflammatory profiles in current and ex - smokers with established copd and to explore the relationship between airway inflammation, smoking intensity, and lung function. current smokers had an increase in sputum mast cell and bal eosinophil numbers compared to ex - smokers. in contrast, ex - smokers with copd had higher total cell counts and absolute neutrophil numbers in sputum, but not in bal or biopsies compared to current smokers. in bal there was an increase in the relative proportions (percentage) of neutrophils, lymphocytes, and epithelial cells in ex- versus current smokers and a trend for bal il-8 levels to be higher in ex - smokers, consistent with the higher percentage of neutrophils in bal. it is quite obvious that the picture that can be obtained will vary considerably depending on sampling modality used, which explains why the results gained in previous reports have varied so much. our study results demonstrate that airway inflammation persists in copd ex - smokers but that the cell patterns differ compared to current smokers, especially in sputum and bal. the picture in airway biopsies, on the other hand, is similar in current and ex - smoker copd patients, suggesting that once copd is established, trafficking of cells through the airway wall into the lumen may be what is different, which may reflect remodeling changes and perhaps changes in vasculature, but this can not be addressed by a cross - sectional study and needs - specific prospective study. notably, for inflammatory cell profiles in general, more historical smoking and poorer lung function were both associated with less marked inflammation, which may imply that cellular inflammation is most prominent in milder physiological disease, and that later, with more significant physiological abnormality, airway remodeling, and perhaps a reduced ability of cells to migrate becomes the more predominant pathological process, but this also needs prospective study of both airway remodeling and cell infiltration after smoking cessation. caution obviously needs to be exercised when attributing cause and effect relationships from cross - sectional studies such as ours, but on the other hand, longitudinal smoking - cessation studies in copd needing several years to complete are unfortunately difficult to recruit for and undertake. from our experience, the majority of recruits with copd to such studies can not stop smoking at all, or if they manage to quit, it is for only such a short time as to make any study of changes in airway pathology unfruitful (unpublished data and personal observation). therefore, inferences from cross - sectional studies such as the current one are necessary and important in trying to gain better understanding of copd pathogenesis, although the relationships between airway inflammation, smoking status, and physiological disease appear complex. willemse compared current smokers with copd using sputum and airway wall biopsy cell profiles to current smokers without copd and then conducted a smoking cessation study in both participant populations.25,26 established copd was associated with increased sputum neutrophils and eosinophilic cationic protein (ecp) levels, but there were no changes within the airway wall that discriminated smokers with copd from those without, although smoking per se was related to an increase in airway biopsy macrophages. interestingly, following one year of smoking cessation there were significant increases in sputum neutrophils, as well as il-8 and ecp levels but only in those participants with copd, whereas inflammatory cell counts and cytokine levels were significantly reduced in the smokers without copd who had successfully quit. once again, there were no changes in cell profiles in bronchial biopsies and willemse and colleagues concluded that airway wall remodeling may be the more important factor and also that the apparent increase in inflammation could reflect an active repair process.26 lapperre from the same research team used pooled data from several centres27 to undertake a cross - sectional comparison of airway biopsies in over a 100 participants with copd and found increased cd3+ve, cd4+ve, and cd8+ve lymphocytes in short term quitters, but an increase in airway wall plasma cell and reduction in cd8+ve numbers in ex - smokers with longer durations of smoking cessation. altogether, these studies are consistent with our findings that inflammation does not seem to reduce following smoking cessation in the setting of established copd and indeed may worsen, but whether this is part of a repair process requires more investigation into which inflammatory changes within the three airway compartments are related to changes of remodeling within the airway wall. a further potential explanation for some of the differences seen in copd between current and ex - smokers, such as a difference in bal macrophages, may be alterations in the balance between inflammatory cell recruitment, apoptosis, and apoptotic cell clearance induced by smoking.28,29 on the other hand, the increase in mast cells in sputum and eosinophils in bal in current smokers compared to ex - smokers may reflect an response to cigarette smoke components, as has been suggested previously and this could be consistent with the higher sputum ecp levels in copd current smokers observed in willemse s study or in the general push of active smoking to produce higher systemic ige levels.30,31 the lack of relationship between cell profiles in sputum, bal, and biopsies indicates that the effects of smoking or smoking cessation are different depending on which airway compartment one studies, suggesting that to get a truly holistic view of the effects of smoking, one needs to sample all compartments. the other important message is that sputum cell profiles do not reflect airway pathology in either more distal airways (sampled by bal), or in more central airway walls, which cautions against overinterpretation of sputum indices when commenting on airway pathology in copd. in summary, in this three compartment airway study we compared inflammatory cell profiles in current and ex - smokers with copd and demonstrated that there is a complex interplay between smoking and airway inflammation in copd, which varies depending on which airway compartment is being assessed. ex - smokers with copd have persisting airway inflammation, which differs qualitatively and quantitatively from current smokers. prospective studies of smoking cessation now need to consider the relationship between inflammation and airway remodeling / repair, which may influence these cell profiles. | rationalesmoking effects on physiological and gross pathology in chronic obstructive pulmonary disease (copd) are relatively well described. however, there is little known in copd about the detailed interrelationships between lung function and inflammatory profiles in different airway compartments from the same individual and whether airway inflammation in these different compartments differs in ex- and current smokers with established copd.objectiveswe compared sputum, bronchoalveolar (bal), and airway wall inflammatory profiles in current versus ex - smokers and related this to smoking intensity and lung function in 17 current and 17 ex - smokers with mild to moderate copd.resultscurrent smokers had more sputum mast cells (% differential and absolute numbers), whereas ex - smokers had increased sputum neutrophils. in bal, there was a significant increase in eosinophils in current smokers, but ex - smokers had significantly increased neutrophils, lymphocytes, and epithelial cells. there were no cell profile differences observed in airway biopsies between current and ex - smokers and there were no correlations between the individual inflammatory cell populations in any of the airway compartments. in current smokers only, smoking intensity was negatively correlated with lung function, and associated with a reduction in overall cellularity of both sputum and bal.conclusionairway inflammation persists in ex - smokers with copd, but differs from copd current smokers. the impact of smoking appears to vary in different airway compartments and any direct relationships between cellularity and lung function tended to be negative, ie, worse lung function indicated the presence of fewer cells. |
previous studies found interactions between the meaning of words and the screen location where the words were presented (i.e., spatial position). for instance, people were faster to decide that a stork flies if the word stork was presented at the top of the screen rather than at the bottom of the screen (eti and domijan, 2007). similar effects were found in a semantic relatedness judgment task (zwaan and yaxley, 2003) and also in a letter identification task in which participants identified a single letter (x or o) presented at the top or bottom of the screen immediately following the name of an object with a typical location (e.g., cowboy boot, estes., 2008). researchers also found interactions between word meaning and spatial position when words refer to more abstract concepts, such as valence or power (richardson., 2003 ; meier and robinson, 2004 ; schnall and clore, 2004 ; schubert, 2005 ; meier., 2007 ; although these abstract concepts have no inherent perceptual spatial positions, they are connected to spatial concepts through metaphorical relations such as good is up and bad is down. these interactions between word meaning and spatial location provide important insight into the underlying mental representations of meaning. the first explanation is that readers understand the meaning of a category by mentally simulating associated sensory - motor information (e.g., barsalou, 1999). thus, a representation of the meaning of flying animal involves simulating looking up at the sky and seeing the animal fly. because such simulations occupy sensory - motor systems, they might interfere with other sensory - motor processing. indeed, interference was found when people simultaneously performed mental visual imagery and a visual perception task (craver lemley and reeves, 1992). (2008) showed a similar interference effect without explicit imagery instructions. in their task, participants viewed word pairs in the center of the screen that referred to objects with typical vertical locations (cowboy hat or cowboy boot). immediately after presentation of the word pair estes. found that letter identification was slower and less accurate if the letter 's position matched the typical location of the word 's referent than if the position mismatched. they did not report the separate effects for items at the top and bottom positions. they presented sentences in which a verb had a horizontal (push) or vertical (sink) orientation, followed by a visual target. the target could appear at one of four locations on the computer screen ; at the top or bottom (horizontally centered), or on the left or right (vertically centered). they found that the orientation of the verb interfered with the position of the visual target. for example, following the vertical word sink, responses to targets presented on the vertical axis (top or bottom) were slower than to targets presented on the horizontal axis (left or right). other studies, using somewhat different designs, found facilitation if word meaning and spatial location were congruent. bergen. (2007) noted that findings of interference or facilitation might be due to differences in timing, but both effects are still explained by the same simulation account. for example, eti and domijan (2007) presented words referring to flying or non - flying animals at the top or bottom of the computer screen. decisions were faster and more accurate for words in a congruent than incongruent position (e.g., performance was better for stork at the top than at the bottom of the screen). additionally, the task itself deciding whether or not animals fly might have directed spatial attention. in order to perform the flying animal task, participants may have systematically directed their mental simulations, and thus their spatial attention, towards the sky. although eti and domijan (2007) did not find a main effect of position, other studies show such main effects (e.g., schubert, 2005). on this account, there may be both a general task - related benefit for words presented at the top of the screen (through spatial attention) as well as a word specific benefit for flying animals presented at the top of the screen (because the mental simulation would be easier for a word in this position). therefore, in the current study, we tested both task congruency (e.g., benefits for all words at the top) as well as word congruency (e.g., additional benefits for flying animals at the top). a second explanation for these congruency effects lies in the response selection process rather than the representation of meaning. proctor and cho (2006 ; see also bar - anan., 2007) proposed that in many binary decision tasks, the speed of response selection is affected by polarity correspondence. stimulus dimensions with binary values are encoded as having a + (plus) polarity or (minus) polarity. in a similar vein, response alternatives are also encoded as + or. response selection is faster when stimulus and response polarities correspond than when they do not correspond. according to proctor and cho, for example, a yes response is typically represented as + and a no response is represented as. up is represented as + and down is represented as. right is represented as + and left is represented as. accordingly, right key presses are coded as + and left key presses are coded as. related to this idea, klatzky. (1973) argued that many conceptual dimensions (e.g., height, valence) also have polarity. furthermore, the adjectives representing the opposite ends of these dimensions consist of a default, positive, or unmarked member (e.g., tall, good) that can also be used to name the dimension in its entirety and a negative, or marked member (e.g., short, bad) that is only used to name one end of the dimension. for example, the question how tall is he ? is neutral as to actual size, whereas the question how short is he ? for example, in judgments of power, powerful may be the unmarked (positive) end and powerless may be the marked (negative) end of the power dimension. alignment of powerful with up therefore leads to faster processing than alignment of powerful with down (schubert, 2005). polarity correspondence can also explain similar results that examined spatial congruency with concepts such as valence or number magnitude (fischer., 2003 ; meier and robinson, 2004 ; santens and gevers, 2008 ; bae., 2009 ; but these polarity correspondences might also explain the results of eti and domijan (2007). in their task, the flying animals always required a yes response and the non - flying animals always required a no response. therefore, in the congruent condition, the polarities of position and response (up - yes and down - no) were aligned, whereas they were misaligned in the incongruent condition (up - no and down - yes). on this account, the results are due to a lack of counterbalancing between the yes / no answer and spatial position. after all, there was no condition in which the task was reversed, such that non - flying animals required a yes - response (e.g., by asking is this a land animal ?). in sum, stimuli in a task may have polarity values based on markedness, on task - specific judgment (yes or no), and on spatial position (up or down). in addition, responses also have polarity values, for example based on the spatial position of the manual responses. thus, rather than supporting a theory of meaning representation through mental simulation, these congruency effects may instead reflect polarity alignment if the role of yes / no response assignment is not counterbalanced or not independently manipulated. to investigate whether the effects of spatial position of a target stimulus are better explained by polarity alignment or by congruence with mental representations, we used two semantic judgment tasks for which the same stimuli required opposite responses. one task was an ocean judgment (is it usually found in the ocean ?) and the other task was a sky judgment (is it usually found in the sky ?). these tasks were chosen because sky and ocean have clear spatial positions but neither one is a linguistically marked or unmarked end of a dimension. while most people will have more experience with looking at things in the sky than in the ocean, our study was run in san diego, which is situated on the pacific coast. therefore, the participants in our study had above average experience with looking at the ocean. because perceptual simulation involves activation of previous perceptual experiences we assumed that simulations of seeing things in the sky and ocean would most likely take the perspective of someone standing on land looking straight ahead, with the sky taking up the top half of visual field and the ocean taking up the bottom half of the visual field. the same set of stimuli was used in the two tasks, and consisted of names of things that are typically found in one of the two locations (e.g., whale, submarine, eagle, helicopter). subjects responded using their two hands, and whether the yes response was given by the left or right hand was counterbalanced across participants. the role of conceptual congruency and polarity alignment can be disentangled by a between task comparison of the interaction between word category and stimulus position. if spatial congruency effects are due to mental simulation, then spatial attention should be directed towards the top of the screen in the sky decision task but towards the bottom of the screen in the ocean decision task. this predicts that in general, reaction times to words at the top of the screen should be faster in the sky task whereas reaction times to words at the bottom of the screen should be faster in the ocean task. beyond this task congruency effect, the mental simulation account also predicts a word congruency effect. for this effect of congruency between a referent 's typical location and the position of the word, performance should be better for sky words presented at the top of the screen as compared to sky words presented at the bottom of the screen. similarly, performance should be better for ocean words presented at the bottom of the screen as compared to ocean words presented at the top of the screen. critically, these effects should occur regardless of the task (i.e., regardless of the yes / no response). if, on the other hand, congruency effects are due to polarity alignment, performance should be better for yes responses to words presented at the top of the screen as compared to yes responses to words presented at the bottom of the screen. furthermore, performance should be better for no responses to words presented at the bottom of the screen as compared to no responses to words presented at the top of the screen. critically, these effects should occur regardless of the typical location (ocean or sky) of the word 's referent (i.e., regardless of the word 's meaning). thus, in the ocean decision task, performance should be better for ocean words at the top and sky words at the bottom than for the opposite positions. moreover, because the right hand is coded as + polarity and the left hand as polarity (according to proctor and cho, 2006) this effect may be restricted or at least most pronounced when yes responses are given with the right hand and no responses are given with the left hand. in summary, the mental simulation account predicts both task congruency effects (task and location) and word congruency effects (word and location), whereas polarity alignment only predicts response congruency effects (response and location). they were randomly assigned to the ocean decision (n = 52) or sky decision task (n = 50). forty words referred to things usually found in the ocean (e.g., whale, submarine) and 40 referred to things usually found in the sky (e.g., eagle, helicopter). the stimuli were selected from a larger set that had been tested in a pilot study. in this pilot study 50 participants made ocean or sky decisions (25 participants in each task) to a larger set of words presented individually in random order in the center of the computer screen. from this larger set, 80 words were selected for which categorization agreement between participants was greater than 75% (m = 92%). the two sets of words were comparable on word length, log word frequency, and number of items from different types of taxonomic categories (e.g., animals, man - made objects, natural objects, persons). participants in the ocean decision task were instructed to decide whether an item could be found in the ocean. participants in the sky decision task were instructed to decide whether an item could be found in the sky. items were presented individually and in random order on the computer screen following the procedure used by eti and domijan (2007). a trial started with a sequence of three consecutive fixation cues (+ + +) presented for 300 ms each, which served to warn participants whether the target word would appear at the top or bottom. the first fixation was presented in the center of the computer screen. in the top condition, the second fixation was presented at 40% from the top of the screen, the third at 30% from the top of the screen, followed by the target word at 20% from the top of the screen. in the bottom condition these positions were at 40%, 30%, and 20% from the bottom of the screen respectively. this sequence of fixations cues did not induce a sense of upwards or downwards motion because the vertical distance between fixations was too great (i.e., there was no apparent motion). the target word was presented immediately after the final fixation cue and remained on the screen until the participant responded or 2,500 ms elapsed. participants pressed the z or m key of the computer keyboard to indicate a yes or no response. feedback was provided for 1,500 ms if the response was incorrect (incorrect) or slower than 2,500 ms (too slow). the next trial started immediately after the response or, in the case of feedback, after the feedback message. every target word was tested twice, with the first occurrence during a first block of trials followed by a second occurrence during a second block of trials. half of the items in a block were assigned to the top position and the other half to the bottom position such that ocean words and sky words were presented equally often at each position. in the second block, the order of blocks was counterbalanced between participants, as was assignment of the m and z keys to the yes and no responses. they were randomly assigned to the ocean decision (n = 52) or sky decision task (n = 50). forty words referred to things usually found in the ocean (e.g., whale, submarine) and 40 referred to things usually found in the sky (e.g., eagle, helicopter). the stimuli were selected from a larger set that had been tested in a pilot study. in this pilot study 50 participants made ocean or sky decisions (25 participants in each task) to a larger set of words presented individually in random order in the center of the computer screen. from this larger set, 80 words were selected for which categorization agreement between participants was greater than 75% (m = 92%). the two sets of words were comparable on word length, log word frequency, and number of items from different types of taxonomic categories (e.g., animals, man - made objects, natural objects, persons). participants in the ocean decision task were instructed to decide whether an item could be found in the ocean. participants in the sky decision task were instructed to decide whether an item could be found in the sky. items were presented individually and in random order on the computer screen following the procedure used by eti and domijan (2007). a trial started with a sequence of three consecutive fixation cues (+ + +) presented for 300 ms each, which served to warn participants whether the target word would appear at the top or bottom. the first fixation was presented in the center of the computer screen. in the top condition, the second fixation was presented at 40% from the top of the screen, the third at 30% from the top of the screen, followed by the target word at 20% from the top of the screen. in the bottom condition these positions were at 40%, 30%, and 20% from the bottom of the screen respectively. this sequence of fixations cues did not induce a sense of upwards or downwards motion because the vertical distance between fixations was too great (i.e., there was no apparent motion). the target word was presented immediately after the final fixation cue and remained on the screen until the participant responded or 2,500 ms elapsed. participants pressed the z or m key of the computer keyboard to indicate a yes or no response. feedback was provided for 1,500 ms if the response was incorrect (incorrect) or slower than 2,500 ms (too slow). the next trial started immediately after the response or, in the case of feedback, after the feedback message. every target word was tested twice, with the first occurrence during a first block of trials followed by a second occurrence during a second block of trials. half of the items in a block were assigned to the top position and the other half to the bottom position such that ocean words and sky words were presented equally often at each position. in the second block, the order of blocks was counterbalanced between participants, as was assignment of the m and z keys to the yes and no responses. correct reaction times were trimmed by removing reaction times that were more than three standard deviations from the participant 's mean for the corresponding response (2.04% of the correct rts). as block did not interact with any other variable, we collapsed the data across blocks. the mean reaction times are presented in table 1 and the error rates are presented in table 2. separate anovas (task category instruction positio) were performed on the rts and error rates. the factors task (ocean vs. sky decision) and instruction (yes - is - right vs. yes - is - left) were between - subject factors, while category (ocean vs. sky word) and position (up vs. down) were within - subject factors. mean reaction times and standard errors in the two semantic decision tasks as a function of word position, word category, and response instruction. mean error scores and standard errors in the two semantic decision tasks as a function of word position, word category, and response instruction. mean reaction times in the two semantic decision tasks as a function of word category and word position. error bars represent confidence intervals for the word category position within - subjects interaction (loftus and masson, 1994). mean error rates in the two semantic decision tasks as a function of word category and word position. error bars represent confidence intervals for the word category position within - subjects interaction (loftus and masson, 1994). next, we report all significant results of both the rt and error rate anovas. we do so in an intermixed fashion, such that for a given effect, both the rt and error rate results are simultaneously reported (if both were significant). in this manner, it can be ascertained whether speed and accuracy traded off against each other (e.g., rt and error rate changing in opposite directions), or whether there was general performance change (e.g., just an rt effect, just an error rate effect, or situations where rt and accuracy went in the same direction). this is because the combination of task and category defines whether yes or no is the correct response to a particular target word. in other words, in the sky task, responses should be easier to sky words at the top of the screen (yes responses), whereas in the ocean task, responses should be easier to ocean words at the top of the screen (also yes responses). however, there was no three - way interaction between task, category, and position either in terms of rt or error rate, f 0.20. conceptual congruency also predicted that performance would be better for sky words at the top and ocean words at the bottom in both tasks, because these are typical positions for the entities referred to by these words. however, there was no interaction at all between word category and position, both fs < 1. thus, the current experiment failed to replicate the word congruency effect reported by eti and domijan (2007). in addition to these results, the anova on rts showed a theoretically uninteresting main effect of category, f(1,98) = 4.08, p = 0.05, = 0.04, and an interaction between category and task, f(1,98) = 73.02, p < 0.001, = 0.43. overall, responses were faster to ocean than sky words. in the ocean decision task this was also the case, but in the sky decision task responses were faster to sky than to ocean words, indicating that yes responses were faster than no responses. this effect provides a manipulation check because it shows that the polarity of the items was reversed by the task. in two semantic decision tasks (ocean decisions and sky decisions) performance was better for words that were presented at a position that was congruent with the task. specifically, performance was better for words at the bottom than at the top of the screen in ocean decision and better for words at the top than at the bottom of the screen in sky decision. this finding is not explained by the polarity principle, but it was expected by the perceptual simulation account if the systematic nature of the task (e.g., a long sequence of sky decisions) caused participants to direct their attention to the task appropriate position of the screen, which is something they might do to properly simulate words in the task indicated location (sky or ocean). task performance is facilitated by alignment of polarities between response and stimulus dimensions (proctor and cho, 2006). in the present study this principle predicted that the yes response, top position of the target word, and the right - hand response would be aligned because they are all coded as + polarity, and the opposites (no response, bottom position, left - hand response) would be aligned because they are all coded as polarity. however, our results showed no advantage when polarity was aligned : there were neither polarity effects when only considering response and screen position nor when considering response, screen position, and response hand. this failure to find polarity effects is consistent with previous studies finding results in one condition that might be interpreted in terms of polarity alignment, but then failing to find these results in a complementary condition (meier and robinson, 2004 ; van dantzig, 2009 ; boot and pecher, 2010). in these studies, effects of congruency between spatial position and concept were observed when considering a sequence of events that went from concept to position (e.g., from power judgment to visual target identification) but not in going from position to concept (e.g., from location decision to power judgment). for example, van dantzig found that power judgments of words (e.g., dictator) had an effect on subsequent identification of a letter presented at the top or bottom of the screen. in a separate experiment, identification of letters (e.g., a p at the top of the screen) thus, power judgment affected spatial attention, but spatial attention did not affect power judgments. in both experiments, position and power had binary polarities. if polarity alignment was the cause of the congruency effects in the concept followed by position experiment, there should have also been congruency effects for the position followed by concept experiment. (2008) noted that the polarity principle is a general principle that should be observed consistently. one complication with previous findings is that the stimulus dimensions had polarities that were mostly fixed. down spatial dimension, up always has + polarity, and in the power dimension, powerful always has + polarity. therefore, it was impossible to disentangle polarity alignment and meaning congruency. in the present study, we gave the same stimuli + or polarity by changing the decision task, and we manipulated response side orthogonally. therefore, the absence of any effect of polarity alignment in the current experiment indicates that, at least for semantic tasks, the polarity principle does not contribute to performance. the semantic congruency account predicted an interaction between word meaning and spatial position, but we did not find such an interaction. previous findings (richardson., 2003 ; eti and domijan, 2007 ; estes., 2008) were explained by congruency between a perceptual simulation of the word 's meaning and the spatial position or orientation of the target. found interference when the visual target 's position or orientation was congruent with the meaning of the preceding word. in contrast, eti and domijan (2007) obtained facilitation rather than interference for congruent stimuli. in the current experiment, we found neither interference nor facilitation between word position and word meaning. rather, we observed a task - wide advantage of the spatial location of the target, regardless of the specific meaning of the target word. thus, the diversity of these findings indicates that the nature of spatial congruency effects is not fully clear yet. some researchers (estes., 2008) have postulated two loci for congruency effects. first, concepts that are associated to typical locations direct spatial attention toward their typical location. this might interfere with processing when simulation and perception occur simultaneously and differ in perceptual details (e.g., simulation of a cowboy boot and identification of the letter x). in contrast, the simulation may facilitate visual processing when simulation and visual target share perceptual details or when the simulation and visual target are presented sequentially (bergen., 2007). therefore, whether spatial congruency results in benefits or deficits and whether this effect is found for individual words or task - wide categories may depend on procedural details. these details may include the timing of spatial target presentation compared to the process of mental simulation, whether the simulated concept is concrete (e.g., cowboy boot) or abstract (e.g., power) (see bergen., 2007), and what task is performed on the spatial target. regardless of these procedural details, our results show that it is unlikely that the polarity principle can explain spatial congruency effects. an explanation for our results that may also explain other findings is that the task directed spatial attention to the location that was congruent with the decision category. when the task was to decide whether a word referred to an entity typically found in the sky, participants directed their attention to the top of the screen, and when the task was to decide whether a word referred to an entity typically found in the ocean, participants directed their attention to the bottom of the screen. this task - specific spatial attention can be explained by mental simulation of the task - relevant location rather than specific entities. while performing the sky decision task, participants might mentally simulate looking at the sky without filling in perceptual details such as objects with specific shapes and colors. because such task - induced spatial attention does not involve a simulation with many perceptual details, it will not interfere with perception of the target word. instead, increased attention at the task - congruent location facilitated processing of the target word, resulting in faster responses. this attentional explanation is consistent with other findings in which lower level perceptual information facilitated higher level conceptual processing (van dantzig., 2008). in van dantzig.s study, participants verified concept - property pairs (e.g., banana - yellow) presented as words. responses were faster for pairs that were preceded by a simple perceptual stimulus from the same modality as the property (e.g., a flashing light) than from a different modality (e.g., a burst of white noise). in this study the correspondence between perception and representation was at a more global level, namely the activation of a sensory modality, rather than at the level of specific perceptual details. in other studies investigating the effect of spatial position of words on conceptual processing (meier and robinson, 2004 ; schubert, 2005 ; meier., 2007), the task - relevant dimension was always congruent with the top position (e.g., valence, power, divinity), which provides an opportunity to assess whether there may have been an attentional effect similar to the current results. close inspection of the results shows that, at least numerically, responses were faster to words presented at the top than at the bottom of the screen. because these studies did not use a second dimension in which the positive exemplars were congruent with the bottom position, it is impossible to say conclusively whether the advantage for stimuli at the top position was due to task - induced spatial attention or a more general advantage for stimuli at the top. however, these previous studies are consistent with the present findings and our explanation. in conclusion our results did show, however, that semantic decision tasks direct spatial attention in a more global way. it may be that people perform a mental simulation of the task - congruent location, which directs spatial attention and facilitates processing of targets in that location. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. | we report an experiment that compared two explanations for the effect of congruency between a word 's on screen spatial position and its meaning. on one account, congruency is explained by the match between position and a mental simulation of meaning. alternatively, congruency is explained by the polarity alignment principle. to distinguish between these accounts we presented the same object names (e.g., shark, helicopter) in a sky decision task or an ocean decision task, such that response polarity and typical location were disentangled. sky decision responses were faster to words at the top of the screen compared to words at the bottom of the screen, but the reverse was found for ocean decision responses. these results are problematic for the polarity principle, and support the claim that spatial attention is directed by mental simulation of the task - relevant conceptual dimension. |
bioresorbable scaffolds (brs) have been designed to overcome problems related to the long - term persistence of metallic stents implanted in coronary arteries. several randomized controlled trials (rcts) comparing polymeric brs with new - generation drug - eluting stents (des) found similar clinical outcomes at 1 year. a recent meta - analysis, though, showed an increased risk of definite or probable scaffold thrombosis for brs (1.3% compared with 0.5%). as the true benefit of brs is expected to ensue later, long - term data of rcts are awaited. absorbable metal scaffolds constitute an attractive alternative to polymeric brs, as they conform more to the technique of percutaneous coronary intervention with des. we assessed a novel second - generation drug - eluting absorbable metal scaffold (dreams 2 g), which has been redesigned iteratively by (i) improving the design of the magnesium backbone of its previous version and (ii) using the same drug polymer combination (sirolimus / poly - l - lactide) as the new - generation biodegradable polymer stent orsiro (biotronik ag, buelach, switzerland). six - month outcomes were recently published, showing favourable angiographic and clinical outcomes with a low rate of target lesion failure (tlf) and no definite or probable scaffold thrombosis. we aimed to evaluate if those results are sustained at 12 months, a time point when the magnesium scaffold is expected to be degraded. a detailed description of the study has been recently published. in brief, biosolve - ii is a prospective, multi - centre, first - in - man study to evaluate the safety and performance of dreams 2 g (biotronik ag, buelach, switzerland). the study is in compliance with the declaration of helsinki, good clinical practice, iso14155, and was approved by the institutional ethics committees at the participating 13 institutions in europe, south america, and asia. eligible patients had stable or unstable angina or documented silent ischaemia, a maximum of two single de novo lesions in two separate coronary arteries, with a reference vessel diameter between 2.2 and 3.7 mm, a lesion length of 21 mm, and a diameter stenosis between 50 and 99%. exclusion criteria included thrombus in the target vessel, severe calcification, three - vessel disease, ostial lesion, bifurcation lesion involving a side branch > 2.0 mm in diameter, target lesion located in or supplied by an arterial or venous bypass graft, and unsuccessful pre - dilatation. the full list of inclusion and exclusion criteria can be accessed at clinicaltrials.gov (nct01960504). clinical follow - up was planned at 1, 6, 12, 24, and 36 months. angiographic follow - up was scheduled at 6 months for all patients, an additional pre - specified imaging follow - up was scheduled at 12 months if subjects consented. a subgroup of 30 patients underwent intravascular ultrasound (ivus), optical coherence tomography (oct), and vasomotion testing at 6 months. at this time point, these patients were also asked to consent for voluntary ivus, oct, and vasomotion assessment at 12 months. only patients without target lesion revascularization (tlr) were asked for additional 12-month imaging assessment. dreams 2 g is a drug - eluting absorbable metal scaffold system comprising of an absorbable magnesium scaffold pre - mounted on a balloon - expandable delivery system. the scaffold is laser - polished and its surface is completely coated with bioresorbable poly - l - lactide, which incorporates sirolimus. the primary endpoint was in - segment late lumen loss (lll) at 6-month follow - up. secondary endpoints at 12 months were tlf, a composite of cardiac death, target - vessel myocardial infarction, and clinically tlr ; scaffold thrombosis ; in - scaffold and in - segment binary restenosis ; diameter stenosis ; and in - scaffold lll. a clinical event committee adjudicated all adverse events, and an independent core laboratory (cardialysis b.v., rotterdam, the netherlands) performed the quantitative coronary angiography (qca), ivus, and oct analyses. the device was available to operators in sizes 2.5 mm 20 mm, 3.0 mm 20 mm, or 3.5 mm 25 mm. pre - dilatation was mandatory ; post - dilatation was left to the operator 's discretion. only one study device per lesion was allowed, although in bailout situations, a second dreams 2 g could be used, and, in case of failure, an orsiro des. the sample size was calculated based on the primary endpoint in - segment lll at 6 months and has been previously reported. the null hypothesis was a mean in - segment lll at 6 months of 0.5 mm. an overall of 121 (101 + 20) subjects were scheduled to be enrolled, assuming an expected in - segment lll mean of 0.45 mm, standard deviation of 0.2 mm, one - sided, significance level of 0.05, and 80% power. the intention - to - treat analysis population was defined as patients for whom an investigational scaffold entered the guide catheter following the diagnostic angiogram. patients not receiving a study device were included in the calculation of procedural success, but excluded from further analysis as defined in the study protocol. for serial imaging analysis, paired data were used. means and standard deviations, medians and interquartile ranges (iqr), and 95% cis were calculated as appropriate. for categorical data, absolute and relative frequencies were calculated and 95% cis for proportions. p - values were calculated using paired t - test, fisher 's exact test, test, and wilcoxon signed - rank test, where applicable. the sponsor was involved in the design and conduction of the study, data collection, monitoring, and data analysis. a detailed description of the study has been recently published. in brief, biosolve - ii is a prospective, multi - centre, first - in - man study to evaluate the safety and performance of dreams 2 g (biotronik ag, buelach, switzerland). the study is in compliance with the declaration of helsinki, good clinical practice, iso14155, and was approved by the institutional ethics committees at the participating 13 institutions in europe, south america, and asia. eligible patients had stable or unstable angina or documented silent ischaemia, a maximum of two single de novo lesions in two separate coronary arteries, with a reference vessel diameter between 2.2 and 3.7 mm, a lesion length of 21 mm, and a diameter stenosis between 50 and 99%. exclusion criteria included thrombus in the target vessel, severe calcification, three - vessel disease, ostial lesion, bifurcation lesion involving a side branch > 2.0 mm in diameter, target lesion located in or supplied by an arterial or venous bypass graft, and unsuccessful pre - dilatation. the full list of inclusion and exclusion criteria can be accessed at clinicaltrials.gov (nct01960504). clinical follow - up was planned at 1, 6, 12, 24, and 36 months. angiographic follow - up was scheduled at 6 months for all patients, an additional pre - specified imaging follow - up was scheduled at 12 months if subjects consented. a subgroup of 30 patients underwent intravascular ultrasound (ivus), optical coherence tomography (oct), and vasomotion testing at 6 months. at this time point, these patients were also asked to consent for voluntary ivus, oct, and vasomotion assessment at 12 months. only patients without target lesion revascularization (tlr) were asked for additional 12-month imaging assessment. dreams 2 g is a drug - eluting absorbable metal scaffold system comprising of an absorbable magnesium scaffold pre - mounted on a balloon - expandable delivery system. the scaffold is laser - polished and its surface is completely coated with bioresorbable poly - l - lactide, which incorporates sirolimus. the primary endpoint was in - segment late lumen loss (lll) at 6-month follow - up. secondary endpoints at 12 months were tlf, a composite of cardiac death, target - vessel myocardial infarction, and clinically tlr ; scaffold thrombosis ; in - scaffold and in - segment binary restenosis ; diameter stenosis ; and in - scaffold lll. a clinical event committee adjudicated all adverse events, and an independent core laboratory (cardialysis b.v., rotterdam, the netherlands) performed the quantitative coronary angiography (qca), ivus, and oct analyses. the device was available to operators in sizes 2.5 mm 20 mm, 3.0 mm 20 mm, or 3.5 mm 25 mm. pre - dilatation was mandatory ; post - dilatation was left to the operator 's discretion. only one study device per lesion was allowed, although in bailout situations, a second dreams 2 g could be used, and, in case of failure, an orsiro des. the sample size was calculated based on the primary endpoint in - segment lll at 6 months and has been previously reported. the null hypothesis was a mean in - segment lll at 6 months of 0.5 mm. an overall of 121 (101 + 20) subjects were scheduled to be enrolled, assuming an expected in - segment lll mean of 0.45 mm, standard deviation of 0.2 mm, one - sided, significance level of 0.05, and 80% power. the intention - to - treat analysis population was defined as patients for whom an investigational scaffold entered the guide catheter following the diagnostic angiogram. patients not receiving a study device were included in the calculation of procedural success, but excluded from further analysis as defined in the study protocol. for serial imaging analysis, paired data were used. means and standard deviations, medians and interquartile ranges (iqr), and 95% cis were calculated as appropriate. for categorical data, p - values were calculated using paired t - test, fisher 's exact test, test, and wilcoxon signed - rank test, where applicable. the sponsor was involved in the design and conduction of the study, data collection, monitoring, and data analysis. a comparison of baseline parameters of the angiographic and ivus / oct subgroups with serial 6- and 12-month evaluation compared with the overall population is provided in the supplementary material online, table s1. in two lesions, dreams 2 g could not be implanted due to insufficient pre - dilatation ; follow - up of these two subjects was consequently not included in this analysis. = 123mean age, years65.2 10.3male gender78 (63.4)hypertension101 (82.1)hyperlipidaemia74 (60.2)diabetes36 (29.3)history of smoking67 (54.5)previous percutaneous coronary interventions44 (35.8)cabg8 (6.5)history of myocardial infarction29 (23.6)renal failure4 (3.3)congestive heart failure8 (6.5)history of stroke or tia7 (5.7)mean lesion length, mm12.61 4.53mean reference vessel diameter, mm2.68 0.40aha / acc classification type b2/c53 (43.4)moderate - to - severe calcification13 (10.6)thrombus3 (2.4)data are shown as mean sd or n (%).aha / acc, american heart association / american college of cardiology ; cabg, coronary artery bypass graft ; tia, transient ischaemic attack.n = 120, in 3 subjects, images were not analysable.n = 122, 1 subject not analysable. two patients who did not receive an implant were used for calculation of device and procedural success only. forty - five angiographic assessments of which two were excluded as they did not have matched projections recorded, one did not have a 6-month but only 12-month angiography. baseline clinical and lesion characteristics data are shown as mean sd or n (%). aha / acc, american heart association / american college of cardiology ; cabg, coronary artery bypass graft ; tia, transient ischaemic attack. two patients who did not receive an implant were used for calculation of device and procedural success only. forty - five angiographic assessments of which two were excluded as they did not have matched projections recorded, one did not have a 6-month but only 12-month angiography. at baseline, 88 (71.5%) patients had stable angina, 17 (13.8%) unstable angina, and 18 (14.6%) silent ischaemia. at 12-month follow - up, 100 (86.2%) patients were symptom - free, 14 (12.1%) had stable angina (9 ccs class i and 5 ccs class ii), 1 (0.9%) had silent ischaemia, and none had unstable angina. fifty - seven patients (48.3%) were still on dual antiplatelet therapy at 12 months. paired qca data could be obtained in 42 patients from seven centres at a mean follow - up time of 181 17 days and 367 17 days for 6- and 12-month assessments, respectively. paired in - segment lll at 6 and 12 months was 0.20 0.21 mm (95% ci : 0.13;0.26) and 0.25 0.22 mm (95% ci : 0.18;0.32), p = 0.117, 0.05 0.21 mm (95% ci : 0.01;0.12), and in - scaffold lll 0.37 0.25 mm (95% ci : 0.29;0.45) and 0.39 0.27 mm (95% ci : 0.31;0.48), p = 0.446, 0.03 0.22 mm (95% ci : 0.04;0.10) (table 2, figure 2). the in - segment and in - scaffold lll of the overall population at 6 months were 0.27 0.37 and 0.44 0.36 mm for the overall population (0.21 0.28 and 0.37 0.28 mm in patients without tlr). there was no statistically significant difference in baseline characteristics between this subgroup and the overall patient population. table 2quantitative coronary angiographic analysis (paired data, n = 42)pre - procedurepost - procedure6 months12 monthsreference vessel diameter in - segment2.74 0.352.75 0.352.60 0.382.60 0.41reference vessel diameter in - scaffoldna2.84 0.372.66 0.342.64 0.44minimum lumen diameter in - segment1.22 0.332.25 0.412.01 0.381.96 0.41minimum lumen diameter in - scaffoldna2.54 0.332.14 0.382.10 0.41acute gain in - segmentna1.00 0.38nanaacute gain in - scaffoldna1.29 0.34nanadiameter stenosis in - segment55.2 10.918.7 6.822.6 9.224.7 10.6diameter stenosis in - scaffoldna10.4 6.019.6 8.420.4 8.6binary restenosis in - segment, % nana0 (0)2 (4.8)binary restenosis in - scaffold, % nana0 (0)0 (0)late lumen loss in - segmentnana0.20 0.210.25 0.22late lumen loss in - scaffoldnana0.37 0.250.39 0.27data are shown as mean sd or n (%). there was no significant difference in outcomes between 6 and 12 months.na, not applicable. figure 2cumulative frequency curves for in - segment (a) and in - scaffold (b) late lumen loss. six- and twelve - month serial analysis of late lumen loss observed in biosolve - ii compared with biosolve - i using the precursor device dreams first generation. quantitative coronary angiographic analysis (paired data, n = 42) data are shown as mean sd or n (%). cumulative frequency curves for in - segment (a) and in - scaffold (b) late lumen loss. six- and twelve - month serial analysis of late lumen loss observed in biosolve - ii compared with biosolve - i using the precursor device dreams first generation. serial ivus and oct analyses at 6 and 12 months were performed in 11 patients (respective data listings are provided in the supplementary material online, tables s2 and s3). paired ivus parameters did not differ significantly between 6 and 12 months (median minimum lumen area of 4.80 vs. 4.69 mm, p = 0.700), except for the number of patients with incomplete strut apposition, which was reduced to zero at 12 months (supplementary material online, table s4 ; figure 3). by oct, the median minimal lumen area decreased from 4.58 mm at 6 months to 4.19 mm at 12 months, p = 0.032. no intraluminal mass figure 4 shows that at 6 and 12 months, strut - like remnants are only visible by ivus but not by oct. only a very small neo - intimal hyperplasia area is visible (7 o'clock). indicates the difference between follow - ups in mm [95% ci ]. figure 4serial angiographic, optical coherence tomography, intravascular ultrasound, and virtual histology of a patient implanted with dreams 2 g. matched images show that at 6 months, struts are hardly discernible by optical coherence tomography and the scaffold strut that had covered the side branch post - procedure (12 o'clock) had disappeared. at 12 months, the vessel surface appears even smoother than at 6 months (2 o'clock). on intravascular ultrasound, assessed by virtual histology, the white colour coding of the scaffold struts disappears over time demonstrating the absorption process. only a very small neo - intimal hyperplasia area is visible (7 o'clock). serial angiographic, optical coherence tomography, intravascular ultrasound, and virtual histology of a patient implanted with dreams 2 g. matched images show that at 6 months, struts are hardly discernible by optical coherence tomography and the scaffold strut that had covered the side branch post - procedure (12 o'clock) had disappeared. at 12 months, the vessel surface appears even smoother than at 6 months (2 o'clock). on intravascular ultrasound, strut remnants assessed by virtual histology, the white colour coding of the scaffold struts disappears over time demonstrating the absorption process. serial vasomotion at 6 and 12 months was tested in 14 patients ; thereof 11 (79%) had a change of > 3% to the mean lumen diameter after infusion or injection of acetylcholine (figure 5). the median percentage change in mean lumen diameter between pre - and post - acetylcholine was 2.6% (iqr : 6.4 to 0.6%, mean 5.1 7.7%) at 6 months and 3.4% (iqr : 9.4 to 3.2%, mean 3.4 11.0%) at 12 months ; the percentage change in mean lumen diameter between post - acetylcholine and nitroglycerine was 3.4% (iqr : 1.19.2%, mean 5.8 6.4%) and 6.7% (iqr : 0.017.4%, mean 8.2 10.6%), respectively. percentage change in mean lumen diameter after acetylcholine and after subsequent intracoronary injection of nitroglycerine at 6 and 12 months in 14 patients. the ends of the boxes represent the first and third quartiles, the band the median, and the crystal the mean. there was no statistical significant difference between 6 and 12 months (p = 0.808 for acetylcholine and p = 0.626 for nitroglycerine). percentage change in mean lumen diameter after acetylcholine and after subsequent intracoronary injection of nitroglycerine at 6 and 12 months in 14 patients. the ends of the boxes represent the first and third quartiles, the band the median, and the crystal the mean. there was no statistical significant difference between 6 and 12 months (p = 0.808 for acetylcholine and p = 0.626 for nitroglycerine). ach, acetylcholine ; nitro, nitroglycerine. target lesion failure at 12 months was observed in 4/118 patients (3.4%, 95% ci : 0.98.4). no events beyond 6 months occurred. specifically, one death (0.8%) of unknown cause on day 134 one target - vessel myocardial infarction (0.8%) was due to temporary no - reflow after scaffold implantation. the main findings of our study are stability of the angiographic and clinical outcomes of the second - generation device dreams 2 g between 6 and 12 months. there was no definite or probable scaffold thrombosis up to 12 months and no tlf beyond 6 months. these findings are relevant as 95% of the magnesium scaffold is expected to be absorbed within 12 months. the importance of this time point for dreams 2 g is reflected in the recently published european society of cardiology european association of percutaneous cardiovascular interventions report on the evaluation on coronary stents, which states that for bioresorbable stents, critical time points of follow - up will depend on the pace of biodegradation and should cover complete resorption. compared with the first generation, dreams 2 g has markedly improved angiographic performance parameters also at 12 months (in - segment lll of 0.25 0.22 mm vs. 0.39 0.33 mm and in - scaffold lll of 0.39 0.27 vs. 0.52 0.39 mm). there are reports that found a significant relationship between lll and tlr even in patients with a lll of 3% was already observed at 6 months, and continued to increase at 12 months. in general, angiographic, ivus, and oct parameters remained stable between 6 and 12 months, yet there was a small, but significant decrease in median minimal lumen area when serially measured by oct in 11 patients (0.25 mm, iqr : 0.64 to 0.09, p = 0.032). given the very small number of patients included in the intracoronary imaging sub - study, these findings should be interpreted with caution. since dreams 2 g was designed to provide a longer scaffolding time compared with dreams 1 g, it may be assumed that late lumen enlargement is postponed and unlikely to be seen as early as after 1 year. additional longer - term follow - up evaluation is required to confirm luminal enlargement, as shown for other polymeric scaffolds or previous versions of the magnesium scaffold implanted in the progress and biosolve - i studies. as with most first - in - man trials, biosolve - ii was not randomized and the clinical follow - up rate was high, but attributed to the fact that it was voluntary per protocol only a relatively small cohort was available for serial qca, ivus, and oct assessments. the demographics of these patients were comparable to those of the overall study cohort, but nevertheless the data were only powered for in - segment lll at 6 month, eventually, a bias is introduced as symptomatic patients may be more prone to agree to 12-month imaging assessments than asymptomatic patients, and12-month imaging assessments were not performed in all participating centres.a further limitation is that long - term data are pending to document lumen enlargement as seen in biosolve - i at 3 years. finally, the favourable outcomes of biosolve - ii need to be confirmed in more complex patient and lesion scenarios. the data were only powered for in - segment lll at 6 month, eventually, a bias is introduced as symptomatic patients may be more prone to agree to 12-month imaging assessments than asymptomatic patients, and 12-month imaging assessments were not performed in all participating centres. as with most first - in - man trials, biosolve - ii was not randomized and included a limited population with simple lesions. consequently, comparisons with other devices have to be interpreted with caution. the clinical follow - up rate was high, but attributed to the fact that it was voluntary per protocol only a relatively small cohort was available for serial qca, ivus, and oct assessments. the demographics of these patients were comparable to those of the overall study cohort, but nevertheless the data were only powered for in - segment lll at 6 month, eventually, a bias is introduced as symptomatic patients may be more prone to agree to 12-month imaging assessments than asymptomatic patients, and12-month imaging assessments were not performed in all participating centres.a further limitation is that long - term data are pending to document lumen enlargement as seen in biosolve - i at 3 years. finally, the favourable outcomes of biosolve - ii need to be confirmed in more complex patient and lesion scenarios. the data were only powered for in - segment lll at 6 month, eventually, a bias is introduced as symptomatic patients may be more prone to agree to 12-month imaging assessments than asymptomatic patients, and 12-month imaging assessments were not performed in all participating centres. this novel absorbable metal scaffold showed a sustained favourable safety profile up to 12 months and no increase in lll between 6 and 12 months in a subgroup of 42 patients. these findings suggest that dreams 2 g can be considered as an alternative to current polymeric brs. m.h. handled funding and supervision, conceived and designed the research, and drafted the manuscript. the open access publication charges for this article was provided by biotronik ag, buelach, switzerland. reports study grants and lecture fees from biotronik, abbott vascular, cardiac dimensions, medtronic, volcano, and lilly. reports institutional research grants from biotronik, medtronic, boston scientific, and astrazeneca ; and personal fees from medtronic, boston scientific, and astra zeneca. w.w. reports grants from abbott vascular, biotronik, and terumo ; and being a non - executive board member and shareholder of argonauts partners, celyad. reports grants from biotronik ; and personal fees and non - financial support from abbott vascular, boston scientific, medtronic, and biotronik. e.e s.t.l. reports institutional research grants from biotronik, medtronic, biosensors, bayer, and actelion ; and non - financial support from abbott vascular, orbus neich, alvimedica, philips, asahi intecc, and terumo. y.o. reports personal fees from biotronik, medtronic, and abbott vascular ; grants and personal fees from astra zeneca and boston scientific ; and grants from the medicines company and edwards lifesciences. | aimsmetal absorbable scaffolds constitute a conceptually attractive alternative to polymeric scaffolds. promising 6-month outcomes of a second - generation drug - eluting absorbable metal scaffold (dreams 2 g), consisting of an absorbable magnesium scaffold backbone, have been reported. we assessed the 12-month safety and performance of this novel device.methods and resultsthe prospective, international, multi - centre, first - in - man biosolve - ii trial enrolled 123 patients with up to two de novo lesions with a reference diameter between 2.2 and 3.7 mm. all patients were scheduled for angiographic follow - up at 6 months, and if subjects consented at 12 months. dual antiplatelet therapy was recommended for 6 months. quantitative coronary angiography (qca) parameters remained stable from 6 to 12 months [paired data of 42 patients : in - segment late lumen loss 0.20 0.21 mm vs. 0.25 0.22 mm, p = 0.117, 0.05 0.21 mm (95% ci : 0.01;0.12) ; in - scaffold late lumen loss 0.37 0.25 mm vs. 0.39 0.27 mm, p = 0.446, 0.03 0.22 (95% ci : 0.04;0.10), respectively ]. intravascular ultrasound and optical coherence tomography findings corroborated the qca results. target lesion failure occurred in four patients (3.4%), consisting of one death of unknown cause, one target - vessel myocardial infarction, and two clinically driven target lesion revascularization. no additional event occurred beyond the 6-month follow - up. during the entire follow - up of 12 months, none of the patients experienced a definite or probable scaffold thrombosis.conclusionthe novel drug - eluting metal absorbable scaffold dreams 2 g showed a continuous favourable safety profile up to 12 months and stable angiographic parameters between 6 and 12 months.clinicaltrials.gov identifiernct01960504. |
swine have served as an important biomedical model for humans for decades ; previous authors have summarized such models in more detail 1 - 5. limitations on reference citations for this review has meant that only the most recent will be cited to cover the latest developments and the broadest range of current pig models. advances using these genomic tools are described in the other reviews in this issue 6, 7. generally speaking, studying animal models of human disease helps scientists understand the mechanisms involved in the disease pathogenesis and thus provides tools for development of gene therapy to cure the disease / condition in humans. to date, the humanized mouse has been widely used to advance our understanding of human hematopoiesis, innate and adaptive immunity, autoimmunity, infectious diseases, cancer biology and regenerative medicine 8. unfortunately, the humanized mouse and many mouse disease models often do not faithfully mimic the relevant human conditions. an example is the well developed swine atherosclerosis model which has facilitated analyses of disease progression and pathogenesis and testing of new therapeutics 9, 10. this review will probe, as detailed examples, genomic studies of melanoma and of infectious disease resistance, highlighting issues to consider in designing such genomic studies. it will end with a short discussion of the potential for genomic approaches to develop new alternatives for control of viral infectious diseases, targeting porcine reproductive and respiratory syndrome virus (prrsv), and the potential for applying knowledge gained with this virus for human infectious disease studies. as outlined in table 1 the swine has been used as a major mammalian model for human biology 11. the similarity in size, particularly for miniature pigs, and physiology, and in organ development and disease progression the ability to deliberately time studies, image internal vessels and organs using standard human technologies, and collect repeated peripheral samples and, at kill, detailed tissue samples, has meant that the pig is an excellent biomedical model for humans. the ability to use pigs from the same litter, or cloned or transgenic pigs, facilitates genetic mapping. availability of numerous well defined cell lines, representing a broad range of tissues, will facilitate detailed testing of gene expression, drug susceptibility, etc. for genomics it is an asset that the pig genome has high sequence and chromosome structure homology with humans. with the swine genome sequence now well advanced there are increasingly improving genetic and proteomic tools for pigs. table 2 highlights some of the broad array of biomedical topics now addressed using swine models. some of these studies already employ genomics approaches, such as the heart, transplantation and melanoma models ; others are still in the early stages of affirming swine physiological parameters and utility as a human biomedical model. each model will be impacted by the availability of the functional genomic tools and swine genome sequence and maps outlined in other reviews in this issue 6, 7. some of the best examples of the impact of swine as a biomedical model are found with atherosclerosis and diabetes, diseases that are increasingly important today as the us faces major problems with obesity 9, 10. research is underway in pigs to determine the role of genetic background and to identify nutritional, exercise, and drug approaches which will alleviate disease progression and prevent pathology. advances in strategies to treat myocardial infarction are being pursued with cellular myoblast repair strategies 12 and tissue engineering of blood vessels 13. testing of emergency room treatment options, such as directed cardiopulmonary resuscitation (cpr) and ventricular fibrillation or cardiopulmonary bypass, which are difficult to assess in humans, are readily testable with pigs. all aspects of reproductive function have been studied in the pig, from the basics of maternal - fetal interactions 14, embryo development 15 - 17 and the impact of transgenesis 18, 19 to sperm function and quality 20, 21. basic sperm biology, such as chromosome positioning during spermatogenesis 22, as well as semen transmission of infectious disease 23, 24 are under active investigation (table 2). major transcriptional genomic and mapping efforts are underway in the pig model 14, 18 and should reveal important pathways regulating reproductive function. because of the size and physiologic similarity of pigs to humans, the pig has become a model of choice for tissue engineering and imaging studies. a range of imaging techniques has been developed with pigs as an early pre - human validation model. for example, sentinel node detection is increasingly important for cancer therapeutics ; the pig model has informed techniques for laparoscopic colon visualization and resection procedures 53, 54. tissue engineering using polymer scaffolds 61 have targeted areas as diverse as alternatives for knee meniscus cartilage and artificial bladder construction 62. swine skin studies have been very important, the swine melanoma model has been particularly informative. targeted studies analyzing percutaneous permeation with different chemicals will influence international biodefense efforts as well as responses to biological toxins 77. gut physiology and intestinal development following probiotics have been pursued in pigs both as a means of decreasing antibiotic usage in pig feed as well as an important human model. assessments have focused on probiotic strain selection, timing and dosing and the effects of these particularly on neonatal gut physiology. the swine melanoma model is a well established spontaneous melanoma model and one of the best developed for genomic approaches. the sinclair melanoma and melanoma - bearing libechov minipig (melim) have been studied in detail using immune analyses, focusing on the role of tumor infiltrating lymphocytes, the potential effects of natural killer (nk) and t cells in tumor regression. imaging and sentinel lymph node (sln) mapping will enhance these studies 54. comparative studies of normal melanocytes with localized tumor cells should reveal tumor specific regulatory pathways 78 ; indeed laser capture microdissection studies revealed loss of the 13q36 - 49 chromosomal region in nodular melanoma cells 79. early mapping studies in the sinclair swine melanoma model determined that a single dose of a specific b swine leukocyte antigen (sla) haplotype on ssc7 was required for tumor initiator 80 ; complex segregation analysis identified a second locus 81. these were followed by more detailed qtl studies using the melim model which identified numerous melanoma candidate loci 40 ; interestingly human candidate genes cdk4 and braf were not susceptibility genes in this model. zhi - qiang 41 continued these studies identifying qtl for the synthetic trait, development of melanoma on ssc1, 2, 13, 15 and 17. their detailed phenotyping of 331 pigs revealed highly significant qtls (p $ 560 million losses in the us each year 84. as an rna virus with an evolving genome, prrsv is particularly problematic due to slow development of protective immunity to homologous challenge and lack of protection against heterologous virus challenge. thus it is a major target for swine research ; information gained from swine studies will inform human infectious disease studies, particularly for analyses of viral persistence and of factors relevant to prevention of congenital and seminal transmission pathways. detailed cellular analyses have assessed gene and protein expression. as with human disease studies analyses of cultured cells, e.g., infected marc cells 85, swine macrophages 86, 87, or samples from infected pigs 88, have expanded our knowledge of the impact of timing and level of viral infection on gene expression and pathway involvement. more detailed gene expression analyses are underway using long oligo and affymetrix arrays, testing both pooled and individual animal samples, as well as mucosal samples at death. results can be disappointing if mucosal samples are collected only after viral levels begin to resolve. the peak of anti - viral immunity (and relevant gene expression) may be much earlier ; however, that may be at a time when the actual viral levels may be difficult to evaluate and thus complicate comparative analyses. therefore, it is important to affirm preliminary results with hypothesis driven repeated analyses and with translation of gene expression and array results into protein expression data. genetic variation does exist in resistance / susceptibility to viral infections and has been proven for swine resistance / susceptibility to prrs 87, 89. although to date there is limited knowledge of host genes determining prrsv resistance ; some candidate genes have been identified (petry., submitted). more detailed studies are required to determine whether naturally disease resistant pigs can be identified and why do [some ] pigs stay healthy even with prrs ? what set of factors (detailed phenotype) truly correlate with lower prrsv burden ? what is the potential for sampling peripheral blood cells, serum or saliva for preinfection predictive studies of genetically determined virus resistance phenotype ? an international prrs genomics consortium, of university, government, and company based scientists, has been established to assess host genetics of prrs resistance / susceptibility. the goal is to develop a large, publicly available disease sample and dataset from thousands of pigs from relevant commercial lines infected with prrsv and from which a detailed phenotype have been collected. the end user performs his / her analysis on the appropriate sample and returns the data to the consortium. it is hoped that the data generated by the consortium will verify the genetic variation in pigs responding to prrsv infection, will reveal factors influencing health, survivability and growth, and will identify the relative importance of different phenotypes, and their heritability, in predicting responses to prrsv infection. overall this data should enable breeders to produce healthier pigs with improved resistance to prrsv and help animal health companies to develop improved vaccines and alternative anti - prrsv therapeutics. this data should help identify new critical control factors in human responses to viral infections. advantages of swine as a biomedical model swine biomedical models lessons learned from swine melanoma studies utilizing genomics for swine models - issues to consider | this review is a short update on the diversity of swine biomedical models and the importance of genomics in their continued development. the swine has been used as a major mammalian model for human studies because of the similarity in size and physiology, and in organ development and disease progression. the pig model allows for deliberately timed studies, imaging of internal vessels and organs using standard human technologies, and collection of repeated peripheral samples and, at kill, detailed mucosal tissues. the ability to use pigs from the same litter, or cloned or transgenic pigs, facilitates comparative analyses and genetic mapping. the availability of numerous well defined cell lines, representing a broad range of tissues, further facilitates testing of gene expression, drug susceptibility, etc. thus the pig is an excellent biomedical model for humans. for genomic applications it is an asset that the pig genome has high sequence and chromosome structure homology with humans. with the swine genome sequence now well advanced there are improving genetic and proteomic tools for these comparative analyses. the review will discuss some of the genomic approaches used to probe these models. the review will highlight genomic studies of melanoma and of infectious disease resistance, discussing issues to consider in designing such studies. it will end with a short discussion of the potential for genomic approaches to develop new alternatives for control of the most economically important disease of pigs, porcine reproductive and respiratory syndrome (prrs), and the potential for applying knowledge gained with this virus for human viral infectious disease studies. |
fabrication of a natural looking restoration is one of the challenges in esthetic dentistry because shade matching with natural teeth is a difficult task due to the complicated optical properties of teeth.1 an esthetic restoration should reproduce morphologic, optical, and biologic characteristics of teeth under varied clinical conditions. switches of ambient light sources and condition cause perceived color shifts of restorations and shade guides.2,3 all - ceramic restorations can be made to match natural teeth in terms of color, surface texture, and translucency4 ; therefore, they address the demand for esthetic restorations.5,6 optical properties of zirconia have introduced new opportunities for achieving superior esthetics.1 based on a clinical evaluation of shade matching maintenance of an all - ceramic system, 97 to 100% of restorations were rated alfa.7 however, one of the clinical problems for all - ceramics is that the allowed thickness for a restoration is limited, which is generally regarded as 1.5 mm.4 shade matching is one of the most pivotal esthetic tasks. although shade matching is usually performed by visual methods, instrumental color taking enhances the validity of visual shade matching.8,9 shade matching performance has been improved through the development of new shade guides and electronic color taking devices for dentistry.1 electronic color taking devices showed excellent repeatability,10 and the use of spectrophotometer (sp) allowed accurate color evaluation of teeth and restorations.11 however, color parameters measured by instruments vary by the measurement protocols.12 perceived color of an object is decided by reflected and transmitted visible light, and an object can only reflect and transmit the spectrum of light that shines on it. since lightings show varied source - dependent spectral power distributions (spds), shade matching performance is highly influenced by the light sources.13 therefore, the impact of illuminating lights on the color of dental substances is a significant clinical concern.3 metameric colors are the color stimuli of identical tristimulus values calculated based on the reflectance values under a particular light source, but have different spectral reflectance values,14 and metamerism is probably the largest single cause of industrial shade matching problems.15 since the spds of popular ambient light sources such as incandescent lamp, fluorescent lamp, and daylight differ, color of dental substances showed changes according to the illuminants used in sp or real light sources.3,16 - 18 it has been confirmed that the instrumental color values of teeth, restoratives, and shade guides vary by standard illuminant used in sp.19 - 24 it was also reported that color shift of all - ceramics by the switch of illuminants in sp was clinically perceptible.18 perceptible color differences were observed in shade guide tabs due to the switch of illuminants in sp.8,23,24 as to the observer factor, shade matching performance was affected by the color temperature of illuminated lights ; lower color temperature light decreased correct shade matching.25 therefore, careful control of lighting conditions is essential to achieve an optically pleasing estoration.26 although the daylight is regarded as an ideal light source, it can not be easily standardized because of its variability by weather, time of the day, and season of the year. cie standard illuminant d65 is defined to represent a phase of the daylight with a color temperature of 6,500k, illuminant a is defined to represent an incandescent light (2,856k), and illuminant f9 is defined to represent a fluorescent lamp light (4,150k).27 if teeth and restorations are opaque, influences of the type of instrument, illuminating and measuring configuration, and the kind of illuminant or light source on the color determination should have been limited.28 however, color taking of translucent substances by sp results in deviated color values compared with the real color perceived by naked eyes.29 these deviations are mainly caused by edge - loss effect due to small measurement aperture of sp,12,30 thickness of translucent layer, and background conditions.31 these distortions in color values measured by sp would decrease when color is taken by a spectroradiometer (sr). sr does not show edge - loss effect, and the illuminating configuration is similar to that of ambient lighting condition ; therefore, simulation of human color vision in this kind of instrument is higher than that in conventionally used sp. light source - dependent color shifts of a shade guide were determined by sr.3 however, properties of light sources used in sr should be further specified,32 - 34 because the cie illuminants are mathematically defined,27 whereas the spds of real light sources vary by the type, brand, and configuration of the source. visual thresholds for color differences although the threshold for acceptability was reported to be 3.5 color difference (eab) units and that for perceptibility was 1.8 eab units based sp readings,35 2.6 eab units was considered the clinically perceptible, while 5.5 eab units was considered the clinically acceptable threshold based on sr readings.36 human color vision is categorized into colorimetry, sensation, perception, and visualization.37 since the instrumental color taking is in the colorimetry domain and the perceptible / acceptable thresholds are in the perception domain, correlating two domains needs careful interpretation. although there have been reports on the influence of illuminants on the sp - based color shifts of dental substances,16 - 18,38 - 40 limitations in sp color taking might have distorted the experimental results of those studies. moreover, the illuminating configuration in the sp instrument is different from that in clinical condition. therefore, the purpose of this study was to determine the influence of the switch of real light sources, simulating the cie standard illuminants d65, a, and f9, on the sr - based color shift of clinically simulated ceramics. the null hypothesis assumed was that the shifts in color and three color coordinates (cie l, a, and b) would not be influenced by the switched light, shade of veneer ceramics, and brand of ceramics. specimens of seven core ceramics were fabricated, 11 mm in diameter, following the manufacturers ' instructions. vita lumin a2 shade (vita zahnfabrik, bad sackingen, germany) was selected. thickness of the specimens was controlled with a polishing machine (am technology, asan, chungnam, korea) to the manufacturers ' recommended thickness required to mask a discolored abutment (table 1). a sintering ceramic (vita vm 7 ; vita zahnfabrik) was used as a reference core material. veneer ceramics were prepared for each core material (table 1 and table 2), with the final thickness of layered specimen of 1.5 mm.4 two shades corresponding to a2 and a3 shades (vita zahnfabrik) were selected. the number of specimens was determined based on previous color studies, in which generally five specimens were investigated.41 - 43 detailed specimen preparation procedures have been reported previously.31 when the color of layered specimens was measured (table 2), corresponding veneer specimen was laid over a core specimen. in this layering procedure, one veneer specimen for each material, representing the mean color value of seven specimens, was used. when layering, a drop of optical fluid (refraction fluid, 1.5 index ; cargille lab, cedar grove, nj, usa) was applied between the veneer and core specimens for an optical connection.31 color of the layered specimens were taken according to the cie lab color scale over a white tile (cie l = 94.4, a = -0.1, and b = 0.6) under each of three light sources. a spectroradiometer (pr-670 spectrascan ; photo research, chatsworth, ca, usa), equipped with a lens (ms-75 macrospectar lens ; photo research), was fixed vertically over the upper part of a light - tight box (color sense ii ; sungjin hitech, gunpo, kyunggi - do, korea) with a vertical distance of 355 mm from the specimen.3 a measurement spot size of 5.25 mm in diameter was selected by setting the automated aperture opening to 1 degree, which was prescribed by the manufacture. two lamps that simulate the illuminant d65 (gretagmacbeth f20t12/65 6500 k lamp ; x - rite, grand rapids, mi, usa), one lamp that simulates the illuminant a (jd 100w / m2 ; iwasaki electric, tokyo, japan), and one lamp that simulates the illuminant f9 (f20t12/cw ; osram, sylvania, mississauga, ontario, canada) were installed on the inner top surface of the lighttight box by the manufacturer to illuminate the inside of the light - tight box with a similar light intensity regardless of the light source.3 spectral reflectance values were obtained from 380 to 780 nm with 2 nm intervals (spectrawin 2.0 ; photo research), which were converted to the cie l, a, and b values. chroma was calculated as cab = (a + b), and color shift was calculated as eab = [(l) + (a) + (b)].27 vectorial shifts of lightness and chroma, and those of cie a and b from the values under d65 simulator to those under a, or f9 simulators were determined. amounts of shifts in color, lightness (cie l), cie a and b, and also chroma, by the switch of lights were calculated. influence of the kind of switched light (a or f9), shade of veneer ceramics (a2 or a3), and brand of core ceramics (n=8) on the shifts in color and color coordinates was evaluated with a three - way analysis of variance (anova, =.05). brand was used as a factor instead of type of ceramics because ceramics in the same type could not be regarded as acting the same pattern by the switch of lights. amounts of shifts by the switch of lights are listed in table 3 and table 4. the range of shifts in color by the switch from d65 to a was 5.9 to 7.7 (mean standard deviation : 6.7 0.6), that of lightness (the value under a simulator minus that under d65) was -1.3 to 1.6 (0.1 0.8), that of cie a was 5.6 to 7.6 (6.5 0.6), that of cie b was -0.1 to 1.3 (0.7 0.4), and that of chroma was 1.1 to 2.6 (1.9 0.4). the range of shifts in color by the switch to f9 was 7.7 to 10.2 (9.2 0.8), that of lightness was 5.9 to 7.0 (6.4 0.4), that of cie a was -0.9 to 0.1 (-0.4 0.2), that of cie b was 4.9 to 7.8 (6.5 0.9), and that of chroma was 4.9 to 7.7 (6.5 0.9).. 2. in fig. 1 to fig. 4, 2m2 (a2) and 2m3 (a3) indicate the corresponding shifts of vitapan 3d - master shade guide (vita zahnfabrik) tabs under the same light switching conditions reported in a previous study.3 the ranges of lightness and chroma for the a2-veneered ceramics under d65 were 81.4 to 83.4 and 18.2 to 24.0, respectively, which shifted to 81.8 to 84.6 and 19.7 to 25.5 under a, and to 87.4 to 89.9 and 23.4 to 30.4 under f9. vectorial shifts of cie a and b are presented in fig. 3 and fig. 4. the ranges of cie a and b for the a2-veneered ceramics under d65 were 0.3 to 1.7 and 18.2 to 24.0, respectively, which shifted to 5.9 to 7.7 and 18.8 to 24.4 under a, and to -0.1 to 1.2 and 23.1 to 30.4 under f9. those for the a3-veneered ceramics were showed similar shifts. based on a three - way anova, the shifts in color and three color coordinates were influenced by the kind of switched light, shade of veneer, and brand of ceramics (p 5.5), which should be considered together with the inconsistencies in light - dependent color shifts among shade tabs, teeth, and restorations. color matching and shade compatibility evaluation should be performed under optimal lighting conditions that simulate the light source, which is most relevant to the patient. | purposeperceived color of ceramics changes by the spectral power distribution of ambient light. this study aimed to quantify the amount of shifts in color and color coordinates of clinically simulated seven all - ceramics due to the switch of three ambient light sources using a human vision simulating spectroradiometer.materials and methodscie color coordinates, such as l, a and b,of ceramic specimens were measured under three light sources, which simulate the cie standard illuminant d65 (daylight), a (incandescent lamp), and f9 (fluorescent lamp). shifts in color and color coordinate by the switch of lights were determined. influence of the switched light (d65 to a, or d65 to f9), shade of veneer ceramics (a2 or a3), and brand of ceramics on the shifts was analyzed by a three - way anova.resultsshifts in color and color coordinates were influenced by three factors (p 5.5). when switched to a, cie a increased (a : 5.6 to 7.6), however, cie b increased (b : 4.9 to 7.8) when switched to f9.conclusionclinically simulated ceramics demonstrated clinically unacceptable color shifts according to the switches in ambient lights based on spectroradiometric readings. therefore, shade matching and compatibility evaluation should be performed considering ambient lighting conditions and should be done under most relevant lighting condition. |
nonvalvular atrial fibrillation (nvaf), a treatable risk factor for ischemic stroke, is considered a worldwide epidemic predicted to increase in the coming decades. indeed, in 2010, the estimated global prevalence of af was 33.5 million.1 oral anticoagulation is the cornerstone treatment for nvaf patients with a moderate or high risk of thromboembolic complication.2 although direct oral anticoagulants (dabigatran, rivaroxaban, apixaban) have been on the market since 2010, vitamin k antagonists (vkas : warfarin, acenocoumarol, fluindione) remain a standard treatment in the management of nvaf. recently, several studies have examined the risks of bleeding and of ischemic stroke and systemic embolism (is / se) associated with perioperative heparin bridging anticoagulation in patients with nvaf.3, 4, 5, 6 however, no studies have investigated bridging risks during vka initiation in outpatient settings. it has been shown that the bleeding risk is highest during the first month of vka initiation.7 this risk may be greatest when a vka is combined with another antithrombotic agent, particularly in patients for whom a bridging therapy is indicated. bridging therapy at the initiation of vka therapy consists of transitioning a bridging agent (lowmolecularweight heparin [lmwh ] or other pentasaccharide, eg, fondaparinux and unfractionated heparin) to a vka. there is an overall consensus in favor of a bridging therapy prior to urgent cardioversion in patients with lifethreatening hemodynamic instability caused by newonset nvaf.8, 9, 10, 11, 12, 13 the recommendation in guidelines9, 10, 12 and summaries of product characteristics14, 15 is less clear for those with stable nvaf who do not require rapid anticoagulation. in reallife conditions, a bridging regimen is commonly used in those with a low stroke risk.16, 17, 18, 19 this practice is not supported by evidence. vka use has declined recently in favor of direct oral anticoagulants ; however, the latest available results have shown that they are still more widely used than direct oral anticoagulants.20, 21, 22, 23, 24 since millions of patients worldwide may be at risk of being unnecessarily exposed to bridging therapy, adverse health outcomes associated with management of vka still warrant investigation. therefore, the purpose of this cohort study was to assess the safety and effectiveness of a bridging regimen during the initiation of vka therapy in nvaf patients in outpatient care. a nationwide and retrospective cohort study was carried out using a french national health insurance database (sniiram).25, 26, 27, 28, 29, 30, 31 this database contains anonymous individual data on all reimbursements for patient health expenditure, including medicinal products and outpatient medical and nursing care, prescribed or provided by healthcare professionals. the sniiram database does not provide any direct information about the medical indication of each reimbursement, but it does contain the patient 's status with respect to full reimbursement of care for a severe and costly longterm condition listed in the international classification of diseases, 10th edition (icd10).32 information from the sniiram database has been crossreferenced with the french hospital discharge database (pmsi), which provides medical information on all patients admitted into the hospital in france, including discharge diagnoses coded in the icd10.33 this study was approved by the french data protection authority (commission nationale de l'informatique et des liberts). since the study was observational and used anonymous data, informed consent from participants was not required. this study included individuals aged 18 years or over who had their first vka (warfarin, fluindione, or acenocoumarol) dispensed between january 1, 2010 and november 30, 2014 for nvaf (inclusion date). a previously used algorithm was applied to identify nvaf individuals managed on an outpatient basis.25, 26 the study consisted of identifying individuals with a nonhospitalized longterm af condition (icd10 code i48) fully covered by insurance, having used a holter monitor or antiarrhythmic drug before or within 3 months after the start of the vka therapy. individuals with a history of heart valve disease or surgery, cancer, dialysis for renal failure, any lesion or condition posing a high risk of major bleeding such as anemia, hepatic impairment or liver disease, current or recent gastroduodenal ulceration, dementia, and recent lower limb surgery were excluded. individuals with a prevalent event related to outcomes of interest were also excluded (is / se, transient ischemic attack, ischemic heart disease, and bleeding). lmwh (tinzaparin, enoxaparin, nadroparin, and dalteparin) and fondaparinux, or an unfractionated heparin (heparin calcium)7 days before or after vka treatment. in order to attribute the use of heparins in the context of bridging therapy in outpatient care, individuals who had a history of heparin use within the year prior to vka initiation were not included. these were identified using previously used algorithms25, 26 from the hospitalization database, which contains discharge diagnoses. the primary outcome was bleeding (icd10 codes for intracranial bleeding : i60, i61, i62, s06.3, s06.4, s06.5, s06.6 ; gastrointestinal bleeding : k25.0, k25.2, k25.4, k25.6, k26.0, k26.2, k26.4, k26.6, k27.0, k27.2, k27.4, k27.6, k28.0, k28.2, k28.4, k28.6, k29.0, k92.0, k92.1, k92.2, i85.0, k62.5 ; and other : n02, r31, d62, r58, j94.2, r04.0, r04.1, r04.2, r04.8, r04.9, k66.1, m25.0, n92.0, n92.1, n92.4, n93.8, n93.9, n92.0, n95.0, h11.3, h35.6, h43.1, h45.0, h92.2, i32.2). secondary outcomes were ischemic stroke (is ; icd10 code : i63 [cerebral infarction except i63.6 ]) or systemic embolism (se ; icd10 code : i74). social deprivation index is a measure of an area 's level of deprivation based on the following : the median household income, the percentage of high school graduates in the population aged 15 years and older, the percentage of bluecollar workers in the active population, and the unemployment rate. this index has been divided into quintiles : the lower quintile (q1) represents the least deprived and the highest quintile (q5) the most deprived.34 vka prescribers were categorized as general practitioner, private practice cardiologist, other private practice specialist, and practitioner in a private institution. occurrence of the following comorbidities before initiation of a vka was considered : heart failure, diabetes mellitus, high blood pressure, chronic kidney disease, chronic hepatitis, peripheral arterial disease, and alcohol and smokingrelated hospitalization and consultation. the cha2ds2vasc score evaluates the risk of is / se in patients with af35 and the hasbled score evaluates the risk of bleeding in patients on oral anticoagulation.36 both scores include some modified items that were originally created from clinical examinations and laboratory tests. using a drug claims database, concomitant medications were defined as a drug of interest dispensed at least once in the 4 months prior to vka initiation, namely, antiarrhythmic agents or digitalis glycosides, lipidlowering agents, nonsteroidal antiinflammatory drugs, oral corticosteroids, gastroprotective agents, benzodiazepines, antiplatelet agents, and antihypertensive agents. bivariate association was examined between bridging status and covariates using and fisher 's exact tests for categorical variables, a cochran mantel haenszel trend test for ordered variables, and a t test and anova for continuous variables. association between outcomes and bridging status was studied during the first and 2 following months of anticoagulation : for crude analysis, a logrank test was used to examine differences between bridged and nonbridged groups in the occurrence of events of interest ; for multivariate analysis, a cox proportional hazards regression model37 was used to estimate hazard ratios (hr) and their 95% ci for bleeding and is / se. regarding bleeding risk, individuals experiencing this event were followed up from date of inclusion to date of the outcome. those without such an event were censored on the date of the following events, whichever came first : is / se (the other end point), death, switch from initial vka to another oral anticoagulant, up to 3 months of followup, or december 2014. the same was carried out for is / se and for each type of outcome of interest (intracranial, gastrointestinal, and other type of bleeding, and ischemic stroke and systemic embolism) (eg, for intracranial bleeding, censored events also included gastrointestinal and other types of bleeding). the following covariates were used in the adjusted models : sex, age, social deprivation index, type of vka therapy, type of vka prescribers, comorbidities, and concomitant medications. three models were studied : model 1 containing no covariate ; model 2, which is adjusted for sex and age ; and model 3, which is further adjusted for all covariates. the main analysis consisted of examining bleeding events occurring within 1 month of followup to be in line with shortterm exposure to a bridging therapy, which is recommended for 5 days on average, and the bleeding risk is highest during this period.7, in addition, a sensitivity analysis was conducted after adjustment for the propensity score for bridging use, built with a logistic regression model including all covariates.39 interactions between bridging status and bleeding events according to sex, age, and modified cha2ds2vasc and hasbled scores were tested. since evidence of interaction was found between bridging status and sex for bleeding (p=0.049), analyses were also performed with stratification according to sex. in addition, to test whether bleeding risk associated with bridging therapy depended on time, we fitted cox proportional hazards regression models with cubic spline functions with four knots at 7, 14, 21, and 28 days.40, 41 a further analysis was conducted to examine the relationship between the risks of is / se and bridging therapy. all analyses were performed with sas software, version 9.3 (sas institute inc, cary, nc). a nationwide and retrospective cohort study was carried out using a french national health insurance database (sniiram).25, 26, 27, 28, 29, 30, 31 this database contains anonymous individual data on all reimbursements for patient health expenditure, including medicinal products and outpatient medical and nursing care, prescribed or provided by healthcare professionals. the sniiram database does not provide any direct information about the medical indication of each reimbursement, but it does contain the patient 's status with respect to full reimbursement of care for a severe and costly longterm condition listed in the international classification of diseases, 10th edition (icd10).32 information from the sniiram database has been crossreferenced with the french hospital discharge database (pmsi), which provides medical information on all patients admitted into the hospital in france, including discharge diagnoses coded in the icd10.33 this study was approved by the french data protection authority (commission nationale de l'informatique et des liberts). since the study was observational and used anonymous data, informed consent from participants was not required. this study included individuals aged 18 years or over who had their first vka (warfarin, fluindione, or acenocoumarol) dispensed between january 1, 2010 and november 30, 2014 for nvaf (inclusion date). a previously used algorithm was applied to identify nvaf individuals managed on an outpatient basis.25, 26 the study consisted of identifying individuals with a nonhospitalized longterm af condition (icd10 code i48) fully covered by insurance, having used a holter monitor or antiarrhythmic drug before or within 3 months after the start of the vka therapy. individuals with a history of heart valve disease or surgery, cancer, dialysis for renal failure, any lesion or condition posing a high risk of major bleeding such as anemia, hepatic impairment or liver disease, current or recent gastroduodenal ulceration, dementia, and recent lower limb surgery were excluded. individuals with a prevalent event related to outcomes of interest were also excluded (is / se, transient ischemic attack, ischemic heart disease, and bleeding). bridging therapy was defined as the receipt of a subcutaneous heparin lmwh (tinzaparin, enoxaparin, nadroparin, and dalteparin) and fondaparinux, or an unfractionated heparin (heparin calcium)7 days before or after vka treatment. in order to attribute the use of heparins in the context of bridging therapy in outpatient care, individuals who had a history of heparin use within the year prior to vka initiation were not included. these were identified using previously used algorithms25, 26 from the hospitalization database, which contains discharge diagnoses. the primary outcome was bleeding (icd10 codes for intracranial bleeding : i60, i61, i62, s06.3, s06.4, s06.5, s06.6 ; gastrointestinal bleeding : k25.0, k25.2, k25.4, k25.6, k26.0, k26.2, k26.4, k26.6, k27.0, k27.2, k27.4, k27.6, k28.0, k28.2, k28.4, k28.6, k29.0, k92.0, k92.1, k92.2, i85.0, k62.5 ; and other : n02, r31, d62, r58, j94.2, r04.0, r04.1, r04.2, r04.8, r04.9, k66.1, m25.0, n92.0, n92.1, n92.4, n93.8, n93.9, n92.0, n95.0, h11.3, h35.6, h43.1, h45.0, h92.2, i32.2). secondary outcomes were ischemic stroke (is ; icd10 code : i63 [cerebral infarction except i63.6 ]) or systemic embolism (se ; icd10 code : i74). social deprivation index is a measure of an area 's level of deprivation based on the following : the median household income, the percentage of high school graduates in the population aged 15 years and older, the percentage of bluecollar workers in the active population, and the unemployment rate. this index has been divided into quintiles : the lower quintile (q1) represents the least deprived and the highest quintile (q5) the most deprived.34 vka prescribers were categorized as general practitioner, private practice cardiologist, other private practice specialist, and practitioner in a private institution. occurrence of the following comorbidities before initiation of a vka was considered : heart failure, diabetes mellitus, high blood pressure, chronic kidney disease, chronic hepatitis, peripheral arterial disease, and alcohol and smokingrelated hospitalization and consultation. the cha2ds2vasc score evaluates the risk of is / se in patients with af35 and the hasbled score evaluates the risk of bleeding in patients on oral anticoagulation.36 both scores include some modified items that were originally created from clinical examinations and laboratory tests. using a drug claims database, concomitant medications were defined as a drug of interest dispensed at least once in the 4 months prior to vka initiation, namely, antiarrhythmic agents or digitalis glycosides, lipidlowering agents, nonsteroidal antiinflammatory drugs, oral corticosteroids, gastroprotective agents, benzodiazepines, antiplatelet agents, and antihypertensive agents. bivariate association was examined between bridging status and covariates using and fisher 's exact tests for categorical variables, a cochran mantel haenszel trend test for ordered variables, and a t test and anova for continuous variables. association between outcomes and bridging status was studied during the first and 2 following months of anticoagulation : for crude analysis, a logrank test was used to examine differences between bridged and nonbridged groups in the occurrence of events of interest ; for multivariate analysis, a cox proportional hazards regression model37 was used to estimate hazard ratios (hr) and their 95% ci for bleeding and is / se. regarding bleeding risk, individuals experiencing this event were followed up from date of inclusion to date of the outcome. those without such an event were censored on the date of the following events, whichever came first : is / se (the other end point), death, switch from initial vka to another oral anticoagulant, up to 3 months of followup, or december 2014. the same was carried out for is / se and for each type of outcome of interest (intracranial, gastrointestinal, and other type of bleeding, and ischemic stroke and systemic embolism) (eg, for intracranial bleeding, censored events also included gastrointestinal and other types of bleeding). the following covariates were used in the adjusted models : sex, age, social deprivation index, type of vka therapy, type of vka prescribers, comorbidities, and concomitant medications. three models were studied : model 1 containing no covariate ; model 2, which is adjusted for sex and age ; and model 3, which is further adjusted for all covariates. the main analysis consisted of examining bleeding events occurring within 1 month of followup to be in line with shortterm exposure to a bridging therapy, which is recommended for 5 days on average, and the bleeding risk is highest during this period.7, 38 the proportional hazards assumption was assessed graphically. in addition, a sensitivity analysis was conducted after adjustment for the propensity score for bridging use, built with a logistic regression model including all covariates.39 interactions between bridging status and bleeding events according to sex, age, and modified cha2ds2vasc and hasbled scores were tested. since evidence of interaction was found between bridging status and sex for bleeding (p=0.049), analyses were also performed with stratification according to sex. in addition, to test whether bleeding risk associated with bridging therapy depended on time, we fitted cox proportional hazards regression models with cubic spline functions with four knots at 7, 14, 21, and 28 days.40, 41 a further analysis was conducted to examine the relationship between the risks of is / se and bridging therapy. all analyses were performed with sas software, version 9.3 (sas institute inc, cary, nc). between january 2010 and november 2014, 163 840 individuals were identified with nonhospitalized nvaf who had a first dispensing of vka. after excluding those who did not meet the inclusion criteria, our study sample was composed of 90 826 individuals, 27 147 (30%) of whom had bridging therapy at vka initiation (figure 1). in the bridging group, 87.8% had a bridging agent and a vka dispensed on the same day ; lmwh represented 80.0%, fondaparinux 16.7%, and heparin calcium 3.6% (the total percentage does not add up to 100%, as 0.3% were dispensed 2 types of heparin). four types of lmwh were used : tinzaparin (45.2%), enoxaparin (29.6%), nadroparin (5.1%), and dalteparin (0.2%). half of them were female (49.9%). bridged individuals were more likely to be younger, female, on fluindione, and have had a first vka prescribed by a cardiologist than their nonbridged counterparts. they were more likely to have heart failure and high blood pressure, and less likely to have chronic renal impairment, chronic hepatitis, and peripheral arterial disease. the bridged group had a slightly lower risk of is / se (mean modified cha2ds2vasc score=2.74 [sd=1.50 ] versus 2.83 [sd=1.40 ]) than the nonbridged group, but the same risk of bleeding (mean modified hasbled score=1.86 [sd=1.00 ]). overall, bridged individuals were less likely to be using concomitant medications, particularly antiplatelet agents, but had higher use of nonsteroidal antiinflammatory agents and oral corticosteroids.. a total of 318 (0.35%) cases of bleeding (57 intracranial, 99 gastrointestinal, and 162 other) and 151 (0.17%) is / se cases were identified during the first month of followup and 231 (0.31%) cases of bleeding (59 intracranial, 57 gastrointestinal, and 115 other) and 122 (0.16%) is / se cases were identified during the 2 following months (table 2 and table s4). number of events according to duration of followup during 1 month of followup, the incidence of bleeding was higher in the bridged group than the nonbridged group (0.47% [n=127 ] versus 0.30% [n=191 ], p<0.001) ; this difference was due to gastrointestinal and other types of bleeding. moreover, no significant difference was observed for is / se (0.16% [n=44 ] versus 0.17% [n=107 ], p=0.840). during the 2 following months, no difference was found between bridged and nonbridged groups for any of the events of interest (table 2). in multivariate analyses, different strategies of adjustment for confounding factors (models 13) generated similar hrs for bleeding (table 3). bleeding and arterial thromboembolism risks according to duration of followup hr indicates hazard ratio. adjustment for age and sex. adjustment for age, sex, comorbidities (heart failure, diabetes mellitus, high blood pressure, chronic kidney disease, chronic hepatitis, peripheral arterial disease, alcohol and smokingrelated hospitalization and consultation), and comedications (antiarrhythmic agents or digitalis glycosides, lipidlowering agents, nonsteroidal antiinflammatory drugs, oral corticosteroids, gastroprotective agents, benzodiazepines, antiplatelet agents, and antihypertensive agents). after adjustment for all confounding factors (model 3), a 60% increased risk was observed in the bridged group compared with the nonbridged counterpart during the first month of followup (hr=1.60 ; 95% ci, 1.282.01). adjustment for the propensity score did not modify hr estimates (hr=1.60 ; 95% ci, 1.272.01). the 1month bleeding risk also increased with bridging duration : when it lasted 7 days or less, the hr was 1.29 (95% ci : 0.951.76) and for 8 days or more, it was 1.93 (95% ci : 1.472.52). moreover, the 1month bleeding risk appeared to be similar according to types of heparin (table s5). however, our study is not sufficiently powered to thoroughly examine differences in bleeding risk between heparins. the 1month bleeding risk was also studied in subgroups defined according to the prescriber 's specialty : in patients managed by cardiologists, this risk was 43% higher in the bridged group compared with the nonbridged group (hr=1.43 ; 95% ci : 1.051.95) and 76% higher in those managed by general practitioners (hr=1.76 ; 95% ci : 1.252.47). when the risk was studied according to type of bleeding, a hr of 1.43 (95% ci, 0.822.47) was found for intracranial, 1.80 (95% ci, 1.202.69) for gastrointestinal, and 1.56 (95% ci, 1.142.15) for other types of bleeding. in addition, to test whether the risk of bleeding associated with bridging therapy depended on time, we fitted cox models with cubic spline functions.40, 41 the hr was highest within the first week after the start of vka and decreased towards 1 around the third week (figure s1). although no significant interaction was observed between bridging status and bleeding according to modified cha2ds2vasc or hasbled scores (table s6), effect modification was, however, found for sex (p value for interaction=0.049) : the risk was twice as high in women (hr=2.04 ; 95% ci, 1.492.80) (figure 2 and table s4). overall, women had a higher baseline risk profile than men : they were 5 years older, had a lower social deprivation index, were less likely to consult a cardiologist, and had higher modified cha2ds2vasc and hasbled scores (tables s7 and s8). study of the interaction between heparin bridging and sex at 1 month of followup (n=90 826). the pvalue is from the test statistic for testing the interaction between bridging status and sex after adjustment for age, comorbidities (heart failure, diabetes mellitus, high blood pressure, chronic kidney disease, chronic hepatitis, peripheral arterial disease, alcohol and smokingrelated hospitalization and consultation) and comedications (antiarrhythmic agents or digitalis glycosides, lipidlowering agents, nonsteroidal antiinflammatory drugs, oral corticosteroids, gastroprotective agents, benzodiazepines, antiplatelet agents, and antihypertensive agents). other bleeding includes the following : acute posthemorrhagic anemia, conjunctival hemorrhage, hemothorax, hemoperitoneum, hemarthrosis, hematuria, postmenopausal bleeding (women), epistaxis, hemoptysis, hemorrhage from other sites in respiratory passages, hemorrhage not classified elsewhere. the bleeding risk was similar in both groups (hr=0.93 ; 95% ci, 0.701.23) when the risk was studied during the second and third months of followup (table 3). regarding the risk of is / se, no significant difference was observed between the bridged and nonbridged groups either at 1 month or later (table 3). moreover, these results did not vary according to modified cha2ds2vasc or hasbled scores (p values for interaction of 0.320 and 0.486, respectively ; table s9). between january 2010 and november 2014, 163 840 individuals were identified with nonhospitalized nvaf who had a first dispensing of vka. after excluding those who did not meet the inclusion criteria, our study sample was composed of 90 826 individuals, 27 147 (30%) of whom had bridging therapy at vka initiation (figure 1). in the bridging group, 87.8% had a bridging agent and a vka dispensed on the same day ; lmwh represented 80.0%, fondaparinux 16.7%, and heparin calcium 3.6% (the total percentage does not add up to 100%, as 0.3% were dispensed 2 types of heparin). four types of lmwh were used : tinzaparin (45.2%), enoxaparin (29.6%), nadroparin (5.1%), and dalteparin (0.2%). half of them were female (49.9%). bridged individuals were more likely to be younger, female, on fluindione, and have had a first vka prescribed by a cardiologist than their nonbridged counterparts. they were more likely to have heart failure and high blood pressure, and less likely to have chronic renal impairment, chronic hepatitis, and peripheral arterial disease. the bridged group had a slightly lower risk of is / se (mean modified cha2ds2vasc score=2.74 [sd=1.50 ] versus 2.83 [sd=1.40 ]) than the nonbridged group, but the same risk of bleeding (mean modified hasbled score=1.86 [sd=1.00 ]). overall, bridged individuals were less likely to be using concomitant medications, particularly antiplatelet agents, but had higher use of nonsteroidal antiinflammatory agents and oral corticosteroids. a total of 318 (0.35%) cases of bleeding (57 intracranial, 99 gastrointestinal, and 162 other) and 151 (0.17%) is / se cases were identified during the first month of followup and 231 (0.31%) cases of bleeding (59 intracranial, 57 gastrointestinal, and 115 other) and 122 (0.16%) is / se cases were identified during the 2 following months (table 2 and table s4). number of events according to duration of followup during 1 month of followup, the incidence of bleeding was higher in the bridged group than the nonbridged group (0.47% [n=127 ] versus 0.30% [n=191 ], p<0.001) ; this difference was due to gastrointestinal and other types of bleeding. moreover, no significant difference was observed for is / se (0.16% [n=44 ] versus 0.17% [n=107 ], p=0.840). during the 2 following months, no difference was found between bridged and nonbridged groups for any of the events of interest (table 2). in multivariate analyses, different strategies of adjustment for confounding factors (models 13) generated similar hrs for bleeding (table 3). bleeding and arterial thromboembolism risks according to duration of followup hr indicates hazard ratio. adjustment for age and sex. adjustment for age, sex, comorbidities (heart failure, diabetes mellitus, high blood pressure, chronic kidney disease, chronic hepatitis, peripheral arterial disease, alcohol and smokingrelated hospitalization and consultation), and comedications (antiarrhythmic agents or digitalis glycosides, lipidlowering agents, nonsteroidal antiinflammatory drugs, oral corticosteroids, gastroprotective agents, benzodiazepines, antiplatelet agents, and antihypertensive agents). after adjustment for all confounding factors (model 3), a 60% increased risk was observed in the bridged group compared with the nonbridged counterpart during the first month of followup (hr=1.60 ; 95% ci, 1.282.01). adjustment for the propensity score did not modify hr estimates (hr=1.60 ; 95% ci, 1.272.01). the 1month bleeding risk also increased with bridging duration : when it lasted 7 days or less, the hr was 1.29 (95% ci : 0.951.76) and for 8 days or more, it was 1.93 (95% ci : 1.472.52). moreover, the 1month bleeding risk appeared to be similar according to types of heparin (table s5). however, our study is not sufficiently powered to thoroughly examine differences in bleeding risk between heparins. the 1month bleeding risk was also studied in subgroups defined according to the prescriber 's specialty : in patients managed by cardiologists, this risk was 43% higher in the bridged group compared with the nonbridged group (hr=1.43 ; 95% ci : 1.051.95) and 76% higher in those managed by general practitioners (hr=1.76 ; 95% ci : 1.252.47). when the risk was studied according to type of bleeding, a hr of 1.43 (95% ci, 0.822.47) was found for intracranial, 1.80 (95% ci, 1.202.69) for gastrointestinal, and 1.56 (95% ci, 1.142.15) for other types of bleeding. in addition, to test whether the risk of bleeding associated with bridging therapy depended on time, we fitted cox models with cubic spline functions.40, 41 the hr was highest within the first week after the start of vka and decreased towards 1 around the third week (figure s1). although no significant interaction was observed between bridging status and bleeding according to modified cha2ds2vasc or hasbled scores (table s6), effect modification was, however, found for sex (p value for interaction=0.049) : the risk was twice as high in women (hr=2.04 ; 95% ci, 1.492.80) (figure 2 and table s4). overall, women had a higher baseline risk profile than men : they were 5 years older, had a lower social deprivation index, were less likely to consult a cardiologist, and had higher modified cha2ds2vasc and hasbled scores (tables s7 and s8). study of the interaction between heparin bridging and sex at 1 month of followup (n=90 826). the pvalue is from the test statistic for testing the interaction between bridging status and sex after adjustment for age, comorbidities (heart failure, diabetes mellitus, high blood pressure, chronic kidney disease, chronic hepatitis, peripheral arterial disease, alcohol and smokingrelated hospitalization and consultation) and comedications (antiarrhythmic agents or digitalis glycosides, lipidlowering agents, nonsteroidal antiinflammatory drugs, oral corticosteroids, gastroprotective agents, benzodiazepines, antiplatelet agents, and antihypertensive agents). other bleeding includes the following : acute posthemorrhagic anemia, conjunctival hemorrhage, hemothorax, hemoperitoneum, hemarthrosis, hematuria, postmenopausal bleeding (women), epistaxis, hemoptysis, hemorrhage from other sites in respiratory passages, hemorrhage not classified elsewhere. the bleeding risk was similar in both groups (hr=0.93 ; 95% ci, 0.701.23) when the risk was studied during the second and third months of followup (table 3). regarding the risk of is / se, no significant difference was observed between the bridged and nonbridged groups either at 1 month or later (table 3). moreover, these results did not vary according to modified cha2ds2vasc or hasbled scores (p values for interaction of 0.320 and 0.486, respectively ; table s9). in patients with nvaf managed in an outpatient care setting at the time of vka initiation, a 60% increase in bleeding risk was found among those who were on a bridging regimen in the first month of oral anticoagulation compared with those who had vka therapy alone. women with bridging therapy were also shown to be particularly exposed to this risk since it was doubled compared with nonbridged counterparts. moreover, a similar risk was observed between these groups with regard to is / se. to our knowledge, this large study represents the first of its kind to examine effectiveness and safety outcomes in nvaf individuals managed in outpatient care who received bridging therapy at the initiation of oral anticoagulation. in this study, the bridging therapy was initiated in 30% of the analytic sample, and the stroke and bleeding risk profile at baseline was similar in the bridged and nonbridged groups. in the literature, two publications were found, reporting that bridging anticoagulation was used in 62% of patients managed in outpatient settings19 and in 19% of hospitalized patients with low stroke risk.16 these results, along with ours, support that bridging therapy is common in clinical practice. first, recommendations in guidelines9, 10, 12 and summaries of product characteristics14, 15 do not strongly advise against bridging therapy. indeed, several health professionals have criticized the lack of clarity of these guidelines.17, 18, 19, 42, 43 second, it may be possible that some physicians continue to believe in the theoretical possibility of a transient hypercoagulable state when starting vka therapy due to the difference in plasma halflife between vitamin k dependent coagulation factors (ii, vii, ix, and x) and proteins c and s, which are important components in coagulation inhibition. a rapid decrease in protein c and s levels may lead to thrombotic occlusions of the microvasculature resulting in necrosis, particularly of the skin.44 unlike our study, results from a nested case control study conducted on af patients using the uk clinical practice research datalink supported this theory, since azoulay found a 71% increased risk of stroke in the first 30 days of warfarin use compared with nonuse, whereas a decreased risk was observed after 30 days of use.45 however, this theory does not justify the routine use of bridging therapy since vkainduced skin necrosis occurs mostly in patients with inherited or acquired thrombophilia, such as protein c or s deficiencies,44 and several case studies have also reported that lmwh can also lead to skin necrosis.46, 47 lastly, there is insufficient evidence justifying systematic use of bridging therapy. this study showed that bridging therapy was associated with an increased risk of bleeding without any beneficial effect, and only 1 study conducted on 5327 patients in hospital settings was found that could put our results into perspective.16 kim reported bleeding and stroke rates of 1.0% and 0.3%, while in our study they were 0.35% to 0.17%, respectively. the lower rates here may lie in the difference between the study settings, (ie, our analytic sample managed in outpatient care may be healthier than hospitalized patients). despite the different incidence rates, kim found results similar to ours for bleeding and stroke risks : they observed an increased bleeding risk associated with bridging therapy (odds ratio 4.44, 95% ci 1.6811.72) and a comparable stroke risk between bridged and nonbridged groups. a statistically significant bridging status sex interaction on 1month bleeding risk was also observed. a subgroup analysis according to sex showed that bleeding risk was higher in bridged women than in nonbridged women, whereas in men no difference was observed. to our knowledge, this finding has not been previously reported. this result may be partially due to differences in pharmacokinetics and pharmacodynamics existing between men and women.48 in addition, sex difference was studied in relation to 1month bleeding risk in the bridged and nonbridged groups : consistent with atria and spaf studies,49 women did not have a statistically significant higher bleeding risk than men either as a whole or in bridged and nonbridged groups (data available on request). since the study population consisted of french healthcare insurance beneficiaries with no history of hospitalization for nvaf or of cerebrovascular accident / transient ischemic attack or ischemic heart disease, our results can not extend to individuals who initiated their oral anticoagulation in the hospital or to those with a high baseline risk of arterial thromboembolism. in order to examine outcomes of interest in stable nvaf patients managed in outpatient settings, we selected those who had no history of hospitalization 3 months before the date of first vka dispensing from a community pharmacy. in doing so, we selected those for whom bridging therapy is not indicated. indeed, bridged and nonbridged groups were quite similar in terms of baseline thromboembolism risk (mean modified cha2ds2vasc score of 2.7 and 2.8, respectively). however, we could have included patients for whom bridging therapy was necessary : a small number of patients were hospitalized within 1 month following the first vka dispensing for electrical cardioversion (2.3% in bridged and 1.0% in nonbridged groups). the hr for 1month bleeding risk associated with bridging therapy remained unchanged (hr=1.60 ; 95% ci : 1.272.01) after excluding these patients. in addition to this sensitivity analysis, we also estimated 1month bleeding risk in patients with only confirmed af with an icd10 code of i48 (68%) : the risk was still 39% higher in the bridged group compared to the nonbridged group (hr=1.39 ; 95% ci : 1.091.77). like other health insurance databases, the sniiram database does not capture certain clinical and laboratory data such as international normalized ratio (inr) values. although inr value is an important covariate, we do not believe our results were significantly affected by the lack of this information since the same target inr range (23) is recommended for bridged and not bridged patients and heparins do not modify inr values. in this study, we could not specifically calculate the sensitivity and specificity of the icd10 diagnoses for outcomes defined from the hospitalization database (pmsi) as compared to medical record review. although these measures are not available, information on hospital stay, in particular the cause of hospitalization, is accurate and precise as it is used to allocate budget to both public and private hospitals ; therefore, the quality of diagnosis codes from these data is regularly checked against patients ' medical records. importantly, as sniiram and pmsi are independent databases, we believe that diagnostic bias is not differential between bridged and not bridged individuals. indeed, crude incidence rates and risk estimates for bleeding and ischemic stroke or systemic embolism calculated here were consistent with those from other studies.16, 25 other variables defined using administrative data, such as nvaf, is / se, other comorbidities, alcohol and tobaccorelated hospitalization, and modified cha2ds2vasc and hasbled scores may be questionable. however, the same algorithms used in a recently published study25, 26 were applied and it was found that incidence rates for bleeding and stroke are close to those reported in a study conducted in hospital settings.16 moreover, expected findings were observed with these derived variables : (1) after 1 month of followup, no bleeding risk difference was observed between bridged and nonbridged individuals, which is consistent with shortterm exposure to a bridging therapy, which usually lasts 5 days38 ; (2) modified cha2ds2vasc score was strongly associated with ischemic stroke and systemic embolism, and modified hasbled score with bleeding events. a causal relationship between bridging anticoagulation and studied outcomes can not be confirmed as these findings are based on an observational study. lastly, although as many confounding factors as possible were taken into account, such as comorbidities and concomitant medications, the confounding effect of unmeasured and/or unknown factors can not be ruled out. however, our results should not be affected by diagnostic biases since databases to define bridging status and end points are completely independent. bridging therapy was widely used at the initiation of oral anticoagulation in a stable nvaf population. this practice did not decrease the is / se risk but increased the bleeding risk. all authors contributed to interpretation of the results and the paper 's revision and approved its final version. all the authors are employees of public institutions (bouillon, drayspira, bertrand, boudali, zureik : french national agency for medicines and health products safety [ansm ] ; ducimetire : french institute of health and medical research [inserm ]). bouillon and zureik had full access to all the data in the study and had final responsibility for the decision to submit the paper for publication. table s1. items included in the modified cha2ds2vasc score table s2. items included in the modified hasbled score table s3. description of bridging agents table s4. description of other types of bleeding during the first month of followup table s5. bleeding risks at 1 month of followup according to types of heparin table s6. study of the interaction between bridging and modified cha2ds2vasc and hasbled scores associated with bleeding risk at 1 month of followup (n=90 826) table s7. demographic and clinical characteristics of the study population according to sex table s8. demographic and clinical characteristics of the study population according to sex and bridging status table s9. study of the interaction between bridging and modified cha2ds2vasc and hasbled scores associated with risk of ischemic stroke and systemic embolism at 1 month of followup (n=90 826) figure s1. effect of bridging therapy on bleeding with time (and 95% pointwise confidence band) estimated by a cubic spline function with knots at 7, 14, 21, and 28 days. | backgroundseveral studies have recently examined the risks of bleeding and of ischemic stroke and systemic embolism associated with perioperative heparin bridging anticoagulation in patients with nonvalvular atrial fibrillation. however, few studies have investigated bridging risks during vitamin k antagonist initiation in outpatient settings.methods and resultsa retrospective cohort study was conducted on individuals starting oral anticoagulation between january 2010 and november 2014 for nonvalvular atrial fibrillation managed in outpatient care and identified from french healthcare insurance. bleeding and ischemic stroke and systemic embolism events were identified from the hospitalization database. adjusted hazard ratios with 95% ci were estimated using cox models during the first and 2 following months of anticoagulation. of 90 826 individuals, 30% had bridging therapy. a total of 318 (0.35%) cases of bleeding and 151 (0.17%) ischemic stroke and systemic embolism cases were identified during the first month of followup and 231 (0.31%) and 122 (0.16%) during the 2 following months, respectively. at 1 month of followup, the incidence of bleeding was higher in the bridged group compared with the nonbridged group (0.47% versus 0.30% ; p<0.001), and this increased risk persisted after adjustment for covariates (hazard ratio=1.60 ; 95% ci, 1.282.01). this difference disappeared after the first month of treatment (0.93 ; 0.701.23). no significant difference in the occurrence of ischemic stroke and systemic embolism was observed either at 1 month of followup or later.conclusionsat vitamin k antagonist initiation for nonvalvular atrial fibrillation managed in ambulatory settings, bridging therapy is associated with a higher risk of bleeding and a similar risk of arterial thromboembolism compared with no bridging therapy. |
illness associated with infection with mers - cov is characterized primarily by mild - to - severe respiratory complaints, most requiring hospital admission for pneumonitis or acute respiratory distress syndrome. as of june 11, 2015, ecdc has reported 1,288 laboratory - confirmed cases, including 498 deaths (1). overall, a large proportion of mers cases is suspected to be a result of zoonotic transmission (1) with growing evidence for dromedary camels (camelus dromedarius) as a reservoir. mers - cov - specific antibodies have been detected in camels across the middle east and the african continent, suggesting a geographically widespread distribution (2). analysis of an outbreak associated with a barn in qatar found dromedaries and humans to be infected with nearly identical strains of mers - cov (3) and further support for camels as reservoir came from a study in saudi arabia (ksa) that found widespread circulation of different genetic variants of mers - cov in camels, with geographic clustering of human and camel mers - cov sequences (4). however, few other studies provided evidence for zoonotic transmission of mers - cov from camels (5). we investigated the rate of mers - cov circulation in dromedaries at the slaughterhouse in qatar, previously linked to two mers cases in qatar. a random group of 105 camels that presented for slaughter in february (n=53) and march (n=52) 2014 were sampled for mers - cov analysis (table 1). animals either had come directly from within qatar or ksa, or had been sold through the central animal market (cm). swabs and lymph nodes were tested for mers - cov rna by internally controlled rt - pcr targeting upe and n genes, as described (3, 6). (7) was obtained from the first group of 53 samples and among others sequences generated from this group have been used to define a general mers - cov typing fragment (8). in total, 59% of the camels showed evidence for virus shedding in at least one type of swab at the time of slaughter (table 1). the percentage positive samples was the highest for nasal samples, followed by oral swabs, fecal swabs, and bronchial swabs. all but one animals with virus shedding from any sample had a positive nasal swab. for saliva (oral), the percentage of positive samples was the highest for animals between 7 and 12 months of age. approximation of the viral loads in the samples using the ct values obtained with the upe target showed no significant differences between types of samples and age groups (fig. 1) it should be noted that viral loads with ct>20 were observed only in the nasal swabs and the nasal swab sample with the highest viral load was found to contain infectious virus (7). mers - cov rna shedding by dromedary camels at the central slaughterhouse, qatar, depicted by sample type (a) and age group for nasal swabs (b). viral loads in samples are approximated using ct values obtained with the up - e target and are expressed as ct (40-ctsample). mers - cov detection in pre- and postmortem samples from camels presented for slaughter in doha, qatar (n=105) percentage positive for mers - cov rna as detected by two rt - pcr targets, followed by (absolute number of samples positive/ total number tested). to obtain further insight in the diversity of the viruses that circulated in dromedary camels at the slaughterhouse, mers - cov strains were sequenced according to a recently developed technique that enables the identification of divergent mers - cov types [sequences and technique in (8) ]. in total, five different sequence types were identified with three different types found at both sampling moments (table 2). camels either came from the large al - shahaniya international racing complex (ash) or from different sources elsewhere in qatar (indicated by the initial arrow for animals 68 and 1012 in (table 2). subsequently, they were either brought to a showing area (al mazad, am), to the barns at the cm for a holding period, or immediately sent to the slaughterhouse (sh). therefore, the sampling for animals 15 and 913 reflects mers - cov sequence diversity as a result of import from other regions in qatar, whereas virus circulation at the cm more likely explains the virus diversity for animals 68. summary of background information from slaughter camels for which sequences could be obtained from nasal swabs ash = al - shahaniya, am = al mazad, sh = slaughterhouse, cm = central market. antibodies to mers - cov s1 were found in 100 out of 103 animals tested by micro - array technology (9). for 53 animals, antibody levels were also determined by virus neutralization assay as described earlier (9). almost all animals had detectable neutralizing antibodies with no obvious age pattern and no significant difference in proportion of animals with low antibody levels (20 were observed only in the nasal swabs and the nasal swab sample with the highest viral load was found to contain infectious virus (7). mers - cov rna shedding by dromedary camels at the central slaughterhouse, qatar, depicted by sample type (a) and age group for nasal swabs (b). viral loads in samples are approximated using ct values obtained with the up - e target and are expressed as ct (40-ctsample). mers - cov detection in pre- and postmortem samples from camels presented for slaughter in doha, qatar (n=105) percentage positive for mers - cov rna as detected by two rt - pcr targets, followed by (absolute number of samples positive/ total number tested). to obtain further insight in the diversity of the viruses that circulated in dromedary camels at the slaughterhouse, mers - cov strains were sequenced according to a recently developed technique that enables the identification of divergent mers - cov types [sequences and technique in (8) ]. in total, five different sequence types were identified with three different types found at both sampling moments (table 2). camels either came from the large al - shahaniya international racing complex (ash) or from different sources elsewhere in qatar (indicated by the initial arrow for animals 68 and 1012 in (table 2). subsequently, they were either brought to a showing area (al mazad, am), to the barns at the cm for a holding period, or immediately sent to the slaughterhouse (sh). therefore, the sampling for animals 15 and 913 reflects mers - cov sequence diversity as a result of import from other regions in qatar, whereas virus circulation at the cm more likely explains the virus diversity for animals 68. summary of background information from slaughter camels for which sequences could be obtained from nasal swabs ash = al - shahaniya, am = al mazad, sh = slaughterhouse, cm = central market. antibodies to mers - cov s1 were found in 100 out of 103 animals tested by micro - array technology (9). for 53 animals, antibody levels were also determined by virus neutralization assay as described earlier (9). almost all animals had detectable neutralizing antibodies with no obvious age pattern and no significant difference in proportion of animals with low antibody levels (< 20) (fig. there was no correlation between antibody levels and the viral load as reflected by ct values (fig. reciprocal mers - cov - neutralizing antibodies titers by age group (a) and correlated with ct (40-ctsample) (b) for 53 camels at central slaughterhouse, qatar. a high proportion of dromedary camels shed mers - cov rna when presented for slaughter on two occasions at the central abattoir in qatar. co - circulation of multiple mers - cov variants demonstrates multiple virus introductions through flow of new animals traded into this group of animals, reflecting the virus diversity in wider qatar, including animals imported from australia, the middle east region and east africa. this suggests that cm is a driver of mers - cov circulation and a high - risk site for human exposure. indeed two cases in qatar were linked to visits to this area, and serology data on the only five workers that exclusively work in camel slaughter in qatar illustrated this potential burden as four of the five slaughterers had igg antibodies specific for mers - cov (10) a study at four slaughterhouses in egypt showed an overall rna prevalence in nasal swabs of 3.6% among 110 camels (11), which is significantly lower than in our study. a comparison of the organization of the meat markets between egypt and qatar could provide insight in the observed differences. the camels that are put together for a holding period of weeks prior to slaughter in doha have a wide variety of origins with varying initial immune status, which might provide a platform for extensive virus circulation. these include nave camels from australia (12) and camels from areas in the horn of africa and the gulf region with known differences in immune status (2, 13, 14). we observed a positivity rate in rectal swabs of 15 out of 103 animals that were analyzed (of which 61 were positive in nasal swabs). other studies observed none to very low numbers of camels shedding mers - cov rna in feces (3, 15). however, the total numbers of animals in these studies were too low to make a significant comparison with the data presented here. in the current views on mers - cov epidemiology, young camels (1year) with primary infections are thought to play a bigger role in mers - cov transmission than older animals for which less frequent shedding is observed (4, 15) and who demonstrate higher rates of seroconversion (reviewed in (ref. 2). however, we observed no significant differences in mers - cov rna shedding between different age groups. moreover, the lack of correlation between viral rna loads and levels of neutralizing antibodies in the animals suggests limited protection and potential for reinfection despite previous exposure, similar to the situation in humans with the four common human covs and as observed in a camel herd in ksa (15). a problem is that the time since onset of infection could not be determined as the animals did not show overt symptoms. therefore, it remains to be determined how the kinetics of infection are. in theory, the observed shedding of virus in the presence of neutralizing antibodies could represent sampling toward the end of an infection cycle. alternatively, the data may reflect limited mucosal immunity as has been shown for other animal coronaviruses (16). the possibility of camel vaccination has been suggested as a possible approach to controlling mers - cov transmission to humans. however, this may prove to be a challenging task in light of the above observations. given the high numbers of animals shedding these viruses in dynamic environments like the doha market and abattoir, potential human health risks need to be considered and the implementation of management alternatives (e.g. separation of nave animals from previously exposed animals and personal protective equipment for employees) might reduce the burden of mers - cov exposure to humans. the authors have not received any funding or benefits from industry or elsewhere to conduct this study. | two of the earliest middle east respiratory syndrome (mers) cases were men who had visited the doha central animal market and adjoining slaughterhouse in qatar. we show that a high proportion of camels presenting for slaughter in qatar show evidence for nasal mers - cov shedding (62/105). sequence analysis showed the circulation of at least five different virus strains at these premises, suggesting that this location is a driver of mers - cov circulation and a high - risk area for human exposure. no correlation between rna loads and levels of neutralizing antibodies was observed, suggesting limited immune protection and potential for reinfection despite previous exposure. |
a common upper airway and digestive tract is a rare congenital anomaly that is usually fatal and exact incidence of which is not known. the diagnosis is assumed to be made if there is inability to perform endotracheal intubation of a newborn with respiratory distress in an expert hand in a neonate not cried at birth. we present a newborn clinically suspected as a case of esophageal atresia with tracheo - esophageal fistula that was diagnosed retrospectively to have laryngeal atresia with absent vocal cords and a common aerodigestive tract continuing distally with trachea. we received a spontaneous vaginally delivered male outborn baby at 35 weeks period of gestation, weighing 1900 grams to a 24-years - old primigravida with antenatal risk factors of gestational diabetes mellitus and polyhydramnios. the baby was born limp without any cry and required immediate resuscitation with bag and mask ventilation. the initial intubation was reportedly difficult, and baby required frequent et suctioning for copious secretions. clinically, a strong suspicion of esophageal atresia (ea) and tracheo - esophageal fistula (tef) was made as feeding tube did not pass into the stomach. the chest x - ray revealed lower end of feeding tube going very low almost to t 6/7 level, and serial x rays revealed the feeding tube going sometimes to left side and sometimes to the right side, in retrospect, we came to know that it was in the trachea and was negotiating to left or right main bronchus. baby also had other associated anomalies at birth i.e. imperforate anus and absent right kidney. the evaluation with ultrasound revealed dilated left renal pelvis with non - visualized right kidney. the newborn was then placed in the left lateral decubitus position and a right posterolateral thoracotomy was performed. tracheo - esophageal fistula was isolated just one cm above the carina and was transfixed and ligated. the feeding tube could not be seen or felt in the operative field on maneuvering by anesthesiologist. a trial of passing a large bore tube i.e. mucus sucker was attempted, which went through but again could not be traced in the operating field as it was going into the trachea along with the ett. a trial of suctioning attempt on the mucus sucker made the right lung deflate with desaturation noticed by the anesthesiologist, which gave us a clue as to the connection of the upper end of esophagus with the trachea. an attempt was made by the otolaryngologist intraoperatively to negotiate the fiber - optic scope through the blind upper pouch, which was unsuccessful. neck exploration for creating a cervical esophagostomy was carried out ; however, the procedure was abandoned due to non - visualization of the upper end of esophagus. peroperatively, we found after neck exploration that there was only one continuous hollow tube containing the et tube. a feeding gastrostomy and sigmoid colostomy was carried out, and baby was shifted back to neonatal intensive care after the completion of surgery. baby was continued on ventilation and other supportive management with antibiotics, iv fluids, and inotropes. however, the baby succumbed on day 4 of life despite aggressive therapeutic and ventilatory support. direct laryngoscopy revealed only aryepiglottic folds behind the epiglottis, no vocal cords, and there was no visible opening for the trachea suggesting a common upper airway and digestive tract. fiber - optic scope was passed through the common opening, which revealed normal hypopharynx with normal development of the epiglottis but no evidence of true vocal cords. there was a collapsing lumen opening into a non - collapsible structure i.e. esophagus (collapsible) with absence of proximal trachea continuing with distal trachea (non - collapsible) [figure 1 ]. a complete autopsy revealed laryngeal atresia with absent vocal cords and a common aerodigestive tract continuing distally with trachea [figure 2 ]. flexible fiber - optic scopy view of the upper airway showing the epiglottis superiorly, an atretic larynx anteriorly, and esophagus (hollow tube) posteriorly postmortem specimen of upper airway showing atretic larynx with no vocal cords and dilator in esophagus very early in the development of a fetus during the third and fourth weeks of gestation, foregut divides to form the esophagus and the trachea. for an unknown reason, sometimes, the wall does not form properly and a tracheal esophageal fistula and/or esophageal atresia may be the result. upper airway agenesis seems to result from the ventral displacement of the tracheo - esophageal septum that forms during this period and a variety of different anomalies can result, including laryngeal and tracheal atresia. the various types of atresia are the result of arrest at different stages in embryonic development. the infant with pure laryngeal atresia presents with a normal color at birth, but cyanosis rapidly develops after clamping of the umbilical cord. about one - half the cases reported in the literature had other potentially fatal malformations. the vast majority of fetuses with complete larynx atresia die because the condition is not recognized and not treated immediately or because of other life - threatening anomalies. laryngotracheoesophageal cleft (ltec) is a rare congenital anomaly that results from failed posterior fusion of the cricoid cartilage associated with incomplete development of the tracheo - esophageal septum. ltec presents with increased secretions, respiratory distress, aspiration, and recurrent pulmonary infections. peroperatively, we anticipated that we might be dealing with a case of ltec, and we had planned for a second stage repair after complete evaluation and optimal stabilization. suspicion for a possible esophageal atresia and trachea - esophageal fistula in our case was based on clinical presentation with copious oral secretions and failure to pass down the feeding tube. at operation we found to have an absent upper blind pouch of esophagus with a common upper airway and digestive tract continuing with trachea distally and lower end of esophagus opening into the trachea. difficulty in locating the upper end of esophagus and deflation of right lung with desaturations while suctioning with a mucus sucker passed for locating the upper end raised a strong possibility of this rare congenital anomaly, which was established in the post - mortem. the diagnosis of congenital defect of a common upper airway and esophagus must be suspected if a newborn infant does not cry and develops severe cyanosis with respiratory distress soon after birth and an attempt for endotracheal intubation is unsuccessful even in expert hand, although such cases are diagnosed peroperatively or in autopsies. antenatal ultrasound findings of fetal ascites, fluid in the upper airway or enlarged and hyperechogenic lungs can raise suspicion of upper airway anatomical defects. these signs may be absent as in our case in the presence of a tracheo - esophageal or a broncho - esophageal fistula because the fluid from the lungs can pass into the stomach via the fistula. hence, in our case, an antenatal ultrasound failed to raise suspicion of a possible laryngeal atresia and tracheal agenesis. first, multiple views of feeding tube through orogastric route on chest x - ray did not show any coiling of the tube but showed passage into left or right side (which we now know was passing into left or right main bronchus). second, inability to locate the upper blind pouch of esophagus in a case of ea with tef should raise the suspicion of a common wall of aerodigestive tract. we aim to familiarize our colleagues with this rare and challenging congenital anomaly for which a high index of suspicion and good team management is required. | a common upper airway and digestive tract is a rare congenital anomaly that is usually fatal and its exact incidence is not known. it is a diagnostic challenge as it requires high index of suspicion. it should be considered in a neonate with respiratory distress in a non - vigorous baby requiring endotracheal intubation, which is difficult even in expert hand. we present a newborn with suspected tracheo - esophageal fistula that was diagnosed intraoperatively to have absent upper blind pouch of the esophagus and on autopsy found to have laryngeal atresia with absent vocal cords and a common aerodigestive tract continuing distally with trachea. the neonate was ventilated with endotracheal tube (ett) placement which in retrospect we came to know that it was in the esophagus. the neonate also had associated multiple congenital anomalies of vacterl association. the importance of teamwork between neonatologist, pediatric surgeon, anesthesiologist, and radiologist is highlighted for diagnosis and management of such rare cases. |
hiv infection induces a deficient immunologic milieu that enables other oncogenic viruses such as ebv to escape immune control in a multifactorial way, which can lead to the onset of lymphoma. the incidence of hiv - related pcnsl has decreased since the widespread introduction of haart. presentation of the disease is more frequent in persons with reduced performance status, cd4-counts below 100/l and high hiv viremia levels [4 - 6 ]. in immunocompetent populations, outcome and toxicity of different therapy regimens are largely agedependent. significantly reduced survival and neurotoxicity rates of up to 90% occur with combined chemotherapy - radiation regimens in the above 60 year age group. in all age groups, combination radiochemotherapies, with mostly intrathecal (it) administration of methotrexate (mtx) and/or cytarabine, prolong overall median survival rates by up to 55 months [7 - 9 ]. however, they can cause increased treatment - associated mortality, with high rates of infection and neurotoxicity. intensive chemotherapy - only regimens show less neurotoxicity with similar survival rates [7 - 10 ]. higher response rates are obtained with intensive autologous stem - cell - rescue protocols [11 - 14 ]. in contrast to therapeutic success demonstrated for immunocompetent patients, the outcome of hiv - associated pcnsl is still considered poor. median survival rates range from four to six weeks with best supportive care alone and 3 to 5 months with cranial radiation treatment [4,15 - 17 ]. most hiv - positive pcnsl patients are in clinically poor condition, and have advanced immunosuppression. moreover, hiv - associated leukoencephalopathy in advanced hiv disease is frequent, considering that neurotoxicity is one of the most frequent debilitating side effects in combined chemo - radiotherapy protocols. haart induced immune recovery improves survival of patients with aids - associated pcnsl and systemic lymphoma, but these patients still have reduced overall survival along with higher treatment - related mortality than hiv - negative patients. the highest survival rates were reported in a german survey with haart and radiotherapy 40 gy (n = 5/6) or hdmtx/40 gy (n = 1/6) of more than 18 months. steroids had no prognostic benefit and most pcnsl - patients (n = 23/29) were not eligible for any treatment. haart has been used alone for hiv - related pcnsl in patients with poor clinical status, patients who have declined other therapies, and in those experiencing failing therapies. the impact of curative haart of hiv - related pcnsl has been reported in five single cases [20 - 23 ]. antiviral and immune - based therapy of hiv - related pcnsl was suggested by raez. : 5 art - naive hiv - positive patients diagnosed with pcnsl were treated with ganciclovir, azt and interleukin-2 for two weeks and ganciclovir maintenance therapy. long lasting remission and a marked response after two weeks of treatment was reported in four out of five patients : two patients were alive and disease - free 22 and 13 months later, one died at month seven due to sepsis, one died after four months with progressive disease, and one patient was lost to follow - up in partial remission after changing to radiation - therapy due to ctc grade 4-neutropenia and thrombocytopenia at week four. aboulafia,. adopted a similar treatment strategy with one out of four patients remaining in complete remission after more than four years of follow - up. we report three patients who were treated at our institution following a modified treatment plan originally published by raez. 250 mg bid from day 15 ; ongoing haart with one additional nrti as haartbackbones, plus one protease inhibitor (lopinavir / ritonavir or nelfinavir according to individual conditions), orally bid in standard doses ; il-2 subcutaneously two million units bid at days 1 - 14 ; foscarnet bid 90 mg per kilogram body weight on days 1 - 14 in conjunction with one litre of saline bid to prevent renal failure. maintenance therapy with foscarnet (alternatively ganciclovir or cidofovir in one patient) was administered according to patient clinical status and individual toxicities. in case of clinical or radiological disease progression, cerebral computerized transaxial tomography (ctt) scan controls were performed on days 7 and 14 after the start of treatment, unless there was clinical deterioration, when cct scans were performed earlier. all patients underwent a brain biopsy that showed large b - cell lymphomas of high malignancy in two patients and lymphoproliferative disease with a large amount of necrotic tissue that was highly suspicious for nhl in the third patient despite lack of proven monoclonality. a treatment plan was adapted and proposed according to each patient 's clinical status, and informed consent was obtained. 250 mg bid from day 15 ; ongoing haart with one additional nrti as haartbackbones, plus one protease inhibitor (lopinavir / ritonavir or nelfinavir according to individual conditions), orally bid in standard doses ; il-2 subcutaneously two million units bid at days 1 - 14 ; foscarnet bid 90 mg per kilogram body weight on days 1 - 14 in conjunction with one litre of saline bid to prevent renal failure. maintenance therapy with foscarnet (alternatively ganciclovir or cidofovir in one patient) was administered according to patient clinical status and individual toxicities. in case of clinical or radiological disease progression, cerebral computerized transaxial tomography (ctt) scan controls were performed on days 7 and 14 after the start of treatment, unless there was clinical deterioration, when cct scans were performed earlier. all patients underwent a brain biopsy that showed large b - cell lymphomas of high malignancy in two patients and lymphoproliferative disease with a large amount of necrotic tissue that was highly suspicious for nhl in the third patient despite lack of proven monoclonality. a treatment plan was adapted and proposed according to each patient 's clinical status, and informed consent was obtained. patient 1 was a 33-year - old msm male, who had been diagnosed hiv - positive since 1995. his cd4-count was 16/l and at the time lymphoma diagnosis, he was art - naive. he also had clinical signs of kaposi - sarcoma with lesions on skin, tongue, palate and glans penis that had been treated for four months with systemic liposomal doxorubicin. he had undergone hospitalization for pneumocystis carinii pneumonia, which had been treated successfully four weeks prior to the lymphoma diagnosis. this was followed by three weeks of ongoing treatment for histologically proven pulmonary tuberculosis and oral candidiasis. subsequently, a 5 cm lesion in the basal ganglia with extensive perifocal oedema was demonstrated by cct scan, which had been performed because of a history of headaches and grand mal seizures. a presumptive diagnosis of cerebral toxoplasmosis was made, and empiric anti - toxoplasmosis therapy was initiated. a stereotactic brain biopsy was performed and revealed a highly malignant, large b - cell lymphoma (lca pos. the patient was treated for 13 days according to the aforementioned treatment plan concomitant to his anti - tuberculosis therapy (isoniazid, rifampicin, ethambutol, pyrazinamide), which was well tolerated. high dose azt, lamivudine and saquinavir boostered by ritonavir were selected for the haart regimen. due to tumour regression could not demonstrated by cct scan on days 7 and 13, so whole brain radiotherapy (wbrt) was initiated. follow - up immune status was not performed. after a cumulative dose of 20 gray, the patient expired due to lymphoma progression after discharge to his home, 50 days after diagnosis and 80 days after onset of signs and symptoms. 1 after 2 weeks of treatment with il-2, azt, haart, foscarnet. patient 2 was a 23-year - old ivdu male (men sex female), who had been diagnosed hiv - positive for two years (cd4-count 50/l, 13%, ratio 0.2, art - nave, hiv - viral load 522.600 c / ml). he was hospitalized for a 2 month exacerbation of the chronic paranoid psychosis and fever of unknown origin. a diagnosis of systemic cryptococcosis, manifested with fever, elevated serum - titre of 1 : 40/1 : 320 and repeated negative csf - findings, was confirmed 2 months before the pcnsl diagnosis. he had also been diagnosed with pneumonia due to s. pneumoniae, 6 weeks before the pcnsl diagnosis, and was successfully treated with iv ampicillin / sulbactam. two weeks later, due to a 2 cm contrast enhancing cerebral focus, empiric toxoplasmosis therapy was initiated for 2 weeks. the patient developed left sided paralysis and cct scan revealed that the cerebral focus had developed into a rapidly growing 4 cm parietal mass with accompanying oedema, and normal cytological, hsvconsensus - pcr and microbiological csf findings. brain biopsy demonstrated an ebv - associated diffuse large b - cell - lymphoma (cd20 +, mib-1 60%, lmp+). the initial tumour growth appeared to stop after one week with the aforementioned treatment plan, which included haart with lamivudine and nelfinavir. however, over the total course of three weeks tumour size progressed to 5 cm with a slight midline shift. his cd4-count increased two weeks after initiation of haart and il-2 to 810/l (52%, cd4/cd8 ratio 1.5), and hiv - viral load decreased to 1840 copies / ml. however, the patient had minimal signs of improvement of the left sided paralysis and severe psychosis. despite support by nurses in hospice care, he continued to worsen clinically, and expired due to lymphoma progression on day 166 after initial lymphoma diagnosis, and 200 days after the onset of signs and symptoms.. pat. 2 after 2 weeks of treatment with il-2, azt, haart, foscarnet. patient 3 was a 44-year old male (men sex male), who had been diagnosed hiv - positive for 7 years (cd4-count 120/l, cd4/8-ratio 0.2, viral load 793.000 copies / ml ; art - nave). he had a history of seasonal allergies, primary syphilis, gonorrhoea, and condylomata accuminata. a cct scan revealed a contrastenhancing temporal mass with a diameter of 1.2 cm accompanied with perifocal oedema. despite one week of empiric toxoplasmosis therapy, the mass had expanded to a diameter of 2 cm. stereotactic brain biopsy showed an ebv induced lymphoproliferation (lmp+, ebna-2 +, cd30-, cd20 +, high proliferation - index ki-67). due to the presence of massive necrosis and high proliferation index, highly malignant lymphoma was suspected, but could not be confirmed with the biopsy specimen material. since a repeat biopsy would have put the patient at further risk, these highly suspicious results were deemed consistent with the clinical presentation, and treatment was initiated with foscarnet, interleukin-2, high dose azt for 18 days, with lamivudine and lopinavir/ ritonavir bid added for haart in standard doses. due to a second seizure on day 7, gabapentin, which had been administered since the first seizure, overall, he tolerated the treatment regimen well except for limb edema, which improved with xipamid 10 mg, and one episode of local induration at an interleukin injection site that resolved without sequelae. a cct scan on day 18 showed a tumor diameter of 2.53 cm with no change in perifocal oedema. his cd4-count on day 11 was 550/l, cd4/cd8 ratio 2.0, and on day 30 it was 180/l, with a ratio of 0.7, and a viral load of 790 copies / ml. haart (azt, 3tc, lpv / r) was continued, and ganciclovir maintenance therapy (once daily 5 mg per bodyweight) was initiated. forty two days after treatment initiation, the patient underwent whole brain radiotherapy with a 30 gy cumulative dose. six months after start of treatment, his hiv viral load was constantly below detectable levels, and after 4 months, cct scan showed cr with leukencephalopathy. after completion of 3 months of ganciclovir maintenance therapy, the patient received cidofovir monthly for one year after the induction period (cumulatively 20 times) against ebv proliferation because of his relatively low cd4-count (nadir 100/l after 6 months). the cidofovir was then discontinued due to a steadily improving immune response (cd4count 306/l). ophthalmological and neurological examinations as well as renal function tests were within normal limits 2 years after the start of treatment. four years from time of diagnosis, cct restaging demonstrated constant marked demyelinization of the cerebral medullary layer, which was most likely radiotherapy - associated. the patient has been clinically and mentally stable 53 months after diagnosis. he remained on haart with azt, 3tc, lpv / r with cd4-counts above 300/l and without a detectable viral load. a year after diagnosis the patient retired from work.. pat. 3 after 5 years of treatment with il-2, azt, haart, foscarnet. patient 1 was a 33-year - old msm male, who had been diagnosed hiv - positive since 1995. his cd4-count was 16/l and at the time lymphoma diagnosis, he was art - naive. he also had clinical signs of kaposi - sarcoma with lesions on skin, tongue, palate and glans penis that had been treated for four months with systemic liposomal doxorubicin. he had undergone hospitalization for pneumocystis carinii pneumonia, which had been treated successfully four weeks prior to the lymphoma diagnosis. this was followed by three weeks of ongoing treatment for histologically proven pulmonary tuberculosis and oral candidiasis. subsequently, a 5 cm lesion in the basal ganglia with extensive perifocal oedema was demonstrated by cct scan, which had been performed because of a history of headaches and grand mal seizures. a presumptive diagnosis of cerebral toxoplasmosis was made, and empiric anti - toxoplasmosis therapy was initiated. a stereotactic brain biopsy was performed and revealed a highly malignant, large b - cell lymphoma (lca pos. the patient was treated for 13 days according to the aforementioned treatment plan concomitant to his anti - tuberculosis therapy (isoniazid, rifampicin, ethambutol, pyrazinamide), which was well tolerated. high dose azt, lamivudine and saquinavir boostered by ritonavir were selected for the haart regimen. due to tumour regression could not demonstrated by cct scan on days 7 and 13, so whole brain radiotherapy (wbrt) was initiated. follow - up immune status was not performed. after a cumulative dose of 20 gray, the patient expired due to lymphoma progression after discharge to his home, 50 days after diagnosis and 80 days after onset of signs and symptoms.. pat. 1 after 2 weeks of treatment with il-2, azt, haart, foscarnet. patient 2 was a 23-year - old ivdu male (men sex female), who had been diagnosed hiv - positive for two years (cd4-count 50/l, 13%, ratio 0.2, art - nave, hiv - viral load 522.600 c / ml). he was hospitalized for a 2 month exacerbation of the chronic paranoid psychosis and fever of unknown origin. a diagnosis of systemic cryptococcosis, manifested with fever, elevated serum - titre of 1 : 40/1 : 320 and repeated negative csf - findings, was confirmed 2 months before the pcnsl diagnosis. he had also been diagnosed with pneumonia due to s. pneumoniae, 6 weeks before the pcnsl diagnosis, and was successfully treated with iv ampicillin / sulbactam. two weeks later, due to a 2 cm contrast enhancing cerebral focus, empiric toxoplasmosis therapy was initiated for 2 weeks. the patient developed left sided paralysis and cct scan revealed that the cerebral focus had developed into a rapidly growing 4 cm parietal mass with accompanying oedema, and normal cytological, hsvconsensus - pcr and microbiological csf findings. brain biopsy demonstrated an ebv - associated diffuse large b - cell - lymphoma (cd20 +, mib-1 60%, lmp+). the initial tumour growth appeared to stop after one week with the aforementioned treatment plan, which included haart with lamivudine and nelfinavir. however, over the total course of three weeks tumour size progressed to 5 cm with a slight midline shift. his cd4-count increased two weeks after initiation of haart and il-2 to 810/l (52%, cd4/cd8 ratio 1.5), and hiv - viral load decreased to 1840 copies / ml. radiation therapy (40 gy) was initiated followed with concomitant dexamethasone. however, the patient had minimal signs of improvement of the left sided paralysis and severe psychosis. despite support by nurses in hospice care, he continued to worsen clinically, and expired due to lymphoma progression on day 166 after initial lymphoma diagnosis, and 200 days after the onset of signs and symptoms.. pat. 2 after 2 weeks of treatment with il-2, azt, haart, foscarnet. patient 3 was a 44-year old male (men sex male), who had been diagnosed hiv - positive for 7 years (cd4-count 120/l, cd4/8-ratio 0.2, viral load 793.000 copies / ml ; art - nave). he had a history of seasonal allergies, primary syphilis, gonorrhoea, and condylomata accuminata. a cct scan revealed a contrastenhancing temporal mass with a diameter of 1.2 cm accompanied with perifocal oedema. despite one week of empiric toxoplasmosis therapy, the mass had expanded to a diameter of 2 cm. stereotactic brain biopsy showed an ebv induced lymphoproliferation (lmp+, ebna-2 +, cd30-, cd20 +, high proliferation - index ki-67). due to the presence of massive necrosis and high proliferation index, highly malignant lymphoma was suspected, but could not be confirmed with the biopsy specimen material. since a repeat biopsy would have put the patient at further risk, these highly suspicious results were deemed consistent with the clinical presentation, and treatment was initiated with foscarnet, interleukin-2, high dose azt for 18 days, with lamivudine and lopinavir/ ritonavir bid added for haart in standard doses. due to a second seizure on day 7, gabapentin, which had been administered since the first seizure, was raised from 1200 mg to 2100 mg daily. overall, he tolerated the treatment regimen well except for limb edema, which improved with xipamid 10 mg, and one episode of local induration at an interleukin injection site that resolved without sequelae. a cct scan on day 18 showed a tumor diameter of 2.53 cm with no change in perifocal oedema. his cd4-count on day 11 was 550/l, cd4/cd8 ratio 2.0, and on day 30 it was 180/l, with a ratio of 0.7, and a viral load of 790 copies / ml. haart (azt, 3tc, lpv / r) was continued, and ganciclovir maintenance therapy (once daily 5 mg per bodyweight) was initiated. forty two days after treatment initiation, the patient underwent whole brain radiotherapy with a 30 gy cumulative dose. six months after start of treatment, his hiv viral load was constantly below detectable levels, and after 4 months, cct scan showed cr with leukencephalopathy. after completion of 3 months of ganciclovir maintenance therapy, the patient received cidofovir monthly for one year after the induction period (cumulatively 20 times) against ebv proliferation because of his relatively low cd4-count (nadir 100/l after 6 months). the cidofovir was then discontinued due to a steadily improving immune response (cd4count 306/l). ophthalmological and neurological examinations as well as renal function tests were within normal limits 2 years after the start of treatment. four years from time of diagnosis, cct restaging demonstrated constant marked demyelinization of the cerebral medullary layer, which was most likely radiotherapy - associated. he remained on haart with azt, 3tc, lpv / r with cd4-counts above 300/l and without a detectable viral load. a year after diagnosis. pat. 3 after 5 years of treatment with il-2, azt, haart, foscarnet. combined first - line therapy with haart, il-2, foscarnet and azt - high - dose was not effective in reducing tumour growth in the 3 patients. therefore, standard radiation therapy was indicated after 2 weeks of therapy in all patients. after 4 months of radiation therapy, patient 3 showed a complete remission of the tumour mass 53 months after diagnosis and a stabilized immune response by haart as well as cidofovir maintenance - therapy (20 cycles). lymphoma was suspected because of the high proliferation index and necrotic tissue masses. retrospectively, based upon the positive response to radiation and absence of an early response to immunomodulating therapy, a), b) diffuse large b - cell lymphoma within the central nervous tissue penetrating a blood vessel. c) the large tumor cells express the b - cell marker cd20 and reveal a high proliverative activity d). e) in a further case h&e stained section shows a diffuse large b - cell lymphoma. h) the proliferation marker mib1 (ki67) labels more than 50% of the tumor cells. i, j) the h&e stained sections reveal necrotic cns tissue with reactive astrocytes, macrophages and inflammatory cells. although these case reports shared comparable clinical characteristics to those treated by raez., they did not have similar results despite having received optimized antiviral medication with foscarnet instead of ganciclovir and demonstrating excellent immune responses in 2 of the 3 patients (patient 1 was not evaluated for an immune response). combined therapy with il-2, azt, foscarnet and haart had no effects on reducing tumour growth in our art - naive hiv - positive patients with pcnsl in the short time of administration. by comparison, this differs from raez, who reported at least partial remission within two to three weeks in 4 out of 5 patients. these case reports suggest that this was probably due to rapid pcnsl tumour growth and that azt, which was the only anti - neoplastic agent, was ineffective against lymphoma cell lines. this finding is concordant to different highly toxic treatment experiences both in hiv - positive and hiv - negative pcnsl patients. haart was effective in immune reconstitution, but patient 2 did not take it regularly from the time of radiation therapy due to poor adherence. replacing ganciclovir by foscarnet during foscarnet has been shown to be effective in ebv - associated lymphoproliferations and is activated independently of viral encoded thymidine - kinase, which is an advantage over ganciclovir. however, it was not effective in halting rapid tumor growth in these patients, which had already become independent from the original multifactorial pathogenesis of polyclonal ebv - and hiv - induced lymphoproliferation. since the herpes - virus consensus pcr was negative in two of the patients ' csf prior to therapy, and not performed in one patient (histological diagnosis), ebv - viral load was not measured. the reason for the reduced effectiveness of foscarnet compared to ganciclovir against ebv could be not be discerned, and it is unclear why raez and aboulafia elected to utilize ganciclovir. interleukin-2 was administered to induce an acceleration of immune response, and to decrease ebvdna. the observed rapid immune reconstitution of cd4 cells by combined antiretroviral and interleukin-2 administration in 2 patients was impressive, nevertheless it had no impact on tumour progression. of note is that this did eventually contribute to stopping lymphoproliferation after radiation in patient 3, which is similar to four reported patients with longer lasting remissions by haart and radiation. even though only lymphoproliferation had been diagnosed in patient 3, the lymphoproliferative polyclonal tumor with impressive necrotic tissue mass, which is pathognomonic for high grade lymphoma in the brain, was highly suspicious for pcnsl. polyclonal lymphoproliferation as a pre - lymphoma stage could not be completely ruled out. additionally, patient 3 was the only patient with a relatively stable immune status and without previous aids - defining illnesses at the time of diagnosis. herpes - consensus pcr in his csf was negative with ebv - positivity in histology, corresponding to a latent infection pattern without active ebv - replication. ritonavir has been reported to have antiangiogenic and radiation sensitizing effects in a mouse carcinoma model, and could eventually have had an influence on response, but only in this patient. however, after responding to radiation he continued haart with long - term antiviral ebv therapy and was clinically stable over the following years haart plus whole brain radiation appears to be the mainstay of treatment for hiv - associated pcnsl patients with highly impaired immune and performance status. in selected cases of hiv - positive pcnsl patients with sufficient performance status, systemic chemotherapy should be considered. haart and systemic chemotherapy, possibly even in patients with impaired immune status, can be considered in an attempt to induce immune reconstitution and thus improving both neurological and overall clinical status. combination chemo - radiotherapies in immunocompetent patients (most of them with intrathecal mtx and/or cytarabine, systemic methotrexate, procarbazine, vincristine, thiotepa, teniposide or carmustine) cause high rates of hematologic toxicity, treatment - related deaths in up to 10%, infection rates up to 19% and leukoencephalopathy rates of up to 30% (without radiation 3%). systemic rituximab alone does not generally seem to be beneficial, but may be so only in selected patients. in a small series with relapsed or progressive disease in hiv - negative persons, intrathecal rituximab was shown to be safe and partially effective [34 - 36 ] and therefore stands as a possible option. systemic methotrexate (mtx) alone, 3 - 8 g / mand delayed radiotherapy are most frequently used in hiv negative patients, and show median survival times of 22 to 47 months with low neurotoxicity rates [7 - 9 ]. select hiv patients are reported to benefit from high - dose mtx alone : seven out of 15 patients treated with 3 g / m2 intravenous mtx without haart showed a complete response after six cycles, with two of 15 patients dying from septicaemia. only a few hiv - positive patients with pcnsl were treated with systemic mtx. in the pre - haart era, combined radio - chemotherapy with procarbazine, lomustine and vincristine (pcv-3) for hiv - related pcnsl patients with more than 200 cd4-cells/l, who had a good performance status and without history of opportunistic infection, showed a median survival of 13 months. autologous stem - cell rescue with high - dose chemotherapy protocols were given successfully in immunocompetent pcnsl patients under the age of 60 years and achieved median survival rates of up to 60 months. hiv - positive patients were excluded from high - dose studies due to safety reasons. temozolomide plus systemic mtx/ prednisone in an elderly patient group showed overall survival rates of 36 months, but severe nephrotoxicity and hematological toxicity occurred in 20% and 13% respectively which is too toxic for aids - patients. a promising schedule for the elderly that utilizes three cycles of rituximab, methotrexate, lomustine, and procarbacine (r - mcp) has been described. the regimen is without radiation, has an acceptable safety profile, and a 12 month overall and disease - free survival of 81% in 16 patients. this treatment modality could eventually become an option for hiv - positive pcnsl patients, considering that the oldest patient was 83 years old and that a reduced performance status was not an exclusion criteria. salvage regimens have been performed with topotecan, pcv (procarbacine, lomustine, vincristine), antracyclines, high - dose cytarabine, etoposide, ifosfamide, thiotepa, temozolomide, temozolomide / rituximab, radiotherapy and surgery. il-2, haart, azt - high - dose and foscarnet were safe, but ineffective when used as the only therapy for hiv - associated pcnsl based on our experience with these 3 patients. the numbers of patients treated with similar regimens have been too few to allow any final conclusion. the deterioration of the overall clinical status in 2 out of 3 patients was rapid, and corresponded to the frequently encountered clinical course of newly diagnosed hiv - associated pcnsl patients, who often have co morbidities. nevertheless, in select patients with advanced disease and without a systemic chemotherapeutic option, this type of regimen could be considered. raez and aboulafia reported similar cases to the 3 patients that we have described. a favourable clinical course may more likely occur in select hiv infected patients with good performance status. two of the 3 patients had severe co morbidities (tbc, kaposi - sarcoma, cryptococcosis, psychosis), so chemotherapy was not a reasonable option., patients within clinical studies who have adequate performance status could qualify for more intensive systemic chemotherapy options combined with haart to avoid late leukencephalopathy caused by radiation. it remains uncertain if interleukin-2, azt high - dose and/or foscarnet can be added or be given sequentially with chemotherapy, especially in severe compromised patients at the beginning of therapy. despite a substantial and sustained increase in the cd4-count, as compared with antiretroviral therapy alone, interleukin-2 plus antiretroviral therapy yielded no clinical benefit in hiv positive persons. if chemotherapy is utilized, multidisciplinary teams including specialists in haematology - oncology and infectious diseases will be needed to manage toxicities, drug - interactions, and specific infectious and other complications that can occur during and after therapy. clinicians should be diligent about detecting and treating cns - lymphoma and other types of cancer in hiv positive patients, and should be encouraged by the improved prognosis due to haart to treat cns - lymphomas more intensively. finally, we emphasize the importance of performing clinical trials in hiv - positive patients with malignancies. | purposecombined immunomodulatory and antiviral treatment was administered to three patients with newly diagnosed hiv - associated primary central nervous system lymphoma (pcnsl) in an attempt to improve outcomes.patients and methodsthree patients from our institution who were recently diagnosed with hiv - associated pcnsl received intravenous azidothymidine (azt) 1.6 gr. bid for two weeks, followed by oral azt 250 mg bid from day 15. in addition, complementary highly active antiretroviral therapy (haart) with a second nucleoside reverse transcriptase inhibitor (nrti) plus one protease inhibitor (pi) and interleukin 2 (il-2) subcutaneously 2 million units twice daily (bid) plus foscarnet 90 mg / kg bid were administered on days 1 - 14. one patient received anti - epsteinbarr virus (ebv)-maintenance therapy with ganciclovir, followed by cidofovir [1].resultsall patients experienced progressive disease while on induction therapy, and switched early to whole - brain radiation therapy (wbrt) as second linetreatment. no grade 3 or 4 toxicities were observed. two patients died on days 50 and 166 respectively due to progressive disease. the third patient with histologically proven lymphoproliferation and only suspected pcnsl remained alive at 53 months. he was on haart and remained clinically and neurologically stable.conclusionalthough il-2, haart, high - dose azt and foscarnet are used for other hiv - related conditions, they did not demonstrate benefit in lymphoma remission for 2 hivassociated pcnsl patients. the third patient went into delayed remission after additional radiotherapy and was in good clinical and neurological health status over 53 months after diagnosis. |
a prospective analysis was carried out in amrita institute of medical sciences, kerala which is a tertiary care hospital, to compare the outcome of hemodialysis patients from different socioeconomic status. patients were allotted in different shifts either in morning, afternoon or evening, based on their preferences. the patient sociodemographics, cost details of dialysis, adverse reactions occurred during dialysis, cost to manage such adverse reactions, regularity, affordability, outcome and patient satisfaction to dialysis, etc. a total of 79 patients were undergoing dialysis per week in three different shifts during the study period. sampling was done and every second patient in the op was selected for the study. for simple analysis, patients undergoing dialysis thrice in a week, without considering the duration of dialysis were included in the study. patients with major illness such as severe cardiac or neurological problems and those who avail reimbursement were excluded. based on the education, occupation and income, patients were categorized into upper, middle, and lower socioeconomic classes. patient perspective was taken for the analysis of cost component and the details were collected by direct patient interview. details of direct medical cost, direct non - medical cost and indirect cost were analyzed. direct medical cost includes cost of hemodialysis, laboratory investigation, erythropoietin, av fistula, blood transfusion, x - ray and medications whereas direct non - medical costs include cost of food, transportation, and extra family care. other costs such as intangible cost and opportunity costs were excluded. from the data obtained, overall costs of hemodialysis per session as well as total cost for a period of 6 months were calculated. the patient data were collected by using the patient data collection form and by the direct patient interview. patient 's demographics, socioeconomic status, comorbid conditions, cost of therapy, regularity, and affordability of hemodialysis, etc. were incorporated in the data collection form. socioeconomic status was also studied using kuppuswamy 's scale since the hospital is located in urban area, and the patients are also from such places. outcomes were compared between patients of different socioecomic status in order to assess regularity, affordability, clinical condition and satisfaction. statistical tests (pearson correlation) were applied to analyze the data using statistical package for the social sciences (spss) version 14. a total of 79 patients were undergoing dialysis per week in three different shifts during the study period. sampling was done and every second patient in the op was selected for the study. for simple analysis, patients undergoing dialysis thrice in a week, without considering the duration of dialysis were included in the study. patients with major illness such as severe cardiac or neurological problems and those who avail reimbursement were excluded. based on the education, occupation and income, patients were categorized into upper, middle, and lower socioeconomic classes. patient perspective was taken for the analysis of cost component and the details were collected by direct patient interview. details of direct medical cost, direct non - medical cost and indirect cost were analyzed. direct medical cost includes cost of hemodialysis, laboratory investigation, erythropoietin, av fistula, blood transfusion, x - ray and medications whereas direct non - medical costs include cost of food, transportation, and extra family care. other costs such as intangible cost and opportunity costs were excluded. from the data obtained, overall costs of hemodialysis per session as well as total cost for a period of 6 months were calculated. the patient data were collected by using the patient data collection form and by the direct patient interview. patient 's demographics, socioeconomic status, comorbid conditions, cost of therapy, regularity, and affordability of hemodialysis, etc. were incorporated in the data collection form. socioeconomic status was also studied using kuppuswamy 's scale since the hospital is located in urban area, and the patients are also from such places. outcomes were compared between patients of different socioecomic status in order to assess regularity, affordability, clinical condition and satisfaction. statistical tests (pearson correlation) were applied to analyze the data using statistical package for the social sciences (spss) version 14. out of 79 patients undergoing hemodialysis during the study period, 30 patients were selected based on the inclusion and exclusion criteria, which is given in detail in the methodology section. the sociodemographics as well as the comorbid conditions of the patients were given in table 1. majority of the patients were in the age group of 5160 with a mean age of 49.72 13.2 and associated with comorbid conditions. diabetes and hypertension were found to be the predominant causes compared to other disease states. out of this duration of renal failure as well as duration of dialysis was also calculated and the result was 4.2 1.6 and 2.8 1.8 years, respectively. sociodemographics data socioeconomic status of the patients was also calculated according to modified kuppuswamy scale based on their educational qualification, occupation, and annual income. it was found that 18 patients were on upper class and 12 were on middle class. lower class patients were not there, that may be due to the fact of non - affordability. all the 18 patients on upper socioeconomic class were regular on dialysis and were able to afford the cost by themselves. the patients from middle class family were also regular on dialysis, but three patients could afford it with the financial help of friends or relatives. clinical outcome was also analyzed to assess whether the patients had better, worse or no change in outcome. by doing a comprehensive survey, we asked the patients to rate their clinical outcome on a visual analog scale from 1 (worsen the condition after treatment) to 5 (improved very much after the treatment). fifteen patients on upper class and eight from middle class experiences better clinical outcome. the correlation between the socioeconomic status and parameters such as regularity, affordability, outcome and patient satisfaction were tested. it revealed that there is no significant correlation between socioeconomic status and regularity, affordability or outcome. analysis of patient satisfaction showed that all the patients from upper class were satisfied with the dialysis modality while two patients from the middle class family were not at all satisfied with the treatment. patient satisfaction scale was used in order to evaluate this and the result showed 26.5 1.2 vs 18.4 2.6 ; p<0.001. the sociodemographic details along with the regularity, affordability, clinical outcome and patient satisfaction were given in table 1. the cost of each session of dialysis and the overall cost for 6 months were calculated and found that the direct cost is more than the indirect costs. details of the categorization of costs were given in the cost analysis section of methodology. fifty six percent of the total cost was contributed by direct medical cost and 19.6% was for the direct non - medical cost. indirect cost was calculated based on the number of missed working hours and the percentage of current income compared to the income that the patients had before they entered the dialysis procedure. cost analysis of hemodialysis treatment since the patients had different co morbidities, the management cost of comorbid conditions were also analyzed. the major complications were muscle cramps, nausea, itching, dyspnoea, vomiting and anemia. these were also properly managed and the additional expenses were provided in table 4. the cost to manage the co morbid conditions may further aggravate the situation. since diabetes and hypertension are the major contributing factor of renal failure that has to be managed properly in its initial stage itself. the lifelong treatment of renal failure along with diabetes and hypertension significantly increase the burden on patients and decreases the outcome also. a study conducted by rao. showed that diabetes is the main cause of poor outcome along with cardiovascular risk. studies revealed that the mean age of esrd patients on hemodialysis in developing countries is lower (32 - 42 years) than the developed countries (6063 years). our study is also consistent with this, with a mean age of 49.72 13.2. the reason for the progression of renal failure may be the delay in detecting the renal disease, late referral, and failure to introduce preventive measures. jha illustrates that 70% of the dialysis centers are in private sector and are accessible for upper income patients only. in this study according to the report of kher, 60% of the hemodialysis patients were lost to follow up within 3 months because of its high cost. another study conducted by mani described that only 4% of people can pay from their own resources, 63% from their employers or charity, 30% will sell the property or jewellery, and 20% will take loan. the study conducted by abraham illustrates that the proportionate hemodialysis cost in low income countries is about five times greater than in developed countries. this may lead to dropout and further complication of the diseased state. according to the socioeconomic status of the patient, only upper and middle class people can afford the hemodialysis service offered by the private sector. patients on low economic status take pain for the regular dialysis and to manage the complications. since they can not afford the costs in private hospitals, they usually approach public hospitals. patients from the middle income family face difficulty in affording the costs for the management of co morbid conditions along with the dialysis cost. this will affect the clinical outcome as well as the satisfaction with the treatment. upper class patients experiences better clinical outcome as well as patient satisfaction when compared to middle class people because they were able to manage the cost without affecting much to their daily life. there is no significant difference in the regularity as well as affordability even though the middle class people face a lot of problems. research is necessary in this area to quantify the cost of therapy at the public sector. studies have to be conducted to evaluate the cost and outcome of hemodialysis patients approaching public vs private hospitals. the quality of life of patients at both the centers also should be evaluated in order to assess the cost effectiveness. if the patients achieve better quality of life at the public sector, government has to take initiative to commence more dialysis centers at the public level. recently various non - governmental organizations as well as charity trusts are coming up in order to provide more cost - effective management. drugs mainly for hypertension as well as diabetes supplying at free of cost will also help the patients to reduce the overall burden of renal failure. this is the first study to analyze the direct as well as indirect cost of hemodialysis in a tertiary care hospital of south india. from this observational study, we could found that 75.6% of the cost was due to direct costs (direct medical and non - medical costs) and 24.4% was due to indirect costs. the dialysis modality for the esrd patients remains the most preferred resources. the non availability or less availability of dialysis unit in public sector compared to private sector, and also lack of reimbursing or insurance scheme to only the upper or upper middle class people can afford hemodialysis due to its high cost. they are also several approaches to reduce the annual cost of renal replacement therapy (rrt). obviously in long term, the most important factor is to reduce the number of patients with esrd. early detection and treatment of these diseases play a vital role in the prevention of progression of renal failure and to postpone the need of rrt to an extent. a clinical pharmacist can be really helpful to prevent this progression of esrd by providing patient counseling about lifestyle modification and educating the risk group and motivating them for regular check up and adhering to the treatment. and also better helping hands from the government sector like more hemodialysis unit, insurance and reimbursement scheme to the patients can provide better patient outcome in our country. long - term studies are needed to assess the cost and consequences of the treatment modalities of esrd patients. | background : in india the incidence of end stage renal disease (esrd) is increasing day by day and the option for the treatment of esrd is dialysis or transplantation. in the present scenario, due to the cost of treatment normal people can afford only hemodialysis rather than transplantation. since the cost of hemodialysis differs across the country, research is needed to evaluate its exact cost.aim:this study is to analyze the healthcare cost of hemodialysis in a private hospital of south india.materials and methods : this is a prospective, observational study carried out in a tertiary care hospital. patients who are undergoing routine hemodialysis in this hospital were selected for the study. patient data as well as cost details were collected for a period of six months. thirty patients were selected for the study and a total of 2160 dialysis sessions were studied. patient perspective was taken for the analysis of cost. both direct and indirect costs were analyzed. this includes cost of dialysis, investigations, erythropoietin, food, transportation, lost wages etc. socioeconomic status of the patient was also studied.result:the total cost per session was found to be around rs. 4500. fifty six percent contributes direct medical cost whereas 20% contributes direct non medical cost. twenty four percent cost was due to indirect costs. since the patients are paying from their own pocket, only the upper or upper middle class patient can undergo hemodialysis regularly.conclusion:these findings are important to find out the impact of cost of hemodialysis on patients suffering from esrd. further studies related to costs and outcome, otherwise known as pharmacoeconomic studies, are needed to analyze the pros and cons of renal replacement therapy and to improve the quality of life of esrd patients. thus pharmacoeconomical studies are needed to realize that government has to take initiative to provide insurance or reimbursement for the common people. |
malignant lymphoma (ml) can involve the central nervous system either primarily or by secondary spread, which tends to occur late in the disease as part of widespread dissemination8,18). the rate of epidural mass formation in all cases of mls is 0.8 - 2.86,14,19,21). owing to this rate of occurrence, a small number of research studies and case reports we report a rare case of primary spinal epidural lymphoma that presented with progressive myelopathy. a 47-year - old man presented with progressive lower extremity weakness and numbness associated with fecal and urinary incontinence over 2 weeks. a physical examination revealed hypoesthesia on the medial aspect of both thighs with bilateral motor weakness (gr 4 - 4 +). there was no abnormality of laboratory findings except the erythrocyte sedimentation rate was elevated (esr-32mm / h ; reference value - less than 20mm / h). preoperative magnetic resonance imaging (mri) of the thora columbar region showed an isointense spinal medulla on t1-weighted images and a slightly hyperintense t2-weighted images of the spinal cord extending from t9-l2. intraoperatively, a grayish epidural mass was identified and resected to decompress the cord and cauda equina. proliferation of small blastic cells, with a lymphoblastic aspect and starry sky pattern, was seen. further immunohistochemical characterization showed that results from tumor testing were positive for cd20, cd3, cd5, bcl-2, cyclin d1 and negative for cd23, cd10 and t - cell markers (fig. further work - up, including computed tomography (ct) scans of the chest and the abdomen, iliac crest bone marrow biopsy, and ultrasound of the lymph nodes showed no extramedullary lymphoma manifestation. the patient had no history of immune disorder, and human immunodeficiency virus (hiv) testing was negative. the treatment regimen consisted of radiotherapy, 40gy administered in 20 fractions, from t9-l2, followed by combination chemotherapy with cyclophosphamide, adriamycin, oncovin, and prednisone (chop). a follow - up mri one year after the surgery showed that the lesions of lymphoma had disappeared (fig. postoperatively, the patient was faring well with no evidence of local recurrence or new lesions at any other site. primary central nervous system lymphoma is a rare form of extranodal lymphoma, particularly isolated primary spinal lymphoma7,8). compression of the spinal cord as the first manifestation of the primary spinal lymphoma is particularly rare, with an incidence of < 5%15). in our report, the patient initially presented with compression of the spinal cord manifesting as hypoesthesia and motor weakness of the lower extremities. although the mechanism of epidural mass formation without systemic lymphoma has not been clarified, the origin of the mass is thought to be bone marrow11). a small amount of lymphocytic cells can migrate to the spinal epidural space directly through the harversian canals of the vertebral bone or hematogenously via the epidural venous plexus, followed by a mass formation even before systemic lymphoma. findings from the ct and mri provided only indirect diagnostic evidence. on t1-weighted mri, primary spinal lymphomas are most frequently isointense to hypointense, while t2-weighted images are commonly isointense to hyperintense, with contrast enhancement4). primary spinal lymphomas are often confused with other spinal tumors, especially metastasis12). in the present case the spinal lesion the mri demonstrated an epidural mass lesion extending from t9 to l2, causing spinal cord compression. the lesion appeared slightly hyperintense on the t2-weighted image and exhibited continuous homogenous enhancement after gadolinium injection. this is similar to the description by negendank of lymphomas elsewhere in the body and li of spinal epidural lymphomas12,17). most primary spinal lymphomas are comprised of diffuse large b - cell lymphomas with a minority being follicular lymphoma, precursor b - lymphoblastic lymphoma, ki - lymphoma, diffuse lymphoblastic lymphoma, small lymphocytic lymphoma or t - cell lymphoma5). small lymphocytic lymphoma is composed mostly of small mature appearing lymphocytes with round nuclei and scant basophilic cytoplasm9). in our case, immunohistochemical studies demonstrated that the tumor cells expressed the b cell markers cd19 and cd20, with coexpression of the t cell marker cd5 (fig. cyclin d1 staining yielded positive results for both the primary lesion and the recurrence1). haddad that patients with the histopathologic subtype of mixed histiocytic and small lymphocytic lymphoma seemed to have a better survival than the other subtypes (61% at 10 years), but a small number (n=17) of patients in this group did not allow statistical confirmation of this apparent observation10). in the study by monnard, out of 52 patients, 48 (92%) underwent a laminectomy, with a partial resection performed in 22 cases (42%) and a complete resection for 7 cases (13%). in surgery, each lesion was brownish or reddish purple and soft to firm in consistency., we performed a hemilaminectomy of t9-l2 and a partial resection of a grayish epidural mass. however, a surgical resection does not have the survival benefits and the reported median overall survival durations with surgery alone are similar to those of untreated patients2,3). some reports suggest that well - established treatments, such as radiotherapy and chemotherapy or a combination of both should remain as the mainstays and that surgery should be used in combination with radiotherapy and/or chemotherapy13). therefore, concurrent chemoradiotherapy treatment was provide (chop ; cyclophosphamide, adriamycin, oncovin, and prednisone+radiotherapy ; 40gy administered in 20 fractions) after pathologic confirmation. monnard observed local control of 88% and a 5-year overall survival of 69% with combined modality treatment. in a multivariate analysis, they found that the combined modality treatment was statistically superior to rt alone16). also, they reported a post - treatment complete neurologic response in 25% of patients who had initial motor deficits. the remaining 75% had partial recovery. in their multivariate analysis, they have found that complete neurologic response was the most significant favorable prognostic finding with regard to overall survival (p=0.001)16). in the present case, because of the manifestations of compression, timely surgical decompression was required to allow for a recovery of nerve function even before the diagnosis had been made. after surgery, the neurologic function in our patient was completely recovered. a primary small lymphocytic lymphoma of the spine should be considered in the differential diagnosis of primary spine tumors. surgical decompression and tumor removal are mandatory to recover neurologic function, if the patient had a neurologic deficit. the combined modality treatment after surgical decompression resulted in improvement of the neurologic symptoms and remission of the lymphoma. | we report the case of a 47-year - old man who presented with progressive paraparesis and sphincter changes over 2 weeks. magnetic resonance imaging revealed a spinal epidural mass from t9 to l2. we performed a decompressive laminectomy and mass removal. the histopathology was consistent with a small lymphocytic lymphoma. no metastatic lesion was noted in the chest and abdomen - pelvic computerized tomography (ct) and positron emission tomography computerized tomography (pet - ct) scan. the final diagnosis was primary spinal lymphoma, so we performed chemotherapy combined with radiotherapy. at one year follow - up, he had no neurological deficit and no recurrence on neurologic and radiologic exams. primary spinal cord lymphomas should be considered in the differential diagnosis of spinal cord tumors. early surgical management is mandatory to achieve a recovery of neurologic function, especially if the patient has a neurological deficit. |
japanese encephalitis virus (jev) is a mosquito - borne microorganism that causes encephalitis in humans and reproductive failure in pregnant sows. jev has been recently recognized as a reemerging pathogen as it has expanded into new territories such as the torres strait and australia [5,10,18 - 20 ]. based on a sequence analysis of the c / prm and e genes, jev has been classified into five genotypes ; their relationships have been characterized. jev genotype 1, which was distributed in limited regions such as thailand and cambodia before the 1990s, has expanded into northeastern asian countries including vietnam, china, japan, and korea. although the exact mechanisms for the expansion of this genotype are unknown, several factors such as bird migration, wind - blown mosquitoes, and the transport of animals infected with jev have been suggested. additionally, climate changes caused by recent global warming and extreme weather patterns may have significantly impacted the transmission of vector - borne diseases such as jev, west nile virus (wnv), and tick - borne encephalitis virus (tbev). this is because rapid weather and climate changes can directly or indirectly affect migratory birds and mosquitoes. sero - epidemiological studies are critical for predicting potentially important viral disease outbreaks and preventing the introduction of new jev genotypes into korea. recently, saito. suggested that wild ducks captured in hokkaido, japan can transmit vector - borne viruses into new territories. migratory birds may serve as viral reservoirs or amplifying hosts, but they do not develop clinical symptoms. although migrating wild birds may be a major jev vector, no epidemiological survey of jev, which could provide valuable information for establishing control measures to prevent jev outbreaks in swine and humans, has been properly conducted for wild birds in korea. thus, we performed a serological survey to determine the prevalence of antibodies against jev in wild birds captured on the korean peninsula. a total of 1,316 blood samples were collected from wild birds in 16 locations of six provinces (gyeonggi - do ; 3 site, gyeongsangnam - do ; 1 site, jeollanam - do ; 4 sites, jeollabuk - do ; 3 sites, chungcheongnam - do ; 3 sites, chungcheongbuk - do ; 2 sites) of korea between april 2007 and december 2009 for our seroprevalence study. all wild birds were lured by rice seed and captured using cannon or mist net. blood sample from wing vein of each bird using sterile syringe of 3 ml was taken. the nine species of wild birds tested in this study were oceanodroma castro (four birds), anas formosa (seven), anas penelope (20), fulica atra (30), anas acuta (89), anas crecca (154), anas platyrhynchos (214), aix galericulata (310), and anas poecilorhyncha (488). clotted blood samples were separated by 3,000 g, and the sera were stored at -20 until use. before performing the hi test, the kv1899 (genotype 1 strain) strain of jev was used as the positive antigen for the hi test. this strain was isolated from korean pig blood in 1999 and has been passed nine times in vero cells after isolation. to estimate the jev antibody prevalence in the wild bird sera, an hi test was performed in 96-well microplates (corning, usa) using slightly modified standard methods. a sucrose - acetone extraction method was used to prepare viral antigens from the brains of suckling mice infected with the korean isolate of jev strain kv1899. briefly, 10 l of the serum samples collected from wild birds and 50 l of 4% bovine albumin were mixed with 40 l of 25% kaolin (sigma, usa) and incubated for 30 min to remove non - specific inhibitors at room temperature. after mixing, the clear supernatant was then mixed with 5 l of packed goose erythrocytes to remove any natural agglutinins. after incubating at 37 for 1 h, the treated sera were separated from the goose erythrocytes by 3,000 g at 4. the sera (25 l) were diluted two - fold from 1 : 20 to 1 : 10,240 in round - bottom 96-well microplates (corning, usa) and incubated with 8 hemagglutinating antigen (ha) units of jev. after incubating at 37 for 1 h, 50 l of 0.33% goose erythrocytes were added and the microplates were incubated for 30 min at 37. to confirm the test reliability, porcine control sera positive and negative for jev were used in all hi tests. hi titer was expressed as the reciprocal of the highest dilution of serum showing complete hemagglutination inhibition. jev prevalence was estimated as the number of positive samples compared to the number of samples tested and expressed as a percentage. a 95% confidence interval (ci) additionally, each titer was classified according to bird species, time (year), and place (capture location). a total of 1,316 blood samples were collected from wild birds in 16 locations of six provinces (gyeonggi - do ; 3 site, gyeongsangnam - do ; 1 site, jeollanam - do ; 4 sites, jeollabuk - do ; 3 sites, chungcheongnam - do ; 3 sites, chungcheongbuk - do ; 2 sites) of korea between april 2007 and december 2009 for our seroprevalence study. all wild birds were lured by rice seed and captured using cannon or mist net. blood sample from wing vein of each bird using sterile syringe of 3 ml was taken. the nine species of wild birds tested in this study were oceanodroma castro (four birds), anas formosa (seven), anas penelope (20), fulica atra (30), anas acuta (89), anas crecca (154), anas platyrhynchos (214), aix galericulata (310), and anas poecilorhyncha (488). clotted blood samples were separated by 3,000 g, and the sera were stored at -20 until use. before performing the hi test, the sera were inactivated by incubating at 56 for 30 min. the kv1899 (genotype 1 strain) strain of jev was used as the positive antigen for the hi test. this strain was isolated from korean pig blood in 1999 and has been passed nine times in vero cells after isolation. to estimate the jev antibody prevalence in the wild bird sera, an hi test was performed in 96-well microplates (corning, usa) using slightly modified standard methods. a sucrose - acetone extraction method was used to prepare viral antigens from the brains of suckling mice infected with the korean isolate of jev strain kv1899. briefly, 10 l of the serum samples collected from wild birds and 50 l of 4% bovine albumin were mixed with 40 l of 25% kaolin (sigma, usa) and incubated for 30 min to remove non - specific inhibitors at room temperature. after mixing, the clear supernatant was then mixed with 5 l of packed goose erythrocytes to remove any natural agglutinins. after incubating at 37 for 1 h, the treated sera were separated from the goose erythrocytes by 3,000 g at 4. the sera (25 l) were diluted two - fold from 1 : 20 to 1 : 10,240 in round - bottom 96-well microplates (corning, usa) and incubated with 8 hemagglutinating antigen (ha) units of jev. after incubating at 37 for 1 h, 50 l of 0.33% goose erythrocytes were added and the microplates were incubated for 30 min at 37. to confirm the test reliability, porcine control sera positive and negative for jev were used in all hi tests. hi titer was expressed as the reciprocal of the highest dilution of serum showing complete hemagglutination inhibition. jev prevalence was estimated as the number of positive samples compared to the number of samples tested and expressed as a percentage. a 95% confidence interval (ci) additionally, each titer was classified according to bird species, time (year), and place (capture location). jev seroprevalence as determined by our study is shown in tables 1~3, and figs. 1 and 2. the overall prevalence of antibodies against jev was 86.7% (95% ci, 84.7~88.5%) for the 1,316 serum samples consisting of nine wild bird species captured in the korean peninsula. prevalence according to year was 68.2% (189 positives/277 tested ; 95% ci, 62.4~73.7%) for 2007, 86.4% (367/425 ; 95% ci, 82.7~89.5%) for 2008, and 95.3% (585/614 ; 95% ci, 93.3~96.8%) for 2009 (fig. the regional seroprevalence of positive hi titers 1 : 20 ranged from 82.9 to 100%. compared to other provinces, the gyeongsangnam - do showed the highest prevalence (100%) ; however, no significant difference in regional prevalence was found. the regional seroprevalences were 100% (80/80) in gyeongsangnam - do, 93.0% (80/86 ; 95% ci, 85.4~97.4%) in jeollanam - do, 91.1% (216/237 ; 95% ci, 86.8~94.4%) in jeollabuk - do, 84.7% (210/248 ; 95% ci, 79.6~88.9%) in gyeonggi - do, 84.6% (176/208 ; 95% ci, 79.0~89.2%) in chungcheongbuk - do, and 82.9% (379/457 ; 95% ci, 79.2~86.3%) in chungcheongnam - do. seroprevalence according to wild bird species were 100% (4/4 ; 7/7 ; 89/89) in oceanodroma castro, anas formosa, and anas acuta (winter visitor) ; 96.7% (29/30 ; 95% ci, 82.8~99.9%) in fulica atra, 90.8% (443/488 ; 95% ci, 87.9~93.2%) in anas poecilorhyncha (migratory breeder), 87.9% (188/214 ; 95% ci, 82.7~91.9%) in anas platyrhynchos, 80% (16/20 ; 95% ci, 56.3~94.3%) in anas penelope (winter visitor), 78.7% in aix galericulata (migratory breeder), and 78.6% (121/154 ; 95% ci, 71.2~84.8%) in anas crecca. the prevalence of wild birds with an hi antibody titer above 1 : 20 or 1 : 1,280 were 86.7% and 21.2%, respectively. wild birds captured in 2009 had a higher jev seropositive rate than those captured in 2007 (fig. 2). among the wild birds captured in 2009 that had an hi antibody titer of 1 : 20 or more, the most frequent hi titer was 1 : 640 (16.2%). climate change, global warming, environmental destruction, and rapid urbanization can alter wild bird migratory paths and increase the incidence of arboviral disease outbreaks. under these conditions, vector - borne diseases caused by jev, wnv, and tbev may be easily introduced into disease - free countries by migratory wild birds. since jev was first isolated in korea in 1946, several korean jev isolates have been reported and characterized based on a c / prm and e gene nucleotide analysis. phylogenetic analyses to determine the genetic relationships between jev isolates found that korean jev isolates changed from genotype 3 to genotype 1 around 1993. however, few studies have addressed the possibility of jev introduction into korea via wild birds. thus, we conducted a nationwide survey of jev antibodies in wild birds, including migratory birds. in this study, the sera of wild birds that were captured in 16 locations of the korean peninsula were tested for the presence of jev antibodies. this hi test result was surprisingly high and suggested that most of the wild birds we captured in korea had been exposed to jev. saito. reported that 85.9% and 5.4% of wild ducks captured in hokkaido, japan have antibodies against jev according to the focus - reduction neutralization test (frnt50 and frnt90), which is more specific than the hi test. additionally, 7.2%, 20.4%, and 28.1% of wild birds in 2007, 2008, and 2009, respectively, had a titer > 1 : 1,280. sugiura and shimada reported that horses experimentally inoculated with jev had an antibody titer of 1 : 1,280 within 2 or 3 weeks after inoculation. therefore, wild birds with titers of 1 : 1,280 or higher may have been infected with wild jev within 3 weeks. although we conducted our serosurveillance study with an hi test, which is known to be less specific than the frnt, our survey results correspond well with those of a previous study in wild ducks in japan. however, cross - reaction between jev and wnv in serodiagnoses is a well - known occurrence in flavivirus - infected animals, including birds. therefore, it is necessary to perform a more specific method, such as a plaque - reduction neutralization test (prnt), for jev serosurveillance in birds. several positive jev serosurveillance results have been reported in different animals including swine, goats, cattle, horses, and wild boars in japan and korea. it has been suggested that goats may serve as good sentinel animals for serological monitoring of jev infection because they are currently not vaccinated against jev in korea. unlike domestic animals, wild birds can not be inoculated with a jev vaccine, but they are easily exposed to several kinds of arthropods such as mosquitoes, ticks, and midges. the high number of positive jev titers in this study does not indicate viral intrusion into korea since jev viremia ends when jev - specific antibodies are detected in ducks. however, wild birds with viremia may carry jev to several countries. according to data obtained from the korean center for disease control and prevention, 45 jev cases in human more than half of these cases occurred in 2010, suggesting that climate change or a high jev infection rate among wild birds may, in part, explain the increasing jev infection rate in the human population. gyeonggi - do had a high incidence rate (42.3%) of jev infection in humans, but no significant relationship was found between the seropositive rates in the capture areas and the jev incidence rate in humans. nevertheless, new jev genotypes may be introduced into korea via wild or migratory birds. it is therefore necessary to monitor jev antibodies using the prnt in wild birds. taken together, these findings lead us to predict that jev infections may appear in domestic animals such as pigs. in conclusion, the results of the present study suggested that wild birds with a high incidence rate (86.7%) of jev antibodies may be introducing new jev genotypes into korea. further studies are needed to isolate all jev genotypes from blood obtained from migratory birds. various research projects examining disease distribution and transmission associated with climate change in the korean peninsula should also be carried out in the near future. | climate change induced by recent global warming may have a significant impact on vector - borne and zoonotic diseases. for example, the distribution of japanese encephalitis virus (jev) has expanded into new regions. we surveyed the levels of hemagglutination - inhibition (hi) antibodies against jev (family flaviviridae, genus flavivirus) in wild birds captured in korea. blood samples were collected from 1,316 wild birds including the following migratory birds : oceanodroma castro (n = 4), anas formosa (n = 7), anas penelope (n = 20), fulica atra (n = 30), anas acuta (n = 89), anas crecca (n = 154), anas platyrhynchos (n = 214), aix galericulata (n = 310), and anas poecilorhyncha (n = 488). all were captured in 16 locations in several korea provinces between april 2007 and december 2009. out of the 1,316 serum samples tested, 1,141 (86.7%) were positive for jev. wild birds captured in 2009 had a higher seroprevalence of ant - jev antibodies than those captured in 2007. wild birds with an hi antibody titer of 1 : 1,280 or higher accounted for 21.2% (280/1,316) of the animals tested. these findings indicated that wild birds from the region examined in our study have been exposed to jev and may pose a high risk for introducing a new jev genotype into korea. |
meta - c h arylation and methylation of 3-phenylpropanoic acid and phenolic derivatives were developed using an easily removable nitrile template. the combination of a weakly coordinating u - shaped template and mono - protected amino acid ligand was crucial for the cross - coupling of c h bonds with organoborons. |
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loss of primary teeth with severe injuries to the surrounding bone will also interfere with the normal growth and development. some severe forms of dentoalveolar injuries associated with the loss of multiple primary teeth would result in large bone defects, healing of which is often complicated. such injuries would require a guide or path for the osteoblasts to regenerate and promote new bone formation. although many methods of bone reconstruction exist, they all have specific indications and limitations. the concept of an induced membrane represents a strategy for bone regeneration, particularly in cases of large bone defects secondary to trauma, infection or tumor excision. this concept of induced bone regeneration with guidance for the bone growth is called as guided bone regeneration (gbr). the principle of gbr remains similar to guided tissue regeneration (gtr) where a biocompatible membrane is placed over bone defect, which keeps the area secluded and help in bone promotion. this article describes the use of concept of gbr for favorable healing of the bone following multiple dentoalveolar fracture associated with intrusion of multiple primary teeth. a 2-year - old boy reported to the pedodontic emergency following trauma due to self - fall and with the complaint of loss of upper front teeth. a thorough history was taken and medical examination was done to rule out any head and neck injuries and other complications. dental examination revealed the de - gloving injury in the region of maxillary anterior teeth with severe intrusion of 51, 52, and 61 [figure 1 ]. radiographic examination confirmed the intrusion of 51, 52, and 61 with 61 impinging on the developing permanent tooth which was in nolla 's stage v (crown almost completed) [figure 2 ]. on examination of the region under relative analgesia (conscious sedation with 60% no2 and 40% o2), multiple vertical fractures in the labial cortical plate were noticed, which extended from the socket crest up to the nasal notch [figure 3 ]. the challenge in treating this condition was to bring about favorable healing of the fractured fragments without any damage to the underlying permanent tooth buds. with such severe and multiple fractures of the labial cortex, the developing tooth buds were left unsupported and literally floating in the mass of blood clot and surrounding damaged connective tissue. reduction of the fragments could well be done, but immobilization of these fragments to promote healing without deformities was a difficult task. the connective tissues would heal fast and the bone would take a longer time to heal. the resulting situation would be that the fast healing connective tissue would occupy the spaces between the fractured fragments and result in mal - union and oro - facial deformities. de - gloving injury in the region of maxillary anterior teeth with severe intrusion of 51, 52, and 61. preoperative radiograph showing intrusion of 51, 52, and 61 with displaced permanent tooth buds. multiple vertical fractures in the maxillary labial cortical plate extending from the socket crest up to the nasal notch. extraction of 51, 52, and 61 was planned as the supporting periodontium was severed. placement of mini plates and screws were contraindicated due to the presence of permanent tooth buds. therefore, for relative immobilization and to promote favorable healing, a resorbable membrane was planned to be placed. the gtr membrane (periocol -gtr : sterile collagen periodontal membrane : eucare pharmaceuticals (pvt., ltd.), india) was trimmed to match the anatomy of the region [figure 4 ] and was placed between the reduced fracture fragments and the injured gingiva [figure 5 ]. this was followed by the placement of sutures and periodontal dressing (reso pac hager werken gmbh & co. the healing was uneventful and at the end of 10 days the sutures were removed. (a) the guided tissue regeneration membrane before trimming and (b) after trimming to match the anatomy of the region. placement of guided tissue regeneration between the reduced fracture fragments and the injured gingiva resulting in restoration of the bony contour and the socket wall shape. thirty days follow - up showed favorable healing of the maxillary anterior labial cortex and the overlying soft tissue [figure 6 ]. postoperative radiograph shows favorable healing [figure 7 ]. there was a need of a space maintainer following teeth loss. hence, an indirect ribbond supported fixed space maintainer was planned [figure 8 ]. the hurdle in treating such children is the lack of cooperation as the children are in the precooperative stage of behavior. when severe injuries occur, it is essential to rehabilitate the structures close to normal to favor normal growth and development. the concept of gbr similar to gtr has been widely used in periodontal pathologies and also in ridge augmentation procedures before the placement of implants. however, its use in the management of the traumatic injuries, especially in children, has not been explored. the regeneration process occurring within the barrier membrane involves angiogenesis and migration of osteogenic cells from the periphery toward the center to create a well - vascularized granulation tissue. initial organization of the blood clot is followed by vascular ingrowth and woven bone deposition, subsequent lamellar bone formation and finally remodeling, resembling bone growth. the role of the biocompatible barrier membrane used in this particular case is : protection of the hematoma from the invasion of non osteogenic cells.stabilization of the hematoma and prevention of its distortion by pressure of the overlying tissue.protection of the underlying developing permanent tooth bud.provision of an anatomically shaped mold for healing of dentoalveolar fracture without any gross facial deformities. stabilization of the hematoma and prevention of its distortion by pressure of the overlying tissue. provision of an anatomically shaped mold for healing of dentoalveolar fracture without any gross facial deformities. the barrier provides an isolated membrane in which the osteogenic process (e.g., osteoinduction, osteoconduction, and osseointegration) can occur undisturbed. based on this principle, the resorbable membrane was used in this case and favorable results have been obtained with the contour of the bone well maintained even after severe crushing injury to the alveolar socket wall and loss of multiple primary teeth. guided tissue regeneration membrane can serve as a vital tool to mold the traumatized dentoalveolar fragments in children. such an approach protects the developing permanent teeth as well, by providing a meshwork to support it and by acting as a mechanical barrier preventing any further dislodging forces which may arise due to dynamic functional status of the para - oral tissues. since the membrane is biodegradable, it resorbs gradually to leave behind well molded, healed normal tissues. | traumatic injuries in the primary dentition pose major challenges for management. this emergency treatment requires proper planning so as to achieve favorable results. trauma causing severe dentoalveolar injuries, especially in children, needs an interdisciplinary approach so as to retain normal functional anatomy for that age. this article describes a clinical innovative technique, which utilizes a resorbable membrane in management of pediatric dentoalveolar trauma. the membrane was shaped to cover the multiple alveolar bone fracture, thereby favoring the healing of the bone defects. the use of this resorbable membrane maintained a secluded space for the bone growth and prevented overgrowth of the soft tissue in the region of the defect. this resulted in uneventful healing leading to well - maintained functional bone contour, which further favored the esthetic rehabilitation as well as protected the underlying permanent tooth buds. |
usually, gartner 's duct cysts are on the anterolateral vaginal wall and its chances of being present on the posterior vaginal wall are relatively rare. its incidence of all the vaginal cysts is 12.5%. as it may arise from remnants of wolffian duct vaginal cyst can be histologically classified as epithelial, inclusion, mullerian, mesonephric, and urothelial in addition to other rare types. these present with symptoms of visible palpable mass, dyspareunia, voiding disturbances, vaginal discharge, and pain. a patient, 54-year - old para 4 live 4, presented herself in the outpatient department of gynaecology, sgrdimsr, amritsar, with chief complaints of mass protruding out from vagina for the last 7 years. on eliciting further history, she narrated that this mass initially was of lemon size which gradually progressed to the size of orange due to which the patient started having discomfort while changing posture / walking. she also gave h / o irregular periods for last 2 years with one episode of profuse bleeding with clots for 10 days prior to visit to the hospital. previous menstrual history was uneventful with menstrual flow of 3 - 4 days/28 days / regular with moderate flow. p / v revealed a pedunculated posterior vaginal wall cyst with smooth pale pink intact surface lining hanging from middle level with 1.5 cm base protruding out of introitus [figure 1 ]. k 90 catheterization in situ, cyst separate from posterior lip of cervix, cyst arising from posterior vaginal wall, and protruding out of introitus polypoidal / rectum (p / r) rectal mucosa was free, base of polypoidal cyst was free, and anterior rectal wall was otherwise smooth and normal. on investigations : hemoglobin 12.4 g%, platelet count 2.9 lacs / cmm, total leukocyte count 6600/cmm, blood urea 24 mg%, serum creatinine 0.7 mg / dl, and cancer antigen 125 - 12 /ml. tvs a well - defined cystic mass lesion of 6.5 cm 4.5 cm 3.7 cm protruding out of the vagina. cyst contained fluid with dense internal echoes with no solid component suggestive of bartholin 's cyst. right ovary had a well - defined anechoic cyst of 3.8 cm 2.2 cm with no solid component in it. left ovary was normal and no adnexal mass was seen in the pouch of douglas. posterior vaginal wall cyst was excised by dissecting posterior vaginal wall from fourchette upward and identifying the base. stalk was ligated, postvaginal wall cyst was removed, and posterior colpoperineorrhaphy was done [figures 26 ]. cervix felt normal, canal was regular, sound was passed up to two and half inches, scanty curettings were obtained, and specimen was sent for histopathological examination (hpe). excision of posterior vaginal wall from fourchette to the base of cyst repaired vaginal wall p / r : rectal mucosa was found to be intact. globular cystic skin covered soft tissue piece measuring 5 cm in diameter. on cut section, unilocular cyst is identified filled with thick jelly such as brownish fluid. photomicrograph showing tall columnar cell lining of cyst wall (h and e, 100) photomicrograph of the same at higher magnification (h and e, 400) postoperative period was uneventful, and the patient was discharged in satisfactory condition. rare histopathological report has aroused the interest to report this case, as mesonephric duct cysts commonly present in anterior / anterolateral wall only on the rarest occasions if residual tissue of wolffian duct persists, then mesonephric cyst may grow from that abnormal site. posterior vaginal wall cyst was excised by dissecting posterior vaginal wall from fourchette upward and identifying the base. stalk was ligated, postvaginal wall cyst was removed, and posterior colpoperineorrhaphy was done [figures 26 ]. cervix felt normal, canal was regular, sound was passed up to two and half inches, scanty curettings were obtained, and specimen was sent for histopathological examination (hpe). excision of posterior vaginal wall from fourchette to the base of cyst repaired vaginal wall p / r : rectal mucosa was found to be intact. globular cystic skin covered soft tissue piece measuring 5 cm in diameter. on cut section, unilocular cyst photomicrograph showing tall columnar cell lining of cyst wall (h and e, 100) photomicrograph of the same at higher magnification (h and e, 400) postoperative period was uneventful, and the patient was discharged in satisfactory condition. rare histopathological report has aroused the interest to report this case, as mesonephric duct cysts commonly present in anterior / anterolateral wall only on the rarest occasions if residual tissue of wolffian duct persists, then mesonephric cyst may grow from that abnormal site. gartner 's ducts are identified in approximately 25% of all adult women, and nearly 1% evolves into gartner 's duct cysts. during embryological development, remnants often remain, however, until they develop a secretory mechanism, cause dilation of surrounding cells, and thus yield a gartner 's duct cyst, most often during and after late adolescence. classically, the cysts are solitary, unilateral, < 2 cm in diameter, and are located in the anterolateral vaginal wall of the proximal, a third of the vagina. gartner 's duct cysts are generally asymptomatic and most commonly diagnosed upon routine gynecologic examination, but patients ' complaints can include that of skin tag, dysuria, pressure, itching, dyspareunia, pelvic pain, or protrusion from the vagina if it enlarges to a detectable size, making it a candidate for surgical removal if large enough to cause obstetrical complications, the cyst can be drained to facilitate delivery. to define the course of the gartner 's duct cyst and differentiate it from other pathologic considerations and structures, magnetic resonance imaging can be a useful tool. histologic examination may be employed to correctly identify the cellular remnants composed of nonmucin secreting low columnar or cuboidal epithelium [figures 7 and 8 ]. the differential diagnosis can be included, but is not limited to bartholin 's gland cyst or abscess, prolapsed urethra, prolapsed uterus, vaginal wall inclusion cyst, endometriosis, leiomyoma, sarcoma botryoides, malignant mass, skene 's gland cyst, or abscess and ureterocele. only in exceptionally rare and isolated cases, large vaginal wall cysts are always symptomatic which compels the patient to visit a gynecologist. they mostly present with discomfort with vaginal discharge on and off, dyspareunia, or urinary complaints. not all patients presenting with mass per vaginum are necessarily a case of uterovaginal prolapse. | cyst of posterior vaginal wall is very rare. this case relates to a patient who presented with polypoidal mass protruding out from vagina which could have been easily mistaken as uterovaginal prolapse, but appropriate clinical evaluation supported with investigations clinched the diagnosis easily. |
nephron - sparing surgery (nss) offers equivalent oncologic control, as does radical nephrectomy in patients with appropriately selected stage t1 renal tumors [1, 2 ]. additionally, nss has been associated with decreased risks for renal impairment [35 ] and cardiovascular events [6, 7 ], which may explain the improved overall survival, documented in partial nephrectomy series. as the benefits of nss become apparent, the indications may continue to expand. in order to establish standards for future comparisons, we must fully appreciate the efficacy, safety, and limitations of nss for renal masses larger than 7 cm. therefore, we reviewed our experience and reported the results in 17 patients with renal masses, which had a maximum diameter of more than 7 cm on preoperative imaging studies, treated with either open or robot - assisted laparoscopic nss during 13 years. between 2001 and 2012, a single surgeon (te) performed 139 nsss (97 open, 35 robot - assisted, and 7 laparoscopic nsss) in one center. we retrospectively reviewed the charts of the patients, whose renal tumors measured more than 7 cm in diameter on preoperative imaging (n = 17, 17.52%). preoperative evaluation consisted of laboratory tests and cross - sectional imaging studies (computerized tomography and/or magnetic resonance imaging). (radius, exophytic / endophytic, nearness, anterior / posterior, location) scoring system. based on preoperative radiologic findings, open nephron - sparing surgery (onss) was performed using the intercostal (between 11th and 12th ribs) extraperitoneal flank approach, as previously described elsewhere in detail. robot - assisted nephron - sparing surgeries (ransss) were performed using the da vinci surgical system (intuitive surgical, inc., sunnyvale, ca, usa) with a 5-port approach, including two 8 mm ports for robotic instruments, one 12 mm port for the robotic scope, and 2 ports for the bedside assistant. ransss were carried out through the transperitoneal route with the patient in the flank position. after demarcating tumor margins with electrocautery, enucleoresection was carried out using cold scissors, leaving a minimal margin of normal parenchyma. the tumor bed was overseen with 30 polyglactin sutures (in case of pelvicalyceal violation), and parenchyma was adapted with the sliding clip technique. operative data consisted of total operative time, estimated blood loss (ebl), warm - ischemia time (wit), and adverse events. all complications within 30 days of surgery were classified according to the clavien - dindo classification system. estimated glomerular filtration rate (egfr) was determined before and after nss using the modified modification of diet in renal disease (mdrd) equation. tumor staging was designated according to the tnm classification based on the 2009 american joint committee on cancer / international union against cancer classification system. tumor recurrence was defined as a new renal mass in the resection bed of the kidney in the absence of distant metastasis based on imaging. followup was calculated from the time of surgery until the last known contact with the patient or until the date of death. student 's t - test and fisher 's exact test were the statistical methods of reference. a total of 17 patients with clinical t2 renal masses (> 7 cm) have undergone nephron - sparing surgery between 2001 and 2012, at our institution. thirteen patients (76.4%) were treated through the open route (figure 1), while 4 patients (23.5%) were managed by ranss (figure 2). mean age of the study population was 49.8 11.3 years (range = 3066). mean asa score of the operated patients was 1.1 0.39 (range = 13). the majority of the tumors were discovered incidentally (n = 11, 64.7%). nss was offered based more commonly on elective indications. the indication to perform nss was imperative (bilateral mass n = 2, solitary kidney n = 1) in 3 patients (17.6%). mean tumor size was 8.24 2.45 (range = 615) cm according to the final pathology reports. diameter of the resected tumor was measured less than 7 cm in 5 patients (29.4%) whose preoperative imaging findings were suggestive of a t2 lesion. mean tumor size in these 5 cases was 6.1 0.14 (range = 66.3). mean estimated blood loss amount were 125 35.4 minutes (range = 75220) and 267.6 117.1 ml (range = 100500), respectively. mean warm - ischemia time was 15.2 4.1 minutes (range = 1021) in the 8 surgeries (47%) during which tumor was enucleoresected after clamping the renal pedicle. scores of the tumors that were managed under ischemic and nonischemic conditions was statistically insignificant (8.6 versus 8.4 p = 0.72). open conversion during robot - assisted surgery was necessary in 2 cases, whose tumors had r.e.n.a.l. patients were hospitalized for a mean duration of 5 5.4 days (range = 326). three patients suffered from a total of 6 grade 2 and higher complications within 30 days of surgery. blood transfusion and bladder recatheterization due to gross hematuria were the recorded grade 2 and 3a complications in 2 patients, respectively. blood transfusions, angioembolization, double - j catheter insertion due to urinary extravasation, and temporary hemodialysis are counted for the grade 2, 3a, 3b, and 4a complications encountered during the postoperative course of another patient who had a 9 cm enhancing mass in his solitary kidney (figure 4). mean preoperative and postoperative egfr was 89.3 20.5 ml / min/1.73 m (range = 60136.1) and 76.9 23.5 ml / min/1.73 m (range = 8.3111.6), respectively. the difference between these 2 mean values was statistically insignificant (p = 0.11). mean duration of follow - up was 35.2 28.1 months. during the followup period, tumor bed recurrence was detected in one patient (5.8%), who underwent radical nephrectomy as a complementary procedure in another center. none of the patients in this cohort was lost either due to rcc or due to other comorbidities. multiple previous studies have shown the oncological equivalency of nss to radical nephrectomy (rn) for t1a and even t1b renal masses [2, 2427 ]. considering the facts that some of these localized renal lesions will be benign and/or low grade, and renal preservation has documented benefits on long - term cardiovascular health [68 ], the guidelines advocate nss as a standard of care for all t1 renal masses. although, there is a paucity of clinical series evaluating the oncologic results of nss for t2 renal lesions, initial results are encouraging (table 2). found that cancer - specific survival and overall survival were similar between nss and rn for t2 tumors or greater. karellas. found that 71% of the patients were alive and free of disease at a median followup of 17 months. becker. reported 5-year overall and cancer - specific survival rates of 88.0% and 97.0%, respectively. conversely, hafez. found a significant increase in recurrence rates as well as decreased survival in their study consisting of partial resections performed for tumors 7 cm in size. it is also important to note that a recent analysis of almost 19.000 localized rccs in the seer database showed that greater than 75% of localized renal cancers > 7 cm in size and more than 65% of those > 10 cm in size are low - grade lesions, suggesting that nss for large renal masses may be oncologically sound. in our series, after a median followup of 33 months, all 17 patients were alive and free of disease. local recurrence was detected only in one patient (5.8%), 10 months after the nephron - sparing procedure. undoubtedly, nss for tumors larger than 7 cm represents a surgical challenge, especially in relatively unexperienced hands. the surgical team has to decide upon the route (open, laparoscopic, or robot assisted nsss) by which resection is going to be carried out. the idea and the method of (clamping the vasculature or manual parenchymal compression) creating ischemic conditions is another tough decision to be made. maintaining hemostasis and reconstructing the pelvicalyceal system over a large parenchymal defect might be technically demanding especially while performing minimally invasive nss. surgical expertise, technologic availabilities, and patient preference will definitely have an influence on the surgical choices. hence, preoperative morphometric data might tailor the surgical approach. in our series, mean r.e.n.a.l. score of the tumors that were resected under perfused conditions was insignificantly lower than that of the tumors operated under ischemic conditions. although being lower in the robotic group, the difference between robot - assisted group (n = 4) and open surgical group (n = 13) in terms of mean r.e.n.a.l. furthermore, none of the patients for whom open surgery was employed had a tumor with low r.e.n.a.l. conversely, one patient (25%) in the robotic group had a tumor in the low r.e.n.a.l. score category. long. evaluated the results of 46 patients with 49 clinical t2 renal tumors and reported a major urological complication in the form of a urinary fistula in six (12.2%) patients. in the other reported series for ct2 nsss, the rate of urinary fistula was in the range of 3.3 to 18.8% [1823 ]. in our series, only one patient (5.8%) suffered from urinary extravasation. furthermore, our transfusion rate was 11.7% (n = 2), which is concordant with the range reported in the literature (030%). as shown by previous studies, almost 1/3 of the clinical t2 tumors were either benign or represented a very low - grade malignancy (chromophobe rcc), emphasizing the importance of renal preservation when technically feasible, regardless of tumor size [17, 18 ]. in our cohort, 23.5% of the tumors were reported to have benign histology, while 46% of the rccs were of either papillary or chromophobe subtype (table 1). it has been reported that, up to 26% of the patients with renal masses have preexisting stage 3 and higher chronic kidney disease (ckd), defined as a glomerular filtration rate (gfr) of 7 cm in size (table 2), renal functional maintenance, which may be considered as the primary advantage of nss, has been highlighted. long. reported advancement in ckd stage in only 11% of their study population. breau. detected a median increase in serum creatinine value by 9.5% compared to 33% for rn. in the studies conducted by becker. and karellas. demonstrated a small increase in mean serum creatinine values at followup in their small cohort of patients. in our study, median egfr value decreased from 89.37 to 79.15 ml / min/1.73 m postoperatively and ckd stage progressed in 5 patients (29.4%). temporary renal replacement therapy, in the form of hemodialysis, had to be initiated for one patient who had a large (9 cm) upper pole mass in his solitary kidney. although the surgery was carried under nonischemic conditions, the extent of the resection may be the factor underlying the need for renal replacement. our study has several limitations such as the retrospective nature with its inherent biases, small sample size, heterogeneous study population, limited follow - up duration, and pathological understaging in 5 patients. nonetheless, our findings are in concordance with the previous studies, encouraging the utility of nss for renal masses larger than 7 cm. nephron - sparing surgery is a feasible option for renal masses, measuring larger than 7 cm on preoperative imaging studies. despite a limited duration of followup in our series renal functional status did not change significantly after nss for large (> 7 cm) renal masses. less than 1/5 of the study population suffered grade 2 and higher complications during the postoperative period. regardless of tumor stage, nephron - sparing surgery may be considered for carefully selected patients, including those with a normally functioning contralateral kidney. | objectives. to document the feasibility of nephron - sparing surgery (nss) for the surgical treatment of renal masses measuring larger than 7 cm (ct2) on preoperative imaging. methods. a total of 139 patients have undergone nss between 2001 and 2012 by a single surgeon in our clinic. of these, we identified 17 patients whose tumors were measuring greater than 7 cm on preoperative imaging studies and were limited to the kidney. their charts were retrospectively reviewed. results. mean age of the study population was 49.8 11.3 years. thirteen patients were managed by open nss, while 4 patients have undergone robot - assisted nss. mean diameter and mean r.e.n.a.l. score of the tumors that were enucleoresected were 8.2 cm and 8.5, respectively. a total of 5 clavien grade 2 and higher complications were recorded within 30 days of surgery. histopathologic examination revealed benign histology in almost 1/4 of the cases. after a median followup of 33 months, all of our patients were alive. only one patient (5.8%) experienced local recurrence. conclusions. nss is a feasible and safe option for large (> 7 cm) renal masses. it may be considered not only for imperative conditions but also for highly selected cases with a normal contralateral kidney. |
in the early days of biological research, mutations that caused discernable phenotypes were the primary tool for understanding how a biological system worked in the absence of a mutation a gene was invisible. today, biologists are armed with a whole arsenal of tools to regulate gene, mrna, and protein abundance and activity, thereby promoting the discovery of mechanisms and how a system gone awry can lead to disease (1). among these are tools for suppressing the activity of a gene or gene product (e.g. site - directed mutagenesis, rna interference, small molecule inhibitors) or enhancing activity (e.g. activating mutations or receptor agonist). for instance, interfering with protein activity using small - molecule inhibitors should have a phenotype similar to reducing the abundance of the corresponding mrna with anti - sense oligonucleotides (2). likewise, similar responses are expected whether increases in intracellular protein concentration are achieved via an inducible promoter or by addition of recombinant protein (3). as such, perturbations form the core of understanding how biological systems work, how diseases arise, and how they can be treated. any serious attempt to analyze a biological process starts by identification and characterization of perturbations that have been used in prior work. this task requires a framework that can be systematically applied and that is amenable to both manual and automatic means. currently, there is no established categorization that sufficiently represents the range of described experimental manipulations beyond high - level semantic and grammatical classifications (4,5) or description of techniques (6). for example, the closest concept we have found is altered expression, defined as altered expression level of a gene / protein (7). we believe that this concept is overly specific and fails to cover important phenomena, among others, changes in protein activity or gene mutations. we propose, instead, taking the existing concept of perturbation and broadening it to comprise the range of terms used in text to indicate changes in the abundance or activity of dna, rna and proteins. perturbations, in this new formulation, would refer to a collection of phenomena in a manner analogous to the way protein protein interactions refer to biological phenomena of different type (e.g. bind, activate, inhibit). since this proposition, like any other, needs to be tested for validity and utility, we have applied it to a case study involving gene phenotype associations in disease and have developed a mining algorithm that detects the diverse forms in which perturbations appear in text. therefore, we are introducing in this work both a new way to understand a crucial part of biology and a new text - mining method tailored to its extraction. design, test and analysis. as initial step, we created the design corpus to develop an analytical framework for annotation. the purpose of this corpus was to identify challenges in the annotation process and to refine guidelines that would help the annotators in choosing their evaluations. annotating perturbations requires at times thorough knowledge of experimental biology, which can only be captured and organized within a solid framework. therefore we sought to perform a preliminary analysis on a test corpus to improve on subsequent annotations. this corpus was limited to sentences that included disease - related gene phenotype relationships. using the semantic relationship nomenclature of tsai. (8), we selected reports in which the agent that deliberately performs an action is represented by a gene or protein, and the patient that is the recipient of the action corresponds to disease phenotypes. the information we sought stands in contrast to associative relationships, such as elevated protein levels correlating with disease activity. to create the design corpus, our initial query matched medline sentences containing ordered triplets of a gene or protein name, a causative verb and a phenotype related to cancer or diabetes. each member of the triplet was separated within the sentence by a maximum of three words. this software package has the ability to retrieve sentences from text that include word patterns established by user queries. semantically, term classes can be defined combining external ontologie and adding term morphological variations and regular expression patterns. syntactically, it recognizes part - of - speech and shallow parsing constructs such as noun phrases, verbs and prepositions. in terms of scope, queries can be confined to different document or text sections, including abstracts, titles or sentences. for example, a sentence - level query may comprise a term class protein, a list of verbs (i2e can automatically generate morphological variants) and a list of phenotype objects. the gene / protein thesaurus was internally developed and based on biothesaurus (9). forty verbs that signal causality (e.g. inhibition, stimulation) were compiled manually, as was a set of disease - related phenotypes relevant to cancer (e.g. tumorigenesis, vascularization) and diabetes (e.g. serum glucose levels, weight gain). a set of 100 retrieved sentences and relationships were annotated by three phd - level evaluators for relevance and direction of perturbation (see table 1 for examples). relevance annotation was used to mark relationships that should be eliminated from the corpus for being irrelevant to the intent of the retrieval query, e.g. if the gene was not acting as an agent. direction indicated the type of perturbation (increased, decreased or unknown) relative to the starting state. for example, if a gene is added back to cells that carry a deletion in that gene to restore the wild - type state, it is noted as an increase, because the abundance of the gene was increased relative to the starting state. unknown if there was no stated perturbation, or direction could not be inferred at the sentence level. it is worth noting that although an unknown direction could be frequently resolved by reviewing the abstract or the full text of the article, we strictly limited our scope to evidence found at the sentence level. table 1.examples of relevance and direction annotationrelevancedirectionsentenceexplanationpmidirrelevantdifferent members of bcl-2 family may promote or inhibit apoptosis by synthesizing anti- and proapoptotic proteinsmembers of a gene family, not a gene, are causing apoptosis phenotype17404013irrelevantthe atf6 and ire1 pathways cooperatively caused apoptosis via induction of chop, activation of xbp1 and phosphorylation of jnk, and the perk - eif2 pathway counteracted the proapoptotic processespathways, not genes, are causing apoptosis phenotype17464326irrelevantthis result suggests that toxic products such as reactive oxygen species and aldehydes liberated by the action of polyamine oxidase on the acetylated polyamines formed by ssat may enhance tumor developmentmetabolites, not proteins that generate them, are causing tumor development phenotype17675337relevantunknownthese results indicate that survivin is important for optimal development of bovine blastocysts and confirm that survivin expression suppresses apoptosis of pre - implantation embryossurvivin expression does not indicate if there is a state change in amount17075833relevantincreasefinally, we showed that after 79 days of incubation, mch also inhibits proliferation of non - stimulated pbmcincubation indicates mch was added exogenously17537530relevantincreasetaken together, ldm induces apoptosis in a p53-dependent manner when given at low doses, but in a p53-independent manner when given at high dosesgiven at low doses indicates ldm was added exogenously17534142relevantdecreasesoluble flt-1 abolished hypoxia / vegf - induced hyperpermeabilitysoluble forms of receptors create inactive complexes17311300 examples of relevance and direction annotation while most sentences contained straightforward descriptions of perturbations (e.g. mutations in gene a or protein b inhibition), the broad range of perturbations in the literature, combined with the complex grammatical structures found in biomedical text, made some sentence relationship pairs difficult to annotate consistently, mainly due to differences in knowledge of experimental descriptions and settings by the evaluators. when inter - annotator agreement proves to be challenging, other groups have adopted strategies to further improve the final gold standard annotation set (10). for this work, sentences deemed difficult to evaluate were set aside for later annotation by discussion and group consensus. therefore, the gold standard was comprised of annotations with three - way agreement from the individual assessment, plus the consensus annotations. when each evaluator 's set of straightforward independent annotations (n = 237 out of 300 annotations, 79%) was compared to the gold standard, agreement averaged 92.9%. sentence relationships for which there was no agreement or only two - way agreement were discussed and annotated by consensus (14%, n = 14). while overall inter - annotator agreement can not be measured with the annotation procedure devised, the agreement metrics described are helpful as proxies to the level of ambiguity of the task. pairs that are only evaluated by consensus differ from the rest largely in the knowledge required from the evaluator to elucidate the annotation, rather than in the factual ambiguity of the sentence. hence, discussion and consensus assessment of pairs yields a better annotation set than individual annotation. only 7% (n = 7) of sentence relationship pairs were marked irrelevant. following the same guidelines, we created the test corpus. each of the three evaluators assessed different sets of 500 sentence sentences deemed not straightforward were left without annotation (n = 126, 8.4%) and later annotated by consensus. n = 95) were considered irrelevant, demonstrating the high precision of the retrieval query. overall, the procedure used to construct the corpus assured high quality to the 1405 sentence relationship annotation. we performed detection of increased, decreased or unknown perturbations using machine learning techniques. for that purpose, we constructed a vector of features for each relationship sentence pair using the test corpus above. one of them represented token presence as a binary vector of token weights wi, d = { w1,,w|t| }, where t is the set of sentence tokens : a weight has value 1 if the token is present in the sentence and value 0 otherwise, an approach called set of words (sow) (11). hyphenated names were considered both as single and separate tokens in the sow in order to capture affixes like since proximity to the gene name can be important in determining a token 's role in describing a perturbation, the sentence was further divided in several sections relative to the gene name 's position. the sections considered were : n tokens before the gene name, where n = { 5, 10, all }, and n tokens after the gene name, where n = { 10, all}. we noted that many perturbation descriptions were adjacent or very close to the gene name (e.g. overexpression of p53). hence, we included a feature with the distance between the gene name and the beginning of the sentence (e.g. distance of 0 or 1 may indicate that the perturbation is unknown, e.g. we also created a set of features using a small perturbation ontology developed independently over a disease - agnostic set of retrieved sentences. if a member of the ontology was present in the sentence a feature was added with value 1, otherwise with value 0. all the feature sets described were integrated in a single feature vector for each sentence. an algorithm based on the principles of maximum entropy (12, http://homepages.inf.ed.ac.uk/s0450736/maxent_toolkit.html) was trained using 80% of the test corpus, randomly sampled from the cancer and diabetes sets, and tested over the remaining 20% (results were averaged over 10 runs). the training allowed the algorithm to predict the presence of a perturbation and its direction. performance measures were evaluated and compared favorably to an svm algorithm (13). to create the analysis corpus, we extended the scope of our methodology used to retrieve the test corpus by eliminating the disease - specific phenotype constraints in the retrieval query (hence, phenotypes were not included in the query). we applied the query to medline diabetes abstracts after 1996 and retrieved 359 385 relationships related to different conditions and phenotypes. this output was run through the machine - learning algorithm to create a wide - scope, disease - agnostic set of 191 240 perturbation predictions. to create a diabetes - specific subset, only sentences from medline abstracts containing the word diabetes in their mesh descriptors were included. there were significant differences both in technique and direction between the cancer and diabetes perturbations in the test corpus, with decreased perturbations more prevalent in cancer. detection of increased and decreased perturbations had a lower f - measure, 72.9% and 71.2%, respectively. when we excluded the perturbation ontology in feature generation, results were only slightly lower, whereas when we exclusively used the perturbation ontology, results were much lower, notably due to reduced recall. straightforward relationships (91.6% of the total) were those that evaluators annotated without consultation with other annotators. these relationships were less challenging for humans, and the algorithm had better performance over this subset than overall. due to absence of previous work in perturbation detection, these results can not be compared, but they fall in ranges typical of other successful biomedical text mining tools. table 2.perturbation extraction performancecountprecision (%) recall (%) f - measure (%) perturbation69079.279.879.4 no ontology76.479.677.6 only ontology77.259.567.0 straightforward83.378.981.0increased43675.371.172.9 no ontology76.669.972.9 only ontology65.750.956.8 straightforward77.768.872.7decreased25479.065.171.2 no ontology78.563.670.0 only ontology79.858.466.4 straightforward80.467.272.9precision, recall and macro - averaged f - measure were calculated using four different combinations of feature sets : full, no ontology, only ontology and straightforward. baseline frequencies were 32.8% for increased perturbations and 18.1% for decreased perturbations. perturbation extraction performance precision, recall and macro - averaged f - measure were calculated using four different combinations of feature sets : full, no ontology, only ontology and straightforward. baseline frequencies were 32.8% for increased perturbations and 18.1% for decreased perturbations. to determine whether the disease impacted the frequency of perturbation types, we compared cancer and diabetes literature. the diabetes literature was significantly enriched in increased perturbations, whereas the cancer literature showed an even distribution between increased and decreased perturbations. in cancer, more perturbations were performed with antisense oligonucleotide (n = 9), antibody (n = 16) and rna interference (n = 26) than in diabetes (antisense, n = 3 ; antibody, n = 2 ; rnai, n = 0). perturbations in diabetes were more frequently described as injections (n = 132) and/or administered by dose (n = 57) than in cancer (dose, n = 3 ; injection, n = 6). perturbations in cancer were also more frequently described as being in vivo (n = 12) or in vitro (n = 14) than in diabetes (in vivo, n = 5 ; in vitro, n = 0). we examined the sentences for frequently occurring terms, grouped by their level of regulation (i.e. protein, mrna or dna), if known. many of the terms related to dosing and routes of administration (e.g. administration, intracerebroventricular, injection) show a strong dominance in the diabetes literature compared to the cancer literature. although these usually indicate an increasing perturbation, there can be exceptions, such as systemic delivery of an inhibitor. table 3.ontology occurrence count in sentences from the test corpus, separated by diabetes and cancer phenotypesgene and protein perturbationscancerdiabetescancerdiabetestotal increasing modifications266586total decreasing modifications267110general increasing modificationsgeneral decreasing modifications activation6612 down - regulated, -ion120 administered, -ion7120 inactivation50 dose, -age, -dependent1186 inhibition307 ectopic70 repression10 enhanced expression100 suppression103 exogenous623dna decreasing modifications i.c.v.011 deficiency, -ent1512 i.p.07 deletion32 increased2618 dominant - negative30 induction82 knockout12 infused, -ion035 loss1213 injected, -ion766 mutant00 intracerebroventricular051 mutated, -ion03 intraperitoneal116mrna decreasing modifications mg / kg,/kg08 anti(-)sense93 oral64 interference51 overexpressed, -ration380 interfering rna20 peripherally07 knockdown120 recombinant710 rna interference40 restoration20 rnai20 subcutaneous02 short hairpin rna10 systemic19 silenced10 treatment2422 sirna150 up - regulation80protein decreasing modificationsdna increasing modifications antagonist728 adenoviral21 anti-254 adenovirus00 antibody, -ies113 gain - of - function20 blockade, -ing128 gene delivery13 deactivation10 transgenic32 decoy20mrna increasing modifications fc-00 inducible20 inhibitor5315protein increasing modifications inverse agonist01 activator100 mabs10 agonist954 neutralization10 analog / analogue27 reduced soluble40 targeting60note that total increased and decreased perturbations exceeds values in table 2 since multiple perturbation terms may appear in a single sentence. ontology occurrence count in sentences from the test corpus, separated by diabetes and cancer phenotypes note that total increased and decreased perturbations exceeds values in table 2 since multiple perturbation terms may appear in a single sentence. the phenotypes in this study were selected based on prevalence in the literature without regard to their use in vivo or in vitro. for instance, in diabetes a change in blood glucose levels signals a change in disease state clearly a phenotype monitored following perturbation in vivo. likewise, in cancer a change in the number of metastases is exclusively described in in vivo systems. in contrast, insulin sensitivity can be used to describe both in vivo and in vitro systems. for cancer, cell proliferation and apoptosis rates if our hypothesis that perturbed genes and proteins form the underpinnings of disease mechanisms, these entities should be well represented in pathway diagrams and among drug candidates. we applied our trained algorithm to a set of 14 345 sentence relationship facts for genes from our analysis corpus belonging to a diabetes pathway (figure 1). predictably, our algorithm found a strong correlation between the number of medline abstracts in which a gene is mentioned and the number of times it is described as perturbed (r = 0.70). the results in figure 1 show the difference in intensity and modality in which each gene is described or pursued experimentally. some genes were typically more increased or decreased, often reflecting their roles in therapeutics. examples of significantly (p < 10) increased were insulin, interleukin 2 or parathyroid hormone. among the decreased were such genes as epidermal growth factor receptor ; caspase 8 and plasminogen activator, urokinase receptor. figure 1.perturbations extracted from genes involved in the diabetes mellitus type ii pathway in the kyoto encyclopedia of genes and genomes (21). differences in perturbation direction are large for some members of the pathway, note that the scale bar is logarithmic. perturbations extracted from genes involved in the diabetes mellitus type ii pathway in the kyoto encyclopedia of genes and genomes (21). differences in perturbation direction are large for some members of the pathway, note that the scale bar is logarithmic. disease associations included in the omim morbid map (omim online reference, http://www.ncbi.nlm.nih.gov/omim/). the average abstract mention and gene perturbation counts were higher for genes with morbidmap associations (t - test, p < 10). this was consistent with our expectation that genes associated to disease would be the subject of deeper study. however, genes that had been perturbed numerous times were not necessarily linked to disease. for example, out of the 100 most perturbed genes, 54 were not linked to disease in omim morbidmap, including at the top such genes as jun oncogene, interleukin 4, fibroblast growth factor 2 (basic), colony stimulating factor 2 (granulocyte macrophage), colony stimulating factor 3 (granulocyte) and mitogen - activated protein kinase 3. perturbations are relevant for areas like the study of gene disease associations, protein protein interactions (ppi) or gene regulatory networks. gene disease association extraction studies have largely focused on simply detecting associations rather than characterizing them (7,14). ppi and gene regulation extraction studies have side - stepped perturbation types, without considering anything further than the experimental technique (15,16). we note that ppi relationships are frequently devoid of perturbations and the focus is on how a ppi was detected, which does not necessarily involve a causative relationship. the proteomics standards initiative molecular interactions (psi - mi) ontology (6) is a comprehensive effort to describe molecular interactions. this ontology, while including a detailed experimental preparation section, lacks expressivity in describing perturbations generically. similarly to bundschus. (7), it includes a section on expression level with entries under expressed, over expressed and physiological. the lack of a general framework for recognizing, characterizing and classifying perturbations is surprising when one considers their importance in phenomena encountered experimentally. researchers with interest in characterizing previous and current perturbation work on a biological system face the challenge of a naturalist trying to deal with animal species without a linnaean taxonomy. this is reflected in the methodological landscape that was set in the early literature in the fields of biomedical ontologies and text mining. for example, the comprehensive text mining tool medlee (17) only considered the state of a gene or protein, where the state has an adjectival role such as mutated in the phrase mutated x. perturbation descriptions, however, should be considered carefully. observe the differences between the sentences (i) x activates y. and (ii) inhibition of x activates y. from the point of view of classic ppi extraction both relationships are equivalent and can be represented with the triplet x activate y. the perturbation in sentence (ii), however, signals that it is likely that protein x is inhibiting protein y instead. we have called this phenomenon reversal. given the results of the present assessment, a review of the relationship data available should be considered under this model. phenotype associations in disease but the principles shown are applicable to other well - known areas, such as ppi, as well as less explored ones such as identification of biomarkers or cellular processes. a perturbation taxonomy, like the one described in table 4, could capture the different dimensions that may be of interest to the inquiring scientist. unknown direction annotation is intended for perturbations whose direction can not be inferred at the sentence level. molecule annotation characterizes the type of molecule being primarily affected by the perturbation : gene, rna or protein. expression annotation is used for a change of expression level without clarifying whether the change is in rna or protein. the following examples illustrate these annotations : a gene mutation is a decrease in gene activity, where the function / activity of a gene is specifically understood as making a wild - type transcript. exogenous addition of a gene via viral transfection, plasmid transformation, etc., is an increase in gene abundance. a gene duplication is an increase in gene abundance while a knockout is a decrease in gene abundance. dominant negative is a mutation in a gene, which indicates that, compared to wild - type, it has defective function. silencing, knock - down and antisense all apply to a decrease in rna abundance. an antibody blocking a protein decreases the protein activity or function. interfering with a protein binding treatment, incubation, recombination, or synthetic refer to exogenous addition of protein. table 4.proposed annotation guidelinesrelevancedirectionmoleculeeffectrelevantincreasegeneactivityirrelevantdecreasernaabundanceunknownprotein expression proposed annotation guidelines perturbations evolve, notably as new techniques are developed and targets are identified. we expect perturbations to be subject to trends and popularity variations similar to those in other aspects of biomedicine (1820). | molecular perturbations provide a powerful toolset for biomedical researchers to scrutinize the contributions of individual molecules in biological systems. perturbations qualify the context of experimental results and, despite their diversity, share properties in different dimensions in ways that can be formalized. we propose a formal framework to describe and classify perturbations that allows accumulation of knowledge in order to inform the process of biomedical scientific experimentation and target analysis. we apply this framework to develop a novel algorithm for automatic detection and characterization of perturbations in text and show its relevance in the study of gene phenotype associations and protein protein interactions in diabetes and cancer. analyzing perturbations introduces a novel view of the multivariate landscape of biological systems. |
pulmonary vein isolation (pvi) has become an important option for treating patients with atrial fibrillation (af). however, a few reports have described esophageal injury after pvi, including the development of lethal atrioesophageal fistulae. in addition, periesophageal nerve (pen) injury after pvi may rarely cause pyloric spasms and gastric hypomotility. the c - acetate breath test has been used to diagnose delayed gastric emptying induced by gastric hypomotility and/or pyloric spasms. the objectives of this study were to clarify the impact of pvi on gastric motility, the prevalence of gastric hypomotility after pvi, and the relationship between de novo esophageal injury and gastric hypomotility following pvi. the c - acetate breath test and esophagogastroduodenoscopy (egd) were performed before and after the procedure to achieve these objectives. thirty - one consecutive patients with symptomatic paroxysmal or persistent af were included in this prospective study. patients with a history of pvi, the maze procedure, gastrostomy, an eating disorder, or episodes of acute coronary syndrome, emboli, or decompensated heart failure within 3 months before enrollment were excluded. we enrolled 20 patients who underwent ablation procedures other than pvi as the control group. egd was performed in all patients within 3 months before the ablation procedure and within 3 days after the procedure. esophageal injury was defined as a de novo lesion detected on the anterior wall of the midthoracic esophageal region after pvi that had not been detected before the procedure. administration of rabeprazole (30 mg / day) was scheduled to start the day after pvi and was re - evaluated on the day of the gastric emptying test after pvi to avoid worsening any esophageal injury. oral medications, other than rabeprazole, that affected gastric peristalsis were initiated or changed in all patients within a month before the ablation and in the hospital. measurements of the gastroesophageal reflux disease symptoms scale and the gastrointestinal symptom rating scale were obtained before and after pvi. these scales are used to evaluate common symptoms of patients with gastrointestinal digestive disorders such as reflux esophagitis,. gastric emptying was evaluated using the c - acetate breath test with a slight modification,,. gastric emptying is conventionally evaluated using the acetaminophen absorption test or technetium-99 m scintigraphy. however, the acetaminophen absorption test requires repeated collection of blood samples, and scintigraphy requires specialized instrumentation. both examination times are approximately 4 h. in contrast, the c - acetate breath test, which is an alternative method to the acetaminophen absorption test or scintigraphy, can be performed at the patients bedside. a strong correlation is observed between gastric emptying time measured by scintigraphy and the time to peak co2 concentration in an expired breath (tmax) as well as half - dose recovery time (t1/2) measured by the c acetate breath test. tmax in patients with functional dyspepsia is significantly delayed compared with that in healthy controls. therefore, we chose the c - acetate breath test, which was modified by not measuring t1/2, as described previously,,. c - sodium acetate administered orally with a test meal moves from the stomach and is absorbed by the intestines, where it is subsequently metabolized to co2, and finally expired by the lungs (fig. if a patient has gastric hypomotility, the duration between swallowing the test meal and the test meal reaching the intestines is longer than that in normal patients. as a result, tmax in a patient with gastric hypomotility is longer than that in a patient without gastric hypomotility. this duration has been validated on the basis of a mean and standard deviation value of 43.910.3 min and a median value of 70 min in patients with advanced parkinsons disease, which is commonly associated with dyspepsia. first, a breath sample was obtained after ingestion of a liquid test meal (racol, 200 kcal/200 ml ; otsuka pharmaceutical co., ltd., otsuka, japan) which contained 100 mg of c - sodium acetate (sigma - aldrich, st. the test meal was consumed in 39 c between the groups (normal tmax to normal tmax vs. normal tmax to abnormal tmax ; table 2). in addition, there was no correlation between tmax and the quantity of radiofrequency energy applied to the left side of the posterior wall of the la (p=0.82) or the number of points of let > 39 c (p=0.30). reflux esophagitis and hernia were detected in seven (23%) and nine (30%) patients, respectively, before pv (table 3). no patients had food residue in the stomach or esophageal mucous membrane lesions before the procedure. furthermore, no patient suffered from digestive symptoms such as nausea, vomiting, bloating, epigastric pain, or dyspepsia after the procedure. food residue in the stomach was detected in another three patients, but only one had an abnormal tmax after pvi. of the other 24 patients with no egd findings, 13 had an abnormal tmax. furthermore, no patient developed a symptomatic pen injury after pvi during their hospital stay. after obtaining informed consent, a thrombus was detected in the laa of one af patient by cct ; this patient was excluded from the study. thirty out of 31 patients with af and all 20 patients in the control group completed the required procedures and were analyzed. the baseline characteristics of both the pvi and control groups are shown in table 1. no significant differences were observed in baseline characteristics between groups, except history of heart failure and stroke. the mean chads2 score was 0.670.76 in the af group, with a maximum score of 2.0. the number of patients with abnormal tmax (75 min) increased from seven (23%) to 13 (43%) after the procedure in the pvi group (fig. no significant differences were observed between the histories of these seven patients and those of the other patients (table 2). the number of patients with abnormal tmax increased from three (15%) to five (25%) after the procedure in the control group (fig. the mean tmax was longer after pvi than before pvi (6414 min vs. 5715 min, p=0.006 ; fig. 2c). on the other hand, the mean tmax in the control group was not significantly different after the procedure (5616 min vs. 5316 min, p=0.35 ; fig. proton pump inhibitor (ppi) administration was initiated between the first and second c - acetate breath test in five patients with af. the mean tmax was significantly longer after pvi than before pvi when the effects of the ppi on gastric emptying were considered, even after excluding these five patients (5816 min vs. 6415 min, p=0.039). the mean difference between tmax before and after the procedure (tmax) was 7.7 min in the af group and 3.3 min in the control group (p=0.27). no significant difference was observed in the quantity of the radiofrequency energy applied to the left side of the posterior wall of the la or the number of points of let > 39 c between the groups (normal tmax to normal tmax vs. normal tmax to abnormal tmax ; table 2). in addition, there was no correlation between tmax and the quantity of radiofrequency energy applied to the left side of the posterior wall of the la (p=0.82) or the number of points of let > 39 c (p=0.30). reflux esophagitis and hernia were detected in seven (23%) and nine (30%) patients, respectively, before pv (table 3). no patients had food residue in the stomach or esophageal mucous membrane lesions before the procedure. furthermore, no patient suffered from digestive symptoms such as nausea, vomiting, bloating, epigastric pain, or dyspepsia after the procedure. food residue in the stomach was detected in another three patients, but only one had an abnormal tmax after pvi. of the other 24 patients with no egd findings, 13 had an abnormal tmax. furthermore, no patient developed a symptomatic pen injury after pvi during their hospital stay. this study demonstrated significantly, although only slightly, prolonged gastric emptying after pvi compared with that before the procedure. however, the average impact of pvi on gastric motility under monitoring of the let was small. the vagus nerve modulates the secretion of gastric acid and gastric peristalsis. in the thoracic cavity, the left vagus nerve forms the mesh - like anterior esophageal plexus after traveling behind the left pulmonary hilum and enters the abdomen through the esophageal diaphragmatic opening (fig. the anterior vagus nerve from the esophageal plexus lies extremely close to the outer muscle of the abdominal esophagus and divides near the esophageal end of the lesser curvature into the gastric and pyloric branches. therefore, the anterior vagal nerve is responsible for coordinated relaxation of the pyloric sphincter and gastric peristalsis during gastric emptying. because radiofrequency energy may injure the esophagus, it is possible that the esophageal nerve plexus situated between the left atrium and esophagus can also be injured however, if the radiofrequency energy injures any part of the plexus, although rare, pen injury may occur. because the plexus surrounds the entire circumference of the esophagus, the nerve rarely becomes completely blocked. they reported that patients with pen injuries were defined as those with symptoms of delayed gastric emptying, such as nausea, vomiting, postprandial fullness, and bloating, associated with gastric hypomotility as assessed by gastrointestinal fluoroscopy. eleven of 3538 patients (0.3%) were diagnosed with a periesophageal vagus nerve injury. the average impact of rf energy on pen and the prevalence of asymptomatic or slight pen injury were previously unknown. this study demonstrates the minimal average impact and shows a very low incidence of severe pen injury. using electrogastrography, lo similar to the c - acetate breath test, electrogastrography is noninvasive and may be a surrogate measure of gastric emptying. we observed a discrepancy between the presence of food residue as detected during egd and abnormal tmax in the gastric emptying test. one reason is that we used a liquid meal for the test ; therefore, the results were representative of liquid gastric emptying. the additive effect may result in the formation of solid residue, even when the pen is slightly affected by rf energy. when a patient complains of gastroparesis - like symptoms but has normal findings of egd after af ablation, the c - acetate breath test may be helpful to diagnose the possible periesophageal vagal nerve injury. in addition to the direct effects of rf energy, mental stress, fatigue, and other factors may also have a considerable influence. in the control group, this finding indicates that not only a direct effect of rf energy but also mental or other stress can influence gastric emptying after the procedure, including pvi. takahashi. reported that rabeprazole may delay tmax to a greater extent than h2 receptor antagonists, although this difference was not significant. rabeprazole suppresses gastric emptying of liquid nutrients after the ingestion of a meal. even after exclusion of patients for whom ppi administration was initiated between the first and second c - acetate breath tests, the mean tmax was significantly longer after pvi than before pvi. showed that dexmedetomidine administered at the upper limit of the recommended dose (0.7 g / kg / h) inhibited gastric emptying, where as memis. reported that doses administered at the lower limit did not affect gastric emptying., we used a low to medium dose of dexmedetomidine and evaluated the effect 13 days after the intravenous injection. because the half - life of dexmedetomidine is extremely short (2 h), we predicted that it would barely influence gastric emptying in our study. before pvi, as already described in table 2, ppi was administered at the time of enrollment in 20% patients with af and may potentially have led to a risk of symptomatic delayed gastric emptying and esophageal injury after pvi. the possibility that the ppi may have influenced gastric emptying in some of the patients with af at baseline also can not be ruled out. however, this was transient and patients recovered by decreasing sedation dose. adaptive servo ventilator or airway devices were not used, but it can not be denied that these patients might have potential sleep apnea syndrome (sas). continuous positive airway pressure (cpap) reduces reflux esophagitis with respiratory failure. respiratory support device such as cpap therefore, it is necessary to evaluate a larger number of patients from multiple centers with a longer follow - up for further clarification of the clinical implications. second, because we used the modified c acetate breath test, we measured excretion only up to 90 min. in patients with peak times of co2 excretion > 90 min, tmax may be considered as 90 min. in our study, two patients out of 30 had a tmax of 90 min before ablation. in both patients, if patients have a greater degree of gastric hypomotility than this, we may overestimate the impact of rf energy. one of them marked 60 min before ablation and the other 75 min. if they had more gastric hypomotility, we may underestimate the impact of rf energy. third, the precise number of patients who underwent transmural ablation in this study remains unknown. some papers have inferred that sas is related to reflux esophagitis. however, another paper has found that sas is not related to reflux esophagitis. in this paper, ju. reported that poor subjective sleep and depressive symptoms are associated with the presence of reflux esophagitis with no association between sas, bmi and reflux esophagitis. this study demonstrated significantly, although only slightly, prolonged gastric emptying after pvi compared with that before the procedure. however, the average impact of pvi on gastric motility under monitoring of the let was small. the vagus nerve modulates the secretion of gastric acid and gastric peristalsis. in the thoracic cavity, the left vagus nerve forms the mesh - like anterior esophageal plexus after traveling behind the left pulmonary hilum and enters the abdomen through the esophageal diaphragmatic opening (fig. the anterior vagus nerve from the esophageal plexus lies extremely close to the outer muscle of the abdominal esophagus and divides near the esophageal end of the lesser curvature into the gastric and pyloric branches. therefore, the anterior vagal nerve is responsible for coordinated relaxation of the pyloric sphincter and gastric peristalsis during gastric emptying. because radiofrequency energy may injure the esophagus, it is possible that the esophageal nerve plexus situated between the left atrium and esophagus can also be injured however, if the radiofrequency energy injures any part of the plexus, although rare, pen injury may occur. because the plexus surrounds the entire circumference of the esophagus, the nerve rarely becomes completely blocked. they reported that patients with pen injuries were defined as those with symptoms of delayed gastric emptying, such as nausea, vomiting, postprandial fullness, and bloating, associated with gastric hypomotility as assessed by gastrointestinal fluoroscopy. eleven of 3538 patients (0.3%) were diagnosed with a periesophageal vagus nerve injury. the average impact of rf energy on pen and the prevalence of asymptomatic or slight pen injury were previously unknown. this study demonstrates the minimal average impact and shows a very low incidence of severe pen injury. using electrogastrography, lo similar to the c - acetate breath test, electrogastrography is noninvasive and may be a surrogate measure of gastric emptying. we observed a discrepancy between the presence of food residue as detected during egd and abnormal tmax in the gastric emptying test. one reason is that we used a liquid meal for the test ; therefore, the results were representative of liquid gastric emptying. the additive effect may result in the formation of solid residue, even when the pen is slightly affected by rf energy. when a patient complains of gastroparesis - like symptoms but has normal findings of egd after af ablation, the c - acetate breath test may be helpful to diagnose the possible periesophageal vagal nerve injury. in addition to the direct effects of rf energy, mental stress, fatigue, and other factors may also have a considerable influence. in the control group, this finding indicates that not only a direct effect of rf energy but also mental or other stress can influence gastric emptying after the procedure, including pvi. takahashi. reported that rabeprazole may delay tmax to a greater extent than h2 receptor antagonists, although this difference was not significant. rabeprazole suppresses gastric emptying of liquid nutrients after the ingestion of a meal. even after exclusion of patients for whom ppi administration was initiated between the first and second c - acetate breath tests, the mean tmax was significantly longer after pvi than before pvi. showed that dexmedetomidine administered at the upper limit of the recommended dose (0.7 g / kg / h) inhibited gastric emptying, where as memis. reported that doses administered at the lower limit did not affect gastric emptying., we used a low to medium dose of dexmedetomidine and evaluated the effect 13 days after the intravenous injection. because the half - life of dexmedetomidine is extremely short (2 h), we predicted that it would barely influence gastric emptying in our study. before pvi, as already described in table 2, ppi was administered at the time of enrollment in 20% patients with af and may potentially have led to a risk of symptomatic delayed gastric emptying and esophageal injury after pvi. the possibility that the ppi may have influenced gastric emptying in some of the patients with af at baseline also can not be ruled out. however, this was transient and patients recovered by decreasing sedation dose. adaptive servo ventilator or airway devices were not used, but it can not be denied that these patients might have potential sleep apnea syndrome (sas). continuous positive airway pressure (cpap) reduces reflux esophagitis with respiratory failure. respiratory support device such as cpap therefore, it is necessary to evaluate a larger number of patients from multiple centers with a longer follow - up for further clarification of the clinical implications. second, because we used the modified c acetate breath test, we measured excretion only up to 90 min. in patients with peak times of co2 excretion > 90 min, tmax may be considered as 90 min. in our study, two patients out of 30 had a tmax of 90 min before ablation. in both patients, if patients have a greater degree of gastric hypomotility than this, we may overestimate the impact of rf energy. one of them marked 60 min before ablation and the other 75 min. if they had more gastric hypomotility, we may underestimate the impact of rf energy. third, the precise number of patients who underwent transmural ablation in this study remains unknown. some papers have inferred that sas is related to reflux esophagitis. however, another paper has found that sas is not related to reflux esophagitis. in this paper, ju. reported that poor subjective sleep and depressive symptoms are associated with the presence of reflux esophagitis with no association between sas, bmi and reflux esophagitis.. however, the average impact of pvi on gastric motility under monitoring of the let was minimal. | backgroundpulmonary vein isolation (pvi) has become an important option for treating patients with atrial fibrillation (af). periesophageal nerve (pen) injury after pvi causes pyloric spasms and gastric hypomotility. this study aimed to clarify the impact of pvi on gastric motility and assess the prevalence of gastric hypomotility after pvi.methodsthirty consecutive patients with af underwent pvi under luminal esophageal temperature (let) monitoring. the 13c - acetate breath test was conducted before and after the procedure for all patients (pvi group). gastric emptying was evaluated using the time to peak concentration of 13co2 (tmax). this test was also conducted in another 20 patients who underwent catheter ablation procedures other than pvi (control group).resultsthe number of patients with abnormal tmax (75 min) increased from seven (23%) to 13 (43%) and from three (15%) to five (25%) after the procedure in the pvi group and control group, respectively. the mean tmax was longer after pvi than before pvi (6414 min vs. 5715 min, p=0.006), whereas there was no significant difference before and after the procedure in the control group. however, no significant difference in tmax was observed between the two groups (p=0.27). no patients suffered from symptomatic gastric hypomotility.conclusionsasymptomatic gastric hypomotility occurred more often after pvi. however, the average impact of pvi on gastric motility was minimal. |
various glomerular diseases can be caused by the deposition of monoclonal immunoglobulin g (igg). renal disease related to the deposition of monoclonal igg containing both heavy and light chains can occur in type 1 cryoglobulinemia, randall - type light- and heavy - chain deposition disease (lhcdd), light- and heavy - chain amyloidosis and immunotactoid glomerulonephritis [15 ]. proliferative glomerulonephritis with monoclonal igg deposits (pgnmid) is a relatively newly described entity mimicking immune complex glomerulonephritis (gn) that does not conform to any of those subtypes. it represents a form of proliferative gn with electron - dense deposits (edds) localized to glomeruli and immunofluorescence (if) findings of monoclonal igg deposits. recurrent pgnmid in the renal allograft has been described in a few case series [68 ]. in this article pathologic features of three cases of pgnmid in the renal allograft showing the variable course and manifestation of the disease. our three cases, like most other reported cases, have no bone marrow involvement and no evidence of monoclonal protein on electrophoresis and immunofixation studies. a 61-year - old caucasian female developed end - stage kidney disease (eskd) secondary to membranoproliferative glomerulonephritis (mpgn) based on a native kidney biopsy in 2001, with no further information about if or electron microscopy (em). in december 2006, her serum creatinine (scr) was 530 mol / l and she underwent a preemptive unrelated living kidney transplantation. in 2014, she was found to have a random urine protein / gram creatinine (upcr) of 452 mg / mmol and an scr of 132 mol / l, up from 106 mol / l. her workup for a lymphoproliferative disorder was negative, as was her serologic workup (see table 1). on biopsy, if showed diffuse granular positivity for igg, c3 and c1q. em showed edds, mostly in the mesangium and subendothelial regions (please see figures 17). she was later switched to carfilzomib, as she developed acute kidney injury with scr up to 247 mol / l ; after completion of therapy her scr was 150 table 1.clinical data of post kidney transplant recipients with pgnmidpatient # 1 # 2 # 3 # 4 # 5 # 6 age (years)617440616868race / sexcaucasian / femalecaucasian / malehispanic / femalecaucasian / malecaucasian / femalecaucasian / femalecause of eskdmpgnpgmidmpgnpgnmidunclear nephrotic range proteinuriapolycystic kidney disease (pkd)transplant typeunrelated living donordeceased donordeceased donordeceased donorrelated living donordeceased donorisfk506, prednisone, mmffk506, prednisone, mmffk506 and azathioprinemmf, rapamycinmmf, rapamycinprednisone, cyclosporinebaseline scr (mol / l)10626496.8114132132scr at biopsy (mol / l)132396114237281228time from transplant to diagnosis of recurrent or de novo pgnmid (months)9861321321158proteinuria (mg / mmol)4001002087050150hematuria1 + 3 + 2 + 2 + 2 + 1+serum complementnormalnormalnormalnormalnormalnormalcryoglobulinnegativenegativenegativen / an / an / ahepatitis bnegativenegativenegativenegativenegativenegativehepatitis cnegativenegativenegativenegativenegativenegativehivnon - reactivenon - reactivenon - reactivenon - reactivenon - reactivenon - reactivemonoclonal protein spikenegativenegativenegativenegativenegativenegativepattern of injuryec on both biopsies with a crescent on the second biopsynative biopsy : 1st atn2nd ectransplant biopsy : mpmpmpgn - likempmpgn - likenumber of glomeruli sclerosednone on first, 3 of 20 on second13/364 of 101 of 91 of 275 of 8interstitial fibrosisnone on first and 15% on second50%15%1 + 3 + 3+igg subtype kappa or lambdaigg3-kappanative : igg lambda (subtype not available)2nd transplant : igg3-lambdaigg3-kappaigg3-kappa, mild lambdaigg3-kappaigg1-kappaelectron microscopy depositsedm, eds on both biopsiesnative biopsy : eds, edmtransplant biopsy : edmedm, edsmpgn - likempmpgn - likebone marrow biopsypositive stain for plasma cells for cd138 5% cellularity ; no staining for kappa and lambda light chain by in situ hybridizationpositive stain for plasma cells for cd138 < 5% cellularity ; no staining for kappa and lambda light chain by in situ hybridization (pre - transplant)not done due to stability of kidney functionnonenonenonetreatmentbortezomib 3 doses, dexamethasone 3 dosespatient opted for noneno changeno changechange to mmf and prednisoneno changeoutcome after allograft biopsy : scr (mol / l)149, proteinuria 600 mg / mmolremains on is as aboveend - stage kidney disease114proteinuria 10 mg / mmolremains on is as abovefatal mi after 20 months of kidney biopsydied with cryptococcal meningitis 15 months after biopsyend - stage kidney diseasefollow - up (months) after biopsy17336201520patient # 7 # 8 # 9 # 10 # 11 # 12 age245560743866race / sexcaucasian / femalecaucasian / malecaucasian / femalecaucasian / femalecaucasian / femalecaucasian / femalecause of eskddiabetic nephropathypgnmidpgnmidpgnmidpgnmidpgnmidtransplant typesimultaneous kidney pancreasunrelated living donordeceased donorunrelated living donorunrelated living donordeceased donorisfk506, prednisone, mmffk506, prednisone, mmffk506, prednisone, mmffk506, prednisone, mmffk506, prednisone, mmffk506, prednisone, mmf stopped at 1 monthbaseline scr (mol / l)15816710612379.260.7scr at biopsy (mol / l)290246326422106176time from transplant to diagnosis of recurrent or de novo pgnmid (months)4312221564318proteinuria (mg / mmol)170807405806330hematuria3+yesyesyesno3+serum complementnormalnormallow c3 and c4low c3 and c4normaln / acryoglobulinn / anegativenegativenegativenot donenegativehepatitis bnegativenegativenegativenegativenegativenegativehepatitis cnegativenegativenegativenegativenegativenegativehivnon - reactivenon - reactivenon - reactivenon - reactivenon - reactiven / amonoclonal protein spikenegativenegativenegativenegativenegativepattern of injurympgn - likefirst two biopsies showed mp, third showed diffuse ec and fourth showed focal proliferativediffuse ec on both with diffuse on first and focal on second biopsyfirst biopsy showed diffuse ec and exudative gn, next two biopsies with mpminimal to mild mpec with crescentsnumber of glomeruli sclerosed8 of 133/37 sclerosed, 16/27 crescentsinterstitial fibrosis2+mildmildmild then moderatemild then moderateigg subtype kappa or lambdaigg3-kappaigg3-kappaigg3-kappaigg3-lambdaigg3-kappakappa (subtype not available)electron microscopy depositsmpgn - likefirst two biopsies showed edm, and edm and eds on last twoedm, edsfirst and third showed eds and second showed edm and edsedm in first two biopsies and edm and eds in others ; third and fifth biopsies showed banff 1aedm and parietal depositsbone marrow biopsynonenonenonenonenonenonetreatmentno changeprednisone, oral cyclophosphamide for 6 monthsmethylprednisolone with prednisone, rituximab 2 doses, lisinoprilmethylprednisolone with prednisone, rituximab 1 dose, with plasmapheresis 4 and hd 3methylprednisolone for acr, rituximab 2 dosesplasmapheresis, ivig 0.5 g / kg / day with methylprednisolone total 850 mg, mmf 1.5 g / dayoutcome after allograft biopsy : scr (mol / l)29020296.811420296.8, transplant nephrectomy in the setting of sepsis 6 months post - treatment with biopsy showing resolution of disease despite nephrectomy, disappearance of ec and cellular crescents, if staining remained kappafollow - up (months) after biopsyshort151183636mpgn, membranoproliferative glomerulonephritis ; pgnmid, proliferative glomerulonephritis and monoclonal immunoglobulin deposits ; mp, mesangial proliferative ; ep, endothelial proliferative ; ec, endocapillary proliferative ; scr, serum creatinine ; mmf, mycophenolate mofetil ; edm, electron - dense mesangial deposits ; eds, electron - dense subendothelial deposits ; fk506, prograf / tacrolimus. the increase in cells includes increased mononuclear elements within the expanded mesangial areas and many mononuclear inflammatory cells within the glomerular capillary lumens. as a result, the capillary tufts have a distinctly lobulated appearance. 3.the peripheral capillary walls are distorted and thickened, often with the formation of double contours or basement membranes with a chain - like appearance. 5.kappa light chains stain strongly positive (3+/4 +) along the peripheral capillary walls and mesangial areas. 6.if staining for the igg subtypes shows intense glomerular positivity for (c) igg3 with negative staining for (a), igg1 (b) igg2 and (d) igg3. 7.glomerular capillaries are greatly distorted and thickened by the presence of numerous, sometimes large and/or confluent subendothelial electron - dense deposits. the electron - dense deposits have a variegated (two - toned) appearance and are finely granular, but they do not show organized substructures clinical data of post kidney transplant recipients with pgnmid mpgn, membranoproliferative glomerulonephritis ; pgnmid, proliferative glomerulonephritis and monoclonal immunoglobulin deposits ; mp, mesangial proliferative ; ep, endothelial proliferative ; ec, endocapillary proliferative ; scr, serum creatinine ; mmf, mycophenolate mofetil ; edm, electron - dense mesangial deposits ; eds, electron - dense subendothelial deposits ; fk506, prograf / tacrolimus. the increase in cells includes increased mononuclear elements within the expanded mesangial areas and many mononuclear inflammatory cells within the glomerular capillary lumens. as a result, the peripheral capillary walls are distorted and thickened, often with the formation of double contours or basement membranes with a chain - like appearance. kappa light chains stain strongly positive (3+/4 +) along the peripheral capillary walls and mesangial areas. if staining for the igg subtypes shows intense glomerular positivity for (c) igg3 with negative staining for (a), igg1 (b) igg2 and (d) igg3. glomerular capillaries are greatly distorted and thickened by the presence of numerous, sometimes large and/or confluent subendothelial electron - dense deposits. the electron - dense deposits have a variegated (two - toned) appearance and are finely granular, but they do not show organized substructures. magnification 12 000, electron micrograph. a 74-year - old caucasian male developed eskd secondary to pgnmid seen on a native biopsy on september 2009. he received bortezomib and dexamethasone ; however, he developed eskd requiring hemodialysis (hd) after three doses. he received a diseased donor transplant (ddt) in may 2013, with his course complicated by biopsy - proven acute tubular injury. his scr improved to 265 mol / l until 6 months post - transplant, at which time his scr was 530 if from his biopsy showed positivity for igg3, c3, c1q and lambda light chains. kappa was negative, as were peritubular capillaries for c4d (see table 1). his kidney disease progressed and he declined therapy and restarted hd 9-month post - transplantation. a 40-year - old hispanic female received a ddt in june 2001 after developing eskd. in 1995 there were no if data for reevaluation. in june 2012, she had hematuria and her scr was 114 mol / l, up from her baseline scr of 97 her allograft biopsy showed a mesangioproliferative pattern of injury by light microscopy (see table 1). if revealed mesangial deposits restricted to igg3-kappa. a 61-year - old caucasian female developed end - stage kidney disease (eskd) secondary to membranoproliferative glomerulonephritis (mpgn) based on a native kidney biopsy in 2001, with no further information about if or electron microscopy (em). in december 2006, her serum creatinine (scr) was 530 mol / l and she underwent a preemptive unrelated living kidney transplantation. in 2014, she was found to have a random urine protein / gram creatinine (upcr) of 452 mg / mmol and an scr of 132 mol / l, up from 106 mol / l. her workup for a lymphoproliferative disorder was negative, as was her serologic workup (see table 1). em showed edds, mostly in the mesangium and subendothelial regions (please see figures 17). she was later switched to carfilzomib, as she developed acute kidney injury with scr up to 247 mol / l ; after completion of therapy her scr was 150 table 1.clinical data of post kidney transplant recipients with pgnmidpatient # 1 # 2 # 3 # 4 # 5 # 6 age (years)617440616868race / sexcaucasian / femalecaucasian / malehispanic / femalecaucasian / malecaucasian / femalecaucasian / femalecause of eskdmpgnpgmidmpgnpgnmidunclear nephrotic range proteinuriapolycystic kidney disease (pkd)transplant typeunrelated living donordeceased donordeceased donordeceased donorrelated living donordeceased donorisfk506, prednisone, mmffk506, prednisone, mmffk506 and azathioprinemmf, rapamycinmmf, rapamycinprednisone, cyclosporinebaseline scr (mol / l)10626496.8114132132scr at biopsy (mol / l)132396114237281228time from transplant to diagnosis of recurrent or de novo pgnmid (months)9861321321158proteinuria (mg / mmol)4001002087050150hematuria1 + 3 + 2 + 2 + 2 + 1+serum complementnormalnormalnormalnormalnormalnormalcryoglobulinnegativenegativenegativen / an / an / ahepatitis bnegativenegativenegativenegativenegativenegativehepatitis cnegativenegativenegativenegativenegativenegativehivnon - reactivenon - reactivenon - reactivenon - reactivenon - reactivenon - reactivemonoclonal protein spikenegativenegativenegativenegativenegativenegativepattern of injuryec on both biopsies with a crescent on the second biopsynative biopsy : 1st atn2nd ectransplant biopsy : mpmpmpgn - likempmpgn - likenumber of glomeruli sclerosednone on first, 3 of 20 on second13/364 of 101 of 91 of 275 of 8interstitial fibrosisnone on first and 15% on second50%15%1 + 3 + 3+igg subtype kappa or lambdaigg3-kappanative : igg lambda (subtype not available)2nd transplant : igg3-lambdaigg3-kappaigg3-kappa, mild lambdaigg3-kappaigg1-kappaelectron microscopy depositsedm, eds on both biopsiesnative biopsy : eds, edmtransplant biopsy : edmedm, edsmpgn - likempmpgn - likebone marrow biopsypositive stain for plasma cells for cd138 5% cellularity ; no staining for kappa and lambda light chain by in situ hybridizationpositive stain for plasma cells for cd138 < 5% cellularity ; no staining for kappa and lambda light chain by in situ hybridization (pre - transplant)not done due to stability of kidney functionnonenonenonetreatmentbortezomib 3 doses, dexamethasone 3 dosespatient opted for noneno changeno changechange to mmf and prednisoneno changeoutcome after allograft biopsy : scr (mol / l)149, proteinuria 600 mg / mmolremains on is as aboveend - stage kidney disease114proteinuria 10 mg / mmolremains on is as abovefatal mi after 20 months of kidney biopsydied with cryptococcal meningitis 15 months after biopsyend - stage kidney diseasefollow - up (months) after biopsy17336201520patient # 7 # 8 # 9 # 10 # 11 # 12 age245560743866race / sexcaucasian / femalecaucasian / malecaucasian / femalecaucasian / femalecaucasian / femalecaucasian / femalecause of eskddiabetic nephropathypgnmidpgnmidpgnmidpgnmidpgnmidtransplant typesimultaneous kidney pancreasunrelated living donordeceased donorunrelated living donorunrelated living donordeceased donorisfk506, prednisone, mmffk506, prednisone, mmffk506, prednisone, mmffk506, prednisone, mmffk506, prednisone, mmffk506, prednisone, mmf stopped at 1 monthbaseline scr (mol / l)15816710612379.260.7scr at biopsy (mol / l)290246326422106176time from transplant to diagnosis of recurrent or de novo pgnmid (months)4312221564318proteinuria (mg / mmol)170807405806330hematuria3+yesyesyesno3+serum complementnormalnormallow c3 and c4low c3 and c4normaln / acryoglobulinn / anegativenegativenegativenot donenegativehepatitis bnegativenegativenegativenegativenegativenegativehepatitis cnegativenegativenegativenegativenegativenegativehivnon - reactivenon - reactivenon - reactivenon - reactivenon - reactiven / amonoclonal protein spikenegativenegativenegativenegativenegativepattern of injurympgn - likefirst two biopsies showed mp, third showed diffuse ec and fourth showed focal proliferativediffuse ec on both with diffuse on first and focal on second biopsyfirst biopsy showed diffuse ec and exudative gn, next two biopsies with mpminimal to mild mpec with crescentsnumber of glomeruli sclerosed8 of 133/37 sclerosed, 16/27 crescentsinterstitial fibrosis2+mildmildmild then moderatemild then moderateigg subtype kappa or lambdaigg3-kappaigg3-kappaigg3-kappaigg3-lambdaigg3-kappakappa (subtype not available)electron microscopy depositsmpgn - likefirst two biopsies showed edm, and edm and eds on last twoedm, edsfirst and third showed eds and second showed edm and edsedm in first two biopsies and edm and eds in others ; third and fifth biopsies showed banff 1aedm and parietal depositsbone marrow biopsynonenonenonenonenonenonetreatmentno changeprednisone, oral cyclophosphamide for 6 monthsmethylprednisolone with prednisone, rituximab 2 doses, lisinoprilmethylprednisolone with prednisone, rituximab 1 dose, with plasmapheresis 4 and hd 3methylprednisolone for acr, rituximab 2 dosesplasmapheresis, ivig 0.5 g / kg / day with methylprednisolone total 850 mg, mmf 1.5 g / dayoutcome after allograft biopsy : scr (mol / l)29020296.811420296.8, transplant nephrectomy in the setting of sepsis 6 months post - treatment with biopsy showing resolution of disease despite nephrectomy, disappearance of ec and cellular crescents, if staining remained kappafollow - up (months) after biopsyshort151183636mpgn, membranoproliferative glomerulonephritis ; pgnmid, proliferative glomerulonephritis and monoclonal immunoglobulin deposits ; mp, mesangial proliferative ; ep, endothelial proliferative ; ec, endocapillary proliferative ; scr, serum creatinine ; mmf, mycophenolate mofetil ; edm, electron - dense mesangial deposits ; eds, electron - dense subendothelial deposits ; fk506, prograf / tacrolimus. the increase in cells includes increased mononuclear elements within the expanded mesangial areas and many mononuclear inflammatory cells within the glomerular capillary lumens. as a result, the capillary tufts have a distinctly lobulated appearance. 3.the peripheral capillary walls are distorted and thickened, often with the formation of double contours or basement membranes with a chain - like appearance. 5.kappa light chains stain strongly positive (3+/4 +) along the peripheral capillary walls and mesangial areas. 6.if staining for the igg subtypes shows intense glomerular positivity for (c) igg3 with negative staining for (a), igg1 (b) igg2 and (d) igg3. 7.glomerular capillaries are greatly distorted and thickened by the presence of numerous, sometimes large and/or confluent subendothelial electron - dense deposits. the electron - dense deposits have a variegated (two - toned) appearance and are finely granular, but they do not show organized substructures. magnification 12 000, electron micrograph. clinical data of post kidney transplant recipients with pgnmid mpgn, membranoproliferative glomerulonephritis ; pgnmid, proliferative glomerulonephritis and monoclonal immunoglobulin deposits ; mp, mesangial proliferative ; ep, endothelial proliferative ; ec, endocapillary proliferative ; scr, serum creatinine ; mmf, mycophenolate mofetil ; edm, electron - dense mesangial deposits ; eds, electron - dense subendothelial deposits ; fk506, prograf / tacrolimus. the increase in cells includes increased mononuclear elements within the expanded mesangial areas and many mononuclear inflammatory cells within the glomerular capillary lumens. as a result, the peripheral capillary walls are distorted and thickened, often with the formation of double contours or basement membranes with a chain - like appearance. kappa light chains stain strongly positive (3+/4 +) along the peripheral capillary walls and mesangial areas. if staining for the igg subtypes shows intense glomerular positivity for (c) igg3 with negative staining for (a), igg1 (b) igg2 and (d) igg3. glomerular capillaries are greatly distorted and thickened by the presence of numerous, sometimes large and/or confluent subendothelial electron - dense deposits. the electron - dense deposits have a variegated (two - toned) appearance and are finely granular, but they do not show organized substructures. magnification 12 000, electron micrograph. a 74-year - old caucasian male developed eskd secondary to pgnmid seen on a native biopsy on september 2009. he received bortezomib and dexamethasone ; however, he developed eskd requiring hemodialysis (hd) after three doses. he received a diseased donor transplant (ddt) in may 2013, with his course complicated by biopsy - proven acute tubular injury. mol / l until 6 months post - transplant, at which time his scr was 530 if from his biopsy showed positivity for igg3, c3, c1q and lambda light chains. kappa was negative, as were peritubular capillaries for c4d (see table 1). his kidney disease progressed and he declined therapy and restarted hd 9-month post - transplantation. a 40-year - old hispanic female received a ddt in june 2001 after developing eskd. in 1995, she had biopsy - proven type 1 mpgn. there were no if data for reevaluation. in june 2012, she had hematuria and her scr was 114 mol / l, up from her baseline scr of 97 her allograft biopsy showed a mesangioproliferative pattern of injury by light microscopy (see table 1). if revealed mesangial deposits restricted to igg3-kappa. pgnmid, a novel type that does not conform to any subtypes, was described initially in 2004 followed by the largest series of 37 cases in 2009. histologic patterns by light microscopy were mostly membranoproliferative with a lobular nodular pattern of injury followed by an endocapillary proliferative pattern. em revealed granular, nonorganized deposits mostly in the mesangium and subendothelium. if demonstrated granular glomerular deposits that stained for a single light - chain isotype and a single heavy - chain subtype, most commonly igg3-kappa [7, 10 ]. more than 40 additional cases were identified by other groups [6, 1119 ]. recently, recurrent pgnmid after kidney transplantation has been reported as case reports or small series of one to four patients [68 ]. the recurrent disease manifested clinically as allograft dysfunction, hematuria and proteinuria. of the patients reported (see table 1), the average age was 57 years. disease recurrence can range from an average of 3.8 months in some series up to 2 years after kidney transplantation [5, 7 ]. the risk of recurrence is difficult to estimate based on the small number of patients and the absence of protocol biopsies in most cases. moreover, the immunosuppressive treatment may have an impact on disease course that may be difficult to isolate. disease recurrence can have a variable outcome, with some patients having persistent renal impairment and some progressing to end - stage renal disease. pgnmid is often missed or underdiagnosed, with diagnoses of mpgn or even light- and heavy - chain deposition disease (lhcdd). some cases like case 1 and case 3 were originally diagnosed as mpgn type 1 before this entity was well established. in many instances, if and em were not available, nor was staining for light chains. pgnmid and lhcdd are similar in many aspects, however, the pathogenesis is different. lhcdd results from the deposition of heavy and light chains in the glomerular and tubular basement membrane, as opposed to pgnmid, where the deposits are intact monoclonal immunoglobulin affecting only the glomeruli. pgnmid is characterized by granular edds, in contrast to lhcdd, where those are mostly powdery deposits. igg3 was the igg isoform identified most often in prior studies to be deposited in the glomeruli despite comprising only 8% of the total immunoglobulin in the serum [6, 7 ]. limitation to igg3 in our three cases confirms the specific predilection for igg3 to precipitate and self - aggregate in the glomeruli given its properties of high molecular weight, positive charge and complement activation capacity. monoclonal gammopathy of renal significance is an increasingly recognized entity in patients who are found to have monoclonal protein with renal involvement. patients who do not fulfill the criteria for multiple myeloma have traditionally been labeled as monoclonal gammopathy of undetermined significance given the limited diagnostic schema, and consequently no therapy has been offered. however, it is more established now that addressing this clonal proliferation may preserve the kidneys and potentially prevent disease recurrence. nevertheless, a thorough workup is strongly recommended as it may impact the therapeutic plan. one patient with polycystic kidney disease was diagnosed with pgnmid 13 years after kidney transplantation. another patient had diabetes nephropathy leading to eskd and developed de novo pgnmid 3 years after transplantation. while having different primary pathology supports that allograft disease is de novo, late occurrence does not exclude recurrence, as kidney transplant recipients may have a different disease course and disease activity due to immunosuppressive drugs. based on a pathogenesis hypothesis, it would seem rational to treat with immunomodulating therapy. bortezomib was tried in one patient with native disease in one series, however, not in any transplant patients. given the role of monoclonal ig in the disease pathogenesis, bortezomib was tried in two of our patients, one with disease in the native kidney prior to transplant (case 2) and one with disease recurrence following transplant (case 1), with no favorable response. pgnmid is becoming a more recognized entity, with more patients being evaluated for renal transplant. evaluating these patients for transplant eligibility may be challenging in the absence of distinct data of remission given that most patients have normal bone marrow biopsy and no monoclonal band on serum and urine testing. more long - term studies will be needed to further investigate disease course and response to therapy. | glomerulopathy due to dysproteinemia can have a wide spectrum of pathologic and clinical features based on specific characteristics of the abnormal protein and the response induced within the parenchymal tissue. monoclonal immunoglobulin g (igg) deposition can manifest as a different glomerular disease. proliferative glomerulonephritis (gn) with monoclonal igg deposits (pgnmid) is a unique entity mimicking immune complex gn that does not conform to any of those subtypes. igg monoclonal granular deposition in the glomeruli with a pattern similar to immune complex disease suggested by c3 and c1q deposition should prompt consideration of pgnmid. literature is scarce in terms of recurrence of disease in renal allografts. in this article we present the clinical pathologic features of three cases of pgnmid in the renal allograft showing the variable course and manifestation of the disease. |
the relationship between inflammation and hypertension has been investigated for many years.1) experimental studies have shown that inflammatory cytokines contribute to hypertension by increasing vascular tone and impairing endothelial function. the relationships between hypertension and high - sensitivity crp (hs - crp), tumor necrosis factor-, interleukin (il)-6, and il-18 are well known,2) but the relationship between il-18 and left ventricular hypertrophy (lvh) is not well studied. inflammatory cells are activated during acute coronary syndromes, and some indexes such as the neutrophil to lymphocyte ratio3) and the white blood cell (wbc) count to mean platelet volume4) ratio are associated with worse prognosis. like inflammatory cells, il-18 was elevated in acute coronary syndromes, and high levels were correlated with worse prognosis.5) in addition, the role of il-18 in carotid intima media thickness (a marker of subclinical atherosclerosis) is well established.6) in experimental animal studies, there is growing evidence regarding the relationship aming il-18, hypertension, and lvh. il-18 knockout mice subjected to pressure overload developed less hypertrophy, but also had worse contractile function.7) daily administration of il-18 in healthy mice induces myocardial hypertrophy and contractile dysfunction.8) these results suggest that il-18 is involved in the hypertrophic response in overload cardiomyopathy. in clinical trials, hypertensive patients tend to have higher il-18 concentrations than normotensive groups,9)10) but the relationship between il-18 and lvh, which is a marker of end - organ damage, is unknown in humans. in this study, we aimed to investigate the relationship between il-18 and left ventricular mass (lvm) in apparently healthy subjects with or without hypertension who were free of atherosclerotic disease. we performed an observational, cross - sectional study in order to examine the relationships between inflammation, hypertension, and lvh. the study population was a sample of subjects admitted to the cardiology department of our institute and who were suspected to have hypertension (n=198). subjects were eligible to participate in the study if they were 20 to 60 years of age, had no history of hypertension or cardiovascular (cv) disease (coronary artery disease, stroke, peripheral vascular disease), were not using antihypertensive medications, were free of any other major systemic illnesses, and were not pregnant. we performed electrocardiogram and a treadmill test to rule out clinically significant coronary artery disease. a positive or non - diagnostic test result according to the modified bruce protocol we recruited 198 subjects (102 women, 96 men) who met these criteria. the local ethics committee assessed and approved the study, and written informed consent for participation in the study was obtained from all individuals whole blood count, total, high - density lipoprotein, and low - density cholesterol, triglycerides, glucose, and serum hs - crp and il-18 concentrations were measured using commercially available enzyme - linked immunosorbent assay (diagnostic products corporation, los angeles, ca, usa). electrocardiogram and two - dimensional echocardiography were performed in all of the subjects participating in the study. the sokolow - lyon index (sli) was calculated as the sum of the amplitudes of v1s and v5r. echocardiographic studies were performed using the acuson sequoia c256 (siemens medical solutions usa inc., mountain view, ca, usa) or the sonos 7500 (phillips, andover, ma, usa). all echocardiographic measurements and the estimation of lv mass were performed according to the recommendations of the american society of echocardiography and the european association of echocardiography.11) the 24 hour ambulatory blood pressure (bp) monitoring was performed with oscillometric equipment (tonoport ge, berlin, germany). the cuff was applied to the non - dominant arm at the end of the visit. the device was set to obtain ambulatory bp and heart rate readings at 30 minutes intervals for 24 hours, during which the subjects were sent home and asked to perform their usual activities. the subjects were asked to keep a diary of daily activities and the time of sleep and to return to the hospital 24 hours later. hypertension was determined based on 24 hour systolic and diastolic bp averages ; a 130/80 mmhg cut - off value was used. statistical calculations were performed using spss 15 for windows (spss inc, chicago, il, usa). continuous and normally distributed variables are presented as meanstandard deviation, and non - normally distributed variables are presented as median (min - max). continuous variables were compared among the groups of patients with student 's t - test for normally distributed data and the kruskal the pearson test for normally distributed data and the spearman test for non - normally distributed data were used for bivariate correlations. the study population was composed of 102 women and 96 men between the ages of 20 - 60 years. patients were divided into hypertensive (n=76) and normotensive (n=120) groups according to average 24 hour bp value. the 24 hour systolic and diastolic bp averages and day and night systolic and diastolic bp averages were significantly higher in the hypertensive group. the frequency of type 2 diabetes was very low in the study population (2.5%) and was not statistically different between groups. lvm, left ventricular mass index (lvmi), sli, wbc count, hs - crp, and il-18 were higher in the hypertensive group than in the normotensive group (table 1). day, night, and 24 hour systolic and diastolic blood pressure averages were all correlated with il-18 level in the total study population. we also found positive correlations between il-18 and body mass index (bmi) lvm, lvmi, triglyceride, and hs - crp and a negative correlation between il-18 and high - density lipoprotein (table 2). the correlation between il-18 and 24 hour systolic bp are shown in fig. 1, and the correlation between il-18 and lvmi is shown in fig. 2. in the hypertensive group, only night systolic bp average and lvm were correlated with il-18 level (table 3). in the normotensive group, no significant correlations were found between bp averages and il-18 ; however, bmi, lvm, and hs - crp level were all positively correlated with il-18 (table 4). in multivariate analysis, the correlations between il-18, lvmi, and hs - crp remained statistically significant in the entire study population. in contrast to these findings, we did not find any independent relationships in normotensive patients. on multivariate analysis, there were no statistically significant relationships between il-18 level and bp parameters (table 5). therefore, il-18 level was independently associated with lvmi and hs - crp level in the whole study population and in newly diagnosed hypertensive patients. bp averages were also positively correlated with serum il-18 level in the entire study population. il-18 level was independently associated with lvmi and hs - crp level in the whole study population and in newly diagnosed hypertensive patients. normotensive subjects do not have clinically significant cv disease or hypertension, both of which are known to alter lvmi and il-18 level. therefore, the absence of these diseases may explain the lack of any relationship between these parameters in this group. we hypothesize that the independent relationships seen in the general population were mainly due to the hypertensive subjects. to our knowledge, our study is the first to show an independent relationship between lvmi and il-18 level in humans. in most previous studies, hypertensive subjects had higher il-18 level than normotensive subjects, and il-18 level was positively correlated with office bp.9)10) these findings are consistent with our study findings. however, unlike out study, the prime study (n=1005) did not find a statistically significant difference in il-18 level between hypertensive and normotensive groups.12) in that study, all participants were males between 50 - 59 years of age, and interleukin level was measured in plasma rather than serum. the narrow range of age and gender might have obscured the relationship between il-18 and hypertension. we used ambulatory blood pressure readings, which are more accurate and have different diagnostic criteria than office measurements.13) several studies have shown that lvh and other markers of organ damage correlate with ambulatory bp more closely than with office bp in hypertensive patients.14) evidence from meta - analyses of published observational studies and pooled individual data has shown that ambulatory bp is a more sensitive risk predictor of clinical cv outcomes, such as coronary morbid or fatal events and stroke, than office bp.15) the superiority of ambulatory bp has been shown in the general population, in hypertensive patients, in patients at high risk, and in patients with cv disease.16)17) these factors make our findings more valuable. left ventricular hypertrophy, especially of the concentric type, is associated with a cv disease risk higher than 20% in 10 years.18) in hypertensive patients with lvh, a therapeutically - induced reduction of lvm was significantly associated with cv event reduction.19) antihypertensive treatment decreases the cv events associated with hypertension ; however, despite successful treatment, risk remains higher than in the general population.20) this finding suggests that hypertension - related cv risk is associated with factors other than high bp. one of these factors might be inflammation ; if so, hypertension treatments targeted to inflammation may result in additional risk reduction. increased vascular oxidative stress is one of the postulated unifying pathways that leads to hypertension. vasoactive hormones that play an important role in the pathogenesis of hypertension (e.g., angiotensin ii, endothelin i), growth factors (e.g., platelet - derived growth factor, transforming growth factor-), and mechanical stimuli (e.g., shear stress and stretch) activate nicotinamide adenine dinucleotide phospate and result in increased levels of reactive oxygen species.21) increased reactive oxygen species damage the vascular wall and impair vasodilation by inactivation of nitric oxide.22) angiotensin ii enhances the expression of il-18 receptor in vascular smooth muscle cells.23) angiotensin ii, along with aldosterone and endothelin-1, increases il-18 messenger ribonucleic acid (mrna) and protein expression in cardiomyocytes.24) the sympathetic nervous system plays an important role in the pathophysiology of hypertension, and evidence supports increased release of epinephrine from the hearts of patients with essential hypertension.25) stimulation of the 2-adrenergic receptor activates il-18 promoter activity, upregulates il-18 mrna, and increases il-18 mrna stability and il-18 protein expression in endothelial cells.26) two strategies have been used to counter the effects of il-18 : il-18 binding protein (il-18bp), a naturally occurring protein, and a neutralizing il-18 antibody. recombinant human il-18bp has been investigated in clinical trials and was found to be safe in healthy and obese volunteers27) and in patients with psoriasis and rheumatoid arthritis.28) evidence from clinical and experimental data suggests that il-18 may be a therapeutic target in acute myocardial infarction,29) but no trials have tested this hypothesis in acute myocardial infarction or hf. based on the scope of the accumulated data in the literature regarding il-18 blockage as a therapeutic intervention, we hypothesize that therapies directed against the inflammatory system may eventually be an option. furthermore, the cross - sectional and observational nature of our study does not allow us to determine cause and effect relationships. the elimination half - life of il-18 in vivo is approximately six hours.30) blood samples were drawn early in the morning, so our measurements did not represent the entire day and might have missed any diurnal variations. this makes it difficult to interpret the relationship between il-18 concentration and bp variation, particularly that occurring at times other than that at which the blood samples were drawn. for instance, variations occurring in the afternoon might have changed il-18 level significantly, but we likely did not detect this effect due to the relatively short elimination half - life. because of this study 's observational and cross sectional design, we can not make any suggestions about patient follow up or treatment strategies. further studies with more participants should be performed to clarify these matters. our study showed that elevated level of il-18 is associated with hypertension in newly diagnosed hypertensive patients and in the general population. we also found that lvmi was independently associated with il-18 in both hypertensive patients and in the general population. these findings comprise a rationale for further larger scale studies that could reveal a cause and effect relationship among il-18, hypertension, and related left ventricular hypertrophy. however, based on our results, it is too early to suggest any therapeutic interventions targeting il-18 for treatment of hypertension and lvh. stronger relaionships supported by future studies may lead to innovations in the treatment of hypertension and lvh in the coming decades. our study showed that elevated level of il-18 is associated with hypertension in newly diagnosed hypertensive patients and in the general population. we also found that lvmi was independently associated with il-18 in both hypertensive patients and in the general population. these findings comprise a rationale for further larger scale studies that could reveal a cause and effect relationship among il-18, hypertension, and related left ventricular hypertrophy. however, based on our results, it is too early to suggest any therapeutic interventions targeting il-18 for treatment of hypertension and lvh. stronger relaionships supported by future studies may lead to innovations in the treatment of hypertension and lvh in the coming decades. | background and objectivesin clinical trials, hypertensive patients tend to have higher interleukin-18 (il-18) concentrations than normotensive groups, but the relationship between il-18 and left ventricular hypertrophy (lvh), which is a marker of end - organ damage, is not well studied. we aimed to investigate the relationship between il-18 and lvh in apparently healthy subjects free of clinically significant atherosclerotic disease.subjects and methodswe enrolled 198 subjects (102 women and 96 men) between may 2006 and march 2007, who were free of cardiovascular or immune diseases, but were suspected to have hypertension. twenty - four - hour ambulatory blood pressure monitoring and two - dimensional echocardiography were performed. lipid profiles, high - sensitivity crp (hs - crp), il-18, and whole blood cell counts were measured for all subjects.resultswhite blood cell count, hs - crp, left ventricular mass, left ventricular mass index (lvmi), and il-18 were higher in the hypertensive group than in the normotensive group (p=0.045, p=0.004, p<0.0001, p=0.001, and p=0.017 respectively). twenty - four hour day and night systolic and diastolic blood pressure averages were positively correlated with il-18 level in the entire study population. in multivariate regression analysis, left ventricular mass index and hs - crp level were independently associated with il-18 level in both the hypertensive group and the entire study population (=0.154, =0.149 p=0.033, p=0.040 and =0.151, =0.155 p=0.036, p=0.032 respectively)conclusionwe found that il-18 level independently predicted lvmi in both the general population and in newly diagnosed hypertensive patients. |
although an increasing number of studies indicate that primary brain tumors and their treatment are often associated with cognitive deficits, there is still limited knowledge about their incidence, nature, severity, and causes. since patients with diffuse infiltrative gliomas (who grade 24) can not be cured, palliation of symptoms and maintenance or improvement of physical functioning and health - related quality of life (hrqol) are important goals of treatment. evaluation of treatment in these patients should thus not only focus on (progression - free) survival, but should also aim at functional outcome and at adverse treatment effects on the normal brain. functional outcome refers to neurological, cognitive, professional, and social performance of an individual, usually abstracted as hrqol. with regard to the effects of tumor and treatment on the normal brain, cognitive functioning is a useful outcome measure for brain tumor patients, since cognitive deficits, even mild, may negatively affect hrqol, professional reintegration, interpersonal relationships, and leisure activities. many potential factors contribute to cognitive functioning. in attempting to determine the isolated effect of resective surgery on cognition, these factors include premorbid level of cognitive functioning, distant mechanical effects on the normal brain by the lesion, epilepsy, medication, and other oncological treatments. cognitive functioning and hrqol assessment are used as secondary outcome measures in several clinical trials and can also serve as an early indicator of disease progression and have prognostic significance [2, 3 ], thereby providing additional arguments in clinical decision making. to illustrate the clinical decision making process, a typical patient is described. this case description allows us to describe the patterns of cognitive functioning related to tumor and/or treatment within an ecological context, stressing the importance of conceptualizing patients cognitive deficits, their families, and their caregivers as an integrated system with all the humanistic and ethical aspects that entails, rather than as cognitive deficits in isolation. a 36-year old female presented with several elementary seizures in the 3 months preceding presentation, characterized by foul smell followed by inattention and dysphasia. otherwise she suffered from fatigue for 10 years which was diagnosed as chronic fatigue syndrome. she was married, had one child and worked as an administrative employee in an international organization. the first mri showed a t2/flair hyperintense lesion of 50 ml anterior in the left insula with no enhancement after gadolinium suggesting a low - grade glioma (fig. information was provided on resective surgery with its presumed beneficial impact on time to progression and survival, the acceptable low risk of permanent neurological deficits when using brain mapping under local anesthesia, and the unknown risk of cognitive decline, as well as alternative treatment options consisting of a biopsy and radiotherapy or chemotherapy and radiological follow - up with delayed treatment. she was highly motivated to undergo resective surgery with language mapping. a baseline assessment for language and neuropsychological examination was obtained showing a score on the boston naming test in the lower normal range.fig. 1rapid transformation from a who grade ii oligodendroglioma at the time of surgery into an anaplastic oligodendroglioma at 22 months a 36-year old female presented with several elementary seizures in the 3 months preceding presentation, characterized by foul smell followed by inattention and dysphasia. otherwise she suffered from fatigue for 10 years which was diagnosed as chronic fatigue syndrome. she was married, had one child and worked as an administrative employee in an international organization. the first mri showed a t2/flair hyperintense lesion of 50 ml anterior in the left insula with no enhancement after gadolinium suggesting a low - grade glioma (fig. information was provided on resective surgery with its presumed beneficial impact on time to progression and survival, the acceptable low risk of permanent neurological deficits when using brain mapping under local anesthesia, and the unknown risk of cognitive decline, as well as alternative treatment options consisting of a biopsy and radiotherapy or chemotherapy and radiological follow - up with delayed treatment. she was highly motivated to undergo resective surgery with language mapping. a baseline assessment for language and neuropsychological examination was obtained showing a score on the boston naming test in the lower normal range. rapid transformation from a who grade ii oligodendroglioma at the time of surgery into an anaplastic oligodendroglioma at 22 months apart from seizures, as presented by the patient in our case description, brain tumor patients may present with headaches, focal neurologic signs, and cognitive impairment. cognitive deficits associated with brain tumors can be induced by compression of normal brain either directly or indirectly by reactive edema. reduction of compression has not only been shown to improve cognitive function after removal of non - invasive lesions such as meningiomas, associated with improvement in attentional functioning, or arachnoidal cysts, associated with better dichotic perception and overall memory performance in patients but even after cranioplasty, where post surgery patients had improved language and reasoning abilities. apart from compression, the invasion of parenchymal glial tumors directly into functional brain regions or indirectly by disconnection of structures can further contribute to cognitive deficits [79 ]. after radiological diagnosis, resective surgery is usually the first of several treatment modalities for patients with brain tumors. resective surgery aims to balance functional outcome (e.g., minimization of neurological deficits) with oncological outcome (e.g., maximization of tumor removal) to improve survival. a vast body of literature exists on the impact of surgery for a variety of brain lesions on neurological outcome, such as motor strength and language. however, due to the limited number of studies including pre- and postoperative cognitive evaluations, the true incidence and extent of cognitive dysfunction specifically related to resective brain tumor surgery is unknown. the patient had a resection in which the brain structures involved in language determined the boundaries of resection (fig. 1b, c). a subtotal resection of 92% (4 ml residual tumor) was obtained (fig. after surgery in the first week a mild dysnomia completely resolved, and her fatigue was increased for 2 months. no adjuvant treatment was advised and she had radiological follow - up every 6 months. the patient had a resection in which the brain structures involved in language determined the boundaries of resection (fig. 1b, c). a subtotal resection of 92% (4 ml residual tumor) was obtained (fig. after surgery in the first week a mild dysnomia completely resolved, and her fatigue was increased for 2 months. no adjuvant treatment was advised and she had radiological follow - up every 6 months. cognitive outcome after resective surgery for temporal lobe epilepsy and for brain tumors will be discussed, respectively. studies in patients with non tumor - related intractable epilepsy showed cognitive improvement (e.g., memory or verbal fluency) with adequate seizure control after temporal resections [1016 ]. less extensive resection of the mesiotemporal structures seems to correlate with better memory outcome compared with more extensive temporal lobectomy according to some groups [1719 ], whereas others have reported conflicting observations [2022 ]. furthermore, dominant temporal lobe resections have been correlated with verbal memory decline in a subset of patients [2329 ], whereas non - dominant temporal lobe resections were correlated with visuospatial memory decline [2935 ]. studies of extra - temporal resective surgery for intractable epilepsy also yielded variable cognitive outcomes. after unilateral removal of frontal cortex cognition was either unchanged [36, 37 ], or specific cognitive domains were impaired, such as reaction time [38, 39 ], impulsivity, advance information utilization, conditional learning, or search and retrieval strategies. furthermore, identification of faces and categorization of emotional facial expression was impaired after either frontal or temporal cortex resection. olfactory identification was impaired following unilateral excision of the temporal lobe or the orbitofrontal cortex on either side. cognitive outcome has not been systematically assessed for resective brain surgery in patients with brain tumors, although several interesting observations have been done in smaller observational cohort studies. long - term improvement of verbal memory compared to preoperative assessment has been reported after low - grade glioma resections in frontal premotor and anterior temporal areas [4648 ], usually after a transient immediate postoperative worsening. additionally, regardless the precise tumor location, patients with low - grade gliomas in the right hemisphere run a lower risk of developing cognitive deficits after surgery. cognitive improvement has also been observed after surgical resection of high - grade gliomas, specifically in word fluency, verbal memory, and visuospatial memory. however, one study also suggests that tumor histology might not be that important in the prediction of cognitive outcome following surgery. for instance, patients with tumors of the third ventricle demonstrated cognitive impairment in memory, executive functioning, and fine manual speed prior to surgery, without worsening of cognition after surgical removal [51, 52 ]. out of several executive tasks, only letter fluency performance was impaired in patients after glioma surgery in left frontal locations compared with right frontal and posterior lesions. visuospatial processing in patients after resective glioma surgery in left and right, frontal and parietal locations was comparable to that of normal subjects according to one study and impaired spatial and positional memory processing was demonstrated in patients with tumors in the right posterior parietal cortex or in the frontal cortex according to others [55, 56 ]. thirdly, a number of studies have demonstrated cognitive deficits in specific domains after brain tumor removal. for instance, some patients demonstrated minor deterioration in attention after resection of parenchymal frontal or precentral tumors [47, 57 ] and resection of the right prefrontal cortex rather than the left was associated with a selective attentional impairment in stroop test performance. after resection of the supplementary motor area, patients exhibited impaired procedural learning and agraphia [59, 60 ]. for instance, impaired sequence ordering of novel material was observed particularly in right - sided lesions, while recognition memory was unaffected, and planning and executive impairment, irrespective of side, site, and size [62, 63 ]. furthermore, severe executive deficits in a reward learning task were observed in patients after bilateral fronto - orbital resections for various tumor types and impaired virtual planning of real life activities after resections in the left and right prefrontal cortex, which could not be explained by memory deficits [65, 66 ]. in the year following surgery she noted more difficulty concentrating, and she was seizure free for 10 months, after which similar seizures reappeared, for which the carbamazepine was increased to 300 mg bid. after a year she divorced from her husband and started working as volunteer in a nursing home. follow - up mris demonstrated slow increase of residual t2/flair hyperintensity of approximately 4 mm per year. at 21 months her clinical condition is unchanged, but now there is a very extensive t2/flair hyperintense infiltration and new multifocal gadolinium enhancement at the genu of the corpus callosum and left prefrontal area (fig. several options were considered : (1) radiotherapy and possibly adjuvant temozolomide at further progression, (2) new histopathology by either biopsy or limited resection, in case who grade 3 would be confirmed participation in a trial, in case who grade 4 would be confirmed chemo - irradiation. because new histopathology would have implications for the radiotherapy plan, participation in a trial or concurrent temozolomide, a new histopathological diagnosis was advised by open biopsy, which would be methionine pet - guided towards the most anaplastic focus which was located prefrontally. the pathologist confirmed anaplastic oligoastrocytoma without necrosis, without 1p/19q loss, and with an extraordinarily high mib1 labeling index. the rapid radiological progression in combination with the high labeling index were the arguments to start radiotherapy plus concomitant and adjuvant temozolomide at this point. again she developed more intense fatigue, lost 10 kg of weight, and developed a grade 4 thrombopenia and fever for which she was hospitalized for 2 weeks. mri at 25 months, after the third cycle of adjuvant temozolomide, demonstrated a partial radiological response. despite a combination of nausea, extreme fatigue, and anxiety for the future she was unable to work and unable to care for her two children most of the time. in the year following surgery she noted more difficulty concentrating, and she was seizure free for 10 months, after which similar seizures reappeared, for which the carbamazepine was increased to 300 mg bid. after a year she divorced from her husband and started working as volunteer in a nursing home. follow - up mris demonstrated slow increase of residual t2/flair hyperintensity of approximately 4 mm per year. at 21 months her clinical condition is unchanged, but now there is a very extensive t2/flair hyperintense infiltration and new multifocal gadolinium enhancement at the genu of the corpus callosum and left prefrontal area (fig. several options were considered : (1) radiotherapy and possibly adjuvant temozolomide at further progression, (2) new histopathology by either biopsy or limited resection, in case who grade 3 would be confirmed participation in a trial, in case who grade 4 would be confirmed chemo - irradiation. because new histopathology would have implications for the radiotherapy plan, participation in a trial or concurrent temozolomide, a new histopathological diagnosis was advised by open biopsy, which would be methionine pet - guided towards the most anaplastic focus which was located prefrontally. the pathologist confirmed anaplastic oligoastrocytoma without necrosis, without 1p/19q loss, and with an extraordinarily high mib1 labeling index. the rapid radiological progression in combination with the high labeling index were the arguments to start radiotherapy plus concomitant and adjuvant temozolomide at this point. again she developed more intense fatigue, lost 10 kg of weight, and developed a grade 4 thrombopenia and fever for which she was hospitalized for 2 weeks. mri at 25 months, after the third cycle of adjuvant temozolomide, demonstrated a partial radiological response. despite a combination of nausea, extreme fatigue, and anxiety for the future she was unable to work and unable to care for her two children most of the time. cognitive deficits are the hallmark of late - delayed encephalopathy, which is an irreversible and progressive complication that may follow radiotherapy by several months to many years through vascular injury causing ischemia of surrounding tissue and demyelination, local radionecrosis, and cerebral atrophy. the severity of cognitive deficits ranges from mild or moderate to dementia with progressive mental slowing and deficits in attention and memory, occurring in at least 12% of patients treated with radiotherapy. in these cases, mri shows diffuse atrophy with ventricular enlargement as well as severe confluent white - matter abnormalities. there is a relation between cognitive status and cerebral atrophy and leukoencephalopathy [70, 71 ]. while short - term follow - up studies show limited or transient effects of radiotherapy, a number of studies in long - term survivors of low - grade glioma (i.e., more than 5 years following radiotherapy) concluded that radiotherapy in these patients poses a significant risk of long - term leukoencephalopathy and cognitive impairment. surma - aho. reported low - grade glioma patients with a follow - up of 7 years to have more memory deficits after early radiotherapy than controls without radiotherapy. moreover, leukoencephalopathy on mri was more severe in the group with postoperative irradiation. a study among low - grade glioma survivors 6 years following diagnosis and initial treatment showed that the use of radiotherapy was associated with poor cognitive function on only a few tests and not restricted to one specific cognitive domain. this finding suggests that cognitive deficits in these patients should not be attributed to radiotherapy, but rather to the tumor itself or other treatment factors, including epilepsy. serious memory deficits, however, are still to be expected when fraction doses exceed 2 gy. study demonstrated that regardless of fraction dose all tumor progression - free low - grade glioma patients that had irradiation showed a progressive deterioration in attentional functioning 13 years after radiotherapy while all patients without irradiation remained stable. then she developed depressive feelings and lack of initiative for which she visited a psychologist. then she developed depressive feelings and lack of initiative for which she visited a psychologist. because this was likely related to the levetiracetam, carbamazepine was reintroduced. the mechanism and pattern of seizures in brain tumor patients is determined by tumor type, tumor location and peritumoral and genetic changes in brain tumor patients. an increased epilepsy burden has been found to adversely affect a broad range of cognitive functions even to a larger extent than radiation therapy. decreased processing speed and attentional and executive deficits are notable sequelae of seizures and antiepileptic drugs (aeds) in patients with brain tumors [73, 74, 77 ]. others did not detect any apparent effect of seizures on cognition across multiple cognitive domains assessed in a postsurgical sample. cognitive side - effects of aeds can add to cognitive decline due to tumor effects, previous surgery, or radiotherapy, and therefore appropriate choice and dose of aed is crucial. the classical aeds (phenytoin, carbamazepine, and valproic acid) are known to decrease cognitive functioning [78, 79 ]. importantly, these drugs may also have pharmacological interactions with chemotherapy [80, 81 ] and thus potentially affect survival. these drugs may result in impaired attention and cognitive slowing, which can subsequently have effects on memory by reducing the efficiency of encoding and retrieval. the importance of the classical aeds as a risk factor for cognitive deficits has been reported in a study on stable disease, long - term low - grade glioma survivors where reduced information processing speed, psychomotor function, working memory capacity, and executive functioning, were significantly related to the use of aeds. as patients in this study who took aeds had cognitive impairment even in the absence of seizures, the use of aeds primarily affects cognitive function. moreover, aed use in low - grade glioma patients may be associated with highly elevated levels of fatigue, which in itself is also associated with poorer cognitive outcome. several new generation aeds, like oxcarbazepine and levetiracetam as add - on therapy, appear to have fewer adverse cognitive effects than the classical agents. of the newer agents, topiramate is associated with the greatest risk of cognitive impairment, although this risk is decreased with slow titration and low target doses [85, 86 ]. it appears to be safe to switch patients from phenytoin to levetiracetam monotherapy following craniotomy for supratentorial glioma. follow - up mris of the patient every 3 months showed a stable response with no gadolinium enhancement and a t2/flair hyperintense region that is progressive at 6 mm / year up to 44 months after initial surgery and 20 months after chemoirradiation. during this time she is still unable to work, and partly able to take care of her children due to concentration problems, fatigue, and depression. follow - up mris of the patient every 3 months showed a stable response with no gadolinium enhancement and a t2/flair hyperintense region that is progressive at 6 mm / year up to 44 months after initial surgery and 20 months after chemoirradiation. during this time she is still unable to work, and partly able to take care of her children due to concentration problems, fatigue, and depression. a considerable number of brain tumor patients have feelings of anxiety, depression, and future uncertainty as psychological reactions to the disease [8890 ]. these mood disturbances may lead to deficits in attention, vigilance, and motivation that subsequently affect several cognitive domains. loss of self confidence, unemployment, and dependency on caregivers may also negatively affect these patients cognitive status. mood changes are more common in brain tumor patients than in patients with other neurological diseases and might be related to tumor location. unilateral surgical removal of prefrontal cortex, including the fronto - orbital or anterior cingulate cortex, has resulted in emotional dysregulation with impaired voice and face expression identification in patients with various brain lesions including brain tumors. furthermore, deficits in recognizing emotional facial expression were observed after surgical removal of brain tumors that involved both heteromodal and limbic / paralimbic cortices. concordantly, impairment of arousal and emotional valence was demonstrated after resective surgery in various brain regions, but particularly in the right temporoparietal region. negative mood changes were observed after brain tumor resection involving heteromodal cortices located either prefrontal or temporoparietal, whereas positive mood changes were observed after lateral frontal resections. mood states did not correlate with laterality of the resection, tumor grading or lesion size. at 44 months after initial surgery and 21 months after chemoirradiation, she experienced headache, a severe decline in cognitive functions, lethargia, and gait instability without seizures. at neurological examination she demonstrated bradyphrenia, disorientation, and a right - sided hemiparesis. she was admitted and a new mri showed clear and sudden progression with extensive t2/flair infiltration, and new gadolinium enhancement at the corpus of the corpus callosum and in the left internal capsula (fig. 1). because of her poor clinical condition and the extent of radiological progression, no further treatment options were considered and she received dexamethasone. she was discharged to a hospice and died at 46 months after initial surgical treatment. at 44 months after initial surgery and 21 months after chemoirradiation, she experienced headache, a severe decline in cognitive functions, lethargia, and gait instability without seizures. at neurological examination she demonstrated bradyphrenia, disorientation, and a right - sided hemiparesis. she was admitted and a new mri showed clear and sudden progression with extensive t2/flair infiltration, and new gadolinium enhancement at the corpus of the corpus callosum and in the left internal capsula (fig. 1). because of her poor clinical condition and the extent of radiological progression, no further treatment options were considered and she received dexamethasone. she was discharged to a hospice and died at 46 months after initial surgical treatment. the potentially neurotoxic effects of corticosteroids are often misdiagnosed and underestimated and corticosteroids may induce behavioral, psychic, and cognitive disturbances, due to functional and, over time, structural alterations in specific brain target areas. corticosteroids may cause mood disturbances, psychosis, and cognitive deficits particularly in declarative memory performance. steroid dementia is a reversible cause of cognitive deficits even in the absence of psychosis. recent data suggest that transiently impaired attention, concentration, and memory are due to neurotoxic effects on both the hippocampal and the prefrontal areas. both short - term and long - term use of steroids has been associated with hippocampal - dependent explicit memory deficits. more likely, however, corticosteroids may improve cognitive deficits because of resolution of edema. apart from seizures, as presented by the patient in our case description, brain tumor patients may present with headaches, focal neurologic signs, and cognitive impairment. cognitive deficits associated with brain tumors can be induced by compression of normal brain either directly or indirectly by reactive edema. reduction of compression has not only been shown to improve cognitive function after removal of non - invasive lesions such as meningiomas, associated with improvement in attentional functioning, or arachnoidal cysts, associated with better dichotic perception and overall memory performance in patients but even after cranioplasty, where post surgery patients had improved language and reasoning abilities. apart from compression, the invasion of parenchymal glial tumors directly into functional brain regions or indirectly by disconnection of structures can further contribute to cognitive deficits [79 ]. after radiological diagnosis, resective surgery is usually the first of several treatment modalities for patients with brain tumors. resective surgery aims to balance functional outcome (e.g., minimization of neurological deficits) with oncological outcome (e.g., maximization of tumor removal) to improve survival. a vast body of literature exists on the impact of surgery for a variety of brain lesions on neurological outcome, such as motor strength and language. however, due to the limited number of studies including pre- and postoperative cognitive evaluations, the true incidence and extent of cognitive dysfunction specifically related to resective brain tumor surgery is unknown. the patient had a resection in which the brain structures involved in language determined the boundaries of resection (fig. 1b, c). a subtotal resection of 92% (4 ml residual tumor) was obtained (fig. after surgery in the first week a mild dysnomia completely resolved, and her fatigue was increased for 2 months. no adjuvant treatment was advised and she had radiological follow - up every 6 months. the patient had a resection in which the brain structures involved in language determined the boundaries of resection (fig. 1b, c). a subtotal resection of 92% (4 ml residual tumor) was obtained (fig. after surgery in the first week a mild dysnomia completely resolved, and her fatigue was increased for 2 months. no adjuvant treatment was advised and she had radiological follow - up every 6 months. cognitive outcome after resective surgery for temporal lobe epilepsy and for brain tumors will be discussed, respectively. studies in patients with non tumor - related intractable epilepsy showed cognitive improvement (e.g., memory or verbal fluency) with adequate seizure control after temporal resections [1016 ]. less extensive resection of the mesiotemporal structures seems to correlate with better memory outcome compared with more extensive temporal lobectomy according to some groups [1719 ], whereas others have reported conflicting observations [2022 ]. furthermore, dominant temporal lobe resections have been correlated with verbal memory decline in a subset of patients [2329 ], whereas non - dominant temporal lobe resections were correlated with visuospatial memory decline [2935 ]. studies of extra - temporal resective surgery for intractable epilepsy also yielded variable cognitive outcomes. after unilateral removal of frontal cortex cognition was either unchanged [36, 37 ], or specific cognitive domains were impaired, such as reaction time [38, 39 ], impulsivity, advance information utilization, conditional learning, or search and retrieval strategies. furthermore, identification of faces and categorization of emotional facial expression was impaired after either frontal or temporal cortex resection. olfactory identification was impaired following unilateral excision of the temporal lobe or the orbitofrontal cortex on either side. cognitive outcome has not been systematically assessed for resective brain surgery in patients with brain tumors, although several interesting observations have been done in smaller observational cohort studies. long - term improvement of verbal memory compared to preoperative assessment has been reported after low - grade glioma resections in frontal premotor and anterior temporal areas [4648 ], usually after a transient immediate postoperative worsening. additionally, regardless the precise tumor location, patients with low - grade gliomas in the right hemisphere run a lower risk of developing cognitive deficits after surgery. cognitive improvement has also been observed after surgical resection of high - grade gliomas, specifically in word fluency, verbal memory, and visuospatial memory. however, one study also suggests that tumor histology might not be that important in the prediction of cognitive outcome following surgery. for instance, patients with tumors of the third ventricle demonstrated cognitive impairment in memory, executive functioning, and fine manual speed prior to surgery, without worsening of cognition after surgical removal [51, 52 ]. out of several executive tasks, only letter fluency performance was impaired in patients after glioma surgery in left frontal locations compared with right frontal and posterior lesions. visuospatial processing in patients after resective glioma surgery in left and right, frontal and parietal locations was comparable to that of normal subjects according to one study and impaired spatial and positional memory processing was demonstrated in patients with tumors in the right posterior parietal cortex or in the frontal cortex according to others [55, 56 ]. thirdly, a number of studies have demonstrated cognitive deficits in specific domains after brain tumor removal. for instance, some patients demonstrated minor deterioration in attention after resection of parenchymal frontal or precentral tumors [47, 57 ] and resection of the right prefrontal cortex rather than the left was associated with a selective attentional impairment in stroop test performance. after resection of the supplementary motor area, patients exhibited impaired procedural learning and agraphia [59, 60 ]. subsets of patients with resections involving the frontal lobe demonstrated a variety of deficits. for instance, impaired sequence ordering of novel material was observed particularly in right - sided lesions, while recognition memory was unaffected, and planning and executive impairment, irrespective of side, site, and size [62, 63 ]. furthermore, severe executive deficits in a reward learning task were observed in patients after bilateral fronto - orbital resections for various tumor types and impaired virtual planning of real life activities after resections in the left and right prefrontal cortex, which could not be explained by memory deficits [65, 66 ]. in the year following surgery she noted more difficulty concentrating, and she was seizure free for 10 months, after which similar seizures reappeared, for which the carbamazepine was increased to 300 mg bid. after a year she divorced from her husband and started working as volunteer in a nursing home. follow - up mris demonstrated slow increase of residual t2/flair hyperintensity of approximately 4 mm per year. at 21 months her clinical condition is unchanged, but now there is a very extensive t2/flair hyperintense infiltration and new multifocal gadolinium enhancement at the genu of the corpus callosum and left prefrontal area (fig. several options were considered : (1) radiotherapy and possibly adjuvant temozolomide at further progression, (2) new histopathology by either biopsy or limited resection, in case who grade 3 would be confirmed participation in a trial, in case who grade 4 would be confirmed chemo - irradiation. because new histopathology would have implications for the radiotherapy plan, participation in a trial or concurrent temozolomide, a new histopathological diagnosis was advised by open biopsy, which would be methionine pet - guided towards the most anaplastic focus which was located prefrontally. the pathologist confirmed anaplastic oligoastrocytoma without necrosis, without 1p/19q loss, and with an extraordinarily high mib1 labeling index. the rapid radiological progression in combination with the high labeling index were the arguments to start radiotherapy plus concomitant and adjuvant temozolomide at this point. again she developed more intense fatigue, lost 10 kg of weight, and developed a grade 4 thrombopenia and fever for which she was hospitalized for 2 weeks. mri at 25 months, after the third cycle of adjuvant temozolomide, demonstrated a partial radiological response. despite a combination of nausea, extreme fatigue, and anxiety for the future she was unable to work and unable to care for her two children most of the time. in the year following surgery she noted more difficulty concentrating, and she was seizure free for 10 months, after which similar seizures reappeared, for which the carbamazepine was increased to 300 mg bid. after a year she divorced from her husband and started working as volunteer in a nursing home. follow - up mris demonstrated slow increase of residual t2/flair hyperintensity of approximately 4 mm per year. at 21 months her clinical condition is unchanged, but now there is a very extensive t2/flair hyperintense infiltration and new multifocal gadolinium enhancement at the genu of the corpus callosum and left prefrontal area (fig. several options were considered : (1) radiotherapy and possibly adjuvant temozolomide at further progression, (2) new histopathology by either biopsy or limited resection, in case who grade 3 would be confirmed participation in a trial, in case who grade 4 would be confirmed chemo - irradiation. because new histopathology would have implications for the radiotherapy plan, participation in a trial or concurrent temozolomide, a new histopathological diagnosis was advised by open biopsy, which would be methionine pet - guided towards the most anaplastic focus which was located prefrontally. the pathologist confirmed anaplastic oligoastrocytoma without necrosis, without 1p/19q loss, and with an extraordinarily high mib1 labeling index. the rapid radiological progression in combination with the high labeling index were the arguments to start radiotherapy plus concomitant and adjuvant temozolomide at this point. again she developed more intense fatigue, lost 10 kg of weight, and developed a grade 4 thrombopenia and fever for which she was hospitalized for 2 weeks. mri at 25 months, after the third cycle of adjuvant temozolomide, demonstrated a partial radiological response. despite a combination of nausea, extreme fatigue, and anxiety for the future she was unable to work and unable to care for her two children most of the time. cognitive deficits are the hallmark of late - delayed encephalopathy, which is an irreversible and progressive complication that may follow radiotherapy by several months to many years through vascular injury causing ischemia of surrounding tissue and demyelination, local radionecrosis, and cerebral atrophy. the severity of cognitive deficits ranges from mild or moderate to dementia with progressive mental slowing and deficits in attention and memory, occurring in at least 12% of patients treated with radiotherapy. in these cases, mri shows diffuse atrophy with ventricular enlargement as well as severe confluent white - matter abnormalities. there is a relation between cognitive status and cerebral atrophy and leukoencephalopathy [70, 71 ]. while short - term follow - up studies show limited or transient effects of radiotherapy, a number of studies in long - term survivors of low - grade glioma (i.e., more than 5 years following radiotherapy) concluded that radiotherapy in these patients poses a significant risk of long - term leukoencephalopathy and cognitive impairment. surma - aho. reported low - grade glioma patients with a follow - up of 7 years to have more memory deficits after early radiotherapy than controls without radiotherapy. a study among low - grade glioma survivors 6 years following diagnosis and initial treatment showed that the use of radiotherapy was associated with poor cognitive function on only a few tests and not restricted to one specific cognitive domain. this finding suggests that cognitive deficits in these patients should not be attributed to radiotherapy, but rather to the tumor itself or other treatment factors, including epilepsy. serious memory deficits, however, are still to be expected when fraction doses exceed 2 gy. a recent follow - up of the klein. study demonstrated that regardless of fraction dose all tumor progression - free low - grade glioma patients that had irradiation showed a progressive deterioration in attentional functioning 13 years after radiotherapy while all patients without irradiation remained stable. then she developed depressive feelings and lack of initiative for which she visited a psychologist. then she developed depressive feelings and lack of initiative for which she visited a psychologist. the mechanism and pattern of seizures in brain tumor patients is determined by tumor type, tumor location and peritumoral and genetic changes in brain tumor patients. an increased epilepsy burden has been found to adversely affect a broad range of cognitive functions even to a larger extent than radiation therapy. decreased processing speed and attentional and executive deficits are notable sequelae of seizures and antiepileptic drugs (aeds) in patients with brain tumors [73, 74, 77 ]. others did not detect any apparent effect of seizures on cognition across multiple cognitive domains assessed in a postsurgical sample. cognitive side - effects of aeds can add to cognitive decline due to tumor effects, previous surgery, or radiotherapy, and therefore appropriate choice and dose of aed is crucial. the classical aeds (phenytoin, carbamazepine, and valproic acid) are known to decrease cognitive functioning [78, 79 ]. importantly, these drugs may also have pharmacological interactions with chemotherapy [80, 81 ] and thus potentially affect survival. these drugs may result in impaired attention and cognitive slowing, which can subsequently have effects on memory by reducing the efficiency of encoding and retrieval. the importance of the classical aeds as a risk factor for cognitive deficits has been reported in a study on stable disease, long - term low - grade glioma survivors where reduced information processing speed, psychomotor function, working memory capacity, and executive functioning, were significantly related to the use of aeds. as patients in this study who took aeds had cognitive impairment even in the absence of seizures, the use of aeds primarily affects cognitive function. moreover, aed use in low - grade glioma patients may be associated with highly elevated levels of fatigue, which in itself is also associated with poorer cognitive outcome. several new generation aeds, like oxcarbazepine and levetiracetam as add - on therapy, appear to have fewer adverse cognitive effects than the classical agents. of the newer agents, topiramate is associated with the greatest risk of cognitive impairment, although this risk is decreased with slow titration and low target doses [85, 86 ]. it appears to be safe to switch patients from phenytoin to levetiracetam monotherapy following craniotomy for supratentorial glioma. follow - up mris of the patient every 3 months showed a stable response with no gadolinium enhancement and a t2/flair hyperintense region that is progressive at 6 mm / year up to 44 months after initial surgery and 20 months after chemoirradiation. during this time she is still unable to work, and partly able to take care of her children due to concentration problems, fatigue, and depression. follow - up mris of the patient every 3 months showed a stable response with no gadolinium enhancement and a t2/flair hyperintense region that is progressive at 6 mm / year up to 44 months after initial surgery and 20 months after chemoirradiation. during this time she is still unable to work, and partly able to take care of her children due to concentration problems, fatigue, and depression. a considerable number of brain tumor patients have feelings of anxiety, depression, and future uncertainty as psychological reactions to the disease [8890 ]. these mood disturbances may lead to deficits in attention, vigilance, and motivation that subsequently affect several cognitive domains. loss of self confidence, unemployment, and dependency on caregivers may also negatively affect these patients cognitive status. mood changes are more common in brain tumor patients than in patients with other neurological diseases and might be related to tumor location. unilateral surgical removal of prefrontal cortex, including the fronto - orbital or anterior cingulate cortex, has resulted in emotional dysregulation with impaired voice and face expression identification in patients with various brain lesions including brain tumors. furthermore, deficits in recognizing emotional facial expression were observed after surgical removal of brain tumors that involved both heteromodal and limbic / paralimbic cortices. concordantly, impairment of arousal and emotional valence was demonstrated after resective surgery in various brain regions, but particularly in the right temporoparietal region. negative mood changes were observed after brain tumor resection involving heteromodal cortices located either prefrontal or temporoparietal, whereas positive mood changes were observed after lateral frontal resections. mood states did not correlate with laterality of the resection, tumor grading or lesion size. at 44 months after initial surgery and 21 months after chemoirradiation, she experienced headache, a severe decline in cognitive functions, lethargia, and gait instability without seizures. at neurological examination she demonstrated bradyphrenia, disorientation, and a right - sided hemiparesis. she was admitted and a new mri showed clear and sudden progression with extensive t2/flair infiltration, and new gadolinium enhancement at the corpus of the corpus callosum and in the left internal capsula (fig. 1). because of her poor clinical condition and the extent of radiological progression, no further treatment options were considered and she received dexamethasone. she was discharged to a hospice and died at 46 months after initial surgical treatment. at 44 months after initial surgery and 21 months after chemoirradiation, she experienced headache, a severe decline in cognitive functions, lethargia, and gait instability without seizures. at neurological examination she was admitted and a new mri showed clear and sudden progression with extensive t2/flair infiltration, and new gadolinium enhancement at the corpus of the corpus callosum and in the left internal capsula (fig. 1). because of her poor clinical condition and the extent of radiological progression, no further treatment options were considered and she received dexamethasone. she was discharged to a hospice and died at 46 months after initial surgical treatment. the potentially neurotoxic effects of corticosteroids are often misdiagnosed and underestimated and corticosteroids may induce behavioral, psychic, and cognitive disturbances, due to functional and, over time, structural alterations in specific brain target areas. corticosteroids may cause mood disturbances, psychosis, and cognitive deficits particularly in declarative memory performance. steroid dementia is a reversible cause of cognitive deficits even in the absence of psychosis. recent data suggest that transiently impaired attention, concentration, and memory are due to neurotoxic effects on both the hippocampal and the prefrontal areas. both short - term and long - term use of steroids has been associated with hippocampal - dependent explicit memory deficits. more likely, however, corticosteroids may improve cognitive deficits because of resolution of edema. comprehensive assessment of neurocognitive function is evidently different from the use of standard hrqol measures, which is considered to be relatively easy and not time - consuming. however, while cognitive deterioration can be predictive of radiologic disease progression in patients with tumor recurrence, measures of hrqol and activities of daily living are only weakly correlated to cognitive decline or to time to tumor progression, suggesting that hrqol measures may not be sensitive enough to detect a change in patient function. it should be noted, on the other hand, that hrqol is an important outcome measure within the context of patient care. self - reported hrqol measures or other methods of informal assessment of cognitive function depend on the patient s report of their cognitive symptoms. self - reports of decreased cognitive functioning au lieu of formal neuropsychological testing not only usually point at feelings of anxiety, depression, or fatigue rather than cognitive deficits [103, 104 ], and use of self - reports is even more problematic in brain tumor patients whose judgment may be severely impaired by the tumor. historically, when a low - grade glioma was diagnosed in a young, healthy adult, a commonly accepted strategy was a wait and see policy because of the presumed indolent nature and variable behavior of these tumors. however, retrospective studies of the kinetics of glioma growth, showed linear growth before anaplastic transformation. the majority of low - grade gliomas are now known to progress to malignant gliomas with time. a better understanding of the natural history of low - grade gliomas has led to an interest in early treatment. the decision as to whether a patient with low - grade glioma should receive resection, radiotherapy, or chemotherapy is based on a number of factors including age, performance status, location of tumor, and patient preference. since low - grade gliomas are such a heterogeneous group of tumors with variable natural histories, the risks and benefits of each of the three therapies must be carefully balanced with the data available from limited prospective studies. since patients with low - grade gliomas can survive in a clinically stable state for several years after diagnosis, the long - term effects of the disease and its treatment on cognitive functioning of these long - term survivors are especially salient. although not class i evidence, numerous studies strongly suggest that more extensive surgical tumor resection is associated with longer life expectancy for both low- and high - grade gliomas. at the same time, the rapid development of operative techniques and technologies, including brain mapping techniques aimed at preserving eloquent brain functions, facilitates the attainment of maximal or radiologically complete tumor resection while minimizing morbidity. recently, supratotal resection extending beyond the radiological boundaries of the tumor, has been postulated to improve both survival and perseveration of function. cognitive functioning of brain tumor patients is an increasingly important outcome measure, because cognitive impairments can have a large impact on self - care, social and professional functioning, and consequently on hrqol. many factors contribute to cognitive outcome, such as direct and indirect tumor effects, seizures, medication, and oncological treatment. review of the literature indicates that neurocognitive outcome in patients with primary brain tumors was assessed systematically in only a limited number of studies, and most involved a relatively small number of patients. due to the absence of pretreatment neurocognitive assessments and treatment randomization the ability to differentiate between tumor effects and treatment effects, including surgery, was limited in the retrospective studies. however, studies among long - term low - grade glioma survivors indicated greater impairments in virtually all neurocognitive domains in patients who had radiotherapy compared to those who had not. although the role of radiotherapy for a long time thought to be the main cause of cognitive deficits in patients with brain tumors has been studied extensively, the adverse effects on cognitive function of other tumor and treatment - related factors remain elusive. as far as resective surgery is concerned, both cognitive improvement and decline have been observed depending on pathology, lesion size, localization and laterality. neurocognitive deficits, if present, are transient in most cases, except for low - grade glioma patients with tumors in the left hemisphere. aeds may result in impairments of attention and neurocognitive slowing, which can subsequently have effects on memory by reducing the efficiency of encoding and retrieval. likewise, intrasurgical cognitive mapping to improve cognitive outcome also has not been systematically applied in these patients. concerted action into studying the costs and benefits of presurgical, intrasurgical, and postsurgical cognitive assessments related to outcome of these patients is thus warranted. since a combination of cortical and subcortical lesions, epilepsy, surgery, radiotherapy, aeds, corticosteroids, and psychological distress is likely to contribute to neurocognitive dysfunctioning in an individually unpredictable way, it would be most pragmatic to choose a core testing battery that gauges a broad range of neurocognitive functions. additionally, the neuropsychological measures have to meet the following criteria : (i) assess several domains found to be most sensitive to tumor and treatment effects ; (ii) have standardized materials and administration procedures ; (iii) retest reliability ; (v) have alternate forms or are relatively insensitive to practice effects, and are therefore suitable to monitor changes in neurocognitive function over time ; (vi) include tests that have been translated into several languages (i.e., dutch, english, french, german, hebrew, italian, turkish) or require translation primarily of test directions ; and (vii) total administration time is 3040 min. the neurocognitive domains deemed essential to be evaluated include attention, executive functions, verbal memory, and motor speed. the test battery that meets most of the afore - mentioned criteria has successfully been used and is still being used in a number of eortc, ncctg, nci - c, rtog, mrc, and hub multisite clinical trials and it has been shown that neurocognitive functioning has independent prognostic significance in patients with low - grade glioma. moreover, neurocognitive deterioration indicates tumor progression before signs of disease recurrence are evident on ct or mri [3, 102, 111 ]. this battery assesses : memory, hopkins verbal learning test, which is a list of 12 words in 3 semantic categories that measures immediate recall across 3 trials, recognition of the words from distractors, and delayed recall ; verbal fluency, controlled oral word association, which requires the production of words beginning over a specific letter for three 1-min trials ; visual - motor scanning speed, trail making test part a, which requires the subject to connect dots in numerical order as rapidly as possible ; executive function, trail making test part b, which requires the subject to connect dots with alternating numbers and letters as rapidly as possible. | compared to classical oncological outcome measures such as time to progression and survival, the importance of cognitive functioning in patients with diffuse infiltrative brain tumors has only recently been recognized. apart from the relatively low incidence and the invariably fatal outcome of gliomas, the general assumption that cognitive assessment is time - consuming and burdensome contributes to this notion. our understanding of the effects of brain surgery on cognition, for instance, is largely based on studies in surgical patients with refractory epilepsy, with only a limited number of studies in surgical patients with gliomas. the impact of other factors affecting cognition in glioma patients such as direct tumor effects, radiotherapy and chemotherapy, and medical treatment, including anti - epileptic drugs and steroids, have been studied more extensively. the purpose of this paper is to provide an overview of cognition in patients with diffuse infiltrative gliomas and the impact of resective surgery as well as other tumor and treatment - related factors. |
an opaque bubble layer is a rare complication of femtosecond laser - assisted in situ keratomileusis (lasik), which occurs during intracorneal femtosecond laser ablation when gas bubbles are produced and trapped in the corneal interface.1 although intraoperative complications concerning the opaque bubble layer are not yet elucidated, femtosecond laser manufacturers have designed specific modifications for lasik flap creation in order to minimize the opaque bubble layer effect.1 fs200 wavelight (alcon laboratories inc., fort worth, tx, usa) provides a venting dissection tunnel - like chimney modification through which the gas bubbles escape from the interface to the hinge side of the limbus.1 diffuse lamellar keratitis (dlk) is an early post - lasik sterile complication located mainly in the flap periphery and sometimes extending to the central area of the flap in more aggressive stages, and could lead to decreased vision.2 risk factors have already been described, ie, epithelial defects, endotoxins released from sterilizer reservoir biofilms, traumatic flap dislocation, atopy, and other external agents (eg, instrument cleaning solutions, povidone - iodine).3 in this case report, we present a patient with dlk located in the femtosecond lasik flap chimney tunnel. uncorrected distance visual acuity was counting fingers in both eyes. corrected distance visual acuity was 20/20 in both eyes, with a manifest refraction of 7.00 to 1.2510 in the right eye and 7.00 to 0.50180 in the left eye. keratometric readings (galilei dual scheimpflug analyzer ; ziemer ophthalmology, biel, switzerland) were 41.58/40.77 diopters and 42.30/40.48 diopters in the right and left eyes, respectively. central corneal thickness was 563 m and 568 m for the right and left eyes, respectively. ophthalmic examination showed clear corneas with mild vascularization in the corneal periphery, especially in the superior part of the cornea ; no other anterior or posterior segment abnormality was detected. lasik using the wavelight fs200 femtosecond laser and the allegretto wave eye - q 400 hz excimer laser (wavelight gmbh, erlangen, germany) was performed ; flap tunnels were created with the same settings in both eyes and orientated superiorly extending from the flap hinge to the limbus. the surgical procedure was conducted uneventfully in both eyes as described in our previous report.4 at the end of the procedure, a drop of ofloxacin 0.3% (exocin ; allergan, irvine, ca, usa) was instilled. the patient was examined 60 minutes postoperatively on a slit - lamp (haag streit 900 ; haag streit ag, bern, switzerland) for flap alignment and adherence. postoperative treatment consisted of nepafenac 0.1% drops (nevanac ; alcon laboratories) four times daily, dexamethasone 1% (maxidex ; alcon laboratories) four times daily, ofloxacin 0.3% drops four times daily, and sodium hyaluronate drops hourly. the patient was advised to wear a plastic shield during sleep. on the first postoperative day, however, slit - lamp examination revealed a dense, white, granular reaction combined with some blood droplets (stage i dlk) in the flap tunnel of the left eye (figure 1a) ; the flap was well positioned and the interface was clear. the remainder of the slit - lamp examination was within normal limits for both eyes. an intensive course (every second hour) of maxidex together with ofloxacin 0.3% was prescribed for both eyes. on the second postoperative day, uncorrected distance visual acuity was 20/20 in the right and 20/25 in the left eye ; there was an improvement in dlk of the left eye. during the following days, dlk responded rapidly to the treatment and by the fifth day it was completely resolved (figure 1b) ; uncorrected distance visual acuity was 20/20 for both eyes. the first published report of post - lasik dlk with microkeratome was from smith and maloney in 1998.5 macrae reported that post - lasik dlk is associated with leakage of red blood cells and other intravascular contents from blood vessels into the interface in the early postoperative period.6 corneal vascularization occurs when blood vessels grow into the cornea between the epithelium and bowman s layer. chronic contact lens wear is the most frequent etiology ; corneal infections and allergy, limbal stem cell dysfunction, blepharitis, and uveitis also induce corneal vascularization.7 wallin proposed that, in refractive candidates with superior corneal vascularization, it is preferred that the hinge of the mechanical microkeratome flap should be placed superiorly in order to avoid the dlk complication through bleeding in the cut.7 femtosecond laser in the flap creation step of lasik raised the incidence of dlk due to higher energy delivery to the corneal stroma compared with flap creation with microkeratome.8 moreover, dlk after femtosecond - assisted lasik was unrelated to hemorrhage or blood vessel leakage, and it has been hypothesized that increased energy and gas bubble formation in the interface may correlate with this granular reaction.810 in this report, we present a case of dlk that occurred in the lasik flap like - chimney tunnel, which is created with the femtosecond laser and facilitates the exit of gas bubbles from the interface to the hinge side of the limbus. the patient had binocular peripheral corneal vascularization due to possible chronic contact lens use and intolerance. thus, we believe that, during flap tunnel creation, blood vessels from corneal vascularization were damaged in the left eye, leaving blood cells and intravascular contents inside this venting chimney causing postoperative dlk. however, the right eye was unaffected ; a possible explanation is that during the cutting process of the femtosecond laser in the right eye, blood vessels were absent or remained intact at the flap tunnel site. moreover, epithelial ingrowth, infectious keratitis, and interface foreign bodies were excluded from the differential diagnosis as the reaction occurred immediately after surgery (first postoperative day) into a confined sterile corneal cavity (the lasik flap tunnel). dlk inflammation was restricted to the tunnel and no reaction was noted in the flap interface ; visual acuity remained stable at 20/20. in addition, dlk was easily managed with corticosteroid treatment and 5 days later was completely resolved. to the best of our knowledge, refractive candidates with chronic contact lens use and evident corneal vascularization should be carefully examined immediately after a lasik procedure and the first postoperative day for possible dlk in the periphery. creation of the femtosecond - assisted lasik flap tunnel could be avoided in these patients ; the flap tunnel could be placed with a different orientation if corneal vascularization is not extended into the entire corneal periphery (eg, superior corneal vascularization). in patients with more severe corneal vascularization (pannus), additional considerations should be given to treating with other refractive modalities, such as photorefractive keratectomy instead of lasik. | here we report a case of a 29-year - old myopic female who underwent femtosecond laser - assisted in situ keratomileusis (lasik) and, on the first postoperative day slit - lamp examination revealed a dense, white, granular reaction with the presence of some blood droplets (stage i diffuse lamellar keratitis [dlk ]) in her left eye, specifically localized into the femtosecond lasik flap tunnel (not extended to the flap interface). the patient received intensive treatment with topical corticosteroids and 5 days later the granular reaction had completely resolved. a new site of dlk, ie, the flap tunnel, in femtosecond - assisted lasik is presented. dlk into the flap tunnel could be managed with corticosteroids if detected early, without affecting the flap interface. |
giant cell tumor (gct) accounts for about 7 to 10% of all cases of primary spinal tumors. patients with gct are usually diagnosed in the third or fourth decade of life, and there is a slight female preponderance152325). gct usually occurs in the metaepiphyseal ends of the long bone, and it rarely occurs in the spine2611). savini.28) reported that only 2.9% of all gct incidences occur in the spine, and goldenberg.14) reported that spine involvement is 1.3% in a study of 218 cases of gct. a research conducted by mayo clinic reported incidence of up to 6.5% of gct in the spine27). gct in the spine usually involves sacrum, but it also occurs in other parts of the spine. gct is classified as benign tumor histopathologically, but it had a locally aggressive tendency. and, if it is incompletely excised, it shows a high recurrence rate. there is a reason for a relatively worse prognosis in gct patients, compared to other benign primary spinal tumor patients7826). many studies reported from 10 to 40% local recurrence rate after spinal gct treatment, depending on surgical protocols46141821). in case of recurrence, excision is conducted when another operation is possible. if re - operation is considered difficult, radiation therapy (rt) is introduced. generally, however, due to the characteristics of spine, there is a possibility of neural or vascular injury, which will result in difficult wide en bloc excision10). some authors reported that rt is not effective in local tumor control and has a risk of malignant transformation of gct15252728). recent studies reported chemotherapy as effective in case of recurrence or when there are complications1591329). the objective of this study is to obtain the results of treatment gct in the spine. in addition, we analyzed whether local recurrence is influenced by surgical protocols, or rt. the subjects of our study were 242 patients treated for gct from 2000 to 2012. patients with involved gct spine were 19 cases, and incidence rate was 7.9%. the median age at their first diagnosis was 31 years (range, 14 to 39 years). fourteen tumors were located in the sacrum, one in cervical, one in thoracic, and three in lumbar spine. all the lesions were single and localized at the original spine except one, which originated in the sacrum extending into the coccyx. the median follow - up period was 92 months (range, 18 to 163 months). main symptom of the patients with gct was pain, and the average visual analogue scale (vas) was 7 (range, 5 to 9). only one out of 19 showed motor weakness initially caused by spinal cord compression at thoracic spine. for initial treatment, 6 out of 19 patients underwent gross total removal (gtr), and 13 patients underwent subtotal removal (str). adjuvant rt was performed in 12 cases, 2 cases in gtr group and 10 cases in str group. we analyzed the recurrence rate and recurrence free period (rfp) for gct in the spine after the treatment. in addition, we analyzed the difference of the recurrence rate according to the surgical protocol and rt. we did not consider the effect of the treatment for the patients with recurrence after the initial treatment. chi - square tests were used for categorical variables, and student 's t - tests were used for continuous and ordinal variables, as appropriately. a p value 0.05 (two - tailed) the data were compiled and analyzed with the software package spss, version 18.0 (spss inc., chicago, il, usa). during the follow - up period, 7 out of 19 patients had local recurrence, at 36.8% rate. an average recurrence free period was 14 months (range, 4 to 34 months) for the patients with recurrence. median recurrence free period of all patients was 84 months (95% ci, from 4 to 163 months) (fig. gtr group did not have any recurring patients, while 7 out of 13 patients who received str had local recurrence (table 2), which is statistically significant (p=0.024). this implies that gtr is the most important method of treatment for controlling the tumor. as for the local control effect of radiation therapy, the average recurrence free period of the group that underwent radiation therapy was 112 months (95% ci, from 74 to 150 months), which was longer than that of the group without radiation therapy, for 65 months (95% ci, from 47 to 83 months) (fig. because there was a statistically significant difference between rt group and non - rt group (p=0.041), we considered that radiation may be effective in local control of tumors. four patients underwent re - operation, 2 patients underwent re - operation and rt, and 1 patient underwent radiosurgery. gtr was possible in only one out of 6 patients who underwent re - operation. all patients reported relieved pain after the treatment according to vas (range, 0 to 5). nine out of 19 patients were pain - free during the follow - up period. the patient with motor weakness showed incomplete recovery after surgery, although the improvement was not functional and bladder dysfunction remained. neurological complication after surgery occurred in 3 out of 19 patients, including bladder or bowel dysfunctions. all the patients who experienced complications had gct in the sacrum. until the final follow - up period, 18 out of 19 patients remained alive without recurrence, and 1 patient with pulmonary metastasis expired due to pulmonary complication. female 31-year - old patient visited our hospital with pain in buttock that lasted for 4 months. magnetic resonance (mr) the patient underwent str, and the pathologic examination revealed to be gct. on computed tomography scan (ct) after the operation showed remnant tumor on the ventral side of s2 spinal canal (fig. four months after the initial operation, mr image showed the progression of the tumor extending to the left side (fig. ninety - four months after re - operation, the tumor did not recur (fig. 33-year - old male patient complained lower back pain and radiating pain into both legs for 4 months. sunnyvale, ca, usa) radiosurgery was done to the remnant tumor located in front of the sacrum (fig. the patient 's pain was relieved and he has been stable for 68 months (fig. female 31-year - old patient visited our hospital with pain in buttock that lasted for 4 months. magnetic resonance (mr) the patient underwent str, and the pathologic examination revealed to be gct. on computed tomography scan (ct) after the operation showed remnant tumor on the ventral side of s2 spinal canal (fig. four months after the initial operation, mr image showed the progression of the tumor extending to the left side (fig. ninety - four months after re - operation, the tumor did not recur (fig. 33-year - old male patient complained lower back pain and radiating pain into both legs for 4 months. sunnyvale, ca, usa) radiosurgery was done to the remnant tumor located in front of the sacrum (fig. the patient 's pain was relieved and he has been stable for 68 months (fig. female 31-year - old patient visited our hospital with pain in buttock that lasted for 4 months. magnetic resonance (mr) image showed a huge enhancing mass involving the sacrum (fig. the patient underwent str, and the pathologic examination revealed to be gct. on computed tomography scan (ct) after the operation showed remnant tumor on the ventral side of s2 spinal canal (fig. four months after the initial operation, mr image showed the progression of the tumor extending to the left side (fig. ninety - four months after re - operation, the tumor did not recur (fig. 33-year - old male patient complained lower back pain and radiating pain into both legs for 4 months. sunnyvale, ca, usa) radiosurgery was done to the remnant tumor located in front of the sacrum (fig. the patient 's pain was relieved and he has been stable for 68 months (fig. the gct usually involves the end of long bone as femur (23%), tibia (21%), and fibula (5%) around knee, and a few other arise from the spine (range, 3% to 7%)24). it is reported that there is a slight female preponderance in spinal gct152325). in our study, the incidence of spinal gct was 7.9%, and the male to female ratio is 1 to 0.9. however, gct in the spine is not easy to remove totally because of lack of accessibility and adjacent important neural and vascular structures10). in case of incomplete excision, rt can be considered, but rt for gct is still controversial1516252728). wide or marginal excision of the tumor or en bloc resections may yield in a lower recurrence rate. liljenqvist.20) stated on malignant tumors of the spine that en bloc spondylectomy enables wide or marginal resection in most cases with acceptable morbidity. compared to a degree of resection, the textbook showed average of 50% (from 35 to 70%) in curettage alone and from 10 to 15% recurrence rate in en bloc resection24). campanacci.6) reported a recurrence rate of 27% after intralesional curettage versus rates of 8% and 0% after marginal or wide resection, respectively. fidler reported successful result of only one recurrence in 9 consecutive patients who underwent en bloc resection12). in a larger series, 10 of 32 patients who had intralesional curettage recurred within 16 months postoperatively and none of 2 patients with en bloc resection recurred15). regardless of its surgical resection, 25 to 28% of recurrence rate showed in other series152728). according to kim.16), 32.4% of recurrence rate in curettage and 11.1% of rate in en bloc resection (overall 27.1%) was16). in our study, gtr was defined as cases in which all the involved structures were removed completely. average recurrence rate of all patients was 36.8%, with 0% recurrence rate in gtr group, and 53.8% in str group. the surgical protocols showed a statistically significant difference (p=0.024), implying gross total resection as more desirable. we performed gtr rather than en bloc wide excision but there was no recurrence case during the median follow up period 92 months. in addition, 14 out of 19 cases located in the sacrum. en bloc resection of the sacrum is much more complicated than that of the thoracolumbar spine. most report about en bloc spondylectomy for gct mentioned above localized in the thoracolumbar spine. in case of the recurrence (case 4), the patient received additional surgery and rt. considering the morbidity of en bloc wide excision in spinal gct, grt is not worse than en bloc wide excision. in the early 2000s, str and rt was used to treat spinal gct in our hospital, which was the reason for high recurrence rate. later, str was considered not effective for local control of gct, which led to a wider use of gtr. however, recent studies insisted that rt is not only a useful adjuvant treatment modality after incomplete removal of tumors but also an effective therapy as a sole treatment of gct of the spine15252728). hart.15) reported that 8 out of 36 patients received rt as an initial treatment before surgery, no case except one recurred. four patients had rt after recurrence, and only 1 patient experienced re - recurrence. kim.16) reported that regardless of surgical protocols, 29 out of 96 cases had received rt and only 3 cases (10.3%) of recurrence occurred. in 67 cases without rt, 23 cases (34.3%) of recurrence occurred, which is significant statistically (p=0.031)16). in our study, although recurrence rate was 42% after rt, average recurrence free period was 112 months for rt group, and 65 months for non - rt group, with statistical significance (p=0.041). even after recurrence, additional rt or radiosurgery after re - operation yielded good results. rt seems to be a good treatment modality for delaying recurrence and locally controlling the tumor. distant metastasis of gct is reported to be about 2 to 9%17). in our study, only 1 out of 19 patients (5%) had pulmonary metastasis. bertoni.3) mentioned that rt or chemotherapy may be useful to treat pulmonary metastasis of gct. other adjuvant therapies, such as a cryotherapy that had cure rate 92% in marcove 's first series and a preoperative embolization that was performed in five patients with result of no recurrence, were mentioned in these series. however a relatively large number of patients is not studied yet22). lee.19) reported that bone cement injection offers an adjuvant strategy that may enhance the efficacy of treatment for gct when complete en bloc spondylectomy is difficult. balke.1) mentioned that most inoperable sacral gcts that had repeatedly recurred did not increase in size with no further recurrence was seen. although the role of bisphosphonates for treatment of gct is still unknown, the administration of bisphosphonates can be considered in complicated cases and metastasis. thomas.29) reported that 35 patients of gct were treated with denosumab, 30 cases were effective for tumor control after 25 weeks. in branstetter.5) study, 17 out of 20 patients were examined at various stages of treatment to distinguish clinical benefits from denosumab such as improved functional status or reduced pain. gct in the spine is difficult to resect completely due to the special structure of the spine and the invasive nature of the tumor. bloc wide excision is a well known a treatment of choice in order to manage gcts. however, en bloc wide excision of the gct in the spine is not easy without damaging neurovascular structures. the authors performed gross total removal rather than en bloc wide excision and obtained results. in case of str, rt was beneficial in delaying tumor recurrence. | objectivethe treatment of giant cell tumor (gct) is mainly performed surgically. however, gct in spine seems difficult to treat because of the limited surgical accessibility and proximity. in this report, we analyzed the outcome of gct treatment in spine.methodsbetween 2000 and 2012, 19 patients received treatment for gct in spine. median age at their first diagnosis was 31 years, 10 patients were male, and 9 female. fourteen tumors were located in the sacrum, 1 in cervical, 1 in thoracic and 3 in lumbar spine. as primary treatment, gross total removal (gtr) was done in 6 patients, and subtotal removal (str) in 13 patients. radiation therapy (rt) as an adjuvant therapy was performed in 2 cases in gtr group and 10 cases in str group.resultsduring the follow - up, 7 patients had local recurrence (36.8%). the average period until recurrence after primary treatment was 14 months. no recurrence was detected in gtr group. recurrence was noted in 7 out of 13 patients who underwent str. these differences were statistically significant (p=0.024). a median of recurrence free period (rfp) was 84 months. also average rfp of the rt group was 112 months, and non - rt group was 65 months. these differences were statistically significant (p=0.041).conclusiontreatment of choice for gct in spine is a complete removal of tumor without neurological deficits. in case of incomplete removal, radiation therapy may be a useful adjuvant treatment modality. |
integrating sensory information and producing the appropriate motor output are the basic functions of the nervous system, and the neural networks underlying these two functions are tightly linked. the basal ganglia are involved in various functions, including motor learning, planning, and execution, as well as in decision making and reward (haber, 2008 ; middleton and strick, 2000 ; schultz., the input layer of the basal ganglia, striatum, receives glutamatergic inputs from multiple cortical areas, including sensory, motor, and prefrontal cortices (alloway., 2009 ; these projections are characterized by a high degree of divergence and convergence (flaherty and graybiel, 1991), enabling striatal neurons to integrate inputs from different cortical areas and modalities (chudler., 1995 ; nagy., 2005, 2006 ; wilson., 1983). corticostriatal projections originate from both hemispheres and are mediated by different subtypes of pyramidal neurons (carman., 1965 ; kress., 2013 ; knzle, 1975 ; lei., 2004 ; wall., 2013), suggesting that striatal neurons may receive bilateral sensory input with different synaptic properties. striatal neurons respond to sensory input from different modalities such as tactile, auditory, and visual input (brown., 1996 ; nagy., 2005 ; schulz., 2009 ; wilson., 1983). due to the high convergence in the corticostriatal pathway and the loose topographical correspondence (kincaid., 1998), individual striatal neurons may be involved in tactile - visual sensory integration ; however, such synaptic integration at the single neuron level has not yet been shown in striatum. striatal projection neurons (msns) are divided into two main subpopulations according to their projection via the direct or indirect pathway (alexander and crutcher, 1990 ; smith., 1998), with direct pathway neurons facilitating motor activity and indirect pathway neurons inhibiting it (albin., 1989 ; kravitz., both subpopulations receive cortical as well as thalamic inputs (doig., 2010) ; however, it has been debated whether contralateral and ipsilateral corticostriatal projections are selective or biased in targeting direct and indirect pathway msns, respectively (kress., 2013 ; lei., 2004 corticostriatal synapses formed onto direct and indirect pathway neurons and interneurons have different properties (calabresi., 1996 ; fino and venance, 2011 ; kreitzer and malenka, 2008 ; surmeier., 2007), suggesting that striatal neurons of different types may also respond differently to sensory input in vivo. msns recorded in vivo are characterized by low discharge frequencies (adler., 2012 ; berke., 2004 ; wilson, 1993), suggesting that a large fraction of their synaptic inputs are subthreshold and do not often contribute to action potential discharge. we therefore used whole - cell striatal recordings in order to study synaptic responses to tactile and visual stimuli. we show that neurons throughout dorsal striatum respond to bilateral whisker stimulation in a type - dependent manner and that neurons in dorsomedial striatum perform multisensory integration. whole - cell patch - clamp recordings were obtained from neurons in dorsolateral striatum (n = 59 neurons), dorsomedial striatum (n = 50 neurons), and layer v of s1 barrel field (n = 20 neurons). of all recorded neurons (n = 129), 45 were stained and morphologically reconstructed (see experimental procedures), three of which were cortical pyramidal neurons and 42 were striatal neurons. striatal neurons were located between 1,854 and 2,613 m below the pia, and the average recording time for all neurons was 48 20 min (minimum = 9 min, maximum = 100 min ; n = 129). the striatal regions targeted for recordings were selected according to the existence of corticostriatal projections from primary somatosensory and visual cortices (s1 and v1, respectively) following anterograde tracing (see experimental procedures ; figures 1a, 1b, 3a, and 3b). as described in previous studies (alloway., 1999, 2006 ; 2001), projections from s1 (barrel field) were found throughout the dorsal striatum, with higher concentration in dorsolateral striatum (figures 1b ; figure s1 available online). projections from visual cortex, on the other hand, were located dorsomedially in proximity to the lateral ventricle (figure 3b), in agreement with previous reports in other species (donoghue and herkenham, 1986 ; faull., 1986 ; norita., 1991 ; serizawa., 1994). all recorded cortical and striatal neurons exhibited slow wave oscillations (figures 2a, 3c, s4, s6, and s7) with bimodal distribution of the membrane potential (figures s4 and s7), as previously described (wilson and kawaguchi, 1996). striatal neurons, however, had longer up state durations than cortical neurons and rarely discharged action potentials, unlike cortical neurons, which discharged several aps during up states (figure s4). the input resistance of recorded neurons was extracted using step current injections and was calculated separately for up and down states (figures s4f, s4h, and s4i). unlike cortical neurons, striatal neurons had higher input resistance at up states and at more depolarized membrane potentials (figure s4f), suggesting that inward rectification in msns (kita., 1984 ; nisenbaum and wilson, 1995) is the dominant factor determining their input resistance as recorded at the soma, even in the presence of the synaptic barrages occurring during the up states. in order to study bilateral sensory integration, we delivered brief air puffs to the whisker pads on both sides (see experimental procedures) and recorded subthreshold responses to ipsilateral, contralateral, and bilateral stimulation in dorsolateral striatal neurons. using the same stimulation protocol, we compared the whisker - evoked responses in striatal msns to cortical regular spiking neurons (putative pyramidal cells) in layer 5 of the barrel cortex (figures 1 and s2 ; table 1). all recorded neurons responded to both ipsilateral and contralateral whisker stimulation (cortical n = 17 ; striatal msns n = 20). sensory responses were classified according to those occurring during up or down states, including cases in which sensory stimulation triggered state transitions (reig and sanchez - vives, 2007) (figure 1 and s2, respectively). response onset delays were significantly longer for ipsilateral than contralateral whisker stimulation in both cortical and striatal neurons (bf ipsilateral : 25.11 6.49 ms, contralateral : 13.32 3.61 ms, p < 0.001, n = 17 ; dorsolateral striatum ipsilateral : 28.45 6.94 ms, contralateral : 19.78 3.42 ms, p < 0.001, n = 20) (figure 1f). corresponding differences were observed also for response peak latencies (bf ipsilateral : 71.13 21.98 ms, contralateral : 37.75 8.52 ms, p < 0.001, n = 17 ; striatum ipsilateral : 66.88 26.07 ms, contralateral : 52.62 15.82 ms, p < 0.01, n = 20) (figure 1 g). onset and peak latencies in response to contralateral and bilateral stimulation were significantly shorter in cortical neurons (figures 1f and 1 g). response amplitudes for both cortical and striatal neurons were always larger when occurring during down states than during up states (figures 1h and s2f, respectively). contralateral whisker stimulation evoked larger amplitudes than ipsilateral stimulation in both cortical and striatal neurons (cortex during down states, ipsilateral : 9.11 3.41 mv, contralateral : 14.07 5.67 mv, p < 0.001 ; striatum during down states, ipsilateral : 10.27 3.27 mv, contralateral : 15.65 6.90 mv, p < 0.01) (figure 1h). however, when stimuli were delivered simultaneously to both sides, a significant increase in amplitude was observed in striatal responses but not in cortical ones (cortex bilateral : 15.72 7.09 mv ; striatum bilateral : 20.37 6.52 mv, p < 0.05) (figure 1h). the increase in amplitude in the striatal msns is also reflected in the slopes of responses to bilateral stimulation (figure 1i), both suggesting a higher sensitivity to bilateral input in striatal msns than that observed in s1 layer 5 pyramidal cells. this difference could be explained by the higher temporal separation between ipsilateral and contralateral responses in cortical neurons compared to msns (response peak latency : cortex = 33.38 21.46 ms ; striatum = 14.26 24.96 ms, p < 0.05, data not shown). sensory and electrically evoked excitation of striatal neurons is accompanied by inhibition (pidoux., 2011), originating from striatal interneurons and msn collaterals (kos and tepper, 1999 ; tunstall., 2002). in order to decompose the excitatory and inhibitory response components, we used a low - chloride intracellular solution (see experimental procedures) enabling us to hold recorded neurons at the reversal potential for excitation (5 mv) or gabaa inhibition (70 mv, figures 2a2d). in all recorded msns, excitation preceded inhibition for ipsilateral, contralateral, and bilateral whisker stimulation (onset delays : ipsilateral excitation 24.63 3.76 ms, inhibition 40.6 9.7ms, p < 0.01 ; contralateral excitation 19.16 2.8 ms, inhibition 28.98 1.93 ms, p < 0.001 ; bilateral excitation 19.71 3.27 ms, inhibition 25.61 3 ms, p < 0.01, n = 8) (figure 2c). regardless of the stimulus condition (ipsilateral, contralateral, or bilateral whisker stimulation), larger excitatory responses were matched by larger inhibitory ones (figure 2d). spontaneous activity was also mediated by mixed excitatory and inhibitory synaptic barrages during up states, as recorded at the respective reversal potentials (figures 2a and s4). we next wanted to assess the stimulus intensity dependence of the different response conditions. to that end, we altered the pressure generating the air puff, resulting in different deflection intensities. the air puff duration remained constant (15 ms), and the air pressure was changed in a range between 0 and 30 psi (0, 2, 5, 10, 20, and 30 psi) (figure 2e). for all three stimulus conditions, onset and peak delays were shortened, and amplitudes and slopes increased when air puff pressure was increased from 2 to 20 psi (figures 2e2i). interestingly, response durations, measured as the width at 75% amplitude, were inversely related to the amplitudes and stimulus strength (figures 2j2l), possibly due to curtailing of responses by the increased inhibitory component (figure 2d). the relationship between ipsilateral and contralateral responses was maintained for different stimulus intensities, with contralateral (and bilateral) stimulation evoking earlier onset and peak response latencies (figures 2f and 2 g), with larger amplitudes and rise slopes (figures 2h and 2i). in order to study responses of striatal neurons to visual stimulus, we obtained whole - cell recordings from neurons in dorsomedial striatum (figure 3). the recording area was selected according to the presence of axonal projections from cortical v1 (figures 3a and 3b, n = 5), which were clustered in dorsomedial portions of the striatum, near the lateral ventricle (figure 3b). visual stimuli were presented to the contralateral eye as brief light flashes from a white led (see experimental procedures) during whole - cell recordings in striatum and lfp recordings from v1. as described above, responses were sorted offline to those occurring during up or down states. the onset delay for contralateral visual responses during down states was 98.43 19.05 ms (ranging between 56.7 and 141.0 ms, n = 25), almost five times longer than the onset response to whisker stimulation (19.78 3.42 ms, figure 1f) and similar to previously reported visual responses (schulz., 2009, 2011). such delays are expected when comparing to the long and variable delays (50130 ms) described for visual responses in mouse visual cortex (niell and stryker, 2008 ; takagaki., 2008). in order to verify the occurrence of cortical visual responses, we obtained extracellular recordings (lfp) in v1 simultaneously with the striatal whole - cell recordings (n = 16, figures 3c and 3d). onset and peak latencies were slightly earlier, although not significantly different between cortical and striatal visual responses (figures 3e and 3f), which may be due to the large response variability. the amplitude of visual responses was 13.28 5.38 mv and slopes 0.14 0.15 mv / ms (data not shown, n = 25). visual responses were evoked in 25 of dorsomedial neurons (66%) and in none of the dorsolateral ones ; however, all neurons in both regions responded to bilateral whisker stimulation (figure 4). figure 4c depicts the position of anatomically reconstructed neurons, in which both tactile and visual stimulation were presented (n = 30). all neurons that responded to visual stimuli were located medially (figure 4c, orange points), close to the area receiving axonal projections from v1 (figure 3b). the differences in onset and peak delays between whisker and visual sensory responses suggested that the strongest responses in striatal neurons would not occur when stimuli are presented simultaneously, but rather when visual stimulation precedes tactile stimulation. to test this prediction, we presented the whisker stimuli at different time intervals with relation to the visual stimuli (figure 4d). indeed, in all cases (n = 7) maximal response amplitudes were evoked when whisker deflections followed the visual stimuli synchronized to the respective response onsets (relative interval 102.11 20.98 ms, figure 4e). multisensory responses did not summate linearly, with maximal response amplitudes smaller and earlier than those predicted by the linear sum of unimodal responses (independent whisker 13.73 3.15 mv, independent visual 13.23 4.66 mv, synchronized onset 18.19 4.54 mv, n = 7) (figures 4d and 4e). this sublinear summation is expected due to the proportional inhibitory component of sensory responses (figures 2a2d) but may also be mediated by activation of common cortical association areas (olcese., 2013). as shown above neurons in both dorsolateral and dorsomedial striatum responded to whisker stimulation ; however, there were notable differences in responses recorded in both striatal regions. dorsolateral msns had stronger and faster responses (figure 4f), as reflected in response amplitudes (contralateral whisker, dorsolateral striatum : 15.6 6.9 mv, dorsomedial 8.4 3.67 mv ; bilateral dorsolateral 20.37 6.52 mv, dorsomedial 10.29 4.42 mv) (figure 4h) and slopes (ipsi - dorsolateral 0.36 0.24 mv / ms, dorsomedial 0.16 0.08 mv / ms ; contralateral dorsolateral 0.63 0.46 mv / ms, dorsomedial 0.17 0.09 mv / ms ; bilateral dorsolateral 1.08 0.63 mv / ms, dorsomedial 0.28 0.18 mv / ms, dorsolateral n = 20, dorsomedial n = 24) (figure 4i). there were no differences in onset delays (ipsi - dorsolateral 28.45 6.94 ms, dorsomedial 31.8 8.35 ms, p = 0.16 ; contra - dorsolateral 19.78 5.09 ms, dorsomedial 18.24 6.68 ms, p = 0.41 ; bilateral dorsolateral 20.49 5.19 ms, dorsomedial 18.39 5.71 ms, p = 0.23, data not shown) ; however, there were significant differences for peak delays in all conditions, reflecting the slower response rising slopes (figure 4 g). these differences in sensory responses correlate to the differences between dorsolateral and dorsomedial striatum in the density of axonal projections from primary somatosensory cortex (figures 1b and s1). in summary, neurons located in the dorsomedial striatum integrate tactile and visual sensory inputs, with maximal responses when the respective response onsets are aligned. dorsolateral msns do not respond to visual stimulation but have larger and faster responses to whisker stimulation than dorsomedial ones. the large majority of striatal neurons are msns, projecting via the striatonigral (direct) and striatopallidal (indirect) pathways. since these projection pathways are believed to have different roles in basal ganglia function, it is important to understand whether and how they differ in their integration of sensory input. to that end, whole - cell recorded and electrophysiological identified msns were subsequently immunostained with d1 antibody (see experimental procedures ; figures 5a, s5, and s6) in order to classify them as direct or indirect pathway msns. the staining allowed us to classify msns into d1-expressing and putative d2-expressing msns, which we refer to below as d2 msns. using a drd2 bac transgenic mouse we verified that the d1 antibody did not stain d2-egfp msns (figure s5), suggesting that recorded msns that were d1 negative are indeed d2 msns. msn subtype identification was obtained for 28 msns : 15 d1 positive and 13 d2 neurons (figures 5a and s6). while similar in most of their electrophysiological properties, d2 msns had higher input resistance than d1 msns, as measured by injection of a depolarizing and hyperpolarizing current step during down states (in mohm, depolarized d1 112.85 25.76 and d2 158.81 28.93, p < 0.001 ; hyperpolarized d1 88.13 27.7 and d2 107.81 39.01, p < 0.05) (figure 5c). similar results were reported for d1 and d2 msns in recent slice studies (gertler. the differences in onset delays between contralateral and ipsilateral responses were significantly larger in d1 msns (d1 ipsi : 31.18 6.49 ms, contra : 17.07 7.97 ms, p < 0.001 ; d2 ipsi : 25.16 3.68 ms, contra : 20.38 5.35 ms, p < this difference was caused by the response onset for ipsilateral stimulation that was longer in d1 than d2 msns (d1 : 31.18 6.49 ms, d2 25.16 3.68 ms, p < peak responses to contralateral and ipsilateral stimulation also had different latencies in d1 msns (ipsi 89.85 44.44 ms, contra 63.08 24.87 ms, p < 0.01) but were similar in d2 msns (ipsi 70.33 23.47 ms, contra 63.71 23.52 ms, p = 0.192) (figures 5d, 5f and 5 g). response amplitudes were overall larger in d1 msns than d2 msns, significantly so for contralateral whisker stimulation (d1 msns 15.14 5.47 mv, d2 msns 10 4.93 mv, p < 0.05) (figure 5h). the two msn subpopulations also had differences in the response slopes, where d1 msns had faster slopes for contralateral versus ipsilateral stimuli (p < 0.05) but not d2 msns (p = 0.13, see table 2 ; figure 5i). both d1 and d2 msns had the fastest slopes for bilateral stimulation (see table 2 ; figure 5i). differences between d1 and d2 msns in their responses to whisker stimulation persisted also when occurring during up states (figure s6) ; however, no differences were observed in spontaneous up states amplitudes or durations (vm down states d1 72.28 5.44 mv, d2 72.66 3.02 mv ; ap threshold d1 46.24 3.31 mv, d2 45.71 3.73 mv ; up state amplitude d1 11.41 3.16 mv, d2 12.24 4.6 mv ; up state duration d1 0.62 0.18 s, d2 0.61 0.26 s ; ap frequency d1 0.16 0.31 hz, d2 0.10 0.13 hz) (figure s6). we also recorded from identified msns in dorsomedial striatum that responded to whisker and visual stimulation (five d1-msns and three d2-msns), thus showing that both msn types integrate multimodal sensory inputs (figures 5, 5j, and 5k). the small sample size of type - identified visually responding msns prevents us from drawing conclusions regarding their respective integration properties. in summary, msns belonging to the direct and indirect pathways exhibited significant differences in their bilateral integration of tactile sensory inputs. direct pathway msns (d1) responded earlier (onset delay), stronger (amplitude), and faster (slope) to contralateral compared to ipsilateral whisker stimulation. in contrast, in d2 msns contralateral and ipsilateral stimulation resulted in more similar response properties, with ipsilateral responses being earlier (onset) and faster (slope). these results suggest that d1 and d2 msns have different roles in their sensory integration, d1 msns tuned to detect differences between ipsilateral and contralateral whiskers than d2 msns, which act as integrators of bilateral inputs. striatal interneurons form a small albeit diverse minority in the striatal microcircuitry ; therefore, our method of blind whole - cell patch - clamping resulted in a rather small number of interneurons. out of 109 recorded striatal neurons, four were classified as interneurons, of which two were fast spiking (figures s7a s7d) and two others were cholinergic interneurons (figures s7e s7h). neurons were initially classified according to their recorded electrical properties and following morphological staining, according to the aspiny dendrites and the large soma size (in the case of the cholinergic interneurons). fs interneurons displayed narrow action potentials, relatively depolarized rest potential, high discharge rate of action potentials during up states, and no apparent inward rectification. cholinergic interneurons were characterized by their voltage sag response to current step injections, depolarized membrane potential, and spontaneous discharge activity. they also displayed spontaneous slow wave activity, although the amplitude range was not as wide as in fs interneurons or msns yet was sufficient to phase - lock spontaneous discharge to the cortical oscillations as recorded simultaneously in s1 and v1 (figure s7). as in all other recorded neurons, interneurons responded to either whisker stimulation with stronger and earlier responses to the contralateral whisker deflection (figures s7c, s7 g, and s7i), and in one interneuron where visual responses were tested, the recorded fs responded to visual input (figure s7c). in this study we used whole - cell patch - clamp recordings to study the integration of bilateral and multimodal sensory information in striatal neurons. we show that individual striatal neurons integrate bilateral as well as multisensory inputs, that both spontaneous and sensory evoked inputs are comprised of overlapping excitatory and inhibitory synaptic input, and that msns of the direct and indirect pathways differ in the way they integrate bilateral sensory input. all neurons recorded in dorsal striatum, including projection neurons and interneurons, responded to bilateral whisker stimulation in a type - dependent manner, and a population of dorsomedial neurons also responded to visual input. recordings were obtained under anesthesia, enabling activation of sensory pathways while avoiding interference with motor related inputs. in future studies it would be of interest to study these sensorimotor interactions in the unanesthetized, behaving animal. all neurons recorded in the dorsal striatum responded to stimulation of whiskers of both sides. the responses differed in latency, slope, and amplitude, with contralateral whisker stimulation inducing larger and earlier responses as seen also in the recordings from cortical neurons. there are, however, notable differences between the striatal and cortical responses, suggesting different integration properties. onset delays for contralateral and bilateral responses in dorsolateral msns were 6 to 7 ms longer than the cortical ones (figure 1f). this result, together with the anatomical tracing data, supports the idea that the responses in striatal neurons under our experimental conditions are generated primarily by cortical inputs without engaging a thalamic shortcut (mowery., 2011). bilateral responses were relatively larger in striatal neurons than in cortical ones (figure 1h), showing that striatal msns act as integrators of bilateral sensory input to a higher degree than cortical neurons. this result can be explained by the time differences between ipsilateral and contralateral onset latencies, which were shorter in striatal neurons, in particular d2 msns (figure 5 g, inset). the sensory responses we observed were almost entirely subthreshold, compared to a larger fraction of suprathreshold responses to contralateral whisker stimulation recently reported in rats (pidoux., 2011). the discrepancy in the measured contralateral responses may be explained by differences in the air puff duration and pressure settings (see experimental procedures), striatal coordinates (medial - lateral or rostro - caudal axes), and species (rat and mouse), but it may also be attributed to differences between intracellular sharp and whole - cell recordings (staley., 1992). neurons in both dorsolateral and dorsomedial striatum responded to bilateral whisker stimulation ; however, these responses differed in several aspects. response amplitudes and slopes were larger in dorsolateral striatum (figures 4f4i) ; however, onset latencies were similar between the two regions, suggesting that both receive monosynaptic inputs from s1. these results together with the anatomical results (figure 1b) suggest that the primary target receiving whisker information is indeed the dorsolateral striatum. a larger striatal area, however, receives the sensory input generating an attenuated and slower response, which, in the case of the dorsomedial striatum, also receives sensory information from a different modality (figures 4a4e). these differences in sensory input to dorsolateral and dorsomedial striatal regions may underlie the differences in their discharge pattern as recorded during task performance (thorn., 2010). sensory - evoked responses in striatal neurons in all tested cases were composed of excitatory and inhibitory components (figure 2). inhibition followed excitation by a few milliseconds, suggesting that it was mediated by intrastriatal gabaergic neurons driven by the same excitatory input. although the inhibitory component was smaller in amplitude than the excitatory input, it could be strong enough to shape striatal output by preventing or delaying msn discharge (kos and tepper, 1999). in vivo studies in neocortex have shown that visual and tactile sensory input induces temporally complex inhibitory inputs mediated by gabaergic interneurons (haider., 2013 ; monier., 2003 is mediated by gabaergic interneurons, in the striatum at least part of the inhibitory component may arise from msn collaterals (tunstall., 2002), in addition to that from gabaergic interneurons. it is not known which interneurons provide the observed inhibition onto msns ; however, likely candidates are parvalbumin - expressing fs interneurons (gerfen., 1985)., 2010 ; kos and tepper, 1999 ; planert., 2010) and are the first neurons to be activated by cortical input, even before neighboring msns (mallet., 2005). a similar form of feedforward inhibition from fs interneurons onto projection neurons exists in the thalamocortical pathway, where an early activation of fs interneurons by thalamic synaptic input provides rapid disynaptic inhibition of excitatory neurons (cruikshank., 2007). inhibition was also present during ongoing activity, in particular during up states (figures 2a and s4), as reported also in cortical up states (haider. this form of inhibition is likely to originate from striatal neurons that are active during up states ; however, the identity of these neurons is not clear. in our recordings, only a small fraction of msns discharged spontaneous action potentials, whereas the small sample of recorded fs interneurons were more spontaneously active (figure s7). striatal inhibition may also arise from external sources such as neurons in globus pallidus, which increase their discharge rate during striatal up states (goldberg., 2003). a particularly robust pallidostriatal inhibitory pathway is mediated by the recently described arkypallidal neurons (mallet., 2012). the source of inhibition during both spontaneous activity and sensory - evoked responses should be addressed in future studies using cell - type - specific manipulations. visual responses were seen in neurons recorded in the dorsomedial striatum in a relatively large yet restricted striatal region that also received axonal projections from visual cortex, thus suggesting that at least part of the response was mediated by excitatory projections from visual cortex (figure 3). visually evoked responses were recently described in neurons from rat dorsal striatum, mediated mainly by subcortical inputs following disinhibition of superior colliculus (schulz. recorded neurons were located more laterally than in the present study, which together with the different species and anesthesia may explain the different visual responsiveness. as in the current study, responses were almost entirely subthreshold and had similar latencies (schulz., 2009), suggesting that the visual responses in our recordings may originate from multiple afferent pathways, both cortical and subcortical. multisensory responses have been described in different basal ganglia nuclei including the striatum using intracellular, extracellular, and optical recordings (chudler., 1995 ; cui., 2013 ; hikosaka., 1989 ; nagy., 2005, 2006 ; wilson., 1983). those studies show that a fraction of striatal neurons changed their discharge rate when presented with sensory input of different modalities, mainly somatosensory and auditory. our findings support these studies and describe the synaptic input underlying such cross modal interactions. the striatal neurons we recorded in dorsal striatum showed clear preference for whisker stimulation, while only a fraction of them responded to both tactile and visual input, depending on their topographic location (figure 4c). responses to whisker stimulation were significantly earlier than those to visual stimulation (figure 4). in the neocortex, whisker responses also show shorter latencies (manns., 2004) the longer latencies in visual responses are in part attributed to retinal processing and may functionally be compensated via retinal motion prediction (berry., 1999). another possible explanation may lie in the nature of these stimuli, with tactile stimulation originating from nearby objects, whereas visual input would originate from more distal objects, before touching the whiskers. these differences in processing time between visual and tactile stimuli suggest that introducing a time lag between tactile and visual stimuli would result in increased responsiveness in the striatum, as observed in cortical multisensory areas (olcese., 2013). in agreement with the cortical studies, we showed that maximal response amplitude occurred when visual and whisker inputs simultaneously impinged onto postsynaptic msns. in the current study we only studied visual and tactile sensory integration ; however, the striatum also integrates other sensory modalities such as auditory (bordi and ledoux, 1992) and olfactory (mcdonald, 1991 ; novejarque., we obtained recordings under anesthesia, enabling the selective activation of sensory pathways, without contamination by motor related interactions. further research addressing multisensory information should also consider other sensory modalities as well as the effects of anesthesia, brain - state, and motor activity on sensory integration (haider. we recorded from several neuronal subtypes and observed differences in their spontaneous activity and response to sensory stimuli. differences in input resistance between direct and indirect pathway msns have been observed in slices (gertler., 2008 ; we found similar results in vivo, showing higher input resistance in d2 msns, which supports the higher excitability and activity rate of this subpopulation (cui. bilateral cortical input has been reported for both direct and indirect pathway msns ; however, there is a debate regarding the bias in target preference for the different cell types (kress., 2013 ; lei., 2004 ; reiner., 2010 ; wall., the difference we observed in the bilateral integration between d1 and d2 msns may reflect differences between the afferent corticostriatal pathways to the two populations. in this case, our results would support the findings describing ipsilateral as well as contralateral corticostriatal projection onto both d1 and d2 msns and a stronger ipsilateral corticostriatal projection to d1 than d2 msns (kress., another explanation may lay in the intrinsic properties of msns subpopulations, in particular the increased excitability of d2 msns and their calcium - mediated dendritic depolarization, which were shown to be different from d1 msns (day., 2008). although our data set is too small to enable a quantitative characterization, a few observations are important to note. all interneurons displayed the slow wave oscillations as was the case for msns and cortical pyramidal cells. as seen in figures s4 and s7, interneurons differed in the oscillation amplitudes and shape, suggesting different connectivity patterns conveying the afferent inputs to these neurons. all interneurons responded to bilateral whisker stimulation, and in a single fs interneuron tested for visual stimulation, such responses were indeed observed, showing that fs interneurons as well as msns perform multisensory integration. further studies should elucidate the functional role of the different interneuron types in sensory integration using the large and growing arsenal of molecular tools (fenno., 2011). all experiments were performed according to the guidelines of the stockholm municipal committee for animal experiments. adult c57bl6 mice of both sexes between 2 and 6 months of age were used to perform the experiments (n = 92). anesthesia was induced by intraperitoneal injection of ketamine (75 mg / kg) and medetomidine (1 mg / kg) diluted in 0.9% nacl. temperature was maintained between 36c37.5c using a feedback - controlled heating pad (fhc inc.). craniotomies were made at five sites for patch - clamp and extracellular recordings : ap 0 mm from bregma, l 2.5 mm (dorsomedial striatum) ; ap 0 mm from bregma, l 3.75 mm (dorsolateral striatum) ; ap 1.5 mm, l 3.25 mm (s1) ; ap 1.5 mm, l 2.0 mm (m1) ; ap 3.5 mm, l 2.5 mm (v1) (following paxinos and franklin, 2001). whole - cell recordings were obtained from dorsolateral striatum between 1,8542,613 m deep and in layer v of cortical barrel field between 617863 m from the pia, in a perpendicular penetration angle. signals were amplified using multiclamp 700b amplifier (molecular devices) and digitized at 20 khz with a ced acquisition board and spike 2 software (cambridge electronic design). patch pipettes were pulled with a flaming / brown micropipette puller p-87 (sutter instruments) and had an initial resistance of 512 m. extracellular recordings were obtained using tungsten electrodes with impedances of 1 to 2 m. the electrodes were placed in infragranular layers in somatosensory (bf), motor (m1), and visual (v1) cortex with an angle between 15 and 25. recordings were amplified using a differential ac amplifier model 1700 (a - m systems) and digitized at 20 khz with ced and spike-2 simultaneously with the whole - cell recording. whisker stimulation was obtained by brief air puffs delivered by a picospritzer unit (picospritzer iii, parker hannifin) via 1 mm diameter plastic tubes placed at 20 mm in front of the whiskers of both sides. air puffs (15 ms duration) were given at least 40 times for each stimulus condition (ipsilateral, contralateral, or bilateral stimulation) in a random order, with 5 s of interstimulus interval. visual stimulation was delivered by a white led positioned 50 mm from the contralateral eye. stimulus duration was 10 ms and was delivered with interstimulus intervals of at least 5 s. the eye was covered with vaseline in order to prevent drying, as previously described (holtmaat., 2009). visual responses were confirmed by monitoring the activation of the contralateral visual cortex using extracellular recordings (figure 3). tracer injections were made using glass pipettes (borosilicate, od = 1.5 mm, i d = 1.18 mm) with a tip diameter of 510 m. a total of 150250 nl of bda 10% (10,000 mw lysine - fixable biotin dextran amine, molecular probes) was dissolved in 0.9% nacl and fast green (to aid visualization of the injected tracer). a single injection was done for each cortical area and animal using the coordinates described above, as taken from paxinos and franklin (2001). three to six days following injections, animals were transcardially perfused with a solution containing 4% formalin and 14% saturated picric acid dissolved in 0.1 m phosphate buffer (pb) (ph 7.4). coronal slices (20 m thick) of both hemispheres containing the entire striatum (from ap 1.7 mm to ap 2.3 mm, following paxinos and franklin, 2001) were obtained using a cryostat and collected on gelatin - coated slides. sections were incubated over night with cy3-conjugated streptavidin (jackson immunoresearch laboratories) and neurotrace 500/525 green fluorescent nissl stain (invitrogen) diluted (1:1,000) in 1% bsa, 0.3% triton x-100 in 0.1 m pb. at the end of each electrophysiological experiment the mouse was perfused and the brain was placed in fixative solution for 1 to 2 hr in order to stain the neurobiotin - filled neurons (same procedures and solution described above). sections (1012 m thick) mounted on gelatin - coated slides were incubated overnight with cy2-conjugated streptavidin (jackson immunoresearch laboratories) diluted (1:500) in 1% bsa, 0.3% triton x-100 in 0.1 m pb. in between all experimental procedures, slices were washed with 0.01 m pbs. the shortest recording duration for a stained neuron was 24 min, and the average was 55.44 17.87 min (n = 45). reconstructed striatal neurons were immunolabeled for the detection of d1 dopamine receptors, where we found msns that clearly expressed d1 or not (d1 n = 15 ; d2 n = 13). primary and secondary antibodies were diluted in 1% bsa, 0.3% triton x-100 in 0.01 m pbs. we used fluorescent and confocal microscopy to recognize the msn receptor expression (d1). in order to control for the efficacy of the d1 receptor expression described above, we stained slices from d2 egfp mice, showing that d2 expressing neurons were not stained by the antibody (figure s5). | summarythe basal ganglia are involved in sensorimotor functions and action selection, both of which require the integration of sensory information. in order to determine how such sensory inputs are integrated, we obtained whole - cell recordings in mouse dorsal striatum during presentation of tactile and visual stimuli. all recorded neurons responded to bilateral whisker stimulation, and a subpopulation also responded to visual stimulation. neurons responding to both visual and tactile stimuli were located in dorsomedial striatum, whereas those responding only to whisker deflections were located dorsolaterally. responses were mediated by overlapping excitation and inhibition, with excitation onset preceding that of inhibition by several milliseconds. responses differed according to the type of neuron, with direct pathway msns having larger responses and longer latencies between ipsilateral and contralateral responses than indirect pathway msns. our results suggest that striatum acts as a sensory hub with specialized functional roles for the different neuron types. |
renal transplantation prolongs survival and improves quality of life for most patients who require renal replacement therapy.. epstein - barr virus (ebv), cytomegalovirus (cmv) and varicella zoster virus (vzv) can all cause serious illnesses in transplanted patients. the spectrum of illness caused by ebv ranges from an acute infectious mononucleosis - like illness to a highly malignant b cell tumour. we describe a patient who developed a lymphoma after her second transplant in whom long - term remission has been achieved by reduction in immunosuppression and the use of rituximab, a monoclonal antibody with activity against b lymphocytes. this prompted us to test for susceptibility to ebv infection in the scottish adult renal transplant pool. a 38-year - old woman presented with left leg, tiredness, sore throat and sweats, 16 months after a second cadaveric renal graft. positive anti - vca igm and negative ebna igg supported a diagnosis of glandular fever. her underlying diagnosis was focal segmental glomerulosclerosis, an early recurrence of which had led to the loss of her first graft. her second transplant was perfectly matched, but highly sensitized, so she had been given basiliximab as induction therapy followed by prednisolone, tacrolimus and mycophenolate. imaging by ultrasound and ct showed a 6 cm soft tissue mass inferior to the transplanted kidney, encircling the femoral vessels. a diagnosis of monoclonal polymorphic high - grade non - hodgkin 's lymphoma was made by a ct - guided biopsy (figure 1). the cells in this tumour were confirmed immunohistochemically as lymphocytes of b - cell origin by their cd20 and cd79a positivity. in situ withdrawal of tacrolimus and mycophenolate followed by infusion of rituximab 375 mg / m once weekly for 4 weeks led to a significant reduction in tumour size. when last seen at the clinic 6 years after her initial presentation with post - transplant lymphoproliferative disorder (ptld), serum creatinine was 137 mol / l with the estimated gfr of 38 mls / min and the urine protein : creatinine ratio of 86.5 mg / mmol. this patient 's case prompted us to test for susceptibility to ebv infection in the scottish adult renal transplant pool. we obtained a list of patients who were active on the renal transplant waiting list in july 2007 through the scottish renal registry and uk transplant, and then tested their most recent stored blood for ebv igg viral capsid antigen and cmv igg vca if not already known. nine (1.8%) of these were ebv igg vca negative and one was equivocal. seven (78%) of the nine patients who were ebv - negative were also cmv negative. our survey showed that 1.8% of scottish patients awaiting renal transplantation are susceptible to ebv infection, and therefore, at risk of ptld. this is comparable to population studies showing ebv seronegativity in up to 5% of european adults and also to a small canadian survey showing 2 ebv seronegative patients amongst 40 adult transplant recipients (5%). the main risk factors for the disease are ebv seronegativity and the degree of immunosuppression. ptld is more common in children than in adults because more children are seronegative, and therefore, susceptible to primary ebv infection at the time of transplantation. the incidence of ptld has increased following the introduction of ciclosporin, tacrolimus and newer immunosuppressive agents such as okt3. the risk of ptld is also 4-fold greater in ebv - negative recipients if they are cmv negative. this is either because cmv acts as a cofactor in the development of ptld or could simply reflect the level of immunosuppression. milder forms of the disease may respond simply to a reduction in immunosuppression although there is no consensus on which drugs to target first [35 ]. some recommend cutting the dose of calcineurin inhibitors by half and stopping antimetabolite drugs while continuing prednisolone at < 10 mg / day. patients with more severe forms of ptld are unlikely to respond to a reduction in immunosuppressive therapy alone. previously, chemotherapy and radiotherapy were used with variable results, but recently it has been shown that treatment with rituximab 375 mg / m by once weekly infusion for 4 weeks may induce complete remission. chemotherapy should now be reserved for patients not responding to antibody treatment. despite these advances in therapy, outcome studies suggest a 5-year patient survival of only 51.4% from time of transplantation in renal patients who develop ptld. what then can be done to prevent the emergence of ptld in high risk (donor ebv positive, recipient ebv negative) patients ? serial ebv monitoring, tailoring of immunosuppression and antiviral prophylaxis have all been reported to reduce the incidence of ptld. the american society of transplantation nevertheless recommended in 2006 that donor and recipient ebv status should be ascertained prior to kidney transplantation and that ebv viral load should be checked monthly for at least 1 year thereafter in patients who are ebv seronegative. the purpose of this is to allow early detection of first - time ebv infections. similar recommendations were made for checking cmv status. in patients at risk by virtue of their ebv seronegativity, reducing the overall burden of immunosuppression and the strong association between ebv infection and ptld risk suggests a possible prophylactic role for antiviral therapies, though this remains controversial as these drugs affect replicating viruses only. in ptld the incidence of ptld is too low to test antiviral therapies by randomized trial, but a large multicentre, case - control study has suggested up to 83% reduction in risk of ptld depending on the antiviral agent. might also be achieved through the use of immunization against ebv prior to the commencement of immunosuppression. a vaccine using the gp350 ebv envelope protein is currently in phase i / ii clinical trials in the uk. | we report a case of high - grade non - hodgkin 's lymphoma following epstein - barr virus (ebv) infection in a 38-year - old renal transplant recipient who was successfully treated with rituximab and remains alive 6 years later with reasonable graft function. we subsequently undertook a survey showing that 1.8% of the scottish adult transplant pool are susceptible to ebv infection. though a vaccine for ebv is currently not yet available, routine screening of potential renal transplant recipients for ebv should help identify those at increased risk of post - transplant lymhoproliferative disorder (ptld), while tailoring of immunosuppression and antiviral prophylaxis with ganciclovir may help reduce the emergence of this potentially life - threatening disease. |
the synthesis of -amino acids has been a subject of great interest and importance for quite some time but especially because it was discovered that -peptides derived from -amino acids have many of the properties of -peptides but are much more proteolytically stable. there has been a decided uptick in the efforts to develop catalytic asymmetric methods for the synthesis of -amino acids in the past decade. our interest in this area follows from our experiences in the development of the catalytic asymmetric synthesis of aziridines. we have found that aziridines can be prepared with a high degree of enantio- and diastereoselection by a three - component coupling of an aldehyde, an amine, and ethyl diazoacetate under the aegis of a borox catalyst (scheme 1). high yields of aziridine-2-carboxylates can be realized starting with aryl, alkyl, or alkynyl aldehydes with a typical selectivity for the cis isomer of 50:1. the enantioselection can depend on the nature of the amine substitutent or on the nature of the ligand ; with the right combination, a minimum of 96% ee can be obtained with aryl, alkynyl, and first, second, and third degree aliphatic aldehydes. the diastereoselection for the aziridine can be switched to trans with the use of a diazoacetamide. the purpose of the present work is to explore the reductive opening of cis - aziridine-2-carboxylates, with the goal of directing opening at the c-2 position to provide for an efficient and highly stereoselective catalytic asymmetric route to -amino esters. a number of methods are known for the reductive opening of aziridine-2-carboxylates to give -amino esters. the nature of the reducing agent can be quite critical when it comes to aziridines with an aryl group in the 3-position. as illustrated in scheme 2, such aziridines are prone to reductive opening, resulting in -amino esters by hydrogenolysis or lewis or brnsted acid - mediated reduction. however, this proclivity for reduction can be reversed by using electron - transfer reduction methods ; because the electron preferentially adds to the carbonyl function, it directs ring opening to the 2-position, resulting in -amino esters. interestingly, the use of manganese(0) to reduce 3-arylaziridine-2-carboxamides occurs with opening at the 3-position to give -amino amides, whereas the same substrates are reduced to -amino amides by samarium diiodide. in a non - electron - transfer process, tributyltin hydride is known to open aziridinyl ketones to give -amino ketones in a radical process. in a distinct and mechanistically different reaction, the addition of silyl anions to 2-aziridinyl ketones leads to -amino ketones via an addition, brook rearrangement, and anionic induced - ring opening. whereas 3-arylaziridine-2-carboxylates can be reductively opened to -amino esters with either samarium diiodide or magnesium(0), all examples in the literature are with trans isomers of the aziridine. because the borox catalyst produces very high selectivities for cis - aziridines, it became imperative to determine if the same regioselectivities observed in the reductive opening of trans - aziridines would translate to cis - aziridines. we also began our studies with fmoc aziridines because this would be the most desirable n - substituent for the purposes of solid - state synthesis of -peptides. fmoc aziridine cis-11a has a phenyl group in the 3-position cis to the ethyl carboxylate, and its reductive ring opening was examined with both magnesium in methanol and samarium diiodide in the presence of n, n - dimethylethanol amine (dmea). the purpose of dmea is to sequester the samarium(iii) that formed and prevent it from opening the aziridine as a lewis acid giving -amino ester product 14a. the reduction with magnesium did not occur under the reported conditions (23 c, fmoc aziridines were not included in the literature study), and high conversion was realized only after prolonged heating at 55 c. however, no -cleavage product 12a, -cleavage product 14a, or c c cleavage product 13a was observed in the crude reaction mixture. the reduction of cis-11a with samarium diiodide was performed under the reported conditions indicated in scheme 3, and the result was that both -cleavage product 12a and c c cleavage product 13a were formed in substantial amounts. the reductive ring opening of cis-11a could be brought to completion with 4 equiv of smi2 and 8 equiv of dmea in thf in 1 h at 0 c (table 1, entry 1). this reaction resulted in the isolation of -cleavage product 12a in 45% yield and c c cleavage product 13a in 46% yield. upon examining the reductive ring opening of the corresponding trans - aziridine trans-11a under the optimized conditions, it became clear that the product distribution greatly depends on the stereochemistry of the aziridine. the cis - aziridine 11a gives a 1:1 mixture of 12 and 13, whereas the trans - aziridine 11a gives a 16.7:1 mixture of 12 and 13 (table 1, entries 1 and 3, respectively). this was not the case with aziridines bearing an alkyl group in the 3-position. both the cis and trans isomers of 3-cyclohexyl aziridine 11b gave exclusive opening at the -position and a highly selective formation of -amino ester 12b (table 1, entries 2 and 4, respectively). unless otherwise specified, all reactions were run with 0.2 mmol of aziridine in thf (0.07 m) with 4 equiv of smi2 and 8 equiv of dmea at 0 c for 1 h and went to completion. yield from the h nmr spectrum of the crude reaction mixture with an internal standard. data previously reported in ref (18). reaction with 5.5 equiv of smi2 and 11 equiv of dmea. reaction with 6 equiv of smi2 and 12 equiv of dmea. in the search for a more general reductive ring - opening method for converting aziridines to -amino esters, a number of different n - protecting groups were examined., it was not surprising to find that the profile for the smi2-mediated reductive ring opening of the boc - protected aziridines closely matched that for the fmoc aziridines, with just slightly lower selectivities. the ring opening of phenyl - substituted cis - aziridine was not selective (1.4:1, table 2, entry 1). phenyl - substituted trans - aziridine 15a was more selective at 6.7:1 (entry 3), and both isomers of cyclohexyl - substituted aziridine 15b were highly selective (table 2, entries 2 and 4). clearly, the most felicitous n - protecting group, with regard to the selectivity of reductive ring opening by samarium diiodide, is the tosyl group. the profile observed is flat, with > 99:1 selectivity for the -amino ester ; cis- and trans - aziridines and phenyl- and cyclohexyl - substituted aziridines were all in very high yields (table 2, entries 58). the ses (trimethylsilyl ethyl sulfonyl) group is an attractive activating group for an amino function because it is easier to remove than tosyl and has excellent selectivity for the -amino ester 21a, as seen with cis - aziridine 17a (23:1, table 2, entry 9). the slightly lower selectivity of the ses group, as compared to that of the tosyl group (table 2, entries 5 and 9, respectively), could be expected for an alkyl sulfonate compared to an aryl sulfonate (vide infra). it was also found that the n - acetyl group is capable of delivering high selectivity for -amino ester 22a over the c c cleavage product in the ring opening of cis - phenyl aziridine cis-18a. however, the isolated yield of -amino ester 22a was only moderate ; the reaction occurs with the formation of 13% of the -amino ester corresponding to 14 in scheme 3 (table 2, entry 10). the latter may result from initial electron transfer to the amide carbonyl followed by the ring opening to a benzyl radical (or anion, vide infra). unless otherwise specified, all reactions were run with 0.2 mmol of aziridine in thf (0.07 m) with 6 equiv of smi2 and 12 equiv of dmea at 0 c for 1 h and were allowed to go to completion. determined from the h nmr spectrum of the crude reaction mixture. yield from the h nmr spectrum of the crude reaction mixture with an internal standard. reaction with 5 equiv of smi2 and 10 equiv of dmea. a small amount (6%) of ses - protected benzylamine was also observed. reaction with 4 equiv of smi2 and 8 equiv of dmea. the ring - opening product from n c3 cleavage to give an -amino ester was obtained in 13% isolated yield. because this is the class of aziridines that the borox catalysts are most efficient at producing, the reductive ring opening of unactivated aziridines by samarium diiodide would be a very useful reaction (scheme 1). kumamoto and coworkers examined the samarium diiodide - mediated ring opening of trans - aziridine-2-carboxylates with an aryl group in the 3-position and a benzyl group on the aziridine nitrogen and found that -amino esters could be obtained only in very low yields. the present work finds that if samarium diiodide is generated from samarium metal and iodine instead of methylene iodide, it results in the isomerization of trans - aziridine to a mixture of cis- and trans - aziridines. there are no other examples of the reductive ring opening of aziridines bearing an alkyl group on the nitrogen with samarium diiodide in which the aziridines have a carbonyl group in the 2-position and either an aryl or alkyl substituent in the 3-position. on the basis of this, we decided to probe the first examples of the reductive ring opening of unactivated cis - aziridine-2-carboxylates with samarium diiodide (table 3). the samarium diiodide used in these studies was prepared from samarium metal and methylene iodide, and no isomerization of the aziridine was observed. if the substituent in the 3-position is a phenyl group, then the only product that is observed is the c c cleavage product. h aziridines, as well as with benzhydryl substituents on the aziridine nitrogen (table 3, entries 1 and 2). a complete switch in the product distribution is seen with cis - aziridines bearing a cyclohexyl group in the 3-position. here c cleavage product ; the yields, however, are quite low (table 3, entries 3 and 4). these reactions produce a complex mixture from which only -amino ester 30b, starting cis - aziridine 29b, and benzhydryl amine 34 could be isolated and characterized. the isolation of 34 suggests that -cyclohexyl ethyl acrylate should also be formed, but it could not be detected in the h nmr spectrum of the crude reaction mixture. unless otherwise specified, all reactions used 0.2 mmol of aziridine in thf (0.07 m) with 5 equiv of smi2 and 10 equiv of dmea at 0 c for 40 min and were allowed to go to completion. determined from the h nmr spectrum of the crude reaction mixture. isolated as a 1:1.4 mixture of 30b and cis-29b (22% + 31%). amine 34 was isolated in 52% yield, and aziridine cis-29b was isolated in 20% yield. the reductive ring opening of stereoisomerically pure trisubstituted aziridine 35(20) occurs with loss of stereochemical information and the formation of two diastereomers in a ratio of 4.8:1 (scheme 4). the major diastereomer was identified as the anti isomer of 36 by chemical correlation to a known compound (supporting information). this loss of stereochemistry is to be expected on the basis of the likely mechanism for this reaction (scheme 5). the ring opening of 35 also occurs with the formation of c c cleavage product 37 in 30% yield. note that the distribution between c n and c c cleavage products is essentially the same as for cis disubstituted n - boc aziridine cis-15a (table 2, entry 1). the generally accepted mechanism for the reductive ring opening of aziridines by samarium diiodide is illustrated in scheme 5. after initial reduction to form ketyl 39 and in the absence of a proton source, this species then undergoes a ring opening to give nitrogen - based radical 40 that, upon further reduction, gives the species 41, containing both a samarium enolate and a samarium amide. the intermediacy of this enolate has been demonstrated, and its utility has been displayed in its alkylation with alkyl halides and aldol reactions with aldehydes. under conditions where a proton source is present, ketyl 39 is thought to be protonated to give neutral radical 43 that, depending on the nature of the aziridine, then undergoes a c subsequent reduction of the resulting nitrogen- or carbon - based radicals 44 and 47 and final protonation would provide -amino ester 46 and/or glycine derivative 49. this mechanistic interpretation accounts for the observations made in the present work. of all of the examples in tables 1 and 2, the aziridines with a cyclohexyl group in the 3-position give a much higher selectivity for the -amino ester over the glycine derivative than do aziridines with a phenyl group in the 3-position. n cleavage for the phenyl aziridines than for the cyclohexyl aziridines, which can be attributed to the greater stability of a benzyl radical compared to an alkyl radical in intermediate 47. conversely, the presence of a radical stabilizing group on the nitrogen facilitates the ring opening with c n bond scission over c c bond scission. this can be seen in the ring - opening reactions of activated aziridines (tables 1 and 2) versus unactivated aziridines (table 3). this is illustrated by the comparison of n - tosyl aziridine cis-16a (table 2, entry 5) to n - benzhydryl aziridine cis-29a (table 3, entry 3) and by the comparison of n - tosyl aziridine cis-16a (table 2, entry 5) to n - boc aziridine cis-15a (table 2, entry 1). the ratio of c n versus c c cleavage is a function not only of the radical stabilizing ability of the substituent on the nitrogen and the substituent on the c-3 position of the aziridine but also of the stereochemistry of the aziridine. the cis - aziridines exhibit a much greater preference for c c bond cleavage than do the trans - aziridines. n - boc aziridine trans-15a gives a 6.7:1 mixture of c n to c c cleavage products, whereas the corresponding cis-15a gives a much greater propensity for the c c cleavage product (1.4:1, table 2, entries 1 and 3, respectively). the same is also true for n - fmoc aziridines cis-11a and trans-11a (table 1, entries 1 and 3, respectively). the greater preference for c c cleavage products with cis - aziridines may be due to a relief in steric interactions between the two cis - substituents in the transition state, allowing the c c bond to begin to lengthen (scheme 6). it is intersting that this relief in steric interaction would not be realized in the trans - aziridine. c cleavage products have been rarely seen in the reductive ring opening of aziridines involving single electron - transfer processes ; this may be due to the fact that cis - aziridines have not been previously evaluated in this reaction. the only example that we are aware of involves an unactivated (n h) aziridine-2-carboxylate with a trans - phenyl group in the 3-position, which gives a 28:16 split between c n and c c cleavage products, as compared to aziridine cis-27a, which gives exclusively the c c cleavage product in 75% yield (table 3, entry 1). from the synthesis point of view, the n - tosyl group is the protecting group of choice for samarium diiodide - mediated reductive ring opening of aziridines if removal of the tosyl group does not cause problems in a later stage. n - tosyl aziridines are completely selective for the c n cleavage product (> 99:1) for both the cis- and trans - aziridines, with both aryl and alkyl substituents in the 3-position (table 2, entries 58). the ses protecting group can be considered to be an alternative to a tosyl sulfonamide, which is notorious for its potential to be troublesome during deprotection. ses - protected aziridine cis-17a gave excellent selectivity (23:1, table 2) for the -amino ester, and the deprotection of ses is known to proceed under much milder reaction conditions. the utility of aziridine-2-carboxylates in the preparation of both - and -amino acids is illustrated in scheme 7, which shows how the same aziridine provides access to both l - dopa and (r)--dopa. l - dopa is the biological precursor to the catecholamine neurotransmitters, a treatment of parkinson s disease, and a key compound in the formation of marine - adhesive proteins.l - dopa became the first commercial pharmaceutical agent to be manufactured by a nonproteinaceous asymmetric catalyst, which was acknowledged in the 2001 nobel prize in chemistry to william s. knowles. the isomeric (r)--dopa has been isolated as an iron(iii) complex from a dark - blue - violet mushroom of the species cortinarius violaceus. both natural products could potentially be obtained from the reductive opening of the same aziridine via controlled reductive ring opening at the c-2 and c-3 positions. of the many synthesis methods of l - dopa and (r)--dopa, no other method can provide access to both isomers from the same asymmetric strategy. our first approach to the synthesis of l - dopa and (r)--dopa began with bis - acetoxy aziridine 54, which was prepared in one step in 98% yield and > 98.5% ee from aldehyde 50, amine 52, and ethyl diazoacetate 53 by a catalytic, asymmetric, multicomponent aziridination with 5 mol % vapol borox catalyst (scheme 8). to set the stage for the regiocomplementary reductive ring opening of aziridine 54, the resulting n h aziridine was not purified but rather directly protected by tscl or sescl to give n - protected aziridines 57 and 58 in 70 and 78% yield, respectively, for the two steps. the samarium diiodide - mediated ring opening of both tosyl aziridine 57 and ses - aziridine 58 gave complex mixtures because of the cleavage of zero, one, or two of the acetoxy groups on the benzene ring. by treating the crude mixtures with acetic anhydride in the presence of triethylamine, -amino esters 61a and 62a were isolated in good yields, although an increase in the yield of the c c cleavage product was observed in both cases compared to the corresponding phenyl - substituted aziridines cis-16a and cis-17a (table 2). this may be due to the electronic effect of the two acetate groups on the benzene ring. despite the effort that was expended to investigate the removal of the tosyl group following many of the standard procedures, 61a could not be deprotected without decomposition. before exploring of ses - deprotection of 62a, we turned our attention to the acetate cleavage problem in the samarium diiodide ring - opening process. this was solved by replacing the acetate groups with the bulky tert - butyl acyloxy (pivaloyl) groups. the catalytic asymmetric multicomponent aziridination worked smoothly with aldehyde 51 and afforded aziridine 55 in 97% yield and 98% ee. interestingly, the medam group could be removed with trifluoroacetic acid in anisole without the cleavage of the pivaloyl groups and then directly reacting the n the formation of -amino ester 63a was smoothly achieved (74% yield) from the reductive ring - opening reaction with no detection of cleavage of the pivolyl groups. the first attempt to remove the ses group from the amine function in 63a following a literature procedure involving heating with csf in dmf at 95 c resulted in the formation of only ethyl 3,4-dihydroxycinnamate 65 in 65% yield (nmr yield). this is probably due to fluoride - mediated deprotonation at the -position of the carbonyl, causing an elimination of the ses - amino group. alternatively, 65 could result from a fluoride - mediated cleavage of the pivaloyl group, followed by a phenoxide - assisted elimination of the ses - amino group and a final rearomatization. it has been previously reported that the elimination of the ses group from ses - protected -amino carbonyl compounds can be a problem during the ses deprotection step. however, there is no example reported for the elimination of the ses - nh2 group from a -amino ester during ses deprotection. it is known that n - acyl - substituted ses groups are much more readily deprotected than simple ses groups. on the basis of the latter, we carried out the acylation of 63a by treating it with (boc)2o and subsequently treating the crude mixture with tbaf in thf at 25 c for 1.5 h to afford the desired -amino ester 64, the protected form of (r)--dopa, in 88% isolated yield. there was some cleavage of the pivaloyl groups by tbaf, and thus a workup with pivaloyl chloride gives 64 as a pure compound. no purification was performed during any of the steps in the conversion of 63a to 64. finally, the reductive ring opening of aziridine 55 with palladium hydroxide - catalyzed hydrogenation in the presence of (boc)2o resulted in the formation of -amino ester 56, the protected form of l - dopa, in 86% isolated yield. the reductive ring opening of 3-substituted aziridine-2-carboxylates with samarium diiodide can be controlled to proceed via c it is necessary to have an activating group on the aziridine nitrogen to achieve selective c aziridines with nonactivating nitrogen substituents (hydrogen or benzhydryl) exclusively form glycine derivatives when there is a phenyl group in the 3-position and, when there is a cyclohexyl group in the 3-position, low yields of -amino esters and other decomposition products. n and c c bond cleavage directly correlates with the electron - withdrawing power of the activating group on the nitrogen. sulfonyl groups give higher selectivity than carbamate groups, and this is especially noticeable with cis - aziridines that have a phenyl group in the 3-position. the lower selectivity with cis - aziridines is thought to be due to a steric release during the c c bond cleavage, leading to glycine products. the utility of this methodology is illustrated in the synthesis of a protected form of (r)--dopa by the reductive opening of aziridine 59 with samarium diiodide to give -amino ester 63a. furthermore, this synthesis features the targeting of (r)--dopa and its regioisomer l - dopa by ring opening of the same aziridine, 55, the former by reductive opening at the c-2 position and the latter by reductive opening at the c-3 position. vapol was prepared according to literature procedures and was determined to be at least 99% optically pure. the preparation of aziridine esters cis-11a, b, cis-15a, b, cis-16a, b, cis-27a, trans-27a, cis-29a, b, and 35(20) has been previously reported. melting points were determined on a capillary melting - point apparatus and were uncorrected. h nmr and c nmr spectra were recorded on a 300, 500, or 600 mhz spectrometer in cdcl3, unless otherwise noted. chcl3 was used as the internal standard for both h nmr (= 7.24) and c nmr (= 77.0). analytical thin - layer chromatography (tlc) was performed on silica - gel plates with an f-254 indicator. visualization was by short wave (254 nm) and long wave (365 nm) ultraviolet light, by staining with phosphomolybdic acid in ethanol, or with the aid of iodine vapor. optical rotations were obtained on a polarimeter at a wavelength of 589 nm (sodium d line) using a 1.0 decimeter cell with a total volume of 1.0 ml. specific rotations are reported in degrees per decimeter at 20 c. to a 100 ml round - bottomed flask were added racemic trans-2-carboxyethyl-3-phenylaziridine 27a (0.296 g, 1.5 mmol, 1.0 equiv), nahco3 (0.252 g, 3.0 mmol, 2.0 equiv), 30 ml of a mixture of acetone, and h2o (3:1). the mixture was stirred at room temperature for 5 min, and then 9-fluorenylmethylchloroformate (0.388 g, 1.5 mmol, 1.0 equiv) was added. the reaction mixture was then stirred at room temperature for 48 h. the acetone was removed by rotary evaporation, and the aqueous residue was extracted with ethyl acetate (10 ml 3). the combined organic layer was washed with sat aq nacl, dried over mgso4, and concentrated by rotary evaporation to afford a yellow oil. purification by silica - gel chromatography (column 1 : 18 mm 250 mm, 4:1:1 hexanes / diethyl ether / ch2cl2 as eluent ; column 2 : 18 mm 250 mm, ch2cl2 as eluent) afforded trans-11a as a colorless oil in 72% isolated yield that solidified upon standing (white solid, mp 9395 c). spectral data for trans-11a : h nmr (500 mhz, cdcl3) 1.25 (t, 3h, j = 7.2 hz), 3.20 (d, 1h, j = 2.4 hz), 3.90 (d, 1h, j = 2.4 hz), 4.18 (q, 2h, j = 7.2 hz), 4.24 (t, 1h, j = 7.2 hz), 4.33 (dd, 1h, j = 10.5, 7.8 hz), 4.48 (dd, 1h, j = 10.5, 7.2 hz), 7.257.31 (m, 2h), 7.327.42 (m, 7h), 7.557.61 (m, 2h), 7.74 (d, 2h, j = 7.5 hz) ; c nmr (150 mhz, cdcl3) 14.0, 44.2, 45.1, 46.8, 62.2, 68.8, 119.96, 119.97, 125.2, 125.3, 126.4, 127.05, 127.06, 127.7, 127.8, 128.6, 128.7, 134.9, 141.25, 141.26, 143.6, 143.7, 159.7, 167.3 ; ir (thin film) 1744 (vs), 1179 (s) cm ; hrms (esi - tof) m / z : [m + h ] calcd for c26h24no4, 414.1705 ; found, 414.1731. the aridizine was prepared according to the procedure described above for trans-11a starting with racemic trans-2-carboxyethyl-3-cyclohexylaziridine (296 mg, 1.50 mmol). purification by silica - gel chromatography (15 mm 250 mm, 8:1 hexanes / etoac as eluent) afforded trans-11b as a colorless oil in 91% isolated yield (572 mg, 1.36 mmol). spectral data for trans-11b : rf = 0.28 (5:1 hexanes / etoac) ; h nmr (600 mhz, cdcl3) 1.161.28 (m, 6h), 1.22 (t, 3h, j = 7.2 hz), 1.621.68 (m, 1h), 1.691.78 (m, 3h), 1.81 (d, 1h, j = 12 hz), 2.66 (dd, 1h, j = 7.2, 2.7 hz), 2.96 (d, 1h, j = 2.7 hz), 4.084.16 (m, 2h), 4.22 (t, 1h, j = 6.9 hz), 4.32 (dd, 1h, j = 10.5, 7.5 hz), 4.46 (dd, 1h, j = 10.2, 7.2 hz), 7.277.31 (m, 2h), 7.38 (t, 2h, j = 7.5 hz), 7.60 (t, 2h, 1h, j = 7.8 hz), 7.74 (d, 2h, 1h, j = 7.8 hz) ; c nmr (150 mhz, cdcl3) 14.0, 25.5, 25.6, 26.0, 29.7, 29.9, 39.2, 39.4, 46.9, 49.0, 61.8, 68.3, 119.90, 119.91, 125.1, 125.2, 127.01, 127.03, 127.66, 127.70, 141.27, 141.29, 143.7, 143.8, 160.2, 168.3 ; ir (thin film) 2928 (s), 2853 (w), 1743 (vs), 1451 (m), 1310 (m), 1177 (s) cm ; mass spectrum, m / z (% rel intensity) 419 m (0.07), 346 (0.15), 178 (100), 165 (62), 84 (34), 49 (56) ; hrms (esi - tof) m / z : [m + h ] calcd for c26h30no4, 420.217 ; found, 420.216. to a flame - dried 25 ml round - bottomed flask filled with argon were added trans-2-carboxyethyl-3-phenylaziridine 27a(28) (150 mg, 0.784 mmol) and 5 ml of meoh followed by the addition of nahco3 (197 mg, 0.450 mmol, 3.00 equiv). the flask was put in an ultrasonic bath for 5 min, and then (boc)2o (428 mg, 1.96 mmol, 2.50 equiv) was added. the mixture was left in the ultrasonic bath for 4 h with a needle in the rubber septum to release the generated co2 gas. after 4 h, additional portions of both nahco3 and (boc)2o in equal amounts were added. the reaction mixture was filtered through celite, and the solid residue was washed with et2o. the cloudy filtrate was filtered again through celite, and then concentrated by rotary evaporation to afford the crude product as a colorless liquid. purification by silica - gel chromatography (15 mm 250 mm, hexanes and then 1:9 ethyl acetate / hexanes as eluent) afforded trans-15a as a colorless oil in 71% isolated yield (163 mg, 0.56 mmol). spectral data for trans-15a : rf = 0.36 (hexanes / etoac = 4:1) ; h nmr (300 mhz, cdcl3) 1.30 (t, 3h, j = 7.0 hz), 1.44 (s, 9h), 3.07 (d, 1h, j = 2.6 hz), 3.79 (d, 1h, j = 2.6 hz), 4.144.36 (m, 2h), 7.247.36 (m, 5h) ; c nmr (150 mhz, cdcl3) 14.16, 27.88, 44.02, 44.92, 61.82, 82.04, 126.43, 128.27, 128.48, 135.33, 158.29, 167.40. to a flame - dried 25 ml round - bottomed flask filled with argon were added racemic trans-2-carboxyethyl-3-cyclohexylaziridine (198 mg, 1.0 mmol, 1.0 equiv), meoh (6.5 ml), and nahco3 (0.84 g, 10 mmol, 10 equiv). the flask was put in an ultrasonic bath for 5 min, and then (boc)2o (1.09 g, 5.00 mmol, 5.00 equiv) was added. the mixture was left in the ultrasonic bath for 4 h with a needle in the rubber septum to release the generated co2 gas, and then it was stirred at room temperature for another 18 h. the reaction mixture was filtered through celite, and the filter cake was washed with diethyl ether. the cloudy filtrate was filtered again through celite and then concentrated by rotary evaporation to afford the crude product as a light - yellow liquid. purification by silica - gel chromatography (15 mm 250 mm, 45:1 hexanes / etoac as eluent until the first fraction came out and then 15:1 hexanes / etoac as eluent) afforded trans-15b as a colorless oil in 89% isolated yield (265 mg, 0.89 mmol). spectral data for trans-15b : rf = 0.42 (5:1 hexanes / etoac) ; h nmr (500 mhz, cdcl3) 1.041.25 (m, 6h), 1.28 (t, 3h, j = 7.2 hz), 1.43 (s, 9h), 1.601.65 (m, 1h), 1.661.76 (m, 3h), 1.801.86 (m, 1h), 2.60 (dd, 1h, j = 6.8, 3.0 hz), 2.83 (d, 1h, j = 3.0 hz), 4.114.28 (m, 2h) ; c nmr (150 mhz, cdcl3) 14.2, 25.5, 25.6, 26.1, 28.0, 29.7, 30.0, 39.3, 39.4, 48.7, 61.5, 81.5, 159.0, 168.4 ; ir (thin film) 2980 (w), 2930 (s), 2855 (w), 1744 (vs), 1728 (s), 1154 (s) cm ; mass spectrum, m / z (% rel intensity) 224 (m 73) (5.6), 196 (21), 124 (85), 95 (73), 57 (100) ; anal. calcd for c16h27no4 : c, 64.62 ; h, 9.15 ; n, 4.71. found : c, 64.88 ; h, 9.27 ; n, 4.72. to a flame - dried 50 ml round - bottomed flask filled with argon were added racemic trans-2-carboxyethyl-3-phenylaziridine 27a(28) (0.335 g, 1.75 mmol, 1.0 equiv) and ch2cl2 (14 ml, freshly distilled). the solution was cooled to 0 c in an ice bath followed by the addition of et3n (0.7 ml, 5.03 mmol, 2.9 equiv, freshly distilled). after the reaction mixture was stirred for 5 min at 0 c, tscl (0.534 g, 2.8 mmol, 1.6 equiv) was added to the mixture at 0 c. thereafter, the ice bath was removed and the mixture was stirred at room temperature for 94 h. the reaction was quenched by the addition of 26 ml of sat aq nh4cl and 5 ml of h2o. the organic layer was separated, and the aqueous layer was extracted with ch2cl2 (30 ml 3). the combined organic layer was washed with the following reagents in the indicated sequence : 5 ml of h2o, 10 ml of aq citric acid, 5 ml of h2o, 10 ml of sat aq nahco3, and 20 ml of sat aq nacl. the combined organic layer was dried over mgso4 and concentrated by rotary evaporation to afford the crude product as an orange oil. purification by silica - gel chromatography (column 1 : 15 mm 250 mm, 40:9 hexanes / etoac as eluent ; column 2 : 15 mm 250 mm, 16:4:1 hexanes / ch2cl2/etoac as eluent) afforded trans-16a as a light - yellowish oil in 54% yield (0.326 g, 0.94 mmol). spectral data for trans-16a : rf = 0.24 (4:1 hexanes / etoac) ; h nmr (300 mhz, cdcl3) 1.32 (t, 3h, j = 7.0 hz), 2.39 (s, 3h), 3.49 (d, 1h, j = 3.9 hz), 4.204.39 (m, 2h), 4.41 (d, 1h, j = 3.9 hz), 7.197.31 (m, 7h), 7.717.80 (m, 2h) ; c nmr (150 mhz, cdcl3) 14.0, 21.6, 47.1, 47.7, 62.4, 127.3, 127.5, 128.6, 128.9, 129.5, 132.7, 137.2, 144.2, 165.7. the h and c nmr data match those previously reported for this compound. according to the same procedure used for trans-16a, racemic trans-2-carboxyethyl-3-cyclohexylaziridine (197 mg, 1.0 mmol, 1.0 equiv) was reacted with tosyl chloride (0.305 g, 1.6 mmol, 1.6 equiv) and et3n (freshly distilled, 0.42 ml, 3.0 mmol, 3.0 equiv) in 1:1(v / v) ch2cl2/chcl3 (8 ml) for 72 h. purification by silica - gel chromatography (column 1 : 15 mm 250 mm, 10:1 hexanes / etoac ; column 2 : 15 mm 250 mm, 6:1:16 ch2cl2/diethyl ether / hexanes as eluent) and recrystallization from hexanes afforded trans-16b as white crystals (mp 7173 c) in 70% isolated yield (246 mg, 0.70 mmol). spectral data for trans-16b : rf = 0.26 (5:1 hexanes / etoac) ; h nmr (300 mhz, cdcl3) 1.001.35 (m, 8h), 1.581.79 (m, 5h), 1.95 (d, 1h, j = 10.5 hz), 2.41 (s, 3h), 3.02 (dd, 1h, j = 9.0, 4.2 hz), 3.18 (d, 1h, j = 4.2 hz), 4.15 (q, 2h, j = 6.9 hz), 7.247.32 (m, 2h), 7.797.83 (m, 2h) ; c nmr (150 mhz, cdcl3) 13.9, 21.6, 25.2, 25.4, 25.9, 30.6, 31.1, 37.5, 43.7, 53.7, 61.9, 127.5, 129.5, 137.1, 144.2, 166.7 ; ir (thin film) 2930 (s), 2855 (m), 1745 (s), 1331 (s), 1101 (s) cm ; mass spectrum, m / z (% rel intensity) 306 (m 45) (2.3), 278 (3.2), 197 (34), 196 (100), 168 (29), 122 (88), 67 (83) ; anal. calcd for c18h25no4s : c, 61.51 ; h, 7.17 ; n, 3.99. found : c, 61.60 ; h, 7.22 ; n, 3.96. to a flame - dried 10 ml round - bottomed flask were added 2-(trimethylsilyl)ethanesulfonyl chloride (82.5 l, 0.639 mmol, 1.28 equiv), et3n (0.7 ml, 5 mmol, 10 equiv), and ch2cl2 (2 ml, freshly distilled). cis-(2r,3r)-2-carboxyethyl-3-phenylaziridine 27a(18) (95.6 mg, 0.5 mmol, 1 equiv, 98% ee) was dissolved in 0.5 ml of ch2cl2, and the solution was added dropwise to the 10 ml round - bottomed flask containing 2-(trimethylsilyl)ethanesulfonyl chloride. the ice bath was removed, and the reaction mixture was stirred at room temperature for 45 h. subsequently, additional portions of 2-(trimethylsilyl)ethanesulfonyl chloride (160 l, 1.26 mmol, 2.5 equiv) and et3n (0.7 ml, 5 mmol, 10 equiv) were added to the reaction mixture. after the mixture was stirred for 24 h at room temperature, the reaction was quenched with 2 ml of sat aq nh4cl and 1 ml of h2o. the organic layer was separated, and the aqueous layer was extracted with ch2cl2 (4 ml 3). the combined organic layer was dried with na2so4, filtered, and concentrated to give a dark - brown oil. purification by silica - gel chromatography (15 mm 180 mm, 9:1 hexanes / etoac as eluent) afforded cis-17a as a light - yellow oil in 83% yield (0.147 g, 0.413 mmol). spectral data for cis-17a : rf = 0.19 (9:1 hexanes / etoac) ; h nmr (500 mhz, cdcl3) 0.04 (s, 9h), 1.00 (t, 3h, j = 7.0 hz), 1.131.24 (m, 2h), 3.163.26 (m, 2h), 3.67 (d, 1h, j = 7.5 hz), 3.934.05 (m, 2h), 4.07 (d, 1h, j = 7.5 hz), 7.267.34 (m, 3h), 7.377.41 (m, 2h) ; c nmr (125 mhz, cdcl3) 2.0, 9.7, 13.9, 43.7, 44.3, 49.5, 61.6, 127.5, 128.3, 128.7, 131.4, 164.5 ; ir (thin film) 2955 (s), 1755 (vs) cm ; hrms (esi - tof) m / z : [m + h ] calcd for c16h26no4ssi, 356.1352 ; found, 356.1351 ; []d20 = 38.0 (c 1.0, ch2cl2) on 98% ee material (the optical purity was assumed to be unchanged from 27a). to a 25 ml flame - dried round - bottomed flask filled with argon were added cis-(2r,3r)-2-carboxyethyl-3-phenylaziridine 27a(18) (38 mg, 0.20 mmol), 1 ml chcl3, et3n (90 l, 0.65 mmol), and acetic anhydride (29 l, 0.31 mmol) at room temperature. the mixture was stirred at room temperature for 4 h, and then the solvent was removed under reduced pressure to give the crude cis-(2r,3r)-18a product as a white solid. purification by silica - gel chromatography (9 mm 250 mm, 1:5 etoac / hexanes as eluent) gave 18a as a white solid in 100% yield (47 mg, 0.20 mmol). spectral data for cis-(2r,3r)-18a : rf = 0.35 (1:5, etoac / hexane) ; h nmr (cdcl3, 300 mhz) 0.92 (t, 3h, j = 7.1 hz), 2.20 (s, 3h), 3.47 (d, 1h, j = 6.6 hz), 3.81 (d, 1h, j = 6.6 hz), 3.853.97 (m, 2h), 7.247.31 (m, 3h), 7.357.39 (m, 2h) ; c nmr (cdcl3, 75 mhz) 13.7, 23.1, 42.0, 43.5, 61.1, 127.3, 128.0, 128.2, 132.5, 165.6, 181.13 ; ir (thin film) 1747 (s), 1713 (s) cm ; mass spectrum, m / z (% rel intensity) 233 m (0.09), 191 (14), 146 (11), 117 (100), 91 (18), 79 (10), 55 (12), 43 (44). anal. calcd for c13h15no3 : c, 66.94 ; h, 6.48 ; n, 6.00. found : c, 66.87 ; h, 6.85 ; n, 5.94. to a flame - dried 10 ml round - bottomed flask filled with argon were added sm (180 mg, 1.20 mmol, 6.00 equiv) and dry thf (1.8 ml, freshly distilled). the vacuum adapter on the flask was changed to a rubber septum, and the suspension was purged with nitrogen under the surface of the solution for 5 min by a needle through the septum. another needle in the septum was used as an outlet for the nitrogen gas. ch2i2 (92.5 l, 1.15 mmol, 5.7 equiv) was then added to the reaction flask, and the reaction mixture was purged with nitrogen for another 1 min. the outlet needle was removed, and the needle used for nitrogen flow was lifted above the surface of the solution. the reaction mixture was stirred at room temperature for 2 h, resulting in a dark - blue slurry. subsequently, to a flame - dried 5 ml round - bottomed flask filled with nitrogen were added cis-(2r,3r)-15a (78% ee, 60 mg, 0.2 mmol, 1.0 equiv), dry thf (1.5 ml, freshly distilled), and n, n - dimethylethanolamine (0.24 ml, 2.4 mmol, 12.0 equiv). the solution was purged with nitrogen under the surface of the solution for 2 min and transferred to the flask containing the smi2 slurry dropwise via cannula. vigorous stirring was maintained during the addition of the aziridine to the smi2 slurry. the 5 ml flask was washed with 0.3 ml of degassed thf, and the rinse was also transferred to the reaction flask containing smi2. the reaction mixture was stirred at 0 c for 40 min to 1 h and then quenched by the addition of sat aq nahco3 (5 ml) at 0 c. the organic layer was separated, and the aqueous layer was extracted with etoac (5 ml 4). the solvent was removed by rotary evaporation to give a light - yellow solid. the h nmr spectrum of the crude reaction mixture showed that it was a mixture of 19a and 23a in a ratio of 1.4:1. purification by silica - gel chromatography (18 250 mm, 1:1:4.6 diethyl ether / hexanes / ch2cl2 as eluent) afforded (s)-19a as a white solid (mp 7577 c) in 55% isolated yield (33.6 mg, 0.11 mmol) and 23a as a colorless oil in 32% isolated yield (19.2 mg, 0.065 mmol). the optical purity of (s)-19a was determined to be 78% ee by hplc analysis (chiralcel od - h column, 98:2 hexanes / iproh at 222 nm, flow rate : 1.0 ml / min) ; retention times of rt = 6.24 min (minor enantiomer (r)-19a) and rt = 7.08 min (major enantiomer (s)-19a). tlc and spectral data for (s)-19a : rf = 0.24 (5:1 hexanes / etoac) ; h nmr (cdcl3, 300 mhz) 1.14 (t, 3h, j = 7.1 hz), 1.40 (s, 9h), 2.702.90 (m, 2h), 4.05 (q, 2h, j = 7.1 hz), 5.09 (brs, 1h), 5.46 (brs, 1h), 7.197.35 (m, 5h) ; c nmr (cdcl3, 150 mhz) 14.0, 28.3, 41.0, 51.2, 60.6, 79.6, 126.1, 127.4, 128.6, 141.2, 155.0, 170.88 ; []d20 = 31.6 (c 1.0, etoac) on 78% ee (s)-19a. tlc and spectral data for 23a (mixture of rotamers) : rf = 0.34 (5:1 hexanes / etoac) ; h nmr (cdcl3, 300 mhz) 1.219 and 1.224 (2 t, 3h, j = 7.1 hz), 1.447 and 1.452 (2s, 9h), 3.75 and 3.89 (2s, 2h), 4.13 and 4.14 (2q, 2h, j = 7.1 hz), 4.49 and 4.52 (2s, 2h), 7.187.35 (m, 5h) ; c nmr (cdcl3, 150 mhz) 14.1, 14.2, 28.2, 28.3, 47.7, 48.1 51.0, 51.5, 60.9, 61.0, 80.4, 80.6, 127.37, 127.43, 127.5, 128.1, 128.5, 137.4, 137.6, 155.6, 155.8, 169.90, 169.94. the reaction was carried out according to the general procedure described above, starting with racemic trans-11a (62.1 mg, 0.15 mmol, 1.0 equiv), smi2 (5.5 equiv), n, n - dimethylethanolamine (0.17 ml, 1.7 mmol, 11.0 equiv), and dry thf (1.5 ml for smi2 and 1.5 ml for aziridine, freshly distilled) held at 0 c for 1 h. the h nmr spectrum of the crude reaction mixture showed that 12a(18) and 13a(18) were obtained in a ratio of 16.7:1. the nmr yield of 12a was determined to be 82% with the aid of ph3ch as an internal standard. this reaction was carried out according to the general procedure described above, starting with racemic trans-11b (62.7 mg, 0.15 mmol, 1.0 equiv), smi2 (6.0 equiv), n, n - dimethylethanolamine (0.18 ml, 1.8 mmol, 12.0 equiv), and dry thf (1.5 ml for smi2 and 1.5 ml for aziridine, freshly distilled) held at 0 c for 1 h. the nmr yield of 12b(18) was determined to be 73% with the aid of ph3ch as an internal standard. the reaction was carried out according to the general procedure described above, starting with racemic trans-15a (58.3 mg, 0.2 mmol, 1.0 equiv), smi2 (6.0 equiv), n, n - dimethylethanolamine (0.24 ml, 2.4 mmol, 12.0 equiv), and dry thf (1.8 ml for smi2 and 1.8 ml for aziridine, freshly distilled) held at 0 c for 1 h. the h nmr spectrum of the crude reaction mixture showed that 19a and 23a were present in a ratio of 6.7:1. purification by silica - gel chromatography (18 250 mm, 1:1:4.6 diethyl ether / hexanes / ch2cl2 as eluent) afforded 19a as a white solid (mp 7577 c) in 85% isolated yield (50.1 mg, 0.17 mmol) and 23a as a colorless oil in 10% isolated yield (5.9 mg, 0.02 mmol). the spectral data for 19a and 23a are the same as those obtained in the reductive ring opening of cis-15a. the general procedure for the reductive ring opening described above was followed, starting with cis-15b, except that 4 equiv of smi2 and 8 equiv of dmea were used. starting from cis-15b (38 mg, 0.12 mmol), purification by silica - gel chromatography (18 mm 150 mm, 1:5 etoac / hexanes as eluent) gave 19b as a white solid in 84% isolated yield (30 mg, 0.1 mmol). the h nmr spectrum of the crude reaction mixture showed the ratio of 19b/23b > 99:1. tlc and spectral data for 19b : rf = 0.25 (1:5 etoac / hexane) ; h nmr (cdcl3, 500 mhz) 0.830.99 (m, 2h), 1.011.17 (m, 3h), 1.21 (t, 3h, j = 7.1 hz), 1.38 (s, 9h), 1.311.40 (m, 1h), 1.611.75 (m, 5h), 2.382.51 (m, 2h), 3.633.75 (m, 1h), 4.08 (q, 2h, j = 7.1 hz), 4.88 (d, 1h, j = 7.1 hz) ; c nmr (cdcl3, 125 mhz) 14.1, 25.98, 26.02, 26.2, 28.3, 28.9, 29.7, 37.0, 41.5, 52.2, 60.4, 79.0, 155.5, 172.0 ; ir (thin film) 3360 (br, s), 2930 (s), 1719 (vs), 1504 (m), 1173 (s) cm. the h nmr spectral data match those previously reported for this compound. the spectral data for 23b has been reported. the reaction was carried out according to the general procedure described above, starting with racemic trans-15b (59.5 mg, 0.2 mmol, 1.0 equiv), smi2 (6.0 equiv), n, n - dimethylethanolamine (0.24 ml, 2.4 mmol, 12.0 equiv), and dry thf (1.8 ml for smi2 and 1.8 ml for aziridine, freshly distilled) held at 0 c for 1 h. -amino ester 19b was obtained in 84% nmr yield with the aid of ph3ch as an internal standard. the h nmr spectrum of the crude reaction mixture showed the ratio of 19b/23b > 99:1. the reaction was carried out according to the general procedure described above, starting with racemic trans-16a (51.9 mg, 0.15 mmol, 1.0 equiv), smi2 (6.0 equiv), n, n - dimethylethanolamine (0.18 ml, 1.8 mmol, 12.0 equiv), and dry thf (1.5 ml for smi2 and 1.5 ml for aziridine, freshly distilled) held at 0 c for 1 h. purification by silica - gel chromatography (18 250 mm, 5:1 hexanes / etoac as eluent) afforded 20a as a colorless liquid in 88% isolated yield (45.9 mg, 0.132 mmol). spectral data for 20a : rf = 0.25 (1:3 etoac / hexanes) ; h nmr (cdcl3, 500 mhz) 1.10 (t, 3h, j = 7.2 hz), 2.35 (s, 3h), 2.72 (dd, 1h, j = 16, 6.0 hz), 2.81 (dd, 1h, j = 16, 6.0 hz), 3.944.05 (m, 2h), 4.70 (q, 1h, j = 6.7 hz), 5.67 (d, 1h, j = 7.5 hz), 7.067.11 (m, 2h), 7.127.19 (m, 5h), 7.567.60 (m, 2h) ; c nmr (cdcl3, 125 mhz) 14.0, 21.4, 41.2, 54.3, 60.9, 126.4, 127.1, 127.7, 128.5, 129.4, 137.5, 139.3, 143.2, 170.6. the h nmr spectrum of the crude reaction mixture showed the ratio of 20a/24a > 99:1. the general procedure for the reductive ring opening described above was followed, starting with aziridine (2r,3r)-16b (82% ee, 53.4 mg, 0.15 mmol, 1.0 equiv), smi2 (5.0 equiv), n, n - dimethylethanolamine (0.15 ml, 1.5 mmol, 10.0 equiv), and dry thf (1.5 ml for smi2 and 1.5 ml for aziridine, freshly distilled) held at 0 c for 1 h. purification by silica - gel chromatography (18 250 mm, 5:1 hexanes / etoac as eluent) afforded (s)-20b as a colorless oil in 97% isolated yield (52.1 mg, 0.147 mmol). the optical purity of (s)-20b was determined to be 84% ee by hplc analysis (chiralcel od - h column, 98:2 hexanes / iproh at 222 nm, flow rate : 1.0 ml / min) ; retention times : rt = 12.73 min (major enantiomer (s)-20b) and rt = 16.94 min (minor enantiomer (r)-20b). the h nmr spectrum of the crude reaction mixture showed the ratio of 20b/24b > 99:1. spectral data for (s)-20b : rf = 0.17 (4:1 hexanes / etoac) ; h nmr (cdcl3, 600 mhz) 0.78 (qd, 1h, j = 12.0, 3.2 hz), 0.87 (qd, 1h, j = 12.0, 3.2), 1.001.17 (m, 3h), 1.18 (t, 3h, j = 7.1 hz), 1.371.45 (m, 1h), 1.541.61 (m, 2h), 1.631.70 (m, 2h), 1.731.81 (m, 1h), 2.25 (dd, 1h, j = 16.0, 5.6 hz), 2.37 (dd, 1h, j = 16.0, 5.6 hz), 2.39 (s, 3h), 3.263.34 (m, 1h), 4.01 (m, 2h), 5.24 (d, 1h, j = 9.3 hz), 7.26 (d, 2h, j = 8.1 hz), 7.72 (d, 2h, j = 8.1 hz) ; c nmr (cdcl3, 150 mhz) 14.1, 21.5, 25.87, 25.93, 26.1, 29.1, 29.3, 35.9, 41.2, 55.5, 60.6, 127.0, 129.6, 138.2, 143.2, 171.6 ; ir (thin film) 3292 (m), 2928 (vs), 2854 (m), 1734 (vs), 1718 (s), 1456 (m), 1324 (s), 1161 (vs) cm ; mass spectrum, m / z (% rel intensity) 354 (mh) (6.4), 271 (31), 270 (100), 224 (50), 198 (42), 155 (86), 91 (86), 41 (12) ; anal. calcd for c18h27no4s : c, 61.16 ; h, 7.70 ; n, 3.96. found : c, 61.30 ; h, 8.12 ; n, 3.88. []d20 = 10 (c 0.4, etoac) on 84% ee (s)-20b. the reaction was carried out according to the general procedure described above, starting with racemic trans-16b (53.3 mg, 0.15 mmol, 1.0 equiv), smi2 (6.0 equiv), n, n - dimethylethanolamine (0.18 ml, 1.8 mmol, 12.0 equiv), and dry thf (1.5 ml for smi2 and 1.5 ml for aziridine, freshly distilled) held at 0 c for 1 h. purification by silica - gel chromatography (18 250 mm, 5:1 hexanes / etoac as eluent) afforded 20b as a colorless oil in 95% isolated yield (51.0 mg, 0.144 mmol). the spectral data of 20b are the same as the product obtained from the reductive ring opening of cis-16b. the h nmr spectrum of the crude reaction mixture showed the ratio of 20b/24b > 99:1 the general procedure for the reductive ring opening described above was followed, starting with aziridine cis-17a (53.4 mg, 0.15 mmol, 1.0 equiv), smi2 (4.0 equiv), n, n - dimethylethanolamine (0.12 ml, 1.2 mmol, 8.0 equiv), and dry thf (2.0 ml for smi2 and 1.5 ml for aziridine, freshly distilled) held at 0 c for 1 h. the h nmr spectrum of the crude reaction mixture showed that 21a and 25a were present in a ratio of 23:1. purification by silica - gel chromatography (18 250 mm, 5:1 hexanes / etoac as eluent) afforded (s)-21a as a white solid in 84% isolated yield (45.2 mg, 0.126 mmol) and 25a as a colorless oil in 4% isolated yield (2.0 mg, 0.0056 mmol). spectral data for 21a : white solid ; mp 6061 c ; h nmr (500 mhz, cdcl3) 0.14 (s, 9h), 0.75 (td, 1h, j = 14, 4.5 hz), 0.84 (td, 1h, j = 14, 4.0 hz), 1.17 (t, 3h, j = 7 hz), 2.52 (td, 1h, j = 14, 4.5 hz), 2.63 (td, 1h, j = 14, 4.0 hz), 2.792.90 (m, 2h), 4.08 (qd, 2h, j = 7.2, 1.5 hz), 4.814.90 (m, 1h), 5.57 (d, 1h, j = 8 hz), 7.247.30 (m, 1h), 7.307.39 (m, 4h) ; c nmr (cdcl3, 125 mhz) 2.2, 10.2, 14.0, 41.9, 49.9, 54.5, 61.0, 126.6, 128.2, 128.9, 140.3, 170.5 ; ir (thin film) 3277 (br, s), 2955 (w), 1736 (s), 1144 (s) cm ; hrms (esi - tof) m / z : [m + h ] calcd for c16h28no4sis, 358.1508 ; found, 358.1509 ; []d20 = 21.7 (c 0.87, ch2cl2) on 98% ee (s)-21a. spectral data for 25a : h nmr (500 mhz, cdcl3) 0.05 (s, 9h), 1.111.16 (m, 2h), 1.24 (t, 3h, j = 7.2 hz), 3.053.10 (m, 2h), 3.90 (s, 2h), 4.16 (q, 2h, j = 7.2 hz), 4.53 (s, 2h), 7.277.36 (m, 5h) ; c nmr (cdcl3, 125 mhz) 2.0, 10.3, 14.2, 46.9, 50.0, 51.8, 61.3, 128.1, 128.5, 128.8, 135.5, 169.6 ; ir (thin film) 2955 (w), 1746 (s), 1333 (s), 1142 (s) cm ; hrms (esi - tof) m / z : [m + h ] calcd for c16h28no4sis, 358.1508 ; found, 358.1530. the general procedure for the reductive ring opening described above was followed, except that 2.5 equiv of sml2 and 5 equiv of dmea were used. starting from cis-18a (47 mg, 0.19 mmol), purification of the product by silica - gel chromatography (18 mm 300 mm, 1:5 etoac / hexanes as eluent) gave 22a as a colorless semisolid in 52% isolated yield (24 mg, 0.1 mmol), along with the -amino ester resulting from the cleavage of the n c3 bond as a colorless semisolid in 13% isolated yield (6 mg, 0.025 mmol). tlc and spectral data for 22a : rf = 0.15 (1:5 etoac / hexane) ; h nmr (cdcl3, 500 mhz) 1.12 (t, 3h, j = 7.1 hz), 1.98 (s, 3h), 2.77 (dd, 1h, j = 15.4, 6.0 hz), 2.87 (dd, 1h, j = 15.7, 6.0 hz), 4.04 (q, 2h, j = 7.1 hz), 5.365.43 (m, 1h), 6.60 (d, 1h, j = 8.0 hz), 7.207.32 (m, 5h). the general procedure for the reductive ring opening described above was followed, except that 5.0 equiv of sml2 and 10 equiv of dmea were used. starting from cis-27a(18) (19 mg, 0.10 mmol), purification of the product by silica - gel chromatography (18 mm 300 mm, 1:1:4 et2o / ch2cl2/hexanes as eluent) gave 31a as a colorless oil in 75% isolated yield (14.4 mg, 0.0746 mmol). spectral data for 31a : h nmr (cdcl3, 300 mhz,) 1.25 (t, 3h, j = 7.1 hz), 3.39 (s, 2h), 3.78 (s, 2h), 4.17 (q, 2h, j = 7.2 hz), 7.207.35 (m, 5h) (n h proton not located). the general procedure for the reductive ring opening described above was followed, except that 5.0 equiv of sml2 and 10 equiv of dmea were used. starting from cis-29a (35 mg, 0.10 mmol), purification of the product by silica - gel chromatography (18 mm 300 mm, 1:1:4 et2o / ch2cl2/hexanes as eluent) gave 33a as a colorless oil in 69% isolated yield (25 mg, 0.069 mmol). spectral data for 33a : h nmr (300 mhz, cdcl3) 1.17 (t, 3h, j = 7.2 hz), 3.25 (s, 2h), 3.82 (s, 2h), 4.04 (q, 2h, j = 7.2 hz), 5.27 (s, 1h), 7.157.51 (m, 15h) ; c nmr (75 mhz, cdcl3) 14.2, 50.3, 54.9, 59.9, 70.3, 127.0, 127.1, 128.28, 128.33, 128.4, 128.7, 139.2, 142.1, 171.6. the general procedure for the reductive ring opening described above was followed, starting with cis-29b (44 mg, 0.12 mmol). purification of the product by silica - gel chromatography (18 mm 300 mm, 1:5 etoac / hexanes as eluent) gave a 1:1.4 mixture of -amino ester 30b (22% nmr yield) and unreacted cis-29b (31% nmr yield). the h nmr spectrum of the crude reaction mixture indicated the formation of amine 34 in 39% yield. extending the reaction time from 40 min to 2 h for the ring - opening reaction of cis-29b (42 mg, 0.12 mmol) at room temperature gave 30b (8.6 mg, 0.024 mmol) in 22% isolated yield and amine 34 in 52% isolated yield. spectral data for 30b : h nmr (600 mhz, cdcl3) 0.901.04 (m, 2h), 1.051.23 (m, 2h), 1.20 (t, 3h, j = 7.1 hz), 1.451.54 (m, 1h), 1.601.80 (m, 6h), 2.34 (dd, 1h, j = 14.5, 7.0 hz), 2.47 (dd, 1h, j = 14.5, 5.3 hz), 2.77 (dt, 1h, j = 7.0, 5.3 hz), 4.034.14 (m, 2h), 4.94 (s, 1h), 7.157.19 (m, 2h), 7.237.28 (m, 4h), 7.357.41 (m, 4h) (n h proton not located) ; c nmr (150 mhz, cdcl3) 14.2, 26.5, 26.6, 26.7, 28.4, 29.5, 36.0, 40.9, 56.8, 60.2, 64.1, 126.89, 126.91, 127.4, 127.6, 128.33, 128.34, 144.2, 144.4, 172.9 ; hrms (esi - tof) m / z : [m + h ] calcd for c24h32no2, 366.2433 ; found, 366.2431. spectral data for 34 : h nmr (600 mhz, cdcl3) 1.84 (bs, 2h), 5.20 (s, 1h), 7.187.23 (m, 2h), 7.267.32 (m, 4h), 7.337.37 (m, 4h) ; c nmr (150 mhz, cdcl3) 59.7, 126.88, 126.92, 128.4, 145.6. the h and c nmr spectral data for 34 match those provided by the source for this compound. the general procedure for the reductive ring opening described above was followed, starting with trisubstituted aziridine 35(20) (76 mg, 0.25 mmol, 99% ee) and 5.0 equiv of smi2. the h nmr spectrum of the crude reaction mixture indicated that a mixture of anti-36, syn-36, and 37 was present in a ratio of 1.28:0.28:1, respectively. purification of the products by silica - gel chromatography (18 mm 200 mm, 1:10 to 1:6 etoac / hexanes as eluent) gave anti-36 as a white solid (mp 5355 c) in 43% isolated yield (32.7 mg, 0.106 mmol), syn-36 as a white solid (mp 8991 c) in 9% isolated yield (6.6 mg, 0.021 mmol), and 37 as a colorless oil in 30% isolated yield (23.2 mg, 0.0755 mmol). spectral data for anti-36 : h nmr (300 mhz, cdcl3) 1.09 (t, 3h, j = 7.1 hz), 1.20 (d, 3h, j = 7.0 hz), 1.39 (s, 9h), 2.87 (brt, 1h, j = 6.5 hz), 4.01 (q, 2h, j = 7.1 hz), 4.81 (brs, 1h), 5.81 (brs, 1h), 7.167.33 (m, 5h) ; c nmr (125 mhz, cdcl3) 14.0, 15.4, 28.3, 45.3, 56.7, 60.6, 79.4, 126.3, 127.3, 128.4, 141.0, 155.4, 174.9 ; ir (thin film) 3355 (br, w), 2978 (m), 1721 (s), 1171 (s) cm ; hrms (esi - tof) m / z : [m + h ] calcd for c17h26no4, 308.1862 ; found, 308.1860 ; []d20 = 40.1 (c 0.5, ch2cl2) on 99% ee material. spectral data for syn-36 : h nmr (600 mhz, cdcl3) 1.11 (t, 3h, j = 7.2 hz), 1.13 (d, 3h, j = 7.2 hz), 1.39 (s, 9h), 2.87 (brs, 1h), 3.964.08 (m, 2h), 4.97 (brs, 1h), 5.27 (brs, 1h), 7.197.25 (m, 3h), 7.267.31 (m, 2h) ; c nmr (150 mhz, cdcl3) 13.1, 14.0, 28.3, 45.4, 56.5, 60.6, 79.6, 126.8, 127.4, 128.4, 140.2, 155.1, 173.7 ; ir (thin film) 3380 (s), 2980 (m), 1728 (s), 1686 (s), 1520 (s), 1173 (s) cm ; hrms (esi - tof) m / z : [m + h ] calcd for c17h26no4, 308.1862 ; found, 308.1855 ; []d20 = 26.0 (c 0.5, ch2cl2) on 99% ee material. spectral data for 37 (compound 37 appeared to be a colorless - oil mixture of two rotamers at room temperature in a ratio of 1.2:1) : h nmr (600 mhz, cdcl3) 1.21 (t, 3h, j = 7.2 hz), 1.271.50 (m, 12h), 3.824.64 (m, 5h), 7.167.37 (m, 5h) ; c nmr (125 mhz, cdcl3) 14.1, 15.4, 15.8, 28.3, 49.7, 50.8, 54.8, 55.4, 60.9, 80.4, 80.6, 127.0, 127.2, 127.3, 128.0, 128.3, 128.6, 138.2, 139.2, 155.4, 155.5, 172.1, 172.3. trifluoroacetic acid (0.240 ml, 357 mg, 3.13 mmol, 35.6 equiv) was added to a solution of anti-36 (27 mg, 0.088 mmol, 1.0 equiv) in ch2cl2 (0.24 ml). after the reaction mixture was stirred at room temperature under nitrogen overnight, it was concentrated and diluted with 0.3 ml of h2o. the ph of the mixture was adjusted to 10 with sat aq nahco3 (ca. 10 ml), and then the mixture was extracted with ch2cl2 (10 ml 3). the combined organic layer was dried over na2so4, filtered, and concentrated. purification of the product by silica - gel chromatography (18 mm 150 mm, 1:1 etoac / hexanes as eluent) gave ethyl 3-amino-2-methyl-3-phenylpropanoate 60 as a colorless oil in 74% isolated yield (13.5 mg, 0.065 mmol). spectral data for ethyl 3-amino-2-methyl-3-phenylpropanoate : h nmr (500 mhz, cdcl3) 0.93 (d, 3h, j = 7.2 hz), 1.26 (t, 3h, j = 7.2 hz), 1.64 (brs, 2h), 2.602.72 (m, 1h), 4.00 (d, 1h, j = 9.4 hz), 4.17 (q, 2h, j = 7.2 hz), 7.217.34 (m, 5h) ; c nmr (125 mhz, cdcl3) 14.2, 15.4, 48.1, 59.1, 60.4, 127.0, 127.5, 128.5, 143.6, 175.9. the h nmr data match those previously reported for the anti isomer but not those of the syn isomer. to a flame - dried 25 ml schlenk flask equipped with a stir bar and filled with nitrogen were added (r)-vapol (54 mg, 0.10 mmol), b(oph)3 (87 mg, 0.30 mmol), amine 52(7d) (599 mg, 2.00 mmol) ; dry toluene (4 ml) was added to dissolve the reagents. the flask was then sealed, and the reaction mixture was stirred at room temperature for 1 h. thereafter, 4 of powdered molecular sieves (600 mg, freshly flame - dried) were added to the reaction flask, followed by the addition of aldehyde 50(39) (467 mg, 2.10 mmol, 1.05 equiv). to this solution was rapidly added ethyl diazoacetate (eda) 53 (0.30 ml, 2.4 mmol, 1.2 equiv). after the resulting mixture was stirred for 20 h at room temperature, it was diluted by the addition of hexane (12 ml). the reaction mixture was then filtered through a celite pad into a 100 ml round - bottomed flask. the reaction flask was rinsed with etoac (6 ml 3), and the rinse was filtered through the same celite pad. the combined filtrate was concentrated in vacuo, followed by exposure to high vacuum (0.05 mmhg) to afford the crude aziridine as a yellow oil. purification of the crude aziridine by silica - gel chromatography (40 mm 210 mm column, 2:1 hexanes / etoac as eluent) afforded pure cis - aziridine 54 as a white solid (mp 6567 c on > 98.5% ee material) in 98% isolated yield (1.15 g, 1.95 mmol). the optical purity of 54 was determined to be > 98.5% ee by hplc analysis (chiralcel od - h column, 85:15 hexane/2-propanol at 222 nm, flow rate : 1 ml / min). retention times : rt = 7.18 min (minor enantiomer, ent-54) and rt = 8.46 min (major enantiomer, 54). spectral data for 54 : rf = 0.19 (1:2 etoac / hexane) ; h nmr (500 mhz, cdcl3) 1.02 (t, 3h, j = 7.1 hz), 2.21 (s, 6h), 2.25 (s, 3h), 2.26 (s, 6h), 2.27 (s, 3h), 2.59 (d, 1h, j = 6.8 hz), 3.09 (d, 1h, j = 6.8 hz), 3.65 (s, 3h), 3.68 (s, 1h), 3.69 (s, 3h), 3.914.03 (m, 2h), 7.047.10 (m, 3h), 7.17 (s, 2h), 7.247.29 (m, 2h) ; c nmr (cdcl3, 125 mhz) 14.0, 16.15, 16.22, 20.59, 20.60, 46.4, 47.2, 59.5, 59.6, 60.8, 76.8, 122.6, 122.9, 126.0, 127.3, 127.7, 130.6, 130.8, 134.2, 137.5, 137.6, 141.2, 141.4, 155.9, 156.2, 167.7, 168.0, 168.2 ; ir (thin film) 2932 (m), 1773 (vs), 1746 (s), 1213 (vs) cm ; hrms (esi - tof) m / z : [m + h ] calcd for c34h40no8, 590.2754 ; found, 590.2769 ; []d20 = 28.2 (c 1.0, ch2cl2) on > 98.5% ee (by hplc) material. the procedure for the synthesis of aziridine 54 was followed, starting with aldehyde 51(40) (643 mg, 2.10 mmol, 1.05 equiv). purification of the crude product by silica - gel chromatography (40 mm 210 mm column, 5:1 hexanes / etoac as eluent) afforded pure cis - aziridine 55 as a white solid (mp 6466 c on 98% ee material) in 97% isolated yield (1.31 g, 1.94 mmol). the optical purity of 55 was determined to be 98% ee by hplc analysis (chiralpak ad column, 95:5 hexane/2-propanol at 222 nm, flow- rate : 0.7 ml / min). retention times : rt = 8.68 min (minor enantiomer, ent-55) and rt = 10.23 min (major enantiomer, 55). spectral data for 55 : rf = 0.21 (1:5 etoac / hexane) ; h nmr (500 mhz, cdcl3) 1.04 (t, 3h, j = 7.0 hz), 1.30 (s, 9h), 1.32 (s, 9h), 2.20 (s, 6h), 2.24 (s, 6h), 2.56 (d, 1h, j = 6.8 hz), 3.08 (d, 1h, j = 6.8 hz), 3.64 (s, 3h), 3.66 (s, 1h), 3.68 (s, 3h), 3.904.03 (m, 2h), 6.98 (d, 1h, j = 8.0 hz), 7.07 (s, 2h), 7.11 (d, 1h, j = 1.5 hz), 7.16 (s, 2h), 7.25 (dd, 1h, j = 8.5, 1.5 hz) ; c nmr (cdcl3, 125 mhz) 14.0, 16.18, 16.22, 27.18, 27.23, 39.0, 39.1, 46.2, 47.3, 59.5, 59.6, 60.7, 76.9, 122.6, 122.8, 125.6, 127.3, 127.7, 130.6, 130.7, 133.7, 137.55, 137.61, 141.7, 141.9, 155.9, 156.1, 167.8, 175.6, 175.8 ; ir (thin film) 2977 (s), 1761 (vs), 1482 (s), 1119 (vs) cm ; hrms (esi - tof) m / z : [m + h ] calcd for c40h52no8, 674.3693 ; found, 674.3694 ; []d20 = 34.2 (c 1.0, ch2cl2) on 98% ee (by hplc) material. to a oven - dried 25 ml round - bottomed flask equipped with a stir bar and filled with nitrogen were added aziridine 55 (67.4 mg, 0.100 mmol, 98% ee), pd(oh)2 (28.0 mg, 0.020 mmol, pd(oh)2 on carbon 20%, moisture 50%), ditert - butyl dicarbonate (33 mg, 0.15 mmol), and methanol (10 ml). the flask was sealed with a rubber septum, and a needle connected to a vacuum line was used to apply vacuum in the flask through the septum. the vacuum was applied for a few seconds with vigorous stirring of the reaction mixture. then the vacuum was stopped, and a hydrogen balloon was connected to the flask by a needle through the septum. the suspension was stirred at room temperature under hydrogen for 17 h and then filtered through a pad of celite. the filter cake was washed with etoac (5 ml) and dcm (3 ml 3). the combined filtrate was concentrated to give a light - yellow oil. purification of the crude product by column chromatography on silica gel (20 mm 160 mm, hexanes / etoac 5:1) gave -amino ester 56 as a colorless oil (42.2 mg, 0.0855 mmol, 86%). spectral data for 56 : rf = 0.20 (1:5 etoac / hexane) ; h nmr (500 mhz, cdcl3) 1.20 (t, 3h, j = 7.0 hz), 1.30 (s, 18h), 1.41 (s, 9h), 3.06 (d, 2h, j = 6.0 hz), 4.074.18 (m, 2h), 4.50 (dt, 1h, j = 7.5, 6.0 hz), 5.01 (d, 1h, j = 7.5 hz), 6.87 (s, 1h), 6.97 (d, 1h, j = 8.2 hz), 7.02 (d, 1h, j = 8.2 hz) ; c nmr (cdcl3, 125 mhz) 14.1, 27.2, 28.3, 37.5, 39.06, 39.09, 54.3, 61.5, 79.9, 123.2, 124.2, 127.0, 134.5, 141.5, 142.3, 155.0, 171.5, 175.6, 175.8 (one sp carbon not located) ; ir (thin film) 2977 (s), 1761 (vs), 1482 (s), 1119 (vs) cm ; hrms (esi - tof) m / z : [m + h ] calcd for c26h40no8, 494.2754 ; found, 494.2751 ; []d20 = + 27.8 (c 1.0, ch2cl2) on 98% ee material. (the optical purity was assumed to be unchanged from that of 55.) to a flame - dried 100 ml round - bottomed flask equipped with a stir bar and filled with nitrogen were added room - temperature aziridine 54 (467 mg, 0.792 mmol) and anisole (4.1 ml). the resulting solution was cooled to 0 c in an ice bath, and trifluoroacetic acid (4.1 ml) was rapidly added. the ice bath was then removed, and the reaction mixture was stirred for 40 min at room temperature. the reaction mixture was quenched by careful addition of sat aq na2co3 (30 ml) and h2o (10 ml), followed by the addition of et2o (30 ml). the organic layer was separated, and the aqueous layer was extracted with et2o (30 ml 3). the combined organic layer was dried with na2so4, filtered, and concentrated in vacuo, followed by exposure to high vacuum (0.05 mmhg) for 4 h to give a yellow oil, to which was added 6.5 ml of ch2ci2/chcl3 (1:1) and et3n (0.33 ml, 2.4 mmol). the resulting solution was cooled to 0 c, and a portion of tosyl chloride (228 mg, 1.20 mmol) was added. the mixture was then stirred at 0 c for 15 h. thereafter, additional portions of both tosyl chloride (228 mg, 1.20 mmol) and et3n (0.33 ml, 2.4 mmol) were added to the reaction mixture at room temperature. after the mixture was stirred for 26 h at room temperature, the reaction was quenched with 12 ml of sat aq nh4cl and 2.5 ml of h2o. the combined organic layer was dried with na2so4, filtered, and concentrated to give a dark - brown oil. purification by silica - gel chromatography (25 mm 160 mm, 2:1 hexanes / etoac as eluent) afforded cis-57 as a light - yellow oil in 70% yield (258 mg, 0.559 mmol). spectral data for 57 : rf = 0.14 (1:2 etoac / hexane) ; h nmr (500 mhz, cdcl3) 0.96 (t, 3h, j = 7.1 hz), 2.236 (s, 3h), 2.240 (s, 3h), 2.43 (s, 3h), 3.66 (d, 1h, j = 7.5 hz), 3.894.02 (m, 2h), 4.03 (d, 1h, j = 7.5 hz), 7.07 (d, 1h, j = 8.3 hz), 7.13 (d, 1h, j = 2.0 hz), 7.17 (dd, 2h, j = 8.3, 2.0 hz), 7.34 (d, 1h, j = 8.0 hz), 7.88 (d, 1h, j = 8.0 hz) ; c nmr (cdcl3, 125 mhz) 13.7, 20.55, 20.60, 21.7, 43.4, 44.3, 61.9, 122.7, 123.3, 125.8, 128.1, 129.9, 130.0, 133.7, 141.8, 142.3, 145.4, 164.1, 167.8, 168.0 ; ir (thin film) 2986 (w), 1773 (vs), 1734 (m), 1210 (vs), 1165 (s) cm ; hrms (esi - tof) m / z : [m + h ] calcd for c22h24no8s, 462.1223 ; found, 462.1234 ; []d20 = + 15.6 (c 1.0, ch2cl2) on > 98.5% ee material. (the optical purity was assumed to be unchanged from that of 54.) to a flame - dried 100 ml round - bottomed flask equipped with a stir bar and filled with nitrogen were added room - temperature aziridine 54 (366 mg, 0.621 mmol) and anisole (3.1 ml). the resulting solution was cooled to 0 c in an ice bath, and trifluoroacetic acid (3.1 ml) was rapidly added. the ice bath was then removed, and the reaction mixture was stirred for 40 min at room temperature. the reaction mixture was quenched by careful addition of saturated aq na2co3 (25 ml) and h2o (10 ml), followed by addition of et2o (30 ml). the organic layer was separated, and the aqueous layer was extracted with et2o (30 ml 3). the combined organic layer was dried with na2so4, filtered, and concentrated in vacuo followed by exposure to high vacuum (0.05 mmhg) for 4 h to give a yellow oil, which was then dissolved in a mixture of ch2cl2 (2 ml) and et3n (0.9 ml). after the solution was cooled to 0 c in an ice bath, 2-(trimethylsilyl)ethanesulfonyl chloride (0.12 ml, 0.93 mmol) was added dropwise to the reaction mixture at 0 c. the ice bath was then removed, and the reaction mixture was stirred at room temperature for 17 h. thereafter, additional portions of both 2-(trimethylsilyl)ethanesulfonyl chloride (0.12 ml, 0.93 mmol) and et3n (0.9 ml) were added to the reaction mixture at room temperature. after the mixture was stirred for 23 h at room temperature, the reaction was quenched with 2.5 ml of sat aq nh4cl and 1 ml of h2o. the organic layer was separated, and the aqueous layer was extracted with ch2cl2 (4 ml 3). the combined organic layer was dried with na2so4, filtered, and concentrated to give a dark - brown oil. purification by silica - gel chromatography (25 mm 160 mm, 3:1 hexanes / etoac as eluent) afforded cis-58 as a light - yellow oil in 78% yield (0.228 g, 0.483 mmol). spectral data for 58 : rf = 0.20 (1:3 etoac / hexane) ; h nmr (500 mhz, cdcl3) 0.05 (s, 9h), 1.03 (t, 3h, j = 7.0 hz), 1.121.20 (m, 2h), 2.26 (s, 6h), 3.163.24 (m, 2h), 3.66 (d, 1h, j = 7.5 hz), 3.974.09 (m, 2h), 4.02 (d, 1h, j = 7.5 hz), 7.15 (d, 1h, j = 8.2 hz), 7.25 (d, 1h, j = 1.9 hz), 7.30 (dd, 1h, j = 8.2 hz, j = 1.9 hz) ; c nmr (125 mhz, cdcl3) 2.1, 9.5, 13.8, 20.57, 20.62, 43.5, 43.6, 49.5, 62.0, 122.8, 123.4, 125.8, 130.1, 142.0, 142.4, 164.2, 167.9, 168.0 ; ir (thin film) 2955 (m), 1777 (s), 1208 (s), 1177 (s) cm ; hrms (esi - tof) m / z : [m + h ] calcd for c20h30no8sis, 472.1461 ; found, 472.1449 ; []d20 = + 27.5 (c 1.0, ch2cl2) on > 98.5% ee material. (the optical purity was assumed to be unchanged from that of 54.) to a flame - dried 100 ml round - bottomed flask equipped with a stir bar and filled with nitrogen were added room - temperature aziridine 55 (674 mg, 1.00 mmol) and anisole (8.9 ml). the resulting solution was cooled to 0 c in a ice bath, and trifluoroacetic acid (8.9 ml) was rapidly added. the ice bath was removed, and the reaction mixture was stirred for 30 min at room temperature. the reaction mixture was quenched by the careful addition of saturated aq na2co3 (68 ml) and h2o (35 ml), followed by the addition of et2o (50 ml). the organic layer was separated, and the aqueous layer was extracted with et2o (100 ml 3). the combined organic layer was dried with na2so4, filtered, and concentrated in vacuo, followed by exposure to high vacuum (0.05 mmhg) for 4 h to give a yellow oil, which was then dissolved in a mixture of ch2cl2 (3.5 ml) and et3n (1.5 ml). after the solution was cooled to 0 c, 2-(trimethylsilyl)ethanesulfonyl chloride (0.20 ml, 1.5 mmol) was added dropwise to the reaction mixture at 0 c. the ice bath was removed, and the reaction mixture was stirred at room temperature for 14 h. thereafter, additional portions of both 2-(trimethylsilyl)ethanesulfonyl chloride (0.20 ml, 1.5 mmol) and et3n (1.5 ml) were added to the reaction mixture at room temperature. after the mixture was stirred for 22 h at room temperature, the reaction was quenched with 4 ml of sat aq nh4cl and 1.5 ml of h2o. the organic layer was separated, and the aqueous layer was extracted with ch2cl2 (6 ml 3). the combined organic layer was dried with na2so4, filtered, and concentrated to give a dark - brown oil. purification by silica - gel chromatography (25 mm 160 mm, 5:1 hexanes / etoac as eluent) afforded cis-59 as a light - yellow oil in 86% yield (0.475 g, 0.855 mmol). spectral data for 59 : rf = 0.20 (1:5 etoac / hexane) ; h nmr (500 mhz, cdcl3) 0.05 (s, 9h), 1.06 (t, 3h, j = 7.1 hz), 1.111.21 (m, 2h), 1.31 (s, 9h), 1.32 (s, 9h), 3.173.26 (m, 2h), 3.66 (d, 1h, j = 7.5 hz), 3.984.07 (m, 2h), 4.02 (d, 1h, j = 7.5 hz), 7.09 (d, 1h, j = 8.3 hz), 7.18 (d, 1h, j = 1.8 hz), 7.27 (dd, 1h, j = 8.3 hz, j = 1.8 hz) ; c nmr (125 mhz, cdcl3) 2.1, 9.5, 13.8, 27.10, 27.14, 39.0, 39.1, 43.2, 43.7, 49.4, 61.9, 122.7, 123.3, 125.4, 129.5, 142.4, 142.9, 164.2, 175.4, 175.6 ; ir (thin film) 2977 (m), 1761 (vs), 1117 (s) cm ; hrms (esi - tof) m / z : [m+nh4 ] calcd for c26h45n2o8sis, 573.2666 ; found, 573.2675 ; []d20 = + 26.9 (c 1.0, ch2cl2) on 98% ee material. (the general procedure for the reductive ring opening described above was followed, starting with aziridine 57 (228 mg, 0.494 mmol), 2.5 equiv of smi2, and 5 equiv of dmea. the h nmr spectrum of the crude reaction mixture indicated a complex mixture of several products due to partial cleavage of the acetate group on the benzene ring. to this mixture after the reaction was stirred at room temperature for 30 min, it was quenched by the addition of etoh (0.1 ml) and h2o (2.5 ml). the combined organic layer was dried with na2so4, filtered, and concentrated to give a yellow oil. purification by silica - gel chromatography (20 mm 160 mm, 1:1.5 etoac / hexanes as eluent) gave 61a as a colorless oil in 76% isolated yield (173 mg, 0.373 mmol) and 61b as a light - yellow oil in 8% isolated yield (17.5 mg, 0.0377 mmol). spectral data for 61a : rf = 0.23 (1:1.5 etoac / hexane) ; h nmr (500 mhz, cdcl3) 1.11 (t, 3h, j = 7.1 hz), 2.22 (s, 6h), 2.33 (s, 3h), 2.68 (dd, 1h, j = 16.2, 6.2 hz), 2.75 (dd, 1h, j = 16.2, 6.4 hz), 3.99 (q, 2h, j = 7.1 hz), 4.70 (dt, 1h, j = 7.5, 6.2 hz), 5.97 (d, 1h, j = 7.8 hz), 6.906.98 (m, 3h), 7.14 (d, 2h, j = 8.0 hz), 7.54 (d, 2h, j = 8.0 hz) ; c nmr (125 mhz, cdcl3) 13.9, 20.5, 20.6, 21.4, 41.0, 53.6, 61.0, 121.8, 123.3, 124.5, 126.9, 129.5, 137.1, 138.0, 141.4, 141.8, 143.4, 167.8, 167.9, 170.4 ; ir (thin film) 3279 (m), 2984 (w), 2930 (w), 1773 (s), 1734 (s), 1211 (s), 1161 (s) cm ; hrms (esi - tof) m / z : [m + h ] calcd for c22h26no8s, 464.1379 ; found, 464.1381 ; []d20 = + 45.2 (c 1.0, ch2cl2) on > 98% ee material. (the optical purity was assumed to be unchanged from that of 54.) spectral data for 61b : rf = 0.38 (1:1.5 etoac / hexane) ; h nmr (500 mhz, cdcl3) 1.13 (t, 3h, j = 7.1 hz), 2.26 (s, 6h), 2.42 (s, 3h), 3.93 (s, 2h), 3.99 (q, 2h, j = 7.1 hz), 4.46 (s, 2h), 7.09 (s, 1h), 7.12 (s, 2h), 7.30 (d, 2h, j = 8.2 hz), 7.73 (d, 2h, j = 8.2 hz) ; c nmr (125 mhz, cdcl3) 14.0, 20.6, 20.7, 21.6, 46.8, 50.5, 61.2, 123.5, 123.6, 126.5, 127.4, 129.6, 134.0, 136.7, 141.9, 142.2, 143.6, 168.1, 168.2, 168.6 ; ir (thin film) 2984 (w), 2934 (w), 1773 (vs), 1213 (s) cm ; hrms (esi - tof) m / z : [m + h ] calcd for c22h26no8s, 464.1379 ; found, 464.1381. the general procedure for the reductive ring opening described above was followed, starting with aziridine 58 (236 mg, 0.500 mmol), 4 equiv of smi2, and 8 equiv of dmea. the h nmr spectrum of the crude reaction mixture indicated a complex mixture of several products due to partial cleavage of the acetate group on the benzene ring. to this mixture after the reaction was stirred at room temperature for 40 min, it was quenched by the addition of etoh (0.32 ml) and h2o (9 ml). the combined organic layer was dried with na2so4, filtered, and concentrated to give a yellow oil. purification by silica - gel chromatography (20 mm 160 mm, 1:2 etoac / hexanes as eluent) gave 62a as a colorless oil in 69% isolated yield (164 mg, 0.346 mmol) and 62b as a light - yellow oil in 19% isolated yield (45 mg, 0.095 mmol). spectral data for 62a : rf = 0.21 (1:2 etoac / hexane) ; h nmr (500 mhz, cdcl3) 0.09 (s, 9h), 0.780.93 (m, 2h), 1.19 (t, 3h, j = 7.1 hz), 2.26 (s, 6h), 2.592.78 (m, 2h), 2.802.89 (m, 2h), 4.064.13 (m, 2h), 4.87 (dt, 1h, j = 8.1, 6.4 hz), 5.70 (d, 1h, j = 8.1 hz), 7.16 (d, 1h, j = 8.7 hz), 7.197.28 (m, 2h) ; c nmr (125 mhz, cdcl3) 2.2, 10.2, 14.0, 20.575, 20.584, 41.6, 50.1, 53.6, 61.2, 121.9, 123.7, 124.6, 139.1, 141.7, 142.2, 167.8, 168.0, 170.5 ; ir (thin film) 3283 (m), 2955 (m), 1773 (vs), 1734 (vs), 1211 (s), 1143 (s) cm ; hrms (esi - tof) m / z : [m+nh4 ] calcd for c20h35n2o8sis, 491.1883 ; found, 491.1894 ; []d20 = + 27.0 (c 1.0, ch2cl2) on > 98.5% ee material (the optical purity was assumed to be unchanged from 54). spectral data for 62b : rf = 0.27 (1:2 etoac / hexane) ; h nmr (500 mhz, cdcl3) 0.04 (s, 9h), 1.081.16 (m, 2h), 1.24 (t, 3h, j = 7.1 hz), 2.26 (s, 6h), 3.043.11 (m, 2h), 3.93 (s, 2h), 4.15 (q, 2h, j = 7.1 hz), 4.52 (s, 2h), 7.127.21 (m, 3h) ; c nmr (125 mhz, cdcl3) 2.0, 10.2, 14.1, 20.59, 20.63, 46.8, 50.0, 50.9, 61.4, 123.2, 123.7, 126.3, 134.4, 141.9, 142.3, 168.1, 168.2, 169.5 ; ir (thin film) 2955 (w), 2930 (w), 1773 (s), 1742 (s), 1211 (s) cm ; hrms (esi - tof) m / z [m+nh4 ] calcd for c20h35n2o8sis, 491.1883 ; found, 491.1897. the general procedure for the reductive ring opening described above was followed, starting with aziridine 59 (111 mg, 0.200 mmol, 98% ee) and 4.0 equiv of smi2. the h nmr spectrum of the crude reaction mixture indicated a mixture of c n cleavage product 63a and c c cleavage product 63b was obtained with a ratio of 4.3:1. purification by silica - gel chromatography (20 mm 160 mm, 1:3 etoac / hexanes as eluent) gave 63a as a yellow oil in 74% isolated yield (81.8 mg, 0.147 mmol) and 63b as a light - yellow oil in 16% isolated yield (18.2 mg, 0.0326 mmol). spectral data for 63a : rf = 0.25 (1:3 etoac / hexane) ; h nmr (500 mhz, cdcl3) 0.11 (s, 9h), 0.750.90 (m, 2h), 1.18 (t, 3h, j = 7.2 hz), 1.29 (s, 9h), 1.30 (s, 9h), 2.552.75 (m, 2h), 2.82 (d, 2h, j = 6.5 hz), 4.07 (q, 2h, j = 7.1 hz), 4.84 (dt, 1h, j = 8.0, 6.5 hz), 5.70 (d, 1h, j = 8.0 hz), 7.09 (d, 1h, j = 8.4 hz), 7.13 (d, 1h, j = 2.0 hz), 7.19 (dd, 1h, j = 8.4, 2.0 hz) ; c nmr (125 mhz, cdcl3) 2.2, 10.2, 14.0, 27.11, 27.13, 39.05, 39.06, 41.6, 50.0, 53.7, 61.1, 121.8, 123.7, 124.3, 138.6, 142.2, 142.6, 170.4, 175.4, 175.5 ; ir (thin film) 3283 (m), 2977 (s), 1763 (vs), 1736 (s), 1117 (s) cm ; hrms (esi - tof) m / z [m+nh4 ] calcd for c26h47n2o8sis, 575.2822 ; found, 575.2842 ; []d20 = + 26.0 (c 1.0, ch2cl2) on 98% ee material. (the optical purity was assumed to be unchanged from that of 55.) spectral data for 63b : rf = 0.39 (1:3 etoac / hexane) ; h nmr (500 mhz, cdcl3) 0.04 (s, 9h), 1.091.16 (m, 2h), 1.24 (t, 3h, j = 7.1 hz), 1.31 (s, 18h), 3.043.13 (m, 2h), 3.92 (s, 2h), 4.15 (q, 2h, j = 7.1 hz), 4.52 (s, 2h), 7.067.11 (m, 2h), 7.17 (dd, 1h, j = 8.4, 1.8 hz) ; c nmr (125 mhz, cdcl3) 2.0, 10.2, 14.1, 27.18, 27.21, 39.10, 39.13, 46.7, 50.0, 50.9, 61.4, 123.4, 123.7, 126.1, 133.9, 142.4, 142.8, 169.5, 175.7, 175.8 ; ir (thin film) 2977 (s), 1761 (vs), 1507 (m), 1337 (m), 1256 (m), 1117 (vs) cm ; hrms (esi - tof) m / z [m+nh4 ] calcd for c26h47n2o8sis, 575.2822 ; found, 575.2834. to a oven - dried 10 ml schlenk flask equipped with a stir bar and filled with nitrogen were added 63a (38 mg, 0.068 mmol, 1.0 equiv), 4-dimethylaminopyridine (0.9 mg, 0.007 mmol, 0.1 equiv), et3n (19 l, 14 mg, 0.14 mmol, 2.0 equiv), and ch2cl2 (0.32 ml). the flask was sealed, and the reaction mixture was stirred for 2 h at room temperature and then stirred at 40 c for 15 h. after the reaction mixture was cooled to room temperature, 0.5 ml of 1 n hcl was added to the flask, followed by the addition of etoac (5 ml). the organic layer was separated, washed with brine (0.3 ml 2), dried with na2so4, and concentrated to give a yellow oil, which was then dissolved in thf (0.65 ml) in a 10 ml schlenk flask filled with nitrogen. to this solution was dropwise added tbaf (0.31 ml, 1 m in thf, 0.31 mmol) at room temperature. after the reaction mixture was stirred at room temperature under nitrogen for 1.5 h, it was concentrated by rotary evaporation and then high vacuum (0.5 mmhg) to give a bright - yellow oil. this oil was dissolved in a mixture of thf (0.2 ml) and et3n (0.2 ml). then trimethylacetyl chloride (0.05 ml, 0.4 mmol) was added to the solution at room temperature. after the resulting reaction mixture was stirred for 15 min at room temperature under nitrogen, it was quenched by the addition of etoh (0.1 ml) and h2o (1.5 ml). the combined organic layer was dried with na2so4, filtered, and concentrated to afford a yellow oil. purification by silica - gel chromatography (20 mm 120 mm, 1:3 etoac / hexanes as eluent) gave 64 as a light - yellow oil in 88% isolated yield (29.5 mg, 0.0598 mmol). spectral data for 64 : rf = 0.27 (1:3 etoac / hexane) ; h nmr (500 mhz, cdcl3) 1.16 (t, 3h, j = 7.2 hz), 1.30 (s, 9h), 1.31 (s, 9h), 1.39 (brs, 9h), 2.622.92 (m, 2h), 4.06 (q, 2h, j = 7.2 hz), 5.06 (brs, 1h), 5.47 (d, 1h, j = 7.5 hz), 7.03 (d, 1h, j = 1.6 hz), 7.06 (d, 1h, j = 8.4 hz), 7.14 (dd, 1h, j = 8.4 hz, j = 1.6 hz) ; c nmr (125 mhz, cdcl3) 14.0, 27.20, 27.22, 28.3, 39.10, 39.12, 40.7, 50.6, 60.9, 79.8, 121.4, 123.5, 124.0, 139.7, 141.8, 142.5, 154.9, 170.7, 175.6, 175.8 ; ir (thin film) 3387 (m), 2978 (s), 2936 (m), 2874 (w), 1761 (vs), 1739 (s), 1723 (s), 1713 (s), 1256 (s), 1119 (vs) cm ; hrms (esi - tof) m / z [m + h ] calcd for c26h40no8, 494.2754 ; found, 494.2758 ; []d20 = + 23.6 (c 1.0, ch2cl2) on 98% ee material. (the optical purity was assumed to be unchanged from that of 55.) | a general study is undertaken to examine the scope of the reductive ring opening of aziridine-2-carboxylates with samarium diiodide. the competition between c c and c n bond cleavage is examined as a function of the nature of the n - substituent of the aziridine, the nature of the substituent in the 3-position of the aziridine, and whether the substituent in the 3-position is in a cis or trans relationship with the carboxylate in the 2-position. the desired c n bond cleavage leads to -amino esters that are the predominant products for most aziridines with an n - activating group. however, c c cleavage products are observed with an aryl group in the 3-position ; this can be particularly pronounced with cis - aziridines where a nearly equal mixture of the two is observed. exclusive formation of the c n cleavage product is observed for all aziridines with the strongly n - activating p - toluene sulfonate group. similarly high selectivity is observed for the 2-trimethylsilylethyl sulfonate group (ses), which is easier to remove. the utility of these methods is illustrated in the synthesis of protected forms of (r)-3-dopa and l - dopa from the same aziridine, the former by smi2-mediated reductive opening at c-2 and the latter by palladium - mediated reductive opening at c-3. |
death by suicide is a leading cause of death,1 and initiatives to advance suicide prevention programmes are a high priority in many countries.2 harmful alcohol use has been recognised as a strong risk factor for suicide death.2 in psychological autopsy studies, alcohol use disorder was strongly associated with suicide death (or=5.2, 95% ci 3.3 to 8.3),3 and heavy alcohol use was also strongly linked with suicide death (or=6.2, 95% ci 5.6 to 6.9 in men).4 in a recent systematic review of the prospective studies, however, the magnitude of the association between alcohol use disorder and suicide death was reported to be moderate (pooled risk ratio=1.74, 95% ci 1.26 to 2.21). the strong associations reported in psychological autopsy studies and case control studies may have resulted, at least in part, from several biases such as surrogate interview bias and recall bias. furthermore, when usual alcohol consumption rather than alcohol use disorder is considered in the general population, evidence of the impact of usual alcohol consumption on suicide death is less than convincing. control studies based on a prospective cohort, have indicated that alcohol consumption was not associated with a higher risk of suicide death, compared to no alcohol consumption, even in the group with the highest consumption in each study,511 with a few exceptions.12 13 overall, the association between usual alcohol consumption and suicide death is unclear. since the majority of adults drink alcohol at some point in their life, a better understanding of usual alcohol consumption and suicide death should inform decision - making for better suicide prevention strategies in the general population. the purpose of this study was to prospectively examine the association of usual alcohol intake - related variables including alcohol intake frequency, average alcohol consumption and quantity of alcohol consumed on a drinking day with suicide death in a community dwelling elderly population in korea. we tried to elucidate questions such as whether greater usual alcohol consumption is associated with a greater risk of suicide death (dose response relationship), and whether moderate alcohol consumption (below 5 drinks / week, or 4 or fewer drinking days a month) has a protective effect against suicide death compared to abstaining completely. the kangwha cohort study was established in march 1985.14 15 among 9378 residents of kangwha county in korea who were 55 years or older as of february 1985, 6372 persons (67.9%) participated in the survey. participants who were lost to follow - up after the initial survey (n=39) were excluded, as were those with missing information about drinking status (n=2), their body mass index (bmi ; n=143), or other covariates (n=25). in the end, a total of 6163 elderly (2635 men ; 3528 women) were included as a study population. the institutional review board of yonsei university (approval no. 4 - 2007 - 0182) no to the question, do you drink alcohol? the drinking frequency was reported as daily, almost daily, 23 times a week, 14 times a month or 412 times a year. the question concerning the type of alcoholic beverage and the amount of alcohol consumption was, how much (in doe (1800 ml), hop (180 ml), bottles or glasses) do you drink of a type of alcoholic beverage? alcohol consumption (alcohol in grams) per week was estimated by multiplying the amount of alcohol consumption per drinking day (ml) by the alcohol content in the particular type of alcoholic beverage, the drinking days per week, and the specific gravity of alcohol (0.8). a different score for drinking days per week was assigned to each drinking frequency (daily : 7.0 ; almost daily : 5.5 ; 23 times a week : 2.5 ; 14 times a month : 0.625 ; 412 times a year : 0.163 ; non - drinking : 0). participants were asked to fill in up to two types of alcoholic beverage they usually consume on one drinking day. the amount of alcohol consumption was calculated for each type and summed. among drinkers, the alcohol content and the bottle volume of each type of alcoholic beverage were determined on the basis of data from the year 1985. soju is a distilled alcoholic beverage, a type of liquor, and makkoli is an unfiltered alcoholic beverage made from rice, both native to korea. soju had 25% alcohol and makkoli 6% alcohol at the time of the survey for the kangwha cohort study in 1985. the amount of ethanol was 10 g in a glass of soju and 9.6 g in a glass of makkoli. the alcohol frequency was merged into four groups in further analysis : daily, 26 days a week (almost daily and 23 times a week combined), 4 or fewer days a month (14 times a month and 412 times a year combined), and non - drinkers. the alcohol intake amount (grams of alcohol) per week was classified into four groups (grams of alcohol per week (drinks per week, with 1 drink as 14 g of alcohol16 ; non - drinkers, 250 g / week) was associated with a non - significantly increased risk of suicide (hr=1.7, 95% ci 1.0 to 2.8) compared to moderate consumption (1100 g / week);17 among 89 299 physicians and 47 654 health professionals in the usa, alcohol consumption was not associated with suicide death;5 7 among 1.3 million korean adults, mostly government and school employees, moderate alcohol intake (124 g / day), but not high alcohol intake (25 g / day), was associated with higher suicide mortality and alcohol intake was not linearly associated with suicide death.6 additionally, among nested case control studies based on prospective cohorts in the usa, the usual amount of alcohol consumption was not associated with suicide death in the community elderly aged 65 years and older, elderly in a retirement community and former college students.810 among prospective studies based on a community cohort, three studies examined the associations in the mainly elderly population with a mean age of 60 years or above.9 10 13 as noted in the previous paragraph, prospective studies have produced inconsistent results on the association between usual alcohol consumption and suicide death. however, results from community - based cohort studies, including the current study, indicated that usual alcohol consumption may be linearly associated with suicide death in the community - dwelling population, at least among current drinkers.1113 additionally, our study does not support a protective effect of moderate alcohol consumption against suicide mortality in accordance with other,12 13 but not all,11 community - based cohort studies. as for occupation - based cohort studies, usual alcohol consumption did not generally show a linear dose, participants were mostly healthier, and had a higher socioeconomic status and potentially easier access to healthcare and other resources than the general population.57 they also had a generally lower alcohol consumption. these factors combined might affect the association between usual alcohol intake and suicide mortality in previous occupation - based cohort studies. prospective cohort studies have seldom evaluated the association between usual alcohol consumption and suicide mortality among men and women separately. in this study, estimates of relative risks associated with alcohol consumption variables a one - drinking - day increase per week, a 70 g (5-drink) increase of average alcohol intake per drinking day, and a 140 g (10-drink) increase of average alcohol intake per week were all higher in women, compared with men. similar results have been observed in a previous study in korean adults, showing that women had a higher relative risk associated with moderate alcohol consumption (124 g / day), compared with men (hr=1.83, 95% ci 1.09 to 3.07 in women ; hr=1.20, 95% ci 0.93 to 1.56 in men).6 p values of the interaction of sex were above 0.05, partly due to the small number of suicide deaths. however, given the lower body weight as well as the lower threshold of alcohol use for better cardiovascular health in women than in men, our findings may support a lower threshold in women than in men for interventions to prevent suicide death. in this study, relative risks associated with alcohol consumption - related continuous variables, unlike depressive symptoms,18 did not decrease during the latter half of follow - up (19972008), compared with the first half (19851996). this finding was concordant with the previous findings in japanese men that relative risk of death by suicide associated with alcohol consumption did not decrease after exclusion of early follow - up.13 this finding suggests that alcohol consumption - related variables may be used as a risk stratification tool for a long - term suicide prevention programme. a prospective design and nearly complete long - term follow - up on suicide using national mortality data are one of the strengths of this study. a homogeneous study population with the same ethnicity and culture, who live in the same regional community, is another of its strengths. yet another strength of our study is that it includes detailed information on alcohol consumption. to the best of our knowledge, only one prospective study examining the association with suicide death collected detailed information on alcohol consumption including drinking frequency, overall average consumption and quantity of alcohol consumed on drinking days.7 this study has limitations. first, we observed only 61 suicide deaths, so statistical power may be lacking in some analyses, including the assessment of interaction by sex. however, the number of suicides in persons with high alcohol consumption such as 30 g / day was comparable to studies with twice the number of all suicide deaths.5 7 12 second, our assessment of alcohol consumption was made using a self - reported questionnaire, and it was not validated separately. however, self - report measures of alcohol consumption are known to have reasonable reliability and validity,19 and they were assessed prospectively before suicide death. therefore, potential measurement errors are most likely non - differential with respect to suicide death, and are unlikely to overestimate the risks. third, depressive symptoms and other psychiatric disorders, strong predictors of suicide death, were not adjusted for.18 however, alcohol use status has been suggested to increase the risk of death by suicide, independently of other psychiatric disorders including depression.20 fourth, other potential confounders such as suicidal ideation, detailed information on health burden, social support and socioeconomic status were not included in the analysis. fifth, our study could not distinguish between never drinkers and ex - drinkers among non - drinkers. since ex - drinkers tended to have higher suicide mortality in some,11 18 but not all,13 previous studies, the risk of suicide death among current drinkers, compared with never drinkers, may be underestimated in this study. sixth, suicide death was defined by death certificates. however, any misclassification of suicide, as mentioned earlier, is likely to have been mostly non - differential with regard to alcohol consumption ; thus, potential misclassifications generally would have been unlikely to overestimate the relative risk. seventh, homogeneity of the participants could also be a limitation of our findings generalisability. the magnitude of association of risk factors with suicide death may differ by ethnicity, culture, age and level of urbanicity.4 18 2124 therefore, some of our findings regarding the korean rural elderly individuals may have limited generalisability to other ethnic, cultural or age groups, or to urban populations. few prospective cohort studies have ever examined the impact of the quantity of usual alcohol consumption on suicide mortality after adjustment for potential confounders.57 1113 17 among community - based cohort studies,1113 in 43 383 japanese men from a public health centre - based cohort, occasional drinkers had the lowest risk of death by suicide, while non - drinkers and heavy drinkers (414 g alcohol per week) had a higher risk;11 in another public health centre - based cohort study in 22 804 japanese men (mean age : 60 years), alcohol consumption was linearly associated with suicide death;13 in 128 934 participants of the northern california medical care programme in the usa, greater alcohol consumption (especially 6 drinks per day) was associated with higher suicide death.12 among occupation - based cohort studies,57 17 in around 49 000 norwegian conscripts, high alcohol consumption (> 250 g / week) was associated with a non - significantly increased risk of suicide (hr=1.7, 95% ci 1.0 to 2.8) compared to moderate consumption (1100 g / week);17 among 89 299 physicians and 47 654 health professionals in the usa, alcohol consumption was not associated with suicide death;5 7 among 1.3 million korean adults, mostly government and school employees, moderate alcohol intake (124 g / day), but not high alcohol intake (25 g / day), was associated with higher suicide mortality and alcohol intake was not linearly associated with suicide death.6 additionally, among nested case control studies based on prospective cohorts in the usa, the usual amount of alcohol consumption was not associated with suicide death in the community elderly aged 65 years and older, elderly in a retirement community and former college students.810 among prospective studies based on a community cohort, three studies examined the associations in the mainly elderly population with a mean age of 60 years or above.9 10 13 as noted in the previous paragraph, prospective studies have produced inconsistent results on the association between usual alcohol consumption and suicide death. however, results from community - based cohort studies, including the current study, indicated that usual alcohol consumption may be linearly associated with suicide death in the community - dwelling population, at least among current drinkers.1113 additionally, our study does not support a protective effect of moderate alcohol consumption against suicide mortality in accordance with other,12 13 but not all,11 community - based cohort studies. as for occupation - based cohort studies, participants were mostly healthier, and had a higher socioeconomic status and potentially easier access to healthcare and other resources than the general population.57 they also had a generally lower alcohol consumption. these factors combined might affect the association between usual alcohol intake and suicide mortality in previous occupation - based cohort studies. prospective cohort studies have seldom evaluated the association between usual alcohol consumption and suicide mortality among men and women separately. in this study, estimates of relative risks associated with alcohol consumption variables a one - drinking - day increase per week, a 70 g (5-drink) increase of average alcohol intake per drinking day, and a 140 g (10-drink) increase of average alcohol intake per week were all higher in women, compared with men. similar results have been observed in a previous study in korean adults, showing that women had a higher relative risk associated with moderate alcohol consumption (124 g / day), compared with men (hr=1.83, 95% ci 1.09 to 3.07 in women ; hr=1.20, 95% ci 0.93 to 1.56 in men).6 p values of the interaction of sex were above 0.05, partly due to the small number of suicide deaths. however, given the lower body weight as well as the lower threshold of alcohol use for better cardiovascular health in women than in men, our findings may support a lower threshold in women than in men for interventions to prevent suicide death. in this study, relative risks associated with alcohol consumption - related continuous variables, unlike depressive symptoms,18 did not decrease during the latter half of follow - up (19972008), compared with the first half (19851996). this finding was concordant with the previous findings in japanese men that relative risk of death by suicide associated with alcohol consumption did not decrease after exclusion of early follow - up.13 this finding suggests that alcohol consumption - related variables may be used as a risk stratification tool for a long - term suicide prevention programme. a prospective design and nearly complete long - term follow - up on suicide using national mortality data are one of the strengths of this study. a homogeneous study population with the same ethnicity and culture, who live in the same regional community, is another of its strengths. yet another strength of our study is that it includes detailed information on alcohol consumption. to the best of our knowledge, only one prospective study examining the association with suicide death collected detailed information on alcohol consumption including drinking frequency, overall average consumption and quantity of alcohol consumed on drinking days.7 this study has limitations. first, we observed only 61 suicide deaths, so statistical power may be lacking in some analyses, including the assessment of interaction by sex. however, the number of suicides in persons with high alcohol consumption such as 30 g / day was comparable to studies with twice the number of all suicide deaths.5 7 12 second, our assessment of alcohol consumption was made using a self - reported questionnaire, and it was not validated separately. however, self - report measures of alcohol consumption are known to have reasonable reliability and validity,19 and they were assessed prospectively before suicide death. therefore, potential measurement errors are most likely non - differential with respect to suicide death, and are unlikely to overestimate the risks. third, depressive symptoms and other psychiatric disorders, strong predictors of suicide death, were not adjusted for.18 however, alcohol use status has been suggested to increase the risk of death by suicide, independently of other psychiatric disorders including depression.20 fourth, other potential confounders such as suicidal ideation, detailed information on health burden, social support and socioeconomic status were not included in the analysis. fifth, our study could not distinguish between never drinkers and ex - drinkers among non - drinkers. since ex - drinkers tended to have higher suicide mortality in some,11 18 but not all,13 previous studies, the risk of suicide death among current drinkers, compared with never drinkers, may be underestimated in this study. sixth, suicide death was defined by death certificates. however, any misclassification of suicide, as mentioned earlier, is likely to have been mostly non - differential with regard to alcohol consumption ; thus, potential misclassifications generally would have been unlikely to overestimate the relative risk. seventh, homogeneity of the participants could also be a limitation of our findings generalisability. the magnitude of association of risk factors with suicide death may differ by ethnicity, culture, age and level of urbanicity.4 18 2124 therefore, some of our findings regarding the korean rural elderly individuals may have limited generalisability to other ethnic, cultural or age groups, or to urban populations. among the korean rural elderly, the frequency and amount of usual alcohol consumption, per drinking day and per week, had a linear dose response relationship with suicide death. when consuming alcohol with the same frequency and amount, compared with men, women might have a higher relative risk of suicide. our findings suggest that there is no protective effect of moderate alcohol consumption on suicide death. our results also support the lower the better policy of alcohol intake, and a sex - specific alcohol threshold for any suicide prevention strategy implemented. what is already known on this subjectin a recent systematic review of the prospective studies, the magnitude of the association between alcohol use disorder and suicide death was moderate (pooled risk ratio=1.74, 95% ci 1.26 to 2.21).evidence that greater usual alcohol consumption is associated with higher suicide death in the prospective studies among the general population is inconclusive. what this study addsthe greater frequency and amount of usual alcohol consumption was linearly associated with higher suicide death, and there was no protective effect of moderate alcohol consumption against suicide death in the elderly persons in a rural community.when consuming alcohol with the same frequency and amount as men, women might have a higher relative risk of suicide. in a recent systematic review of the prospective studies, the magnitude of the association between alcohol use disorder and suicide death was moderate (pooled risk ratio=1.74, 95% ci 1.26 to 2.21). evidence that greater usual alcohol consumption is associated with higher suicide death in the prospective studies among the general population is inconclusive. the greater frequency and amount of usual alcohol consumption was linearly associated with higher suicide death, and there was no protective effect of moderate alcohol consumption against suicide death in the elderly persons in a rural community. when consuming alcohol with the same frequency and amount as men, women might have a higher relative risk of suicide. | backgroundthe evidence from prospective studies on whether greater usual alcohol consumption is associated with a higher risk of death by suicide in the general population is inconclusive.methods6163 participants (2635 men ; 3528 women) in a 1985 survey among rural residents in korea aged 55 years and above were followed until 2008. a cox model was used to calculate hrs of suicide death after adjustment for demographic, socioeconomic and health - related confounders.results37 men and 24 women died by suicide. elderly persons who consumed alcohol daily, 70 g alcohol (5 drinks) or more per drinking day, or 210 g alcohol (15 drinks) or more per week had higher suicide mortality (p<0.05), compared with non - drinkers. an increase of one drinking day per week (hr=1.17, 95% ci 1.05 to 1.31), 70 g (5 drinks) additional alcohol intake per drinking day (hr=1.38, 95% ci 1.13 to 1.70), and 140 g (10 drinks) additional alcohol intake per week was associated with a 17%, 38% and 12% higher risk of suicide death, respectively. women had a higher relative risk of suicide death associated with alcohol consumption, compared with men.conclusionsa greater frequency and amount of usual alcohol consumption was linearly associated with higher suicide death. given the same amount of alcohol consumption, women might have a higher relative risk of suicide than men. our findings support the lower the better for alcohol intake, no protective effect of moderate alcohol consumption, and a sex - specific guideline (lower alcohol threshold for women) as actions to prevent suicide death. |
purulent conditions of the mediastinum and lungs spread by continuity through the interfascial spaces of the neck are among the rarest complications of odontogenic inflammatory processes of the oral cavity. descending necrotizing mediastinitis (dnm) is a noteworthy example of such infection, originating from inflammatory conditions within the oral cavity and teeth. descending necrotizing mediastinitis develops in patients between 30 and 50 years old and occurs six times more frequently among men. the symptoms characteristic of dnm include strong retrosternal pain (intensifying during breathing and coughing), rapid breathing, tachycardia, increased body temperature, and inflammatory infiltration of the neck and chest. treatment consists in combining broad - spectrum antibiotic therapy with radical dissection and drainage of the purulent foci located in the chest and mediastinum. despite intensive treatment, dnm mortality remains high, amounting to as much as 20 - 50% of cases. the aim of this study was to present the case of a 26-year - old woman treated at the clinical department of maxillofacial surgery and the department of anesthesiology and intensive care of the regional specialist hospital in rzeszw, in whom, in consequence of odontogenic infection and phlegmon of the face and neck, dnm developed with numerous lung and mediastinal abscesses, resulting in the patient 's death. descending necrotizing mediastinitis as a complication of odontogenic infections is rarely encountered in everyday practice ; therefore, it appears purposeful to report the case of this patient who was recently treated at our department. the 26-year - old female patient was admitted to the clinical department of maxillofacial surgery of the regional specialist hospital in rzeszw due to a swelling of the left cheek (which spread to the mental region before involving bilaterally the submandibular region), difficulties with swallowing, and limited jaw opening. several days before the appearance of the swelling, the patient had experienced tooth pain on the left side of the mandible, which she found difficult to locate precisely. a clinical examination was performed, revealing a massive swelling of the left cheek and the submandibular and mental regions. neck lymph nodes on both sides were unavailable for examination due to the swelling as well as significant local pain. intraoral examination revealed insufficient oral hygiene, numerous tooth roots and teeth with their pulp in a state of gangrenous decay, and pain on percussion of teeth 36 and 46. the mucous membrane of the floor of the oral cavity had undergone inflammatory changes, and a hard, painful infiltrate was present. the patient 's life parameters were within normal ranges : body temperature 31.1c, heart rate 80 bpm, arterial pressure 140/80 mmhg. laboratory investigation revealed high leukocytosis (43 100/mm), electrolyte impairments reduced levels of sodium and chlorides (na 130 mmol / l and cl 95.0 mmol / l), glucose concentration increased to approximately 180 mg / dl, and crp 44.3 mg / dl. the remaining parameters, i.e. morphology, coagulation parameters, biochemical tests, urea, and creatinine, were within the normal ranges. ultrasound examination of the neck revealed a hypoechogenic area of irregular shape located in the mental region at the depth of 1.5 to 4 cm probably an inflammatory infiltrate. lymph nodes around the salivary gland and the right corner of the mandible were of irregular shape, hypoechogenic, and swollen to 22 13 mm. panoramic radiography revealed extensive damage to the hard tissue of teeth 18, 46, and 47, reaching the pulp chambers of the teeth ; presence of tooth roots 14, 26, and 34 ; and thinned bone structure characteristic of chronic periapical periodontitis in the vicinity of the apexes of teeth 34, 46, and 47 (fig. panoramic radiograph image day of admission the patient underwent pharmacological treatment, receiving antibiotics (clindamycin), analgesics, and steroids (dexamethasone) ; subsequently, under general anesthesia, she underwent the removal of teeth deemed to constitute the foci of inflammation (14, 26, 34, 46, 47) ; the abscess forming in the mental and submandibular regions was incised and drained., sensitive to the following antibiotics : penicillin g, amoxicillin, piperacillin, amoxicillin / clavulanic acid, metronidazole, clindamycin, and cefotetan., new purulent foci were incised and drained in the superolateral part of the neck ; a small amount of fetid, brown, bloody material was obtained. interfascial spaces were connected and, subsequently, rinsed multiple times with solutions of metronidazole and sodium bicarbonate. after the incision and evacuation of the abscess, the patient reported a slight improvement in her well - being and an improvement in her ability to swallow. laboratory investigation revealed a drop in leukocytosis to 15 800/mm ; the high level of crp persisted (53.7 mg / dl), as did the electrolyte impairments reduced potassium (k 2.6 mmol / l) and high glycemic values (up to 220 mg / dl). potassium supplementation was maintained, and, additionally, a second antibiotic was introduced (amoxicillin with clavulanic acid). during the 5 day of hospitalization, a stinging pain appeared in the patient 's interscapular area, intensifying during deep inhalation and cough. in the respiratory tract, a dense, mucous secretion was present, which the patient was unable to expectorate, mostly due to strong pain. the dimensions of the cheek infiltrate decreased ; a dough - like swelling of the neck persisted, but did not show a tendency for abscess formation. plain chest x - ray revealed the presence of fluid in the left pleural cavity reaching rib vii in the posterior axillary line, an obscured contour of the right dome of the diaphragm and the right costophrenic angle, small atelectatic consolidations in the right lower lung field, and an increased cardiac silhouette (fig. 2). photograph of the patient 's chest 4 day of hospitalization at the department of maxillofacial surgery left thoracocentesis was performed, but no fluid was obtained. from the 6/7 day of hospitalization, an internist consultation confirmed the suspicion of pneumonia and pleuritis as a complication of an odontogenic infection ; additionally, steroid diabetes was revealed. the subsequently performed chest x - ray demonstrated an increased amount of fluid in both pleural cavities. moreover, in the lower field of the right lung, a well - delineated shadow area (5 2.5 cm in size) appeared, indicating the development of inflammation in this lobe. bilateral thoracocentesis was performed ; in total, over 200 ml of fluid was evacuated and passed on for bacteriological examination. as no significant improvement of the patient 's general condition was achieved, a chest and neck ct was performed, revealing spaces with dense fluid (probably pus) in the left side of the neck and air - filled spaces reaching the level of the mandibular ramus. the fluid - filled spaces were located in the masseter muscle area, down to the parapharyngeal and retropharyngeal spaces, reaching laterally to the supraclavicular area and entering the chest the mediastinum was enlarged, reaching the size of 95 75 mm, up to the level of the cardiac waistline. singular abscesses were present in lung parenchyma ; their largest sizes were 80 mm in the right lung and approximately 45 mm in the left. fluid was present in the pleural cavities : up to 20 mm in width in the right cavity and up to 20 mm in the left. subcarinal and paratracheal lymph nodes had increased in size to approximately 23 mm. there were no focal lesions in the bone structure of the examined area (fig. 3). after a thoracic surgeon was consulted, the patient was qualified for urgent surgery due to mediastinal phlegmon and the suspicion of empyemas in both pleurae. bilateral drainage of the parapharyngeal spaces and the mediastinum was performed via cervical access ; right - sided thoracotomy with mediastinal incision and right lung decortication were conducted and followed with left - sided videoassisted thoracoscopic surgery (vats). opening the chest revealed the presence of organizing purulent material in the pleural cavity as well as necrotic lesions in the tissues of the mediastinum, extending to the pericardial sac and secreting purulent material. irrigation drainage was introduced to the mediastinum and pleura. during the left - sided vats, cloudy fluid was evacuated, adhesions were freed, fibrin was partially removed from the supradiaphragmatic area, and irrigation drainage was applied. chest ct 8 day of hospitalization (preoperative image) after the surgery, the patient, exhibiting symptoms of sepsis and postoperative respiratory failure, was transferred directly from the operating theater at the department of maxillofacial surgery to the department of anesthesiology and intensive care (daic). at the daic, in severe condition and under analgosedation (midazolam, morphine sulfate) chest x - ray performed during the 1 day of the patient 's stay at the daic showed a regression of lesions. samples of material from the bronchial tree (bronchofiberoscopy bal) were collected for bacteriological testing, along with pus drained from the pleural cavity and mediastinum, as well as blood and urine. broad - spectrum antibiotic therapy was introduced (cefotaxime, amicacin, metronidazole), along with steroid therapy, anti - fungal therapy, anticoagulative treatment, fluid therapy, and parenteral feeding. during the 1 day after thoracotomy, the patient 's circulation was stable : sap 110 - 120 mmhg, hr 100 - 115/min, sato2 98 - 99% with fio2 at 40%, diuresis 1500 ml / day, normal body temperature. tests for the presence of hbvag, anti - hcv, and hiv were negative. klebsiella pneumoniae esbl a microorganism sensitive to the antibiotics already administered to the patient, was isolated from culture material collected from the bronchi. wide access to the interfascial spaces of the neck was maintained ; they were rinsed regularly in order to prevent premature occlusion of the draining duct. in accordance with the postoperative recommendations of the thoracic surgeon, irrigation drainage of both pleural cavities and, the patient 's condition did not change : it was severe, but stable. inflammation markers were reduced. due to the long - lasting secretion of purulent material from the drains, moreover, a new fluid - filled lesion appeared in the right pleural cavity, along with changes suggesting pneumonia (fig. bleeding was stopped pharmacologically, and the air leak, despite mechanical ventilation, was small and subsided on its own. during the 8 day at the daic, the patient 's condition deteriorated. her body temperature rose rapidly to 39.2c, and ventilation conditions worsened, requiring the increase of fio2 to 0.5. during the 10 day at the daic, the patient 's general conditioned continued to deteriorate significantly. with fio2 at 50 - 100% a non - depolarizing blocking agent (vecuronium) was added to the therapy, achieving a slight improvement in terms of ventilation conditions. the results of cultures from the right pleural cavity indicated the presence of acinetobacter baumannii, a multiresistant bacterial strain resistant to the antibiotics previously administered to the patient. in accordance with the antibiogram a multiresistant strain of enterococcus faecium, sensitive to linezolid, was cultured ; linezolid was subsequently added to the therapy. during the 11 day, pressor amines (norepinephrine) were added to the therapy. despite intensive treatment, signs of multiple organ dysfunction syndrome intensified. the therapy was unsuccessful : the patient died during the 12 day of treatment at the daic. abscesses and odontogenic inflammatory conditions of the head and neck are often encountered in clinical practice and are usually not difficult to diagnose and treat. in rare cases, such infections may spread through the interfascial spaces of the neck to the chest and mediastinum [1, 2 ]. the dnm case of a 26-year - old woman described above is an example of a complication which posed a significant diagnostic and treatment challenge and resulted in the patient 's death. most authors list odontogenic infections related to pulp infections of the lower molars as the primary cause of dnm [3, 4 ]. according to statistical data, such infections are responsible for 50 - 80% of dnm occurrences. in the present case, the purulent complications also originated from dental neglect and the fact that the pulp of numerous lower molars of the patient was in a state of gangrenous decay. the literature underscores that the typical bacterial flora of the upper airway and the gastrointestinal tract plays an important role in the etiopathogenesis of dnm [5, 6 ]., streptococcus anginosus, candida albicans, klebsiella pneumoniae, enterococcus faecium, and acinetobacter baumannii. descending necrotizing mediastinitis is a rare complication ; according to freeman, even thoracic surgeons rarely encounter this disease during their professional careers. it is, therefore, understandable that this complication constitutes a significant problem in the practice of various specialized fields, including maxillofacial surgery. knowledge of the pathogenesis and symptoms of dnm is of cardinal significance in diagnosing, treating, and predicting the course of the disease. the clinical symptoms of dnm observed in our patient were similar to those described in the literature [79 ] ; however, it appears that they were erroneously interpreted as the signs of concomitant respiratory tract infection, which delayed the correct diagnosis. deterioration of the patient 's general condition, growing leukocytosis, and rising crp and pct values are indications for supplementing the diagnostic investigation with head, neck, and chest ct with contrast. literature data demonstrate the superiority of ct over conventional chest x - ray examinations in the early diagnosis of dnm. the present case confirms the above, as the initial changes visible on the x - ray image were non - specific, and dnm was only diagnosed after the performance of a ct scan. in terms of surgical treatment, it is of fundamental importance to remove any necrotic tissue as well as bacteria and their toxins and, subsequently, employ irrigation drainage. in conclusion, it should be underscored that chest pain, cough, and lack of improvement in the general condition of a patient after the drainage of abscesses around the jaw and neck should be indicative of remote lung complications, including dnm. our observations confirm that, in the treatment of complicated inflammatory conditions of the head and neck, multi - specialist cooperation of maxillofacial surgeons, thoracic surgeons, and intensive care specialists, as well as, in some cases, otolaryngologists, is indispensable. | this paper presents the case of a 26-year - old female patient in whom descending necrotizing mediastinitis (dnm) developed as a complication of an odontogenic purulent infection of the mouth. despite the efforts of a multidisciplinary treatment team, the patient died with symptoms of septic shock and multiple organ failure. according to the literature, and as confirmed by our own observations, successful treatment requires early tomographic diagnosis, radical surgery, combination antibiotic therapy, and intensive care. |
cervical cancer represents the third most common cancer in women worldwide, with 86% of all deaths occurring in developing countries, where there is lack of access to prevention, early diagnosis and treatment (jemal., 2011). in brazil, cervical cancer ranks as the second most frequent cancer among women between 15 and 44 years of age (who / ico, 2010). most cervical cancers can be detected early through a routine exfoliative cytology test (papanicolaou test), a screening which has been used for more than 50 years in an effort to decrease the number of deaths from cervical cancer (stack, 1997). dna testing has also been reported as an important tool for detecting hpv (mayrand., 2007) and is being introduced in some countries as an adjunct to the papanicolaou test or as the primary screening test (castle., 2012). in developing countries, this introduction has been controversial, mainly because cytology - based screening programs suffer from lack of basic healthcare infrastructure (bradford and goodman, 2013). in a meta - analysis study, hpv types 16, 18, 31, 33 and 45 were shown to be the most prevalent types associated with cervical carcinomas in latin america (ciapponi., 2011). chromosomes or chromosome segments that fail to be incorporated into nuclei during cell division configure micronuclei (mn). the mn test, readily performed via microscopy, has been successfully used to screen population groups at risk for cancer, representing a sensitive indicator of genetic damage (samanta and dey, 2012). the frequency of mn appears to increase in carcinogen - exposed tissue before there are any clinical symptoms (gayathri., 2012). considering the fact that oncogenic hpv viral proteins may induce cytogenetic changes in exfoliated cells of the uterine cervix, a positive correlation between micro - nucleated cells and dysplasia in papanicolaou smears has been reported (gandhi and kaur, 2003 ; guzmn., 2003 ; corts - gutirrez., 2010 ; aires., 2011). taking this into account, the aim of the present study was to investigate the association between hpv - dna and mn in women from southern brazil with normal cervical cytology in order to contribute additional information to cervical cytology analysis. additionally, some of the most prevalent high - risk hpv types were investigated. a total of 158 normal cervical smears were analysed cytologically for hpv - dna and mn comparisons. the smears were collected through the use of a cytobrush in a gynecology clinic at a healthcare medical centre (posto de sade modelo) in porto alegre, rio grande do sul, southern brazil from march to december 2009. all women who agreed to participate signed an informed consent form. this study was approved by the ethics committee of universidade luterana do brasil (number 2008 - 601h). two slides were made, one for the conventional papanicolaou exam and another for mn analysis. after the slides were prepared, the cytobrush was placed in a tube containing 2 ml te 1x and stored at 20c for later dna extraction. for the papanicolaou test, the analyses were based on the bethesda system (solomon., 2002). the slides for detecting and counting mn were stained with giemsa, and evaluated four times, twice by each of two observers independently. a total of 1000 cells were counted under a light microscope at a 1000 magnification following the criteria described by tolbert. a non - organic method developed in our laboratory was used to extract the dna from the clinical specimens (michelon., 2011). the resulting sediment was resuspended in 50 l of 1x te and incubated at 99 c for 10 min. the dna was then purified using 5 l of a glass matrix (concert extraction systems ; life technologies, rockville, md, usa). the amplification of a 150 bp fragment of the l1 region of the hpv genome was performed using the gp5+/biogp6 + consensus primers (de roda husman., 1995). the pcr was carried out in a final volume of 50 l containing 50 mm kcl, 10 mm tris - hcl ph 8.5, 2.5 mm mgcl2, 0.1 mm of each deoxynucleoside triphosphate, 50 ng of each primer, 2.5 u of taq dna polymerase (invitrogen) and 10 l of extracted dna. a negative control (no dna) was included in each pcr run to ensure that there was no cross contamination. samples were amplified under the following conditions : initial denaturation step at 95 c for 5 min, 40 cycles at 95 c for 1 min, 52 c for 1 min and 72 c for 1 min, and a final elongation step at 72 c for 10 min. dna of caski (hpv-16 positive) cells were used as a positive control in all pcr reactions. the final pcr product (10 l) was visualized in electrophoresis agarose gel (1.5%) under uv light. hpv dna genotyping of high - risk types 16, 18, 31, 33, 39, 45 and 59 was carried out using a microplate colorimetric hybridization assay (mcha) developed in our laboratory (barcellos., 2011). seven dna plasmids containing the l1 gene of a high - risk hpv type (hpv16, 18, 31, 33, 39, 45 and 59) were used as positive controls. the absolute and relative frequencies of the categorical variables and the distribution of the continuous variables were evaluated to define the profile of the population under study. chicago, il, usa). the test was used to study differences in categorical parameters the mean age of the 158 women was 42.7 13.9 years, the mean age of menarche was 11.6 1.7 years and the mean number of children per woman was 1.4 1.2 children. in terms of other baseline characteristics, 107 (65.5%) had a stable marital status, 119 (73%) were caucasian, 25 (25.2%) smoked, 58 (35.6%) used oral contraceptive, and 78 (47.9%) were post - menopausal. the mean age of hpv - dna positive women (34.2 12.6) was significantly lower than the mean age of hpvdna negative women (43.9 13.7) (p = 0.003). the positive samples were tested for the presence of hpv 16, 18, 31, 33, 39, 45 and 59. fourteen of the 20 positive samples (70.0%) were positive for hpv 16, with 7 out of 20 (35.0%) being positive for hpv 31 ; 4 out of 20 (20.0%) for hpv 33 ; 2 out of 20 (10.0%) for hpv 39 ; 3 out of 20 (15.0%) for hpv 45 ; and 2 out of 20 (10.0%) for hpv 59. infection by one and multiple hpv types was found in 11 out of 20 (55.0%) and 9 out of 20 (45.0%) samples, respectively. the cellular changes consistent with the presence of mn were observed in 47 (29.7%) of 158 slides, of which 11 were hpv - positive and 36 were hpv negative. the presence of mn was significantly associated with hpv - dna positivity (p = 0.016) (table 1). on the other hand, the absence of mn observed in a considerable number of hpvdna negative samples (102) may be of great value in predicting the absence of hpv. in developed countries, the variability of performance in cytology - based screening has led to the development of more effective alternatives, such as reliable molecular testing. several large randomized controlled trials have concluded that hpv testing is more sensitive than cytology for cervical cancer screening (mayrand., 2007 ; castle., 2012). despite the importance of hpv testing, its use requires advanced technology and still has a high cost, limiting the possibility of its introduction in developing countries. considering the importance of cervical cancer control programs and the limited number of studies on mn scoring in normal cervical cytology (aires. 2012), the present study was intended to evaluate whether mn is a suitable test to complement the results of the papanicolaou smear in a low - resource setting, analyzing the association between hpv - dna and mn in normal cervical cytology of women from southern brazil. mn scoring on the cytology smear is easy, cheap, reproducible and can be performed on routinely stained papanicolaou smears as a biomarker of chromosomal damage (gayathri., 2012 ; samanta and dey, 2012). in the present study, the prevalence of hpv - dna was 12.7% and was similar to that found in a meta - analysis study, in which the estimated global hpv prevalence in women with normal cytology was 11.7% (bruni., brazil has the highest incidence of cervical cancer, with a prevalence of hpv infection among women with normal cervical cytology of 21.1%, being the northern and northeastern regions with higher mortality than eastern and southern states (dutra., 2012). in another study developed in rio grande do sul state, the prevalence of hpv - dna found in women with normal cytology was of 32.7% (coser., 2013). it is reasonable to assume that these differences in hpv prevalence can be due to risks factors associated to the characteristics of populations in different countries, sensibility of the study method used to detect dna, and/or the number of women included in each study (sample size). this finding supports previous research, which concludes that hpv prevalence is higher in women younger than 35 years of age, decreasing in women of more advanced age (sanjos., 2007). this result corroborates earlier findings that hpv 16 is the most prevalent type of hpv woldwide (guan., 2012). in the present study, the occurrence of mn was associated with the presence of hpv - dna in normal cervical cytology, thus corroborating data obtained by other authors (aires., 2011 ; this finding suggests that the presence of mn, even with no cytological signs of hpv, can be used by the physician as a criterion for repeating the test within a shorter interval of time. on the other hand, the high number of samples with negative results in both tests (mn and hpv - dna) suggests that these women are at low risk of hpv infection and can be called in for a new round of screening at the normal interval. other authors have used the micronucleus test as a biomarker for chromosome instability and malignancy, observing higher frequencies of micronucleated cells in samples with cervical cancer than in controls (majer., 2001 ;, 2008 ; corts - gutirrez., 2010 ; gayathri., finally, there is a consensus that the most clinically effective and cost - effective methods for reducing the incidence of cervical cancer are those that limit the number of required visits (hoppenot., 2012). since cervical screening is usually based on cytology alone, the study of mn in papanicolaou smears can improve the quality of diagnosis and screening. | the aim of the present study was to investigate the association between hpv - dna and micronucleus (mn) frequency in women with normal cervical cytology. a total of 158 normal cervical smears were analyzed cytologically. the hpv genome was amplified using the gp5+/biogp6 + consensus primers. hpv - dna of high - risk types 16, 18, 31, 33, 39, 45 and 59 were also investigated. of the 158 samples, 20 (12.7%) and 47 (29.7%) were positive for hpv - dna and mn, respectively. evidence for mn was found in 11 out of 20 (55%) hpv - dna positive samples and in 36 out of 138 (26.1%) hpv - dna negative ones. mn presence was significantly higher in hpv - dna positive samples (p = 0.016). on the other hand, the absence of mn observed in a considerable number of hpv - dna negative samples (102) may be of great value in predicting the absence of hpv. the mean age of hpv - dna positive women (34.2 12.6) was significantly lower than the mean age of hpv - dna negative women (43.9 13.7) (p = 0.003). infection by one or multiple hpv types was found in 11 out of 20 (55.0%) and 9 out of 20 (45.0%) samples, respectively. the evaluation of mn using cervical smears collected for cytology tests could, thus, be used as additional information to monitor a population s exposure to hpv. |
hypercalcemia, syndrome of inappropriate secretion of antidiuretic hormone, cushing 's syndrome, and skeletal - connective tissue syndromes are common in patients with lung cancer, especially those with small cell lung cancer (sclc). approximately two thirds of sclc patients present with metastatic stage at the time of diagnosis. lambert - eaton syndrome occurs in 3%percnt ; of sclc patients as neurological paraneoplastic syndrome. however, the association of malignancy and guillain - barr syndrome (gbs) is not well known. it is a rare disease that occurs at a rate of 1.11 cases per 100,000 person - years. however, once infected, up to 20%percnt ; of patients develop severe disability, and approximately 5%percnt ; die. there have been reports of paraneoplastic gbs in different cancers. to the best of the present authors knowledge, there are 6 published reports of gbs in sclc [2, 3, 4, 5, 6, 7 ]. here, we report a case of a 52-year - old man who was diagnosed with gbs in sclc with chemotherapy. he was diagnosed with sclc, limited disease, at another hospital 5 years ago. he had been recommended concurrent chemoradiotherapy at that time but had wanted to be treated with chemotherapy only. after 6 cycles, complete remission was noted, and prophylactic cranial irradiation was given. a year later, he had relocated residence and visited our hospital, presenting with relapsed lung cancer but refusing treatment. after 7 months, further disease progression and pancreatic metastasis was noted on computed tomography (ct) scanning. meanwhile, superior vena cava syndrome occurred, and radiation therapy was delivered in the middle of chemotherapy. a ct scan for the response showed disease progression. he was started on cisplatin, doxorubicin, and cyclophosphamide (cap) and also received palliative brain radiotherapy due to brain metastasis during the chemotherapy. however, in his fifth year of cancer diagnosis, he presented with lower - limb weakness and was admitted. he noticed weakness in both legs 2 weeks prior to admission and developed numbness throughout the lower extremities. a neurological examination revealed no nuchal rigidity or kernig 's sign as well as absence of deep tendon reflexes. magnetic resonance imaging (mri) of the brain showed some unidentified bright objects (fig. however, there was little possibility of brain metastasis or embolic infarction when comparing the patient 's symptoms and physical examination with the mri lesions. to evaluate neurologic problems, it revealed a fracture of the second lumbar vertebra that seemed to be benign (fig., the ascending paralysis, from the lower extremities to the upper extremities, did not correlate with the cord compression lesion either. nerve conduction studies showed decreased amplitudes and slow velocities of compound muscle action potential and sensory nerve action potential in the extremities. a lumbar puncture yielded cerebrospinal fluid protein 94 mg / dl, albumin 61.3 mg / dl, glucose 173 mg / dl, and white blood cells 1/l. these were assessed as being due to albuminocytological dissociation, and there were no signs of meningitis. the patient was diagnosed with gbs and was recommended treatment with intravenous immunoglobulin (ivig), which he refused. it could be caused by adverse effects of chemotherapy or by other underlying diseases that have nothing to do with malignancies, such as diabetes. however, these sensorimotor neuropathies almost always result in mild to moderate dysfunction and usually do not affect patients survival. it is characterized by both limbs and muscles innervated by cranial nerves being weakened. approximately 70%percnt ; of gbs cases a progressive bilateral, symmetric weakness of the extremities manifests over a period of 12 h to 28 days from disease onset. the nerve conduction study result was consistent with acute peripheral neuropathy, a cerebrospinal fluid analysis demonstrating an elevated protein level and absence of leukocytosis due to albuminocytological dissociation will be helpful to confirm the gbs diagnosis. gbs should take into account various diseases, such as brain or leptomeningeal metastasis from cancer, and spinal cord compression. described neuropathy occurring after treatment with a combination of oxaliplatin and cisplatin ; nardone. in addition, when patients with gbs are not able to walk independently, immunotherapy is required. in immunotherapy, either ivig or plasmapheresis can be used. these treatments have been observed to be effective when started within the first 2 weeks after disease onset. gbs occurred in a patient receiving long - term antineoplastic therapy, whereas gbs had occurred at the initial point of presentation in the 6 other cases [2, 3, 4, 5, 6, 7 ]. this is the first reported case of paraneoplastic gbs that developed in an sclc patient with long - term therapy. to the present authors knowledge, in the 5 cases of gbs with sclc, there was no relationship between chemotherapy and gbs. in 4 of the 5 reports, patients received chemotherapy ; however, in all 4 of these reports, the patients underwent chemotherapy after gbs had been diagnosed and ivig had been given. in addition, in 3 of these 4 reports, a combination of etoposide and carboplatin was used, whereas the other report did not indicate the chemotherapy regimen [2, 3, 4, 5, 6 ]. immunotherapy was recommended at the time of diagnosis, but he refused the treatment because of his advanced cancer stage and poor general condition. patients with cancer are vulnerable to infection and can have problems with the immune system. as illustrated by this report, paraneoplastic gbs in sclc is a life - threatening disease. thus, it should be monitored and treated when acute progressive peripheral neuropathy occurs in patients with sclc. the authors submitted a waiver from their institutional review board stating that this case report does not require institutional review board approval or oversight. | guillain - barr syndrome (gbs) is defined as an acute, autoimmune polyradiculoneuropathy. it is a rare disease that occurs at a rate of 1.11 cases per 100,000 person - years. however, once infected, up to 20%percnt ; of patients develop severe disability, and approximately 5%percnt ; die. there have been reports of gbs in different cancers. among them, there are 6 previous reports of gbs in small cell lung cancer. here, we report a case of a 52-year - old man who was diagnosed with gbs in the setting of small cell lung cancer with chemotherapy. |
calcinosis cutis is the pathologic deposition of insoluble calcium salt in the skin and subcutaneous tissue. it is classified into four main subtypes : dystrophic, metastatic, idiopathic, and iatrogenic. dystrophic calcinosis cutis follows inflammation, infection, connective tissue diseases, and so on. complications of acne vulgaris such as calcinosis cutis are rarely reported previously in the literature. a 55-year - old man who is a diagnosed case of obstructive sleep apnea (osa) presented with symptoms of sinusitis for a period of 15 days. a computed tomography (ct) scan was done to confirm the diagnosis of sinusitis. however, ct scan showed multiple soft tissue calcification distributed all over the face mainly in the region of distribution of acne vulgaris suggestive of calcinosis cutis [figure 1 ]. the patient was given antibiotics and supportive treatment for sinusitis after which he recovered completely. he reported that he had severe acne vulgaris in his adolescence for which he did not take any treatment. on retrospective questioning however, the patient also had multiple skin - colored papules of various sizes ranging from pin head to maximum of 5 mm over the face predominantly on cheeks [figure 2 ], nose, and forehead for a period of 25 years. the plasma calcium, phosphate, vitamin d, and parathormone levels were within normal range. however, all other hematological and biochemical investigations including complete blood count and peripheral smear, liver, kidney function tests, and urine microscopy were normal. antinuclear antibody, rheumatoid factor, p- and c - anca, anti - topoisomerase, anticentromere antibodies, and hiv serology were negative. computed tomography scan showing multiple soft tissue calcification distributed all over the face mainly in the region of distribution of acne vulgaris suggestive of calcinosis cutis a punch biopsy from the acne scars on the face showing areas of small calcific deposits in the upper - to - mid dermis a punch biopsy was done from the acne scars on the face, which showed areas of small calcific deposits in the upper - to - mid dermis. multiple skin - colored papules of various sizes ranging from pin head to maximum of 5 mm over the left cheek it is caused by abnormal follicular hyperkeratinization and overproduction of sebum by the sebaceous gland. acne vulgaris affects nearly 80% of the population between 12 and 25 years without gender, ethnicity, or race prevalence differences. the pathophysiology of acne vulgaris includes increased sebum production, follicular hyperkeratinization, proliferation of propionibacterium acnes, and production of inflammation. usually calcinosis cutis presents as multiple, hard, whitish papules, plaques, or nodules. it is known to occur in a variety of disorders and classified into four subtypes according to its etiology : dystrophic, metastatic, iatrogenic, and idiopathic. the term dystrophic calcinosis is used for calcification associated with infection, inflammatory processes, cutaneous neoplasm, or connective tissue diseases. it is the most common type of ectopic calcification and develops around localized tissue damage, with no alterations of calcium or phosphate metabolism. in contrast, metastatic calcification is characterized by abnormal calcium and/or phosphate metabolism that leads to the precipitation of calcium in cutaneous and subcutaneous tissue. medical intervention can cause tissue damage or disturbances in calcium and phosphate metabolism, leading to soft tissue calcification in some cases known as iatrogenic calcinosis cutis. in a normal tissue, ectopic deposits of calcium salts develop when the calcium phosphate product in plasma exceeds 70 mg / dl. however, in a damaged tissue the following pathogenic phenomena may play a role increased intracellular calcium concentration, denaturation of proteins that preferentially bind phosphate, genetic mutations of elastic fibers and collagen, and increased g - carboxyglutamic acid. calcinosis cutis is a rare disease, so there are no controlled clinical trials on its treatment. various medical and surgical modalities have been found to be beneficial but none has been accepted as standard. medical management include warfarin, bisphosphonates, minocycline, ceftriaxone, diltiazem, aluminum hydroxide, probenecid, intralesional corticosteroids, and intravenous immunoglobulin. surgical modalities include curettage, surgical excision, carbon dioxide laser, and extracorporeal shock wave lithotripsy. our patient had a history of acne lesions during puberty, following which scar lesions were present on his face. however, despite an extensive search no relationship was found between osa and calcinosis cutis. thus, it is concluded that dystrophic calcinosis cutis can develop rarely secondary to acne vulgaris. | acne vulgaris is a common dermatological disease commonly affecting the adolescent and young adults. it is characterized by the presence of pleomorphic skin lesions such as comadones, papules, pustules, and nodules. the common complications are postacne hyperpigmentation and scarring causing psychological impact. calcinosis cutis is the pathologic deposition of insoluble calcium salt in the skin and subcutaneous tissue. calcinosis cutis following acne vulgaris is rarely reported in the literature. we report a case of calcinosis cutis in acne vulgaris in a 55-year - old man. |
dental caries and periodontal diseases occur in nearly 95% of the population and are bacterially mediated processes.1 in children at moderate to high risk for dental caries the predominantly surgical approach needs to be combined with an antimicrobial agent to ensure adequate control of the caries process. several antibiotic or antimicrobial agents have been tested including chlorhexidine, povidone - iodine, and fluoride mouthrinses.2 another approach to bacterially- mediated diseases has been the use of probiotics which has been defined as bacterial cultures or living micro - organisms which upon ingestion in certain numbers, exert health benefits beyond inherent general nutrition and support a good healthy intestinal bacterial flora. the idea was that the harmless bacteria in the fermented products competed with pathogens injurious to health. a wide range of probiotic products containing different bacterial strains are commercially available such as lozenges, sucking tablets, chewing gums, dairy products such as milk, ice cream, cheese, yoghurt, etc.1 the archetypical probiotic food is yoghurt (curd) and daily consumption of dairy products seems to be most natural way to ingest probiotic bacteria.3 dairy yoghurt is produced using a culture of lactobacillus delbrueckii subsp. in addition, lactobacillus acidophilus, lactobacillus bifidus and lactobacillus casei are also sometimes used in culturing yoghurt. recent studies have shown that certain gut bacteria including lactobacilli and bifidobacterium may exert beneficial effects in the oral cavity by inhibiting cariogenic streptococci and candida species.1 in this study, the probiotic yogurt (curd) containing lactobacillus acidophilus has been used to investigate its effects on salivary ph and streptococcus mutans counts. this study was carried out in 40 healthy caries - free school children aged 10 - 12 years. a complete oral examination was also carried out using the who format after obtaining an informed consent. using simple random sampling (lottery method) the samples were allocated to either test or control groups. forty healthy children in the age group of 10 - 12 yearscaries free children (d m f t = 0) forty healthy children in the age group of 10 - 12 years caries free children (d m f t = 0) children with decayed teethchildren under antibiotic treatment during the course of the studychildren on any other probiotic supplements during the course of the studychildren on use of any xylitol products for three weeks before and during the course of the study children with decayed teeth children under antibiotic treatment during the course of the study children on any other probiotic supplements during the course of the study children on use of any xylitol products for three weeks before and during the course of the study basic examination kit consisting of disposable mouth mask and gloves, mouth explorer, tweezers and sterile cottonnestle dahi (curd) with probioticnestle fresh n natural dahi (curd)ependorff tubesdigital ph metermitis salivarius bacitracin (msb) agar (hi - media)calibrated inoculating loop and bunsen burnermicro slides1ml and 10 ml pipettesincubatorautoclave basic examination kit consisting of disposable mouth mask and gloves, mouth explorer, tweezers and sterile cotton nestle dahi (curd) with probiotic nestle fresh n natural dahi (curd) mitis salivarius bacitracin (msb) agar (hi - media) calibrated inoculating loop and bunsen burner 1ml and 10 ml pipettes paraffin stimulated whole saliva was collected from all forty children in the morning on the day 1 (baseline sample) and 30 days after the intervention period. the saliva was expectorated directly into ependorff tubes during a 5 min chewing period after a thorough rinse with water. a digital ph meter analyzed the collected saliva. a serial dilution of the collected salivary sample the diluted salivary sample was streaked on mitis salivarius bacitracin agar (msb) selective media for s. mutans. the plates were incubated at 37c for 36 - 48 h and the number of colonies was counted based on the colony characteristics [figure 1 ] and confirmed by gram staining. [figure 2 ] msb agar with streptococcus mutans growth gram stained streptococcus mutans the children were then randomly divided into two groups of 20 each. operators were blinded, they were not aware of which yoghurt was given to them. control group : consisted of children who were given normal curd. both the groups were given curds (probiotic or normal) for 1 month. after 30 days of consumption of curds, salivary samples were collected and tested for salivary ph and salivary s. mutans count was estimated. excel and spss (spss inc, chicago) software packages were used for data entry and analysis. the results were averaged (mean + standard deviation) for each parameter and are presented in tables 1 & 2 and figures 3 & 4. comparison of the ph between test group and control group at baseline and at 30 days interval comparison of ph between test group and control group at 30 days interval mean ph values in the test group and control group at baseline and 30 days interval mean number of colonies in test group and control group at baseline and 30 days interval forty healthy children in the age group of 10 - 12 yearscaries free children (d m f t = 0) forty healthy children in the age group of 10 - 12 years caries free children (d m f t = 0) children with decayed teethchildren under antibiotic treatment during the course of the studychildren on any other probiotic supplements during the course of the studychildren on use of any xylitol products for three weeks before and during the course of the study children with decayed teeth children under antibiotic treatment during the course of the study children on any other probiotic supplements during the course of the study children on use of any xylitol products for three weeks before and during the course of the study basic examination kit consisting of disposable mouth mask and gloves, mouth explorer, tweezers and sterile cottonnestle dahi (curd) with probioticnestle fresh n natural dahi (curd)ependorff tubesdigital ph metermitis salivarius bacitracin (msb) agar (hi - media)calibrated inoculating loop and bunsen burnermicro slides1ml and 10 ml pipettesincubatorautoclave basic examination kit consisting of disposable mouth mask and gloves, mouth explorer, tweezers and sterile cotton nestle dahi (curd) with probiotic nestle fresh n natural dahi (curd) mitis salivarius bacitracin (msb) agar (hi - media) calibrated inoculating loop and bunsen burner 1ml and 10 ml pipettes basic examination kit consisting of disposable mouth mask and gloves, mouth explorer, tweezers and sterile cottonnestle dahi (curd) with probioticnestle fresh n natural dahi (curd)ependorff tubesdigital ph metermitis salivarius bacitracin (msb) agar (hi - media)calibrated inoculating loop and bunsen burnermicro slides1ml and 10 ml pipettesincubatorautoclave basic examination kit consisting of disposable mouth mask and gloves, mouth explorer, tweezers and sterile cotton nestle dahi (curd) with probiotic nestle fresh n natural dahi (curd) mitis salivarius bacitracin (msb) agar (hi - media) calibrated inoculating loop and bunsen burner 1ml and 10 ml pipettes paraffin stimulated whole saliva was collected from all forty children in the morning on the day 1 (baseline sample) and 30 days after the intervention period. the saliva was expectorated directly into ependorff tubes during a 5 min chewing period after a thorough rinse with water. a digital ph meter analyzed the collected saliva. a serial dilution of the collected salivary sample the diluted salivary sample was streaked on mitis salivarius bacitracin agar (msb) selective media for s. mutans. the plates were incubated at 37c for 36 - 48 h and the number of colonies was counted based on the colony characteristics [figure 1 ] and confirmed by gram staining. [figure 2 ] msb agar with streptococcus mutans growth gram stained streptococcus mutans the children were then randomly divided into two groups of 20 each. operators were blinded, they were not aware of which yoghurt was given to them. control group : consisted of children who were given normal curd. both the groups were given curds (probiotic or normal) for 1 month. after 30 days of consumption of curds, salivary samples were collected and tested for salivary ph and salivary s. mutans count was estimated. paraffin stimulated whole saliva was collected from all forty children in the morning on the day 1 (baseline sample) and 30 days after the intervention period. the saliva was expectorated directly into ependorff tubes during a 5 min chewing period after a thorough rinse with water. a serial dilution of the collected salivary sample was done in saline. using inoculating loop the diluted salivary sample was streaked on mitis salivarius bacitracin agar (msb) selective media for s. mutans. the plates were incubated at 37c for 36 - 48 h and the number of colonies was counted based on the colony characteristics [figure 1 ] and confirmed by gram staining. [figure 2 ] msb agar with streptococcus mutans growth gram stained streptococcus mutans the children were then randomly divided into two groups of 20 each. operators were blinded, they were not aware of which yoghurt was given to them. control group : consisted of children who were given normal curd. both the groups were given curds (probiotic or normal) for 1 month. after 30 days of consumption of curds, salivary samples were collected and tested for salivary ph and salivary s. mutans count was estimated. excel and spss (spss inc, chicago) software packages were used for data entry and analysis. the results were averaged (mean + standard deviation) for each parameter and are presented in tables 1 & 2 and figures 3 & 4. comparison of the ph between test group and control group at baseline and at 30 days interval comparison of ph between test group and control group at 30 days interval mean ph values in the test group and control group at baseline and 30 days interval mean number of colonies in test group and control group at baseline and 30 days interval in the control group, the mean ph values were higher at baseline compared to the mean ph at 30 days. a statistically significant reduction in the mean ph from baseline to 30 days the mean ph values were higher at baseline compared to the mean ph at 30 days. a statistically significant reduction in the mean ph from baseline to 30 days the mean ph value in test group was slightly higher compared to the control group but this difference was not statistically significant (p>0.05) [table 2 ]. the mean colony count in control group at baseline was higher when compared with the mean colony count at 30-day time interval. but this reduction in mean colony count was not statistically significant (p>0.05) [table 3 ]. comparison of the number of colonies between test group and control group at baseline and at 30 days interval the mean colony count in test group at baseline was higher when compared with the mean colony count at 30-day time interval. this reduction in mean colony count was found to be statistically significant (p<0.01) [table 3 ]. at 30 days time interval, the mean number of colonies was found to be higher in control group compared to test group and this difference was found to be statistically significant (p<0.05) [table 4 ]. comparison of the number of colonies between test group and control group at 30 days interval dental caries being a multifactorial disease process often requires a multimodal approach to treatment and control. one of the antimicrobial approaches has been the use of probiotic - enriched products such as milk, ice cream, lozenges, cheese and yoghurt. several of these normal dietary constituents contain microorganisms, which can function as probiotic agents. however, these specially formulated probiotic products contain around 10 to 10 colony forming units of microorganisms per milliliter. the hypothesized mechanisms of action include (a) direct interactions in the dental plaque with interference of biofilm formation, plaque ecology, competing with oral microbes for the available substrate and production of antimicrobial substances and (b) indirect actions including modulation of systemic immune function, local immunity, effect on nonimmunologic defense mechanisms, regulation of mucosal permeability and oral colonization by less pathogenic species.4 the present study was conducted to investigate the inhibitory effect of short term consumption of probiotic curd containing lactobacillus acidophilus on salivary streptococcus mutans counts, its effect on salivary ph value and to compare this with normal curds in caries - free children. n natural dahi) was selected as it is a common dietary constituent of the indian population, is readily available, inexpensive and can be used in several different combinations that are acceptable to the paediatric age group. unlike milk and milk products, curd being semisolid may be retained in the oral cavity for a longer period of time with an extended beneficial effect. further, this study aims to compare the relative effects of regular curd with a specially formulated probiotic curd (nestle dahi with probiotic) that contains 10 colony forming units per milliliter of lactobacillus acidophilus. several studies have shown that lactobacilli may inhibit the growth of other bacteria including streptococci5 and lactobacilli6 by producing low molecular weight bacteriocins.7 there have been no documented studies correlating the effect of probiotic containing curd on salivary ph. in the present study, salivary samples were used to determine any change in ph and s. mutans counts on the day before onset (baseline) and 30 days after the intervention period (consumption of probiotic curds). the assessment was therefore of the impact of probiotic, as well as normal curd on the general ecology of the oral cavity rather than a specific plaque evaluation. on comparison of mean salivary ph in the control group, as well as the test group, a statistically significant lowering of mean ph value was seen at 30 days interval as compared to baseline [table 1 ]. at 30 days interval, the mean ph value in test group was slightly higher as compared to the control group but this difference was not statistically significant [table 2 ]. this decrease in salivary ph on consumption of both probiotic containing curd and normal curd was primarily due to the acidic nature of the curds. however, the ph levels were still above the critical ph (5.2 - 5.5), which otherwise could have been considered as harmful for the causation or progression of dental caries. this further proves that curd though acidic in nature does not pose any risk for caries active children. conversely it may prove beneficial due to its calcium, phosphorous, protein and vitamin content. streptococcus mutans has been implicated as one of the major and most virulent of the caries - producing organisms. therefore, the effect of probiotic curd containing lactobacillus acidophilus on s. mutans count was looked into. in this study, the s. mutans colony counts in the test group at 30-day time interval was significantly lower than at baseline. in the control group the results are in accordance to other studies which support the use of probiotics against salivary streptococcus mutans, oral malodour and other oral infections like candida albicans.816 this effect against streptococcus mutans has been attributed to the general antimicrobial activity of curd rather than a specific competitive activity of curd microorganisms.67 due to the paucity of studies using curd / yoghurt a definite conclusion on the possible mechanism of action in reducing cariogenic bacteria could not be drawn from the present study. further studies need to be conducted to investigate the effect of probiotic agents on oral ecology. however, the use of lactobacillus acidophilus based milk product has been justified due to the good buffering capacity of milk, presence of tooth protective minerals including calcium.17 however, the use of these products has been contraindicated in children with open carious lesions.3 also in patients on long term use of probiotics monitoring of oral health becomes important along with lactobacilli counts.18 most antimicrobial strategies are effective only as long as the patient is on the treatment. following cessation of the intervention the oral bacterial counts rapidly return to the original counts. in this regard probiotics may prove to be beneficial especially when combined with a modification in dietary patterns along with effective restorative treatments. further studies need to be done in the use of these probiotic products in children with varying degrees of caries risk over a longer period of time. monitoring of plaque microbial counts may provide additional indicators of the ecological changes that may occur with use of these products. the following conclusions can be drawn from the present study : probiotic curds containing lactobacillus acidophilus was found to have an inhibitory effect on the salivary streptococcus mutans countsthe consumption of normal curd and probiotic curd caused a small decrease in salivary ph that is still above the critical ph levelprobiotic curds containing lactobacillus acidophilus may be used as adjuncts, for prevention of dental caries especially as a part of dietary modification in children at varying risk for dental caries. probiotic curds containing lactobacillus acidophilus was found to have an inhibitory effect on the salivary streptococcus mutans counts the consumption of normal curd and probiotic curd caused a small decrease in salivary ph that is still above the critical ph level probiotic curds containing lactobacillus acidophilus may be used as adjuncts, for prevention of dental caries especially as a part of dietary modification in children at varying risk for dental caries. | background : antimicrobial methods of controlling dental caries that include probiotic agents can play a valuable role in establishing caries control in children at moderate to high risk for developing dental caries. several studies have demonstrated the beneficial effects of use of various probiotic products including curd. the objective of this study was to compare the effect of short - term consumption of probiotic curd containing lactobacillus acidophilus and normal curd on salivary streptococcus mutans counts, as well as salivary ph.materials and methods : forty, caries - free, 10 - 12 years old children were selected and randomly allocated to two groups. test group consisted of 20 children who consumed 200ml of probiotic curd daily for 30 days. control group consisted of 20 children who were given 200ml of regular curd for 30 days. salivary ph and salivary streptococcus mutans counts were recorded at baseline and after 30 days and statistically compared using the student 's t-test.results:consumption of probiotic curd resulted in a statistically significant reduction in s. mutans colony counts (p0.05) in both the groups.conclusion:short-term consumption of probiotic curds can reduce oral s. mutans counts. however, this caused a slight reduction in salivary ph. |
we initially studied 90 patients (age 21 years) who had newly diagnosed all and were enrolled on the st., we subsequently studied an additional 170 patients with all enrolled on the same treatment protocol, using publicly available snp data we had previously reported. jude children s research hospital, and informed consent was obtained from patients, their guardians, or both before enrollment. leukemia cells were isolated by applying a ficoll - hypaque gradient to bone marrow aspirates obtained at diagnosis (median, 97% blast cells). normal leukocytes were isolated from peripheral blood samples obtained after the successful completion of remission induction therapy (on days 4548 after the start of treatment). to assess gene copy number loss in msh2-l leukemia cells, dna was extracted from leukemia cells and normal peripheral blood leukocytes (obtained when patients were in complete remission) and genotyped for 600k snps using the affymetrix genechip human mapping 50k - hind-240, 50k - xba-240, 250k - sty and 250k nsp snp arrays. dna was restriction enzyme digested, pcr - amplified, purified, labeled, fragmented and hybridized to the arrays according to the manufacturer s instructions. snp array data were analyzed using dchip for chromosomal abnormalities. to improve the accuracy of copy number inference using dchip, we applied a normalization procedure that uses snps exclusively from regions shown to be diploid in the normal leukocytes and maps signals from those snps to a common target probability distribution. the snps with an estimated copy number lower than 1.40 were considered as evidence of deletions. we initially performed snp analysis on prkcz, because it is known to regulate msh2 stability. this led us to subsequently perform a broader pathway analysis, to interrogate additional genes upstream of prkcz (figure 1c). for each snp, we evaluated the over - representation of deletions in msh2-l leukemia cells compared to msh - h leukemias, using fisher s exact test. the significance of each snp was then adjusted for multiple testing using 100,000 permutations. at each permutation, we recorded the smallest p - value among all the 122 snps based on randomly assigned msh2 status. the adjusted p - values were computed as the proportion of permutations whose smallest p - values were lower than or equal to the observed. the human t - lineage leukemia cell line ccrf - cem was obtained from the american type culture collection. the human pre - b leukemia cell lines 697 and nalm-6 were obtained from the german collection of microorganisms and cell cultures. cells were cultured in rpmi-1640 medium containing 2 mm glutamine and 10% fetal bovine serum at 37 c with 5% co2. ccrf - cem cells were infected with mission lentiviral transduction particles (sigma - aldrich) produced from a library of sequence - verified shrnas targeting human pik3c2b, frap1/mtor, herc1 or prkcz transcripts. non - target shrna control particles (shc002v) were also purchased from sigma - aldrich. fisher s exact test was used to test the overrepresentation of losses among msh2 low patients versus msh2 positive patients. a linear regression model was used to test the correlation between the levels of msh2 mrna and protein. the expression levels of the probe sets were analyzed by applying a general linear model in which the effect of msh2 status (positive vs. negative) was adjusted for the all genetic subtypes. the cox proportional hazards model was used to identify independent prognostic effect of msh2 status. for patients who achieved complete remission, cumulative incidence of hematological relapse were constructed by the method of kalbfleisch and prentice, and compared with gray s test. we initially studied 90 patients (age 21 years) who had newly diagnosed all and were enrolled on the st., we subsequently studied an additional 170 patients with all enrolled on the same treatment protocol, using publicly available snp data we had previously reported. jude children s research hospital, and informed consent was obtained from patients, their guardians, or both before enrollment. leukemia cells were isolated by applying a ficoll - hypaque gradient to bone marrow aspirates obtained at diagnosis (median, 97% blast cells). normal leukocytes were isolated from peripheral blood samples obtained after the successful completion of remission induction therapy (on days 4548 after the start of treatment). to assess gene copy number loss in msh2-l leukemia cells, dna was extracted from leukemia cells and normal peripheral blood leukocytes (obtained when patients were in complete remission) and genotyped for 600k snps using the affymetrix genechip human mapping 50k - hind-240, 50k - xba-240, 250k - sty and 250k nsp snp arrays. dna was restriction enzyme digested, pcr - amplified, purified, labeled, fragmented and hybridized to the arrays according to the manufacturer s instructions. snp array data were analyzed using dchip for chromosomal abnormalities. to improve the accuracy of copy number inference using dchip, we applied a normalization procedure that uses snps exclusively from regions shown to be diploid in the normal leukocytes and maps signals from those snps to a common target probability distribution. the snps with an estimated copy number lower than 1.40 were considered as evidence of deletions. we initially performed snp analysis on prkcz, because it is known to regulate msh2 stability. this led us to subsequently perform a broader pathway analysis, to interrogate additional genes upstream of prkcz (figure 1c)., we evaluated the over - representation of deletions in msh2-l leukemia cells compared to msh - h leukemias, using fisher s exact test. the significance of each snp was then adjusted for multiple testing using 100,000 permutations. at each permutation, we recorded the smallest p - value among all the 122 snps based on randomly assigned msh2 status. the adjusted p - values were computed as the proportion of permutations whose smallest p - values were lower than or equal to the observed. the human t - lineage leukemia cell line ccrf - cem was obtained from the american type culture collection. the human pre - b leukemia cell lines cells were cultured in rpmi-1640 medium containing 2 mm glutamine and 10% fetal bovine serum at 37 c with 5% co2. ccrf - cem cells were infected with mission lentiviral transduction particles (sigma - aldrich) produced from a library of sequence - verified shrnas targeting human pik3c2b, frap1/mtor, herc1 or prkcz transcripts. non - target shrna control particles (shc002v) were also purchased from sigma - aldrich. fisher s exact test was used to test the overrepresentation of losses among msh2 low patients versus msh2 positive patients. a linear regression model was used to test the correlation between the levels of msh2 mrna and protein. the expression levels of the probe sets were analyzed by applying a general linear model in which the effect of msh2 status (positive vs. negative) was adjusted for the all genetic subtypes. the cox proportional hazards model was used to identify independent prognostic effect of msh2 status. for patients who achieved complete remission, cumulative incidence of hematological relapse were constructed by the method of kalbfleisch and prentice, and compared with gray s test. | dna mismatch repair enzymes (e.g., msh2) maintain genomic integrity, and their deficiency predisposes to several human cancers and to drug resistance. we found that leukemia cells from a substantial proportion of patients (~11%) with newly diagnosed acute lymphoblastic leukemia (all) have low or undetectable msh2 protein levels (msh2-l), despite abundant wild - type msh2 mrna. msh2-l leukemia cells contained partial or complete somatic deletions of 14 genes that regulate msh2 degradation (frap1, herc1, prkcz, pik3c2b) ; these deletions were also found in adult all (16%) and sporadic colorectal cancer (13.5%). knockdown of these genes in human leukemia cells recapitulated the msh2 protein deficiency by enhancing msh2-degradation, leading to significant reduction in dna mismatch repair (mmr) and increased resistance to thiopurines. these findings reveal a previously unrecognized mechanism whereby somatic deletions of genes regulating msh2 degradation result in undetectable levels of msh2 protein in leukemia cells, mmr deficiency and drug resistance. |
for well over a decade, the cost of in vitro and in vivo screening of absorption, distribution, metabolism, excretion, and toxicity (adme / tox) properties of molecules has motivated efforts to develop various in silico methods to efficiently pre - filter candidates for actual physical testing. by relying on very large, internally consistent datasets, large pharmaceutical companies have succeeded in developing highly predictive but ultimately proprietary models. at one pharmaceutical company, for example, many of these models (e.g., volume of distribution, aqueous kinetic solubility, acid dissociation constant, distribution coefficient, microsomal clearance, cyp3a4 time - dependent inhibition) as well as other endpoints have achieved such high accuracy that they have essentially put the experimental assays out of business. it is likely that most large pharmaceutical companies can now perform experimental assays for a small fraction of compounds pre - filtered through the proprietary adme / tox and physicochemical property computational models, thus improving cost efficiency while minimizing in vitro and animal experimentation. extra - pharma computational efforts have not been so successful, largely because they have, by necessity, drawn upon considerably smaller datasets, in many cases trying to combine information from the literature. this situation, however, has improved with larger datasets publicly available in pubchem, chembl, cdd, and others, and some drug companies depositing their data (e.g., the recently deposited astrazeneca data in chembl), which can be useful for model building. adme / tox properties have been modeled by us and many other groups using an array of machine learning algorithms such as support vector machines, bayesian modeling, gaussian processes, and many others. a more exhaustive review of the different machine learning approaches is outside the scope of this work. these combined efforts at adme / tox model building have likely resulted in hundreds of published models which are, unfortunately, inaccessible to anyone but their authors in most cases. this limited access problem for published models is also likely the case with computational models for bioactivity or other physicochemical properties of interest. the ability to share such models freely still remains a major challenge when dealing with issues of proprietary samples or data, as repercussions for such for - profit pharmaceutical companies could be severe. the current development of technologies for open models and descriptors builds on established methodologies. datasets for quantitative structure activity relationships (qsar) have previously been represented in a reproducible way via qsar - ml. these methods also come with a reference implementation for the bioclipse workbench, which provides a graphical interface. there have been several early efforts at cheminformatics web services ; e.g., indiana university provides access to cheminformatics methods (fingerprints, 2d depiction, and various molecular descriptors) and statistical techniques. in addition, there are web tools for the prediction of bioactivities and physicochemical properties, like the chemistry activity predictor (gusar). also, the open notebook science (ons) project has developed models for solubility and melting point using web services based on open descriptors and algorithms. these tools all enable parties to collaborate publicly but do not facilitate private or selective collaboration. we have previously demonstrated a proof of concept using open descriptors and modeling tools to model very large adme datasets at pfizer. models were constructed with open descriptors and keys (cdk + smarts) using open software (c5.0) and performed essentially identically to expensive proprietary descriptors and models (moe2d + smarts + rulequest s cubist) across all metrics of performance when evaluated on human liver microsomal stability (hlm), rrck passive permeability, p - gp efflux, and aqueous solubility datasets. pfizer s hlm dataset, used in this study, contained more than 230,000 compounds and covered a diverse range of chemistry space as well as addressing many therapeutic areas. the hlm dataset was split into a training set (80%) and a test set (20%) using the venetian blind splitting method. in addition, a newly screened set of 2310 compounds was evaluated as a blind dataset. all the key metrics of model performance, e.g., r, rmse, kappa, sensitivity, specificity, and positive predictive value (ppv), were nearly identical for the open source approach vs proprietary software (e.g., ppv = 0.80 vs 0.82). our goal is to enable extra - pharma drug discovery projects to exploit in silico machine learning methods that have, until now, been confined in practice to pharma and to a few knowledgeable academics. these methods better exploit limited screening resources and will enable such projects to cover more unexplored chemical space and to address adme / tox earlier in the discovery process. extra - pharma projects represent a growing trend for commercial drug discovery to be the principal efforts to find cures for many neglected diseases (e.g., tuberculosis, malaria, chagas disease, visceral leishmaniasis, etc.), and thousands of orphan indications will require more collaborations and data, and therefore model sharing. this approach has the potential to accelerate the discovery of promising drug - like lead compounds with acceptable properties in vivo and ultimately yield a significant impact on global health. we now describe the creation of a reference implementation of a bayesian model - building software module, which we have released as an open source component that is now included in the chemistry development kit (cdk) project and incorporated using the fcfp6 descriptors in the cdd vault, which was also recently made open source. we make use of the cdd public database, which has over 100 public datasets that can be used to generate community - based models, including extensive neglected infectious disease sar datasets (malaria, tuberculosis, chagas disease, etc.), and admedata.com datasets that are broadly applicable to many projects. an accompanying paper uses this software to develop models on a much larger scale. all molecules for malaria, tuberculosis, and cholera datasets from table 1 are available in cdd public (https://app.collaborativedrug.com/register), and the models from table 2 are available from http://molsync.com/bayesian1. bayesian models have been a useful part of computer - aided drug discovery for many years and were popularized in pipeline pilot. the statistical method is particularly useful for correlated structure - derived fingerprint bit strings with an activity measurement that has been classified as active or inactive on the basis of a selected threshold. variants on the original bayes theorem can be used to produce an estimate of the likelihood of activity for proposed compounds. for reproducing binary classifications, bayesian methods the model creation process is typically very fast and can be implemented in o(n) time, which means that an ordinary desktop computer can build and evaluate models with hundreds of thousands of compounds with minimal delay. when general purpose structure - derived fingerprints are used, the methods tend to be quite robust, which is in contrast to methods such as qsar, which require some expertise to select appropriate descriptors, avoid overtraining, and ensure domain applicability. unlike many other applications of bayesian methods, the use of chemical structure fingerprints as inputs means priors often numbers in the thousands, which produces scale distortions. even if the prior probabilities are approximately 0.5 in each case, multiplying thousands of such values together tends to warp the distribution of the posterior probabilities to being asymptotically close to 0 or 1, which introduces numerical precision issues. by summing the logarithms of the ratios rather than multiplying the fractions, and incrementing the numerator and denominator, the precision issues are eliminated, and the resulting predictions tend to follow a linear distribution. the main drawback with this particular bayesian variant is that the resulting prediction is not a probability, but rather an arbitrary number that has no particular upper or lower bound. the results can be converted into a two - state classification by selecting a threshold, or into a probability - like value by picking a linear transformation function, but these are post - bayesian calibrations that must be made by applying judgment criteria that are not an intrinsic part of the method. we have used our work on the reference extended connectivity ecfp and fcfp fingerprints to create a software class that allows bayesian models to be created from a collection of molecules and activity data, used to predict probabilities for new molecules, and to serialize / deserialize the model as a structure text string that can be saved to a file or shared with any other package that implements the same functionality. the laplacian - modified nave bayesian (which we call bayesian models for simplicity) formula uses a simple definition, which pre - supposes that each molecule has been described by enumerating a list of fingerprints that applies to it, and has a determination of whether it is active or inactive. for each fingerprint code in the entire dataset : where ci is the contribution associated with the presence of a fingerprint hash code i, which is in turn derived from ai and ti, which are respectively the number of active molecules with the fingerprint and the total number of molecules with the fingerprint, while r is the overall fraction of actives. building the bayesian model is a simple matter of determining the total set of fingerprints in the dataset and, for each of them, calculating the value of ci. any fingerprint that is theoretically possible but not encountered in the training set has an implied value of 0. any fingerprint hash code that is observed equally often in active and inactive molecules (or not at all in either) has a ratio of 1, for which the log value is zero. when making a prediction for an incoming molecule, the value is determined by adding up the contributions for each fingerprint hash code for the molecule : the resulting prediction, pm, is an uncalibrated value : unlike for the conventional bayes theorem, the result is not a probability and is generally not directly interpretable, meaning that there is no significance to either the scale or offset. creating a bayesian model using this method is very fast and has favorable scalability properties, because it requires just two passes through the input collection : the total number of actives and inactives needs to be summed, and after that, each compound needs to be considered only individually. the total memory required to build the bayesian model is bounded by the theoretical number of fingerprints. for each possible unique fingerprint hash code, it is necessary to store two integers (ai, ti) and derive one floating point value per fingerprint (ci). for small, relatively dense fingerprint schemes, these can be stored in a flat array (e.g., when folding fingerprints into 1024 possible values), but for larger schemes with sparse occupancy it is better to use a dictionary object (e.g., when the full 32-bit range of ecfp6 or fcfp6 fingerprints is allowed). the pseudocode for the model building is as follows : let t = empty dictionary (key : hash code i, value : total t) let a = empty dictionary (key : hash code i, value : actives a)for m in all molecules in training set : determine list of fingerprints f for molecule mfor fingerprint hash code i in f : increment tiif molecule m is active : increment ailet r = total actives / total moleculeslet c = empty dictionary (key : hash code i, value : contribution v)let l = unique list of keys for tfor i in l : put ci = log([ai + 1]/[tir + 1])for making a prediction for an incoming molecule : let m = molecular structuredetermine list of fingerprints f for molecule mlet pm = 0for i in l : if i is one of the fingerprints in f : let pm = pm + ci let t = empty dictionary (key : hash code i, value : total t) let a = empty dictionary (key : hash code i, value : actives a) for m in all molecules in training set : determine list of fingerprints f for molecule mfor fingerprint hash code i in f : increment tiif molecule m is active : increment ai determine list of fingerprints f for molecule m for fingerprint hash code i in f : increment tiif molecule m is active : increment ai if molecule m is active : increment ai let r = total actives / total molecules let c = empty dictionary (key : hash code i, value : contribution v) let l = unique list of keys for t for i in l : put ci = log([ai + 1]/[tir + 1 ]) put ci = log([ai + 1]/[tir + 1 ]) let m = molecular structure determine list of fingerprints f for molecule m if i is one of the fingerprints in f : implementing these algorithms using a flat array rather than a dictionary object is analogous and differs only in the way indices are looked up. the method described in this article is implemented in the chemistry development kit (cdk) project and made available under the terms of the lesser gnu public license (lgpl). the latest version of the project can be obtained from its sourceforge host and underlying git repository (http://sourceforge.net/p/cdk/code/ci/master/tree). the bayesian modeling capabilities are available within the tools section, the main class for which is org.openscience.cdk.fingerprint.model.bayesian. using the cdk library to create a new bayesian model from a collection of molecule objects and boolean activity values for example, given the filename for an mdl sdfile with a field called pic50, for which any molecule with a value of 6 or greater is considered active, the following java code snippet can be used to create a serialized model : the resulting serialized form can be stored for future use. if it is stored in a file, it can be easily retrieved and used to apply to a different sdfile, e.g. : the first step is to read the model from the pre - existing file (activity.bayesian). the second step iterates over the input sdfile, while writing to another sdfile with two extra fields appended : rawprediction, which contains the uncalibrated outcome from the modified bayesian method, and scaledprediction, which contains the prediction that has been scaled using metrics originally derived from the internal cross - validation. these two examples demonstrate the create and consume use cases and can be easily adapted to scenarios besides reading and writing from files. serialized bayesian models can be embedded in any kind of text - friendly data structure, e.g., xml documents, json messages, sql tables, etc. use of models to provide predictions can be applied to a variety of invocations, such as command line tools, incorporation into modeling packages with a graphical interface, web services accessible via api, etc. for saving models for subsequent reuse, the information necessary to apply the model to make predictions for new molecules can be stored in a text - based file format. the molecules that were used to build the model are not included in the serialized form, nor are the fingerprints that were generated from them. this means that sharing a serialized model allows the recipient to make inferences on the basis of the original data without explicitly having access to it. for confidentiality purposes, sharing models without the underlying data is useful in a number of situations, but it should be noted that this can not be considered as entirely foolproof : a determined hacker with some context would likely be able to make a well educated guess as to the actives contained in the training set. the default file extension is.bayesian, and the mime type is chemical / x - bayesian. the text should be encoded as utf-8 unicode, for which all of the content is limited to the ascii subset, except for the freeform text notes. end of line should be encoded unix - style, and floating point numbers can be encoded with a decimal point (e.g., 1.23, with a period symbol for the separator, invariant of localization) or scientific notation (e.g., 1.23 10). the format is case- and whitespace - sensitive. the body of the format consists of individual lines, each of which encodes a discrete property, and is of arbitrary length. the default file extension is.bayesian, and the mime type is chemical / x - bayesian. the first line contains the header, which consists of the recognition sequence and essential information about the model. the first nine characters are always set to the ascii characters for the string bayesian ! (hex : 42 61 79 65 73 69 61 6e 21), which can be used as a recognition sequence. this is useful for situations such as embedding in streams, within whitespace - padded subfields such as xml elements, or when the file extension or mime type is unavailable or unreliable. the recognition sequence is followed by four comma - separated fields : fingerprint type, folding length, calibration minimum, and maximum. only the first two fields are mandatory, and parsers should ignore additional fields, in case the format is subsequently extended. the fingerprint type must be one of ecfpn or fcfpn, where n is 0, 2, 4, or 6. these correspond to the eight different permutations of circular fingerprints that are implemented in the cdk library. when a parser encounters a fingerprint type that it does not recognize, it should invoke an error pathway if there is any intention of applying the model to new molecules, since the ability to produce the exact same fingerprints is a pre - requisite. the folding length should either be 0 (no folding, i.e., full range of 32-bit integers) or a power of 2 (e.g., 512, 1024, 2048, etc.). the parser should fail for invalid folding lengths. the calibration minimum and maximum values this is calculated by analyzing the cross - validation metrics, which is an optional step, the information may not be available. if not included, then the model can only be used to generate raw prediction values. note that sometimes the minimum and maximum values are equal, which can occur for datasets that are small or trivial. in this case, the degenerate value should be treated like a simple threshold, giving results of 0 or 1, rather than a probability - like transform. the model specification ends with a line beginning with the string ! end. this should be considered as the terminator sequence regardless of trailing characters or whitespace. all lines in between the header and footer can be examined out of order. lines that match the template { bit index}={contribution }, where bit index is an integer and contribution is a floating point number, make up the payload of the model. the contributions - per - bit are typically stored in a dictionary object, since the bit coverage is sparse, i.e. usually not all of the possible bits are represented. if the fingerprints are folded, then the bit indices range from 0 to folding-1. if not folded, the indices are represented as signed integers : approximately half of the values will be negative. for generating raw bayesian predictions, all that is needed is fingerprint type, folding, and contribution list. all remaining lines are optional and must follow the general format of { category}:{key}={value }, whereby category and key must be plain ascii without whitespace, category must begin with an alphanumeric character, and value may contain any unicode characters, except for end - of - line. when software needs to parse, modify then write a model file, any optional lines that are not understood should be preserved as - is. the optional data that are currently used by the cdk implementation include the following:training : size and training : actives : the total number of compounds in the training set, and the number of actives, respectivelyroc : auc : integral of the receiver operator characteristic, which is a number between 0 and 1roc : type : the method used to partition the data for internal cross - validation, which is one of leave - one - out, three - fold, or five-fold.roc:x and roc : y : two comma - separated lists of numbers from 0 to 1 which can be used to recreate the roc curve visually. note that for large datasets, the total number of points may be reduced in order to limit the impact on file size. this means that while the resolution is indistinguishable for graph plotting purposes, recalculating the integral from these points is less precise than using the stored roc : auc value.note:title : ideally a short free - text description of the model that communicates to a scientist what data were being modeled. it should be expected to be displayed in a single line.note:origin : a short free - text description that provides information about the provider of the model, be it the software algorithm or the source of the data, or both.note:comment : a completely freeform field, which may be of any length. since newlines are disallowed, multiple paragraphs of comments should be encoded by having multiple comment lines. training : size and training : actives : the total number of compounds in the training set, and the number of actives, respectively roc : auc : integral of the receiver operator characteristic, which is a number between 0 and 1 roc : type : the method used to partition the data for internal cross - validation, which is one of leave - one - out, three - fold, or five - fold. roc : x and roc : y : two comma - separated lists of numbers from 0 to 1 which can be used to recreate the roc curve visually. note that for large datasets, the total number of points may be reduced in order to limit the impact on file size. this means that while the resolution is indistinguishable for graph plotting purposes, recalculating the integral from these points is less precise than using the stored roc : auc value. note : title : ideally a short free - text description of the model that communicates to a scientist what data were being modeled. note : origin : a short free - text description that provides information about the provider of the model, be it the software algorithm or the source of the data, or both. note : comment : a completely freeform field, which may be of any length. since newlines are disallowed, multiple paragraphs of comments should be encoded by having multiple comment lines. the file size for a serialized model depends on the size and diversity of the molecules. one of the main reasons for opting to fold the fingerprints is that it places a reasonable maximum limit on the file size. for example, a collection of 7000 molecules with experimentally determined activity against mycobacterium tuberculosis using ecfp6 with no folding produced a file size of 1.4 mb. folding the fingerprints into 32,768 bits reduced the file size to 646 kb, and into 2048 down to 67 kb. the cdd vault product makes use of the fingerprinting functionality in the cdk to provide bayesian model - building capabilities, which we have termed cdd models. while the bayesian implementation is proprietary, the underlying algorithm for model generation is equivalent to the method described in this article. models can be created and used within the cdd vault environment, and at any time they can be exported using the format described above, which means that they can be utilized by any software that either implements the requisite algorithms described in this article or makes use of the cdk library. a model is created by separating a set of molecules into two collections : those that could be considered inactives. these classes are then used to train the model, after a series of steps are taken to ensure logical consistency. these include ensuring that duplicates do not appear in either collection, that there is no overlap between the collections, and that each collection contains at least two molecules. the standard inchikey of each molecule is used as the criteria for detecting and removing duplicates. these precautions are addressed in a series of pre - processing steps in the cdd ruby on rails application, where the modeling process and molecule management system is hosted, wherein the training sets are algorithmically curated via optimized raw sql code. once the training set has passed these checks and pre - processing, the model is generated. this machine learning model is stored as a special type of protocol (category = machine - learning model), which provides an roc plot generated by stratified three - fold cross - validation. this roc plot is interactive, allowing the user to explore the sensitivity, specificity, and corresponding score cutoff at each point along the curve (figure 2a). after the model has been created, each molecule in the user s selected project receives a relative score, applicability number (fraction of structural features shared with the training set), and maximum similarity number (maximum tanimoto / jaccard similarity to any of the good molecules). the model can be subsequently shared with both other users and the user s other projects to score any molecule of interest. the model can also be exported from cdd vault by making use of the aforementioned.bayesian file format (figure 2b). to render a serialized version of a model, cdd vault feeds the training set structures into the serialization implementation described in the previous section. the connection between the ruby code in cdd vault and the java - based serialization code is accomplished using rjb (ruby - java bridge). (a) model derived from whole - cell datasets from antimalarial screening across four cdd public datasets (mmv, st. jude, novartis, and tcams), 20,000 ec50 values, cutoff 0.80) and comparable to those obtained from proprietary datasets. several of these datasets (blood brain barrier permeability and pxr) were used recently for a comparison across svm and bayesian methods, and the three - fold cross - validation roc values were similar to those obtained with five - fold cross - validation in this study. the use of the roc value in this way is a reasonable method to evaluate the utility of the computational models. however, ideally the use of an additional external test set would provide further confidence. the roc values for the m. tuberculosis models are comparable to those published recently using a commercial tool. for example, in this study mlsmr single - point model three - fold roc = 0.87 (figure s1, five - fold roc 0.87, leave out 50% 100 response model three - fold cross - validation roc = 0.75 (leave out 50% 100 cross - validation roc = 0.73), m. tuberculosis efficacy in mouse three - fold roc = 0.73 (five - fold roc = 0.73), and ames mutagenicity three - fold roc = 0.83 (five - fold roc = 0.84). the models developed using the same underlying code in mobile apps used the ecfp6 descriptors, and all eight models described had five - fold roc values > 0.75 (table 2). several of these datasets have previously been used to generate svm and bayesian methods with fcfp6 descriptors using other software. for example, the three - fold roc for the probe - like dataset in this study was 0.76 (five - fold roc = 0.73), the three - fold roc for the herg dataset was 0.85 (five - fold roc = 0.84), and the three - fold roc for the kcnq1 dataset was 0.84 (five - fold roc = 0.86). the models derived with fcfp6 (table 1) and ecfp6 descriptors (table 2) can be compared ; e.g., the three - fold roc for the malaria dataset using fcfp6 was 0.97 (table 1) (using ecfp6 for the same dataset five - fold roc = 0.98, table 2). these examples of models generated previously and now with open source descriptors and algorithms suggest they are likely comparable (based on roc values) and will be evaluated prospectively in future studies. we have also made the models in the mobile app freely accessible via the link http://molsync.com/bayesian1, which is summarized in figure 4. screenshots summarizing the roc plots and active and inactive compounds for eight models implemented in mmds. we have recently suggested how providing computational models tightly integrated in software used for storing and sharing chemistry and biology data will be useful for decision making. some resources exist such as qsardb.org and ochem.eu for public model sharing and development, while another, chembench, provides a resource for creating and using models and other cheminformatics tools privately. our work, proposing that open source descriptors and algorithms are comparable to commercial software in performance, will ideally lead to more sharing of computational models. at approximately the same time, qsar - ml was developed to enable standards for interoperability of qsar models. we now build on this prior work as the current study sets the stage for being able to generate a model in proprietary software such as cdd vault and export a model in a format that could be run in open source software using cdk components. this is a significant advance, because it means that a shared bayesian model can in principle be used by anyone, regardless of which commercial software packages they have licenses to, since the model capabilities are implemented by an open source toolkit that runs on essentially every desktop platform (cdk is written in cross - platform java). the creation of additional products that implement the same identical reference algorithm, e.g., mobile apps, makes use of shared models increasingly convenient. none of the existing web sites for creating or storing qsar models appear to offer this capability. in terms of willingness to share models, sharing with collaborators is one thing, while sharing models openly with the community at large is another, but we have at least removed the main technology hurdle for fingerprint - based bayesian models in this study. as previously noted, the shared models do not contain chemical structures or the fingerprints corresponding directly to them. however, the direct correlation between structural features and fingerprint does provide clues as to what active molecules an organization may have been using to build their models, and so this caveat must be taken into account when trustworthiness can not be assumed. while additional security measures are appropriate for the world of proprietary high - value disease targets, this is much less of an issue for rare or neglected diseases, which is where we believe that open model sharing will have the greatest impact. there has been considerable research and discussion on efforts to securely share chemistry data, and some of these approaches could be implemented to encrypt models in future. we have now described how implementation of bayesian models with fcfp6 descriptors generated in the cdd vault enables the rapid creation of machine learning models from public datasets or the user s own proprietary data. we also enable the resultant models to be selectively shared (or not) within cdd without having to disclose the underlying data this represents a practical middle ground, where a trusted broker (cdd) allows a research group to share some of the benefits of their results, but not necessarily full access to the raw data, nor sufficient detail to reverse engineer it. since sharing is not mandatory, and the option exists to export a model in an open format that can be used by anyone, this means that the full spectrum of model sharing options is available. providing researchers with greater flexibility to designate and share models with specified collaborators, over particular time intervals, and with clear rights encourages data exchange by allowing researchers to share on terms they control. more fine - grained access control will expand the boundary of what models can be shared to fit the comfort levels of scientists (and their management and lawyers). from having been involved in a number of collaborations large and small, we have observed considerable variation in the need for security and desired degree of openness. the possibility of using such models to further drug discovery for neglected diseases is of considerable interest, since the available software has traditionally catered to the proprietary market that provides most of the funding. there is now a significant amount of sar data for rare and neglected diseases that is publicly available, and following up on open data with open source modeling algorithms is an important step. for example, pharmaceutical companies and other research groups have performed high - throughput screens on likely millions of compounds in the search for antimalarials, but they have generally only offered up the active compounds, some of which are available in cdd public. by selecting a cut - off for activity for the antimalarial data that is very stringent (e.g., < 10 nm) in cdd models one can construct a bayesian model with a three - fold roc = 0.97 upon combining four public datasets (table 1). this model may be useful for virtual screening of future compound libraries and complements our other efforts at machine learning models for antimalarial research. ideally having access to the millions of other inactive compounds would also be useful, although one could imagine a company could just make a model available by selecting a cut - off for inactives as we have demonstrated herein. any efficiencies that can be gained in drug discovery would be highly desirable as it is widely known it is both time - consuming and very costly. therefore, the use of tools like computational models that can point out drug candidate liabilities earlier will have considerable value. with a considerable percentage of drug failures attributed to adme / tox issues, it is still important to assess these qualities early in the drug development process. running experimental adme / tox assays on each compound for initial screening of chemical libraries is cost- and labor - intensive, while computational approaches that rapidly and reliably predict these qualities are gaining more acceptance in the drug discovery community. it is therefore possible to exclude compounds that are most likely to exhibit undesirable adme or toxicity problems sooner. we present an approach to drug discovery using computational methods for predicting whole - cell activity as well as adme / tox and physicochemical properties that can be broadly applied and do not have to be restricted to large companies with sophisticated software and big budgets. for example, modeling of microsomal metabolism has been used with large datasets, and such models are now more accessible through availability of public data. the results summarized in tables 1 and 2 suggest that reliable bayesian models for various bioactivity and adme / tox models can be generated with simple fingerprint descriptors (fcfp6 and ecfp6) and the same bayesian algorithm. this is enabled in such a way that experience in building computational models, while valuable, may not be essential to facilitate model generation, compound scoring, and interpretation. the models described in table 2 which are available in mmds are now also freely accessible (http://molsync.com/bayesian1). our main motivation for creating and disseminating this work is to enable the sharing of bayesian models between a diverse set of toolkits and computing platforms. we have previously described our open source implementation of ecfp6 and fcfp6 fingerprints, inspired by the original commercial implementation that was partially reported in the literature without the disclosure of key details, which remain a trade secret. while there are several other examples of the general approach, our intent was to create a reference implementation and document it so that identical results could be reproduced. the algorithm herein is explicitly documented in a stepwise fashion, and the reference method is available publicly in source code, and hence can be used to compare against when re - implementing in another environment. we believe that taking such care to ensure that the algorithms can be implemented in a way that is 100% compatible with the formal reference removes a major barrier to scientific progress, since building and using models is no longer an isolated activity. we have deliberately taken a two - prong approach : by releasing a fully functional implementation as part of a popular open source toolkit, and also taking the effort to document the algorithm in fine grained detail, to encourage creators of commercial software to consider the advantages of interoperability within their own proprietary products. because the source code is a part of the cdk, the modeling functionality that we describe can be used in a variety of scenarios as - is. any software environment that is capable of linking to a java virtual machine (either directly or through a pipe) can make use of this functionality. since the cdk is made available under the lgpl license, it can be incorporated into proprietary products as long as it is linked as a separate library, but for internal projects, back - end services for which the software is not distributed, or open source projects with a compatible license, it can be used essentially without restrictions. for wholly closed - source products, and platforms that are not compatible with the java virtual machine, the methodology can be re - implemented without difficulty. the exact implementation of bayesian model building and subsequent calibration is straightforward, and we have represented it in pseudocode form (see the accompanying paper for details of algorithms for additional analysis). the cdk version is readily available to verify literal compatibility and can be used as a limitless source of validation data for direct comparison. thus far, the method has been ported to objective - c, in order to enable the use of bayesian models within several different mobile apps (figure 3) and cdd vault as cdd models. the use of cdd models online in the cdd vault data sharing platform to create bayesian models, the use of mobile apps to apply them to small collections of proposed compounds, and integration into other products and scripts via the cdk library present a number of opportunities for making computational modeling potentially more useful and widespread. currently structure activity models are generally only able to be created and used by one specific platform, or if they have some portability, they often suffer from serious compatibility issues due to differences in the underlying technology (e.g., aromaticity models, ylide representations, smarts implementations, partial charge models, etc.) by releasing a well - documented reference implementation as open source and building powerful and useful functionality on top of it, we hope to encourage computational chemists and software creators to make use of this increased inter - operability. future work related to this project will include the implementation of further measures to assess model quality and the applicability of a model to a test compound. in the accompanying paper, we describe several additional algorithms, including calibration of raw bayesian results to a probability - like scale, the effects of folding fingerprints into a smaller range, methods for extracting suitable validation test sets from large public datasets, automated determination of thresholds for active / inactive, and the impact of training set selection on internal cross - validation metrics. as others begin to use the new cdk functionality, cdd models, and bayesian functionality implemented in various mobile apps, we will expect to see further prospective and retrospective testing of the underlying technology and descriptions of the utility and limitations. | on the order of hundreds of absorption, distribution, metabolism, excretion, and toxicity (adme / tox) models have been described in the literature in the past decade which are more often than not inaccessible to anyone but their authors. public accessibility is also an issue with computational models for bioactivity, and the ability to share such models still remains a major challenge limiting drug discovery. we describe the creation of a reference implementation of a bayesian model - building software module, which we have released as an open source component that is now included in the chemistry development kit (cdk) project, as well as implemented in the cdd vault and in several mobile apps. we use this implementation to build an array of bayesian models for adme / tox, in vitro and in vivo bioactivity, and other physicochemical properties. we show that these models possess cross - validation receiver operator curve values comparable to those generated previously in prior publications using alternative tools. we have now described how the implementation of bayesian models with fcfp6 descriptors generated in the cdd vault enables the rapid production of robust machine learning models from public data or the user s own datasets. the current study sets the stage for generating models in proprietary software (such as cdd) and exporting these models in a format that could be run in open source software using cdk components. this work also demonstrates that we can enable biocomputation across distributed private or public datasets to enhance drug discovery. |
interictal depressive symptoms, and interictal major depressive episodes, are quite common in epilepsy in general but appear to be particularly linked to tle. this link between these two disorders has been a source of great interest to both neurologists and psychiatrists for many years and has generated an expansion of knowledge in both fields that has been used to better understand not only these two disorders, but the relationship of mood, cognition, and temporolimbic function in other related conditions as well. despite this, a review of the literature reveals that there is limited cohesive guidance regarding the prevalence of depression in tle patients and the clinical features by which diagnosis can be made, nor are there universally accepted guidelines for the management of depression in this population. difficulties in study design, variable sample populations, and the challenges of confirming temporal lobe focus in some patient populations are all possible contributors to this gap. however, many established authorities in this field have been making significant efforts to overcome these issues and move towards a cohesive approach to diagnosis and management of depression in the tle population. although there have been many studies examining the frequency of depression in temporal lobe epilepsy patients, it has been difficult to establish a clear pattern with respect to prevalence, particularly in comparison to other types of epilepsy. historically, depressive symptoms have been considered to be more frequent in epilepsy with a temporal lobe focus than in extra - tle or generalized epilepsy [38 ]. however, several other studies have not been able to document any such differences [2, 917 ]. firstly, concomitant or additive mechanisms may play a role in the development of depressive symptoms in sufferers of tle. rodin. noted that many patients with tle have more than one seizure type, and that the number of seizures rather than the location of the focus may be more relevant. two later studies confirmed that frequency of seizures were more significant in predicting depressive symptoms than focus location [18, 19 ]. other risk factors have also been identified, such as age of onset and laterality of temporal lobe focus that may also place patients at higher or lower risk than location of focus alone [20, 21 ]. similarly, frontal lobe dysfunction in addition to temporal lobe dysfunction may be an important risk factor in developing depression in tle patients. described significant methodological differences among the various studies examining prevalence rates of depression in epilepsy, including small sample size, lack of control groups, variable and often nonstandardized diagnostic instruments, and variability in the study population (inpatients, outpatients, surgical patients, etc.). finally, adams. observed that many studies assessing psychiatric symptoms in epilepsy predate the advent of technology such as video electroencephalogram monitoring and magnetic resonance imaging, which may make characterization of the underlying lesion more unreliable. all of the above may be contributing to the fact that although there is a plethora of studies dedicated to the assessment of prevalence of depression in temporal lobe epilepsy, consensus has yet to be achieved. nevertheless, a handful of recent studies have attempted to rectify some of the above issues and have provided some interesting data. studied 308 patients that were carefully classified as having epileptiform foci that was temporal or extratemporal in localization. these patients were then administered the structured interview for dsm - iv axis i psychiatric disorders (scid - i), and results were compared using a multivariate analysis. of the tle group, 22% had a lifetime prevalence of major depressive disorder, and 14.6% had previous one - year prevalence of this disorder. extra - tle sufferers had a lifetime prevalence of 14.6% and a previous one - year prevalence of 6.5%. of all the psychiatric disorders assessed (mood disorders, psychotic disorders, somatoform disorders, substance abuse disorders, and anxiety disorders), only previous - year prevalence of major depressive disorder was significantly different between the two groups, with tle sufferers having a higher rate of major depression. adams. followed 319 individuals with focal epilepsy over an 11-year period. the epileptic site, laterality, and type of the lesion were confirmed with video electroencephalogram monitoring and mri scans. the patients were assessed by the study neuropsychiatrist using dsm iv diagnostic criteria prior to their epilepsy diagnosis being made and were reviewed by a second neuropsychiatrist to confirm diagnostic concordance. no significant differences were found in the prevalence of depression between tle patients (31.2%) and extra - tle patients (37.9%), nor between left or right - sided lesions. of note, patients with nonlesion focal epilepsy exhibited a higher rate of depression (41.6%) compared to those with a lesion on mri, regardless of temporal or extratemporal focus.. assessed 67 tle patients and 64 extratemporal lobe focus patients for depression using the beck depression inventory and the composite international diagnostic interview. no significant differences were noted between the two localization groups, but rates of psychiatric symptoms were higher in patients with more frequent and prolonged seizures. in summary, the question of whether a temporal lobe focus of epilepsy assigns a higher prevalence of depression to patients is not yet fully answered. earlier studies were divided on this issue, but variability in methodology, patient samples, accuracy of identification of epileptic focus, and in diagnostic tools used for assessment of depression all made comparison of the data difficult. additionally, this variability in results may also represent multiple factors at play in the development of depression beyond simply localization of epileptic focus, such as frequency and length of seizures, age, and comorbid dysfunction across multiple areas. although this survey of the available literature did not uncover studies of the natural history of depressive symptoms in tle patients specifically, most studies examining the relationship between depressive symptoms and age of onset of epilepsy or duration of epilepsy do not identify clear association with risk of depression [8, 22, 23 ]. one study did find a potential link between depression and development of seizures in later life. one of the questions that has been raised historically is whether depressive symptoms in the epilepsy and tle population represent a comorbid mood disorder, with diagnostic characteristics and a natural history similar to major depressive disorder as seen in the general population, or whether these symptoms instead represent a collection of emotional and cognitive disabilities similar but not equal to a major depressive disorder [2527 ]. the concept of interictal dysphoric disorder was proposed to describe the latter in response to studies suggesting that a significant portion of epilepsy patients with depressive symptoms would not have met criteria for major depressive disorder as typically described in diagnostic schedules [28, 29 ]. for example, kanner. examined patients with refractory seizures and depressive symptoms and found only 29% met dsm iv criteria for major depressive disorder. initially proposed by kraepelin, then later bleuler, this concept originally described a pattern of symptoms consisting of prominent irritability, euphoria, anxiety, anergia, insomnia, and pain. these symptoms are described to have a chronic, relapsing and remitting course, but to respond well to antidepressants. a more specific range of symptoms had been described by blumer. in their neurobiological inventory for epilepsy, which was a reformulation of earlier inventories that were meant to define the tle personality characteristics [25, 29 ]. two categories of symptoms have been described : depressive - somatoform symptoms (depressed mood, anergia, pain, and insomnia) and affective symptoms (irritability, euphoric mood, fear, and anxiety). anhedonia has also been proposed as a better marker for depression in patients with epilepsy, in part secondary to its independence from physical symptoms associated with medications and chronic illness. however, there have also been many proponents of the belief that there is not sufficient evidence to support a model of psychopathology unique to temporal lobe epilepsy. kanner and nieto proposed that the symptoms described in these tle- or epilepsy - specific psychopathology inventories are quite similar to a stable mood disorder with marked depressive and anxiety features rather than a de novo condition. lishman also concurred that the depressive symptoms described as specific to the tle population were an artifact of sampling and selective reporting by patients in institutional settings. reilly. reasoned that the latent variable factors observed to be impacted in depressive disorders (negative attitude, performance difficulty, and somatic elements) could be measured in tle patients to compare the level of dysfunction across these domains to known quantities in major depression. tle patients manifested difficulties across these domains that were very similar to that seen in major depressive disorder, suggesting that these symptoms represented a major depressive disorder, rather than a condition unique to tle. jones. documented the validity of the scid and mini (mini international neuropsychiatric interview) by comparing to patient self - report of symptoms of major depressive disorder, and finding very high concordance. in summary, there has been some suggestion historically that depressive symptoms in the tle population may represent not depression but rather a condition unique to these patients. however, there now also have been studies that appear to confirm that the dsm iv criteria for major depression are valid in the tle population, and that the depressive symptoms they experience can be understood as a stable mood disorder. the various structures of the limbic system have been a focus of interest in understanding both depression and tle for quite some time. regions of particular interest for both these disorders include the temporal lobes (particularly the hippocampus, amygdala, entorhinal, and neocortical cortex), the frontal lobes, and important limbic subcortical structures such as the basal ganglia and thalamus, as well as the circuits connecting all these structures [1, 3740 ]. although the hippocampus and amygdala have been the major focus of attention, all of the above structures have been found to share association across these two disorders. in both depression and tle, hippocampal volumes have been found to be abnormal. in depressed patients with and without tle, hippocampal volumes are reduced, usually bilaterally or occasionally left sided only [37, 4145 ]. in tle patients, volumes are usually reduced on the side of the epileptic focus [40, 42, 45 ]. in depressed tle patients, interestingly, in patients with a left - sided tle focus, cognitive problems with memory and learning are more marked. other studies have confirmed this, linking depression and verbal learning impairments in tle patients. the suspected cause behind this is dysfunction within the larger language representation in the left hemisphere [46, 47 ]. the amygdala has also been a source of intense study in the combined field of depression and tle, given its key role in fear and associated emotions. this structure appears to change as an acute depressive episode becomes chronic, initially becoming enlarged bilaterally, then shrinking bilaterally as the mood disorder becomes chronic. two studies have found a relationship between escalating amygdala volume and severity of symptoms of depression in the tle population [49, 50 ]. left - sided volume increases of the amygdala and severity of depression symptoms in tle patients seemed particularly associated with each other. the suspected mechanism for this is hyperactivity of the amygdala in the acute phase of depression in tle and non - tle patients, resulting in an increase in volume secondary to increased regional blood flow [1, 49, 50 ]. the two commonest lesions for the development of temporal lobe epilepsy are mesial sclerosis and the more rare neocortical temporal lesions. patients with mesial temporal sclerosis have significantly higher rates of depression than those with neocortical temporal lesions, regardless of lateralization. additionally, patients with mesial temporal sclerosis have a greater frequency of cognitive side effects and mood problems with antiepileptic drugs. interestingly, salgado. found significantly more widespread grey matter volume loss in tle patients with depression as compared to their nondepressed fellow patients. this leads to the suggestion that there is a bidirectional relationship between these two disorders. in summary, various important structures of the limbic system have been found to be significantly different in depressed tle patients in comparison with nondepressed fellow patients. there may be a bidirectional relationship between depression and tle influencing these structures. surgery for intractable epilepsy has become increasingly available for patients, resulting in more individuals becoming seizure - free, often thus dramatically improving quality of life [35, 54 ]. however, there is an emerging recognition that psychiatric complications can occur in the postoperative period, including de novo symptoms of depression. the strongest risk factor for depression in the postoperative course is, perhaps not surprisingly, preoperative depression [5557 ] and has been reported in approximately 2030% of patients undergoing surgery [5860 ]. rates of de novo depression in tle patients in the postoperative period range from 5 to 25%. other risk factors identified include older patients at time of surgery [61, 62 ], male gender, strong family history of psychiatric illness, and poor seizure outcome postoperatively. the highest risk period appears to be in the first 3 months following surgery, with slow improvement at the 12- or 24-month mark [54, 63 ]. lateralization has been the focus of many studies, but no clear, cohesive pattern appears to have emerged yet. several studies suggest that right temporal lobe resections represent a greater risk of postoperative depression [57, 64 ], while several more support left temporal lobe resection as higher risk for this complication [56, 57 ], and yet others report no evidence of laterality at all. these ambivalent results are echoed in the literature examining rates of depression following tumor resections [65, 66 ]. however, some studies have documented what appears to be a bidirectional risk in the relationship between postoperative seizure control and depressive symptoms. metternich. documented significantly lower beck depression inventory scores in patients that were seizure - free postoperatively. observed that postoperative tle patients improved significantly with respect to depressive symptoms in comparison to medically managed tle patients, but only if seizure control was significantly improved. this led them to suppose that depressive symptoms were associated with epileptic activity rather than structural changes. finally, wrench. recruited 60 patients undergoing two types of surgery (mesial temporal lobe resection and nonmesial temporal lobe resection) and followed them longitudinally. preoperatively, 43% of these patients had a lifetime prevalence of depression, with no difference between the surgical groups. however, in the postoperative phase, the mesial temporal resection group experienced a significantly higher rate of depression, both recurrence and de novo, suggesting that perhaps disruption of these structures carries a higher risk of depression as a complication postoperatively. in summary, recurrence and de novo development of depression is a risk in tle patients undergoing surgery, particularly in the first 3 months postoperatively. although no clear pattern is emerging regarding the relevance of laterality, there is significant evidence to support a bidirectional relationship between depression and postoperative seizure control, where the presence of one can exacerbate the other. this echoes the findings previously described earlier in this chapter, in which each condition may operate as a significant risk factor for the other. antiepileptic drugs (aed 's) have been known to have positive psychotropic effects beyond their antiseizure effect for quite some time. indeed, many aed 's have separate indications for the treatment of psychiatric disorders, including roles as mood stabilizers, anxiolytics, and in the management of withdrawal syndromes. it is also equally true that many aed 's have negative psychotropic effects that can complicate the management of both epilepsy and depression in patients. the aed 's associated with the highest risk of occurrence of depressive symptoms in patients with epilepsy are those which act at the benzodiazepine - gaba receptor complex. levetiracetam and felbamate appear to represent an intermediate risk of depressive symptoms, leaving the other aed 's as either low risk or unknown (table 1). unfortunately, a clear understanding of which populations may be at particular risk of developing depressive symptoms while on aed 's has yet to be developed, and this review of the literature did not produce any studies that endeavored to identify this in the tle population. however, mula and schmitz suggests a general approach of monotherapy if possible, with introduction of any new aed 's with slow, careful titration, and careful histories of premorbid and family psychiatric disorders being collected regularly in this patient population. depression has been acknowledged by the world health organization as one of the most significant sources of burden of disease and suffering globally. the impact of this disorder on mortality, morbidity, quality of life, social function, and occupational function have been well described. similarly, the additive burden of depression in chronically medically ill people has also been well described, both for epilepsy as well as other medical conditions as diverse as copd, esrd, cancer, and diabetes. thus, it is expected that there would be a consequence to be borne by those tle patients that carry the comorbidity of these two disorders. however, as previously noted in this chapter, there is strong support for a bidirectional risk existing between these two disorders. in other words, the presence of depression may have a direct impact on tle symptom severity, control, and possibly even onset. for example, forsgren and nystrom found that there was a seven - fold increase in rates of depression being diagnosed prior to the onset of the seizure disorder in patients with newly diagnosed epilepsy when compared to age- and sex - matched controls. this remarkable finding was further raised to a 17-fold increase when patients with a localized onset were studied. another study found a 3.7-fold increase in frequency of diagnosis of depression preceding the first seizure in older adults with new onset epilepsy. a study of icelandic children and adults with new onset epilepsy found a similar increase in rates of depression preceding seizure onset (1.7 fold), as well as a 5.1-fold increase in a premorbid history of attempted suicide. in a study of children with new onset seizures, psychopathological symptoms (including anxiety, depression, attention disorders, thought disorders, and somatic disorders) were present at higher rates than controls for 32% of the newly epileptic children. finally, recent studies have also suggested that psychopathology could be a significant risk factor for infants developing nonfebrile seizures or epilepsy in childhood. perhaps the most interesting finding of the pattern of mood symptoms predating epilepsy is alper 's. patients on the medications had significantly lower rates of seizures when compared to their matched fellow patients receiving placebo. this last study in particular is suggestive of the potentially exacerbating role that untreated depressive symptoms may have on seizure control. completed suicide is one of the most tragic and feared outcomes of a depressive episode and is always a concern to clinicians when working with patients with significant psychiatric comorbidity. unfortunately, in epilepsy, the rate of suicide is approximately two to five times that of the general population, and this is further elevated to a 25-fold increase among patients with tle [79, 80 ]. not surprisingly, the rate of completed suicide is further elevated up to 32-fold by the presence of a comorbid depressive disorder. this shocking increase has been considered to be primarily influenced by the psychosocial consequences of living with chronic epilepsy. however, recent data would suggest that the situation is more complex than psychosocial consequences of chronic illness alone, and that a part of understanding completed suicide risk in epilepsy may lie in the examination of suicide attempts. a study comparing suicide attempts among patients with epilepsy to comparably handicapped controls with other chronic disabilities found that 30% of patients with epilepsy had attempted suicide as compared to 7% of controls [80, 81 ]. this is relevant given the fact that suicide attempts are complex behaviors involving many factors, including impulsivity and executive dysfunction, which can be associated with temporolimbic function [8286 ]. jones. found a lifetime prevalence of suicide attempts of 20.8% among 139 outpatients followed at epilepsy centers in the united states. in this sample, the highest rates of attempts were among patients with a lifetime history of a major depressive episode or manic episode, and higher rates of suicidal ideation were also associated with a lifetime history of mood or anxiety disorders. major depressive disorder was the most frequent psychiatric disorder identified among patients with a history of suicide attempt (51.7%), while anxiety disorders were more strongly associated with suicidal ideation (58.8%) in this sample. this suggests that there are characteristics specific to epilepsy, in particular tle, which may place patients at elevated risk of completed suicide and suicide attempts, particularly in the context of comorbid depression. espinosa. studied 42 patients with newly diagnosed tle over a 1-year period and assessed suicide risk via the plutchik risk of suicide scale. they found that 57.1% of their sample scored > 7 on this scale, which is the highest risk category for suicide, 28.6% had a past history of suicide attempts, and 45.2% had experienced suicidal thoughts. the study authors also assessed for multiple associated factors including psychiatric comorbidity, past medical and psychiatric history, and neuropsychological deficits. they discovered a significant relationship between higher suicide risk and a higher rate of suicide attempts in patients with a family history of psychiatric diseases, left - sided tle, current major depressive episode, and higher perseverative responses on neuropsychological testing via wisconsin card sorting test (wcst). they also found a strong correlation between poor wcst performance and severity of depressive symptoms, implying that the presence of depression likely exacerbates executive dysfunction in a population that already has documented difficulty in this area. once again, this suggests a bidirectional relationship between depression and epilepsy in the manifestation of suicide risk. there is an added layer of complexity in understanding suicidality in tle patients. on december 16, 2008, the us food and drug administration issued a warning about an increased risk of suicidal ideation and behavior among people taking aed 's. the agency had performed a review of 119 clinical trials of 11 aed 's and noted a 1.8-fold increase in suicidal behavior or ideation in patients taking aed 's in comparison to those taking placebo. although many methodological issues have been identified with this study, it raised concerning questions regarding the used of these drugs in a population known to suffer significant psychiatric comorbidity. as described previously in this chapter, aed 's have been documented to produce both positive and negative psychotropic effects, and the fda 's announcement produced a flurry of epidemiologic work to attempt to confirm this increased risk of suicidality and hopefully suggest mechanisms. these questions are still being answered, and the studies ' findings have been quite mixed, but results thus far suggest that if there is an elevated risk of suicidality with aed 's, it appears to be very low, and that no clear pattern is emerging regarding risk stratification across the various aed 's [69, 90 ]. additionally, none of these studies have focused on tle in particular, but rather have included samples that tend to be quite broad both in type of epilepsy and comorbidity of psychiatric illness, which in part may explain the variability of findings. wen. noted an interesting trend in their retrospective analysis of patients in the comprehensive epilepsy research program (cerp) database over a 32-month period. briefly, they noted that the strongest predictors for the development of suicidality over time were the presence of depressive symptoms or suicidality prior to aed treatment, and that those patients started on new or multiple aed 's had less improvement of suicidality over time in comparison with those who had no changes made to their aed regimen. this led the authors to suggest that perhaps the findings of the original fda study were generated by the artifact of patients on aed 's improving less than placebo controls with respect to their suicidality symptoms over time. multiple studies in epilepsy in general and tle specifically have made efforts to examine the relationship between various factors associated with living with epilepsy and the impact on quality of life [9198 ]. kanner reviewed 5 studies in particular that consistently demonstrated that depression was the most powerful predictor of health - related quality of life across multiple domains, even when controlling for factors such as seizure frequency, severity, and other psychosocial variables. studied 106 patients with drug - resistant unilateral temporal lobe epilepsy, administering various standardized quality of life instruments as well as the beck depression inventory and an anxiety scale. they also found depression to be consistently the strongest predictor of lower scores on all qol domains except seizure worry. this effect was independent of socioeconomic status, gender, lateralization of seizure focus, seizure frequency and severity, and anxiety. additionally, comorbid depression appears to be associated with a greater likelihood of adverse events associated with antiepileptic drugs, more frequent visits to physicians, and higher cost of medical care related to the seizure disorder, rather than any cost associated with the psychiatric disorder and its treatment [9799 ]. this is an interesting finding in light of the recurring theme woven through the literature of the bidirectionality of depressive symptoms and epilepsy symptoms. again, the presence of one appears to make the management of the other more of a challenge. in general, evidence for treatment strategies of mood disorders in epilepsy are lacking, and development of management approaches tend to rely on clinical experience rather than evidence - based trials favoring one treatment over another. the paucity of data is even more pronounced when examining the literature for tle - specific depression treatment studies. not surprisingly then, there are no widely accepted guidelines for the treatment of depression in tle patients. however, clinicians can turn to a body of literature that, while lacking in large, double - blinded, and placebo - controlled rtc 's, still includes several smaller open label trials, case series reports, and a handful of comparative studies. the bulk of this data does not limit itself to tle although a recently published comparative study in tle patients in particular is included in the following discussion. there are three main considerations when initiating an antidepressant trial in a patient with epilepsy : exacerbation of seizure control, potential for interaction with aed 's, and efficacy of the antidepressant in depression symptom resolution. there appears to be variability between and also within the various antidepressant drug classes although some generalizations can be made from the available literature. the potential for antidepressants to provoke seizures has been a source of concern and possibly a barrier to treatment of depression in patients with epilepsy. the data documenting seizures secondary to antidepressants is derived largely from psychiatric populations, in vitro or animal model studies, or from samples which were not specifically patients with epilepsy [36, 100 ]. often, seizures associated with antidepressants are described in cases of toxicity, such as accidental or intentional overdose [36, 100 ]. this makes it difficult, if not impossible, to generalize findings to epilepsy patients. the situation is further complicated by the fact that animal and human studies suggest that some antidepressants may have an anticonvulsant effect, while some may have a proconvulsant effect, and yet others may have a biphasic effect, in which they are anticonvulsant at lower doses and proconvulsant at higher doses. mechanisms proposed for the proconvulsant effect include the anticholinergic effect of many antidepressants (particularly at higher doses), as well as the elevation of serotonin and noradrenalin neurotransmission. anticonvulsant effect may be mediated by the interaction of the antidepressant with other factors, including aed 's. for example, fluoxetine has been noted to enhance the anticonvulsant effect of phenytoin and carbamazepine via selective inhibition of serotonin uptake. tricyclic antidepressants (tca 's) have a wide variety of neurotransmitter related effects, many of which are dose dependent as well. overall, they are considered proconvulsant, in large part secondary to their significant anticholinergic effect, which is known to lower seizure threshold. as stated previously, some selective serotonin reuptake inhibitors (ssri 's) in general, clinical and research experience suggests that the risk of seizures with ssri 's is very low and perhaps not different from placebo, and certainly lower than with tca 's. a special consideration with ssri 's is the fact that they can promote hyponatremia, which can represent a risk in the precipitation of seizures. other antidepressants which are considered to be high risk for seizures in the general population or in toxicity studies (e.g., bupropion) have been found overall to have an acceptably low risk when prescribed correctly. overall, the incidence of seizures with antidepressants is less than 0.5%, particularly when used within the recommended therapeutic range and when other risk factors are excluded (table 2). given the significant consequences described earlier in this chapter associated with depression in tle, it would therefore seem that concerns about exacerbating seizure control should not be enough to rule out antidepressants in most tle patients. tca 's have, by nature of their longevity, perhaps the most collective clinical experience in many medical conditions, epilepsy included. amitriptyline, clomipramine, and imipramine are extensively metabolized by cyp 1a2, 2d6, and 3a4 (table 3). nortriptyline and desipramine, the metabolites of amitriptyline and imipramine respectively, are metabolized mainly by cyp 2d6. all aed 's with enzyme - inducing properties (barbiturates, carbamazepine, and phenytoin) all induce the metabolism of tca 's. however, carbamazepine also reduces protein binding of imipramine and desipramine, resulting in increased free fraction of these drugs, and thus resulting in little need to alter dosing of these two tca 's when coadministered with carbamazepine. valproate can inhibit the metabolism of tca 's resulting in increased plasma levels at comparable dosage. overall, coadministration of tca 's and aed 's can be done safely and effectively as long as dosing is done slowly and carefully and is accompanied by regular monitoring of blood levels of both drugs. ssri 's are generally more tolerable and safe than tca 's, due in part to their reduced anticholinergic effect and inability to block sodium channels, even in overdose. as a result fluoxetine and paroxetine are metabolized by cyp 2d6, sertraline by cyp 3a4, fluvoxamine by cyp 1a2, and finally citalopram by cyp 2c. paroxetine is an inhibitor of cyp 2d6, fluvoxamine inhibits cyp 1a2, and fluoxetine moderately inhibits cyp 2d6 and 3a4. fluoxetine has a high risk of interaction with phenytoin, but less clearly with carbamazepine [36, 106, 107 ]. paroxetine and sertraline seem to have low risk of interaction with phenytoin [36, 106 ]. however, carbamazepine seems to induce citalopram 's metabolism significantly, thus reducing plasma concentrations and possibly efficacy of this drug. however, there is a paucity of evidence on the safety of fluvoxamine coadministration with valproate or phenytoin. finally, sertraline was found to have no significant effect on carbamazepine levels in a double - blind randomized, placebo - controlled trial of 14 healthy volunteers. snri 's (venlafaxine and duloxetine) are primarily metabolized by cyp 2d6, but interactions with aed 's have yet to be studied. mirtazepine is a noradrenergic and specific serotonergic reuptake inhibitor (nassa) and is primarily metabolized by cyp 2d6. studies are lacking in this population on interactions with aed 's, but this medication has been noted to bind histaminic receptors, resulting in sedation, increased appetite, and weight gain. given that these are side effects of many aed 's, the potential for an additive effect is present. carbamazepine is a potent inducer of the metabolism of bupropion, significantly reducing plasma levels. again, there is a dearth of evidence - based, controlled trials that attempt to study efficacy of antidepressants in epilepsy in general, and the number of trials specific to tle found in this review of the literature was a single one. the earliest controlled trial in patients with epilepsy involved amitriptyline and an antidepressant that no longer exists (nomifensine). at 6 weeks, improvement of depressive symptoms was documented in both drugs, but at 12 weeks, nomifensine was superior to amitriptyline. overall, data about tca 's efficacy in the treatment of depression in epilepsy is largely uncontrolled or anecdotal but appears to support efficacy and tolerability. ssri 's have become the first line of pharmacotherapy in most depressive disorders due to their proven efficacy and benign side - effect profiles. a series of open label studies support some efficacy and tolerability for sertraline, citalopram, mirtazepine, and fluoxetine. in general, ssri 's seem to be effective and well tolerated, but the response rates have been quite mixed across studies, likely due to great variability in sample populations, limited control of comorbid psychiatric disorders, and the occasional presence of cognitive disorders or brain damage in the samples. however, khn. produced a prospective study of safety and efficacy of citalopram, mirtazepine, and reboxetine (not available in north america) in tle patients. they performed a post hoc analysis of 75 tle patients with depression who received standard treatment with one of the above drugs. in general, they found that all the antidepressants were effective in treating the symptoms of depression, and that there were no serious adverse events or drug interactions. however, the dropout rate was significantly higher for mirtazepine that the other two agents, perhaps again because of the tendency of this drug to cause sedation, increased appetite, and weight gain in a population already often prone to these complications secondary to aed 's. although most known for its efficacy in bipolar disorders, lithium is also used as an augmentation strategy for treatment resistant unipolar depression. unfortunately, even less has been published on the safety and efficacy of lithium in epilepsy than for antidepressants. coadministration of lithium carbonate and carbamazepine may have a benefit in terms of mood stabilization but appears to be associated with multiple interactions, including hematologic, thyroid, and electrolyte disregulation. however, lithium appears to be relatively tolerable when administered with valproate, but once again the additive aspects of sedation, weight gain, and tremor were noted. lamotrigine and lithium appeared to be well tolerated together, though topiramate and lithium was associated with toxicity in at least one case. lithium also is known to be proconvulsive at higher doses, but this does not seem to be a significant concern at lower doses in epilepsy patients on aed 's. however, as lithium is usually administered as an augmentation agent in unipolar depression, the additive risk of serotonin syndrome and lowered seizure threshold of both an antidepressant and lithium being administered to an epilepsy patient must be considered. once again, very little data has been collected on psychological therapies in the epilepsy population, tle. however, the few studies that exist seem to support the efficacy of cognitive behavioral therapies as useful in the treatment of depression in epilepsy. | depression in temporal lobe epilepsy has been established as a frequent occurrence, and various possible mechanisms for this significant comorbidity have been posited. however, there is still little to guide a clinician in the recognition and management of depression in patients with temporal lobe epilepsy. this is in part due to the lack of consistent findings in earlier studies, which was likely partly due to variabilities in methodology, sampling, and diagnosis of both temporal lobe epilepsy and depression. however, in recent years, significant effort has been made to address these issues and provide a framework for diagnosis and management of depression in this population. the following is a review of the literature, with special emphasis on clinical phenomenology of depressive symptoms, described bidirectional risk between depression and temporal lobe epilepsy, and treatment strategies in the context of potential drug interactions with antiepileptic drugs. |
timely immunization receipt is commonly assessed by up - to - date (utd) rates for a combination of vaccine series, such as the 4 : 3 : 1 : 3 : 3 : 1 series recommended between birth and 18 months of age by the american academy of pediatrics (aap) and the advisory committee for immunization practice (acip). the 4 : 3 : 1 : 3 : 3 : 1 series contains 4 dtap (diphtheria / tetanus / pertussis), 3 ipv (polio), 1 mmr (measles / mumps / rubella), 3 hbv (hepatitis b), 3 hib (haemophilus influenzae), and 1 varicella vaccines. the need for four early dtap immunizations is due to the continual reoccurrence of pertussis in the us, its risk to infants, and the difficulty in building adequate disease protection to pertussis. children who are not complete for their early childhood shots generally lack only one visit or shot, and the commonly missing component is the fourth dtap due between 15 and 18 months of age [2, 3 ]. the lack of a fourth dtap frequently holds down series utd rates even when all other vaccines are well delivered. the utd rate for completing four dtaps as measured by the national immunization survey by 19 to 35 months of age lags behind most other early childhood immunizations. immunization utd rates are used as measurable proxies for the overall receipt of appropriate early medical care and well - child visits. the dtap series by itself is also recommended as a proxy measure for receipt of all other early childhood immunizations. as the only shot directly due by schedule at the 1.5-year - old well - baby visit, a lack of a timely fourth dtap may imply a lack of appropriate well - child visits and services in this period. the purpose of this study is to assess whether children who miss a timely fourth dtap are also missing timely provider encounters during the 15- to 18-month period or are failing to have immunization eligible visits during this period. a challenge that this study addresses is that the usual data sources used to assess immunization utd rates such as surveys or immunization registries typically do not contain information on provider visits where immunizations were not delivered. missed opportunities are defined in immunization evaluation as a visit where some but not all recommended immunizations were delivered. however, visits where no recommended immunizations are delivered are invisible to the usual methods of calculating missed opportunities and utd rates. in order to address this problem, the present study used a joined set of immunization records from oregon 's alert immunization system (alert iis), and medical encounter records for children enrolled in the oregon health plan, so that children who missed a timely 4th dtap could be assessed against their full provider visit record. secondary questions for this study include whether having a usual source of care or medical home is related to higher rates of fourth dtap completion and if the number of provider encounters during the 15- to 18-month period is also associated with more fourth dtap receipt. immunization records for two - year olds in 2007 were extracted from the oregon alert iis and joined to broader medical encounter records and enrollment information from the oregon health plan (ohp). the ohp is administered by oregon 's division of medical assistance programs (dmap) and consisted during the study period of traditional medicaid populations and an expanded schip population of children in families below 185% of the federal poverty level. health plans and providers are required to submit detailed encounter records on ohp - enrolled children for all services received. all ohp children are eligible for free vaccines through the vaccines for children (vfc) program. the alert iis receives immunization records from 95% of oregon private healthcare providers seeing children and from 100% of public providers. from the merged dataset, a study population of children was selected for those eligible to receive a fourth dtap according to the acip schedule at 15 to 18 months of age, and for whom provider encounter records during the 15- to 18-month period were expected to be submitted to the ohp. possible confounders to testing the study question include the effects of parental vaccine hesitancy and gaps in either medicaid or private insurance coverage. to minimize the effect of such confounders, the study population was limited to children who had a record of timely receipt of their first three dtap immunizations and a known public insurance source with free vaccine availability covering the period of 15 to 18 months of age. specific selection criteria included having received a third dtap by nine months of age according to alert ; not having an early fourth dtap prior to 15 months of age ; active ohp enrollment covering the 15 to 18 months of age period when the fourth dtap is due. potential vaccination eligible encounters that occurred between 15 and 18 months of age were counted for members of the study population, based on a review of international classification of diseases (icd-9) and current procedural terminology (cpt) coding in ohp encounter records. a potential vaccination - eligible encounter was defined as an encounter occurring in a nonemergent or noninpatient setting with a medical provider eligible to deliver immunizations, and with either a cpt procedure code for routine care or evaluation, or a principle icd-9 diagnostic code indicating that the purpose of the encounter was consistent with routine care and immunization evaluation. a medical home measure was created by counting the number of providers in alert who administered the first three dtap doses to each child, so that those who received all of their prior dtaps from a single source were defined as having a consistent medical home or usual source of care. the study population consisted of 9,539 children who met the criteria of having three dtaps by nine months of age, no early fourth dtap, and ohp enrollment between 15 through 18 months of age. the study population represents 47% of the 2005 birth cohort with any length of enrollment in the oregon health plan and 20% of the total oregon 2005 birth cohort. overall 8,113 children, or 86.7% of the study population, had at least one vaccination - eligible provider encounter between 15 and 18 months of age. a fourth dtap was received between 15 and 18 months of age by 7,547 children, or 68.4% of the study population., 13.3% did not have vaccination eligible encounters between 15 and 18 months of age, while 18.3% had vaccination - eligible encounters without receiving a fourth dtap. of those children who did not receive a fourth dtap, 57.9% had at least one missed opportunity for the fourth dtap between 15 and 18 months of age. among those who received a timely 4th dtap, the average number of provider encounters between 15 and 18 months of age was 2.45 (95% c.i. of 2.42 to 2.49). among those who did not receive a 4th dtap but who also had at least one encounter, the average number of encounters was 2.22 (95% c.i. the chances of receiving a 4th dtap significantly increased for those with two encounters (average rate of 78.9%, 95% c.i. of 72.8% to 76.7%) versus those with one encounter (average rate of 74.8%, 95% c.i. of 76.8% to 80.9%) otherwise, while the trend was for increasing 4th dtap receipt with increasing numbers of encounters, the differences were not statistically significant (figure 2). the majority of the study population also presented evidence of having a usual source of care. for 91% of the study children, alert records contained sufficient information to identify a primary provider by clinic for each of their first three dtap doses. the remaining 9% included at least one dtap report from secondary sources such as billing or administrative data that could not be tied to a specific clinic. of those with identified providers for all of their first 3 dtaps, 76.1% had only a single provider, 8.1% had two providers, and 15.8% had three providers. however, as presented in table 1, there was no significant difference in fourth dtap rates between those with only a single provider versus those who used two or three providers. the principal finding of this study was that the majority of children who missed a timely fourth dtap had provider encounters between 15 months and 18 months of age that were potentially eligible for vaccine administration. this result was observed in a population where cost, access, and parental vaccine hesitancy were not substantial barriers due to the design of the study. parents in this population were compliant with immunization recommendations for the first three dtaps ; and due to their enrollment in the ohp, access to vaccines without cost was assured. prior work with this population has demonstrated that provider encounters without immunizations are also common across a larger age span from birth to the age of two. in addition, one other study linking immunization registry and medical record data systems has shown similar problems of special population children visiting providers but not receiving needed immunizations. the number of encounters children had during the period when the fourth dtap was due was only mildly associated with their receipt of the 4th dtap. while 75% of the study population with only one encounter received a fourth dtap, the rate increased slightly to 84% for those with six or more encounters. this is consistent with prior findings that missed opportunities generally stay missed and are not readily caught up. the small increases in utd rate for more than one visit may indicate that providers were not routinely checking for missed shots beyond the first visit in the period, or that missed 4th dtaps for reasons such as illness or parental preference will not be made up in subsequent visits. it is possible that a substantial number of 4th dtaps were missed due to illness at the time of the provider encounter, even if the encounter was for a well - baby check. in a prior survey of parents, a child 's illness was the most cited reason for delaying receipt of dtap immunizations. similarly, provider reluctance to give immunizations during sick visits is a recognized barrier to timely immunizations. as a measure of medical home, in this study there was no evidence to suggest that children with only a single source of immunizations were more likely to receive a fourth dtap. this result varies from at least one previous finding that increases in the number of providers in early childhood were related to lower fourth dtap rates. as the 4th dtap is the only immunization routinely due at 15 to 18 months, it is likely that most of the children not receiving a 4th dtap in this study would be classified by usual methods of assessing immunizations based solely on the immunization record as lacking provider visits. this could potentially misdirect intervention efforts to focus solely on bringing children in for more encounters. in this study, the majority of the children without a 4th dtap had one or more timely encounters where an immunization likely could have been delivered. the definition of immunization - eligible encounters based on a routine evaluation code in the medical record was broad and includes sick visits in various medical settings, with potential bias by site. however, mild illness with or without fever, such as for the most common reasons for sick visits including otitis media, is not valid contraindication to giving a fourth dtap. additionally, the study population was selected specifically to look at missing fourth dtap immunizations, instead of any missing immunization, and so the results may not be generalizable to other vaccines. the study design was selected to reduce potential biases such as lack of access to care or previous reluctance to receive dtap. completion of other missing vaccines, such as the third polio dose, may be influenced by other factors, such as hesitancy to receive many injections at a time on each visit. children with access barriers or whose parents opt for alternate vaccination schedules also will likely possess different patterns of immunizations at 15 to 18 months. the ohp population used in this study, while a large component of all oregon infants, contains subgroups with higher medical and social risks which may influence provider and parent behavior. findings may therefore not be generalizable to all oregon children and point to the need for further analysis on the relation of provider type, setting, and reason for visit to the observed patterns of encounters without immunizations. improving immunization series rates in early childhood depends on taking advantage of existing opportunities to give missing dtap immunizations, along with ensuring that children have appropriate and timely encounters for immunizations. | the successful completion of early childhood immunizations is a proxy for overall quality of early care. immunization statuses are usually assessed by up - to - date (utd) rates covering combined series of different immunizations. however, series utd rates often only bear on which single immunization is missing, rather than the success of all immunizations. in the us, most series utd rates are limited by missing fourth dtap - containing immunizations (diphtheria / tetanus / pertussis) due at 15 to 18 months of age. missing 4th dtap immunizations are associated either with a lack of visits at 15 to 18 months of age, or to visits without immunizations. typical immunization data however can not distinguish between these two reasons. this study compared immunization records from the oregon alert iis with medical encounter records for two - year olds in the oregon health plan. among those with 3 valid dtaps by 9 months of age, 31.6% failed to receive a timely 4th dtap ; of those without a 4th dtap, 42.1% did not have any provider visits from 15 through 18 months of age, while 57.9% had at least one provider visit. those with a 4th dtap averaged 2.45 encounters, while those with encounters but without 4th dtaps averaged 2.23 encounters. |
substrate proteins were expressed with c - terminal his6 tags, and assayed in 50 mm tris, 50 mm k acetate, ph 8.0, with 100 m sam as the methyl donor. all reactions were incubated 1 h at 30c and analyzed for methylation by immunoblot. for elisa assays, reactions were incubated as above then transferred to protein a - coated elisa plates pretreated with anti - me2-spk. after 1 h, the reaction was removed and wells were treated with 1% sds for 30 min. efficient removal of the initiating met is dependent on the identity of the second residue. to avoid this problem, we created fusions from residues 210 of rcc1 with his6-tag plus factor x cleavage site at the n - terminus, and c - terminal gfp. xpk - egfp substrate proteins were expressed in e.coli and purified on ni - nta beads, then cleaved using factor x. on - bead endoproteinase glu - c and asp - n digestions were used to identify the immunoprecipitated proteins. the retained n - termini were then eluted from the beads and analyzed for methylation (supplementary fig. substrate proteins were expressed with c - terminal his6 tags, and assayed in 50 mm tris, 50 mm k acetate, ph 8.0, with 100 m sam as the methyl donor. all reactions were incubated 1 h at 30c and analyzed for methylation by immunoblot. for elisa assays, reactions were incubated as above then transferred to protein a - coated elisa plates pretreated with anti - me2-spk. after 1 h, the reaction was removed and wells were treated with 1% sds for 30 min. efficient removal of the initiating met is dependent on the identity of the second residue. to avoid this problem, we created fusions from residues 210 of rcc1 with his6-tag plus factor x cleavage site at the n - terminus, and c - terminal gfp. xpk - egfp substrate proteins were expressed in e.coli and purified on ni - nta beads, then cleaved using factor x. on - bead endoproteinase glu - c and asp - n digestions were used to identify the immunoprecipitated proteins. the retained n - termini were then eluted from the beads and analyzed for methylation (supplementary fig. | the post - translational methylation of -amino groups was first discovered over 30 years ago on the bacterial ribosomal proteins l16 and l3312, but almost nothing is known about the function or enzymology of this modification. several other bacterial and eukaryotic proteins have since been shown to be -n - methylated310. however, the ran guanine nucleotide - exchange factor, rcc1, is the only protein for which any biological function of -n - methylation has been identified3, 11. methylation - defective mutants of rcc1 have reduced affinity for dna and cause mitotic defects3, 11, but further characterization of this modification has been hindered by ignorance of the responsible methyltransferase. all fungal and animal n - terminally methylated proteins contain a unique n - terminal motif, met-(ala / pro / ser)-pro - lys, indicating they may be targets of the same, unknown enzyme3,12. the initiating met is cleaved, and the exposed -amino group is mono-, di-, or trimethylated. here we report the discovery of the first -n - methyltransferase, which we named n - terminal rcc1 methyltransferase (nrmt). substrate docking and mutational analysis of rcc1 defined the nrmt recognition sequence and enabled the identification of numerous new methylation targets, including set / taf - i / phapii and the retinoblastoma protein, rb. knockdown of nrmt recapitulates the multi - spindle phenotype seen with methylation - defective rcc1 mutants3, demonstrating the importance of alpha - n - methylation for normal bipolar spindle formation and chromosome segregation. |
the following genetically modified mice and crosses between them were used for electrophysiological analysis : nestin - gfp (cb57bl/6 background), pv - cre (jax laboratory ; stock number : 008069 ; stock name : b6;129p2-pvalb / j), sst - cre (jax laboratory ; stock number : 013044 ; stock name : sst / j), vip - cre (jax laboratory, stock number : 010908 ; stock name : vip / j). the following mice were used for neurogenesis analysis : wild - type (c57bl/6), nestin - creer;z / eg;2 (ref ; c57bl/6) and nestin - creer;z / eg (c57bl/6), pv - cre (b6;129), sst - cre (b6;129), and vip - cre (b6;129). socially isolated animals were individually housed right after weaning for at least 6 weeks before tamoxifen or edu injection, and had free access to food and water. a single dose of tamoxifen (tmx ; 62 mg / kg) was i.p. injected into 6- to 10-week - old mice as previously described. for optogenetic manipulations, cre - dependent recombinant aav vectors were used based on a dna cassette carrying two pairs of incompatible loxp sites with the opsin genes (chr2-h134-mcherry, chr2-h134-yfp, or enphr3.0-yfp) inserted between lox sites in the reverse orientation as described previously (supplementary fig. the recombinant aav vectors were serotyped with aav2/9 for chr2 (packaged at the upenn vector core), and aav9 for enphr3.0 (packaged at university of north carolina vector core). the following final viral concentrations were used for aav viruses (x10 particles / ml) : 7.4 (chr2-yfp), 36 (chr2-mcherry), and 8 (enphr3.0-yfp), respectively. aav was stereotaxically delivered into the dentate gyrus using the following coordinates (in mm) : anterioposterior = 2 from bregma ; lateral = 1.5 ; ventral = 2.2. were implanted at the same injection sites right after aav injection with the dorsal - ventral depth of 1.6 mm from the skull. animals were then allowed to recover for at least 3 weeks from surgery. for analysis of rgl activation at the population level after optogenetic manipulations, littermates of animals were used and an in vivo light paradigm was administered 8 hrs per day for 5 consecutive days (supplementary fig. 5a, 6e and 7b). for chr2-yfp stimulation, blue light flashes (472 nm ; 5 ms at 8 hz) through the dpssl laser system (laser century co. ltd., shanghai, china) were delivered in vivo every 5 minutes for 30 s / per trial. for enphr - yfp stimulation, continuous yellow light (593 nm), animals were injected with edu (41.1 mg / kg body weight) 6 times with an interval of 2 hrs. animals were sacrificed 2 hrs after the last edu injection and processed for immunostaining as previously described. briefly, brains were quickly removed into the ice - cold solution (in mm : 110 choline chloride, 2.5 kcl, 1.3 kh2po4, 25.0 nahco3, 0.5 cacl2, 7 mgso4, 20 dextrose, 1.3 sodium l - ascorbate, 0.6 sodium pyruvate, 5.0 kynurenic acid). slices (300 m thick) were sectioned using a vibratome (leica vt1000s) and transferred to a chamber containing the external solution (in mm : 125.0 nacl, 2.5 kcl, 1.3 kh2po4, 1.3 mgso4, 25.0 nahco3, 2 cacl2, 1.3 sodium l - ascorbate, 0.6 sodium pyruvate, 10 dextrose, ph 7.4, 320 mosm), bubbled with 95% o2/5% co2. gfp rgls located within the sgz in adult nestin - gfp mice were visualized by dic and fluorescence microscopy. a whole - cell patch - clamp configuration was employed in the voltage - clamp mode (vm = 65 mv) or current - clamp mode. microelectrodes (46 m) were pulled from borosilicate glass capillaries and filled with the internal solution containing (in mm) : 135 cscl gluconate, 15 kcl, 4 mgcl2, 0.1 egta, 10.0 hepes, 4 atp (magnesium salt), 0.3 gtp (sodium salt), 7 phosphocreatine (ph 7.4, 300 mosm). all rgl recordings were carried out in the presence of kynurenic acid (5 mm). data was collected using an axon 200b amplifier and acquired with a digidata 1322a (axon instruments) at 10 khz. for measuring gaba - induced responses from gfp rgls, focal pressure ejection of 200 mm gaba or muscimol through a puffer pipette controlled by a picospritze (2s puff at 35 psi) was used to activate gabaars under the whole - cell voltage - clamp. a bipolar electrode (world precision instruments) low frequency (0.1 hz) and theta bursts (8 hz with a train of 100 stimuli) were delivered. the stimulus intensity (50 a) the following pharmacological agents were used : diazepam (1 m), no-711 (10 m), flumazenil (10 m), midazolam (10 m), etmd (10 m), l-655708 (50 m), and vigabatrin (100 m). all drugs were purchased from sigma except bicuculline (50 or 100 m ; tocris). rgl recordings under optogenetic manipulation in acute brain slices were performed at least 4 weeks after aav injection. to stimulate chr2 in labelled interneurons, light flashes (5 ms at 1, 8 or 100 hz) generated by a lambda dg-4 plus high - speed optical switch with a 300w xenon lamp and a 472 nm filter set (chroma) were delivered to coronal sections through a 40x objective (carl zeiss). to stimulate enphr in labelled interneurons, continuous yellow light generated by dg-4 plus system with a 593 nm filter set were delivered to coronal sections across a full high - power (40x) field. for immunostaining with anti - nestin and anti - mcm2, an antigen retrieval protocol was performed by microwaving sections in boiled citric buffer for 7 minutes as previously described. for 2 immunostaining, a weak fixation protocol using live tissues was adopted as previously described. for characterization of different interneuron subtypes, following antibodies were used : anti - pv (swant ; mouse or rabbit ; 1:500), anti - gad-67 (millipore ; mouse or rabbit ; 1:500), ant - sst (millipore ; rat ; 1:200), and anti - vip (immunostar ; rabbit ; 1:200). for clonal analysis, coronal brain sections (40 m) through the entire dentate gyrus were collected in a serial order, and immunostaining was performed using following primary antibodies as previously described : anti - gfp (rockland ; goat ; 1:500), anti - nestin (aves ; chick ; 1:500), anti - mcm2 (bd ; mouse ; 1:500), anti - gfap (millipore ; mouse or rabbit ; 1:1000), anti - psa - ncam (millipore, mouse lgm ; 1:500). for quantification of gfp clones at 2 and 7 dpi, a single gfp rgl was scored as a quiescent clone. clonal analysis at 30 dpi was carried out exactly as previously described. for experiments with diazepam (5 mg / kg body weight ; once daily for 5 days), coronal brain sections (40 m) through the entire dentate gyrus were collected in a serial order. for optogenetic manipulations, sections within a distance of 1.0 mm anterior and 1.0 mm posterior to injection sites were used for quantification, given the estimated light spread in vivo. edu labelling was performed using click - it edu alexa fluor imaging kit (invitrogen). images were acquired on a zeiss lsm 710 confocal system (carl zeiss, thornwood, ny, usa) with a 40x objective using a multi - track configuration. stereological quantification of cells positive for various molecular markers was assessed in the dentate gyrus using a modified optical fractionator technique. for quantification of edu or mcm2 rgls, an inverted y shape from anti - nestin staining superimposed onto edu or mcm2 nucleus was scored double positive for nestin and edu or mcm2. statistic analysis was performed with student t - test. for generation of movie files, images were serially reconstructed in reconstruct (john c. fiala, the national institutes of health), normalized, and deconvolved with autoquant x2 (media cybernetics). images were then segmented in matlab (the mathworks) using custom code and imported into imaris (bitplane). surface renderings and movies were made using the surface and animation functions, respectively, in imaris (supplementary movies 13). the following genetically modified mice and crosses between them were used for electrophysiological analysis : nestin - gfp (cb57bl/6 background), pv - cre (jax laboratory ; stock number : 008069 ; stock name : b6;129p2-pvalb / j), sst - cre (jax laboratory ; stock number : 013044 ; stock name : sst / j), vip - cre (jax laboratory, stock number : 010908 ; stock name : vip / j). the following mice were used for neurogenesis analysis : wild - type (c57bl/6), nestin - creer;z / eg;2 (ref ; c57bl/6) and nestin - creer;z / eg (c57bl/6), pv - cre (b6;129), sst - cre (b6;129), and vip - cre (b6;129). socially isolated animals were individually housed right after weaning for at least 6 weeks before tamoxifen or edu injection, and had free access to food and water. a single dose of tamoxifen (tmx ; 62 mg / kg) was i.p. injected into 6- to 10-week - old mice as previously described. for optogenetic manipulations, cre - dependent recombinant aav vectors were used based on a dna cassette carrying two pairs of incompatible loxp sites with the opsin genes (chr2-h134-mcherry, chr2-h134-yfp, or enphr3.0-yfp) inserted between lox sites in the reverse orientation as described previously (supplementary fig. the recombinant aav vectors were serotyped with aav2/9 for chr2 (packaged at the upenn vector core), and aav9 for enphr3.0 (packaged at university of north carolina vector core). the following final viral concentrations were used for aav viruses (x10 particles / ml) : 7.4 (chr2-yfp), 36 (chr2-mcherry), and 8 (enphr3.0-yfp), respectively. aav was stereotaxically delivered into the dentate gyrus using the following coordinates (in mm) : anterioposterior = 2 from bregma ; lateral = 1.5 ; ventral = 2.2. were implanted at the same injection sites right after aav injection with the dorsal - ventral depth of 1.6 mm from the skull. animals were then allowed to recover for at least 3 weeks from surgery. for analysis of rgl activation at the population level after optogenetic manipulations, littermates of animals were used and an in vivo light paradigm was administered 8 hrs per day for 5 consecutive days (supplementary fig. 5a, 6e and 7b). for chr2-yfp stimulation, blue light flashes (472 nm ; 5 ms at 8 hz) through the dpssl laser system (laser century co. ltd., shanghai, china) were delivered in vivo every 5 minutes for 30 s / per trial. for enphr - yfp stimulation, continuous yellow light (593 nm), animals were injected with edu (41.1 mg / kg body weight) 6 times with an interval of 2 hrs. animals were sacrificed 2 hrs after the last edu injection and processed for immunostaining as previously described., brains were quickly removed into the ice - cold solution (in mm : 110 choline chloride, 2.5 kcl, 1.3 kh2po4, 25.0 nahco3, 0.5 cacl2, 7 mgso4, 20 dextrose, 1.3 sodium l - ascorbate, 0.6 sodium pyruvate, 5.0 kynurenic acid). slices (300 m thick) were sectioned using a vibratome (leica vt1000s) and transferred to a chamber containing the external solution (in mm : 125.0 nacl, 2.5 kcl, 1.3 kh2po4, 1.3 mgso4, 25.0 nahco3, 2 cacl2, 1.3 sodium l - ascorbate, 0.6 sodium pyruvate, 10 dextrose, ph 7.4, 320 mosm), bubbled with 95% o2/5% co2. gfp rgls located within the sgz in adult nestin - gfp mice were visualized by dic and fluorescence microscopy. a whole - cell patch - clamp configuration was employed in the voltage - clamp mode (vm = 65 mv) or current - clamp mode. microelectrodes (46 m) were pulled from borosilicate glass capillaries and filled with the internal solution containing (in mm) : 135 cscl gluconate, 15 kcl, 4 mgcl2, 0.1 egta, 10.0 hepes, 4 atp (magnesium salt), 0.3 gtp (sodium salt), 7 phosphocreatine (ph 7.4, 300 mosm). all rgl recordings were carried out in the presence of kynurenic acid (5 mm). data was collected using an axon 200b amplifier and acquired with a digidata 1322a (axon instruments) at 10 khz. for measuring gaba - induced responses from gfp rgls, focal pressure ejection of 200 mm gaba or muscimol through a puffer pipette controlled by a picospritze (2s puff at 35 psi) was used to activate gabaars under the whole - cell voltage - clamp. a bipolar electrode (world precision instruments) low frequency (0.1 hz) and theta bursts (8 hz with a train of 100 stimuli) were delivered. the stimulus intensity (50 a) the following pharmacological agents were used : diazepam (1 m), no-711 (10 m), flumazenil (10 m), midazolam (10 m), etmd (10 m), l-655708 (50 m), and vigabatrin (100 m). all drugs were purchased from sigma except bicuculline (50 or 100 m ; tocris). rgl recordings under optogenetic manipulation in acute brain slices were performed at least 4 weeks after aav injection. to stimulate chr2 in labelled interneurons, light flashes (5 ms at 1, 8 or 100 hz) generated by a lambda dg-4 plus high - speed optical switch with a 300w xenon lamp and a 472 nm filter set (chroma) were delivered to coronal sections through a 40x objective (carl zeiss). to stimulate enphr in labelled interneurons, continuous yellow light generated by dg-4 plus system with a 593 nm filter set were delivered to coronal sections across a full high - power (40x) field. for immunostaining with anti - nestin and anti - mcm2, an antigen retrieval protocol was performed by microwaving sections in boiled citric buffer for 7 minutes as previously described. for 2 immunostaining, a weak fixation protocol using live tissues was adopted as previously described. for characterization of different interneuron subtypes, following antibodies were used : anti - pv (swant ; mouse or rabbit ; 1:500), anti - gad-67 (millipore ; mouse or rabbit ; 1:500), ant - sst (millipore ; rat ; 1:200), and anti - vip (immunostar ; rabbit ; 1:200). for clonal analysis, coronal brain sections (40 m) through the entire dentate gyrus were collected in a serial order, and immunostaining was performed using following primary antibodies as previously described : anti - gfp (rockland ; goat ; 1:500), anti - nestin (aves ; chick ; 1:500), anti - mcm2 (bd ; mouse ; 1:500), anti - gfap (millipore ; mouse or rabbit ; 1:1000), anti - psa - ncam (millipore, mouse lgm ; 1:500). for quantification of gfp clones at 2 and 7 dpi, clonal analysis at 30 dpi was carried out exactly as previously described. for experiments with diazepam (5 mg / kg body weight ; once daily for 5 days), coronal brain sections (40 m) through the entire dentate gyrus were collected in a serial order. for optogenetic manipulations, sections within a distance of 1.0 mm anterior and 1.0 mm posterior to injection sites edu labelling was performed using click - it edu alexa fluor imaging kit (invitrogen). images were acquired on a zeiss lsm 710 confocal system (carl zeiss, thornwood, ny, usa) with a 40x objective using a multi - track configuration. stereological quantification of cells positive for various molecular markers was assessed in the dentate gyrus using a modified optical fractionator technique. for quantification of edu or mcm2 rgls, an inverted y shape from anti - nestin staining superimposed onto edu or mcm2 nucleus was scored double positive for nestin and edu or mcm2. statistic analysis was performed with student t - test. for generation of movie files, images were serially reconstructed in reconstruct (john c. fiala, the national institutes of health), normalized, and deconvolved with autoquant x2 (media cybernetics). images were then segmented in matlab (the mathworks) using custom code and imported into imaris (bitplane). surface renderings and movies were made using the surface and animation functions, respectively, in imaris (supplementary movies 13). | adult neurogenesis arises from neural stem cells within specialized niches13. neuronal activity and experience, presumably acting upon this local niche, regulate multiple stages of adult neurogenesis, from neural progenitor proliferation to new neuron maturation, synaptic integration and survival1, 3. whether local neuronal circuitry has a direct impact on adult neural stem cells is unknown. here we show that in the adult hippocampus nestin - expressing radial glia - like quiescent neural stem cells49 (rgls) respond tonically to the neurotransmitter gaba via 2 subunit - containing gabaa rs. clonal analysis9 of individual rgls revealed a rapid exit from quiescence and enhanced symmetric self - renewal after conditional 2 deletion. rgls are in close proximity to gad67 + terminals of parvalbumin - expressing (pv+) interneurons and respond tonically to gaba released from these neurons. functionally, optogenetic control of dentate pv+, but not somatostatin- or vasoactive intestinal polypeptide (vip)-expressing, interneuron activity can dictate the rgl choice between quiescence and activation. furthermore, pv+ interneuron activation restores rgl quiescence following social isolation, an experience that induces rgl activation and symmetric division8. our study identifies a niche cell - signal - receptor trio and a local circuitry mechanism that control the activation and self - renewal mode of quiescent adult neural stem cells in response to neuronal activity and experience. |
the mdc cohort is a population - based prospective cohort including 28,449 participants, with baseline data collection conducted throughout the years 199196 (22). the study population includes individuals born during 192350 (23) and living in malm, the third - largest city in sweden, with about 295,000 citizens. the participation rate was approximately 40% (24). among mdc participants recruited from november 1991 to february 1994 (n = 12,445), a random 50% was invited to further participate in a carotid artery disease study, the mdc cardiovascular cohort. in total, 6,103 individuals underwent a review of their medical history, a physical examination and a laboratory assessment of cardiovascular risk factors (22, 24, 25). information on ldl, hdl, and triglyceride fasting blood concentrations was available in 5,363 individuals. totally, information on diet, fads genotype, and blood lipids was available in 4,943 individuals. after excluding individuals with diabetes mellitus (self - reported diagnosis or using anti - diabetes medication, n = 123), users of lipid - lowering medication (n = 117), and those with a history of cardiovascular event (coronary event or stroke, n = 117), the study sample included 4,635 individuals (4568 years, 60.3% female). information about the history of cardiovascular event (coronary event or stroke) was taken from the national swedish hospital discharge register and the local register of stroke (24). all individuals provided written, informed consent, and the ethics committee of lund university approved the mdc study protocols. (t / c) was performed by matrix - assisted laser desorption ionization time - of - flight mass spectrometry on the sequenom mass - array platform (san diego, ca). genotyping was successful in 5,806 (96%) of the 6,055 individuals with dna available and the rs174547 was in hardy - weinberg equilibrium (p = 0.92). in addition, 5,490 of the 6,055 individuals, of whom we still have dna available, were additionally genotyped by the taqman allelic discrimination on abi 7900 with a concordance rate for the two methods of 99.2%. dietary intake was measured by a modified diet history methodology combining a 168 item dietary questionnaire, a 7 day menu book and a 1 h diet history interview, especially designed for the mdc study (26). the 168 item dietary questionnaire covered food items regularly consumed during the past year. the participants were asked to fill in the frequency of food intake and estimate the usual portion sizes using a booklet with photographic aids. the 7 day menu book covered cooked lunch and dinner meals, cold beverages (including alcoholic beverages), medications, natural remedies, and dietary supplements used by the participants. to complete the dietary data, the participants were interviewed, for 1 h about their food choices, food preparation practices, and portion sizes (by using a more - extensive booklet of photos) of the food reported in the menu book. the trained interviewers checked the menu book and questionnaire for very high reported intakes and overlapping information. the average daily food intake (grams per day) was calculated based on the information from the menu book, interview, and questionnaire, and converted into nutrient and energy intakes by using the mdc food and nutrient database, developed from the pc kost-93 of the swedish national food administration (26). the different pufa intakes were converted into the percentage contributed by the specific pufas to total energy intake (e%). the pufa dietary variables included in this study were : ala (c18:3-3) ; long - chain -3 pufa (epa [c20:5-3 ], docosapentanoic acid [dpa, c22:5-3 ], and dha [c22:6-3 ]) ; total -3 pufa (ala, epa, dpa, and dha) ; total -6 pufa (la [c18:2-6 ], -linolenic acid [c18:3-6 ], and aa [c20:4-6 ]) ; ala to la ratio (ala / la) ; total -3 to total -6 pufa ratio (-3/-6 pufa). the relative validity of the modified diet history method was examined in 105 women and 101 men. as the reference method, a total of 18 days of weighed food records was collected during 3 consecutive days, every second month for 1 year. the energy - adjusted correlation coefficients between the modified diet history method and the reference method were in men : 0.22 for ala, 0.23 for la, 0.55 for aa, 0.24 for epa, 0.37 for dpa, and 0.20 for dha. in women, the coefficients were 0.58 for ala, 0.68 for la, 0.44 for aa, 0.38 for epa, 0.40 for dpa, and 0.27 for dha (27). hdl, triglyceride, and total cholesterol concentrations were determined in overnight fasting blood samples by standard methods at the department of clinical chemistry, university hospital of malm. ldl concentration was calculated by the friedewald formula : ldl = total cholesterol hdl (triglycerides/2.2) (24). for individuals with a triglyceride concentration of more than 400 mg per deciliter (4.5 mmol / l), ldl was defined as missing (25). body mass index (bmi) was calculated as weight in kilograms divided by square of height in meters (kg / m). a self - administered questionnaire was used to determine lifestyle factors including cigarette smoking, alcohol intake, and physical activity habits. three categories of smoking status were used : current (including irregular smoking), former, and never smokers. individuals reporting no alcohol consumption during the last year in the questionnaire, which also were zero reporters of alcohol in the 7 day menu book, were categorized as zero consumers of alcohol. we divided the other study participants alcohol consumption (grams per day) into categories with different cutoffs according to gender. the cutoff levels for females were 5, 10, and 20 g of alcohol per day and the cutoff levels for males were 10, 20, and 40 g of alcohol per day. the leisure - time physical activity level (pal) was calculated from a list of 17 different activities in the questionnaire. the time spent on each activity was multiplied by an intensity factor, creating a leisure - time physical activity score. the leisure - time physical activity score was then divided into quintiles, with the same cutoffs for both genders. separate categories for smoking, alcohol intake, and leisure - time physical activity were constructed for the subjects with missing data. statistical significance was set at p 0.28 e%). when examining within - genotype categories, the high long - chain -3 pufa intake was associated significantly with higher ldl concentration among the cc - genotype (p 0.28 e%) intakes (p = 0.98 and p = 0.86, respectively). a low intake of long - chain -3 pufa was associated with 0.20 mmol / l and 0.06 mmol / l higher hdl concentration among individuals with cc - genotype (p = 2.7 10) and tc - genotype (p = 0.04) but not among those with tt - genotype (p = 0.17). association between ala / la and hdl in strata of rs174547 among 4,635 individuals. a high ala / la was associated with 0.04 mmol / l and 0.02 mmol / l higher hdl concentration among individuals with cc - genotype (p = 0.046) and tc - genotype (p = 0.02) but not among those with tt - genotype (p = 0.96). there was no association between the cc genotype and hdl in individuals with low, medium, or high ala / la (p = 0.07, p = 0.35, and p = 0.35, respectively). we observed a significant interaction of the fads genotype categories and long - chain -3 pufa on ldl concentrations in men (p = 0.01), but not in women (p = 0.39). however, these interactions did not significantly differ between the genders (p = 0.08), and there was no significant difference in gender distribution across the tertiles of long - chain -3 pufa. no significant interactions of fads genotype categories and ala / la on the levels of hdl were detected, or any of the different pufa intakes on triglycerides levels, either in men or in women. there were significantly more males than females in the highest tertiles of total -3 and -6 pufa intake compared with the mid- and lowest tertiles (p < 0.001). because the gender distribution across the tertiles of -3 and -6 pufa intakes was not equal, we repeated all of the interaction analyses using gender - specific cutoffs for the long - chain -3 and -6 pufa tertiles. however, the above - reported results remained essentially the same when using gender - specific cutoffs. in sensitivity analyses, when excluding suspected misreporters of energy (19.3% of the study sample), the interaction between the fads genotype categories and long - chain -3 pufa intakes on ldl remained significant (p = 0.04). the interaction between the fads genotype categories and ala / la intake levels on hdl cholesterol was slightly attenuated after excluding misreporters (p = 0.06). interactions between fads genotype categories and the different pufa intake levels on triglyceride remained nonsignificant. this study indicates that the dietary intake levels of long - chain -3 pufas may modify the association between fads rs174547 and ldl concentration. additionally, the intake levels of ala / la may modify the associated effect of fads genotype categories on hdl concentrations. although several earlier studies, similar to our results, have found an association between the minor allele of rs174547 (or an snp in high allelic association) and decreased ldl concentrations (6, 13, 2931), some inconsistency between the reported associations with lipids and lipoproteins remains. (4) demonstrated that the minor c - allele of rs174547 associated with lower hdl and higher triglyceride concentrations in caucasians and similar associations were observed in a japanese population (21). however, because the same c - allele was found to associate significantly with decreased ldl, but not with hdl or triglycerides in a mongolian population, it was suggested that differences in dietary fish intake between japanese and mongolians could provide an explanation of the dissimilar results (21). the mongolian diet is mainly based on livestock products, with a very low intake of fish compared with the japanese diet, which, in general, includes a very large amount of fish products. consistently, japanese individuals had higher plasma concentrations of -3 pufa and a higher -3/-6 pufa compared with mongolians (32). in our study, the rs174547 c - allele was associated with lower ldl concentration only in the lowest intake tertile of long - chain -3 pufa. because the dietary source of long - chain fatty acids mainly comes from fatty fish, our finding is in agreement with the suggestion of nakayama. (21) that among individuals with low fish intake, in our study represented by those in the lowest intake tertile of long - chain pufas, the association between the fads genotype categories and ldl concentrations may be accentuated. (13), who reported a significant association between the minor allele of rs174546 (corresponding to the c - allele of rs174547 of our study) and lower hdl concentration among individuals with high consumption of -6 pufas (5.26 e%), we observed no significant interaction between -6 pufa intakes and the fads genotype on hdl concentration. however, because low intake of -6 pufa could indirectly reflect a high ala / la intake, our observation of interaction between the fads genotype categories and ala / la intakes on hdl cholesterol concentration, as well as association between high ala / la intake and higher hdl concentration among the c - allele carriers, could be in line with findings by lu. fads1 is responsible for the desaturation of ala and la into epa and aa (9, 10). (4) demonstrated that the minor allele of rs174547 (in our study associated with lower ldl concentration) was associated with lower gene expression of the fads1 and fads3 transcripts. tanaka. (31) showed that individuals homozygous for the minor allele of an snp in high allelic association with fads rs174547 had lower plasma concentrations of aa compared with individuals homozygote for the major allele. the potential mechanism for association between high ala / la intake and higher hdl among the minor allele carriers could be that the minor allele, which has been associated with a lower desaturase activity (14), may affect the availability of long - chain -3 pufas differently between the genotypes. the metabolic derivatives, eicosanoids, of these long - chain -3 pufas are potent activators of peroxisome proliferator - activated receptors (9, 20), which can regulate the expression of genes directly involved in hdl production (13). therefore, our result may reflect a limited long - chain pufa availability among rs174547 minor allele carriers and thereby association between hdl concentrations and ala / la intake. the strengths of our study include a relatively large sample size and detailed information on dietary intakes based on a 168 item dietary questionnaire, a 7 day menu book, and a 1 h interview. however, the estimated dietary intakes were based on self - reports, and the limitation of a short - term diet measurement to reflect the habitual intake may have introduced misclassification of dietary intakes and attenuation of the associations. although dietary data from the mdc is in general of very high relative validity (33), the relative validity of some pufas is still rather low, especially that of long - chain -3 pufas in men, which is a weakness of the current study. one reason for the low validity may be the infrequent consumption of fatty fish (major source of long - chain -3 pufas) among many of the study participants. because the fish consumed at main meals was only registered during a limited number of 7 days, misclassification may be a problem. furthermore, this is a cross - sectional study, which limits our possibility to investigate causality. finally, we performed multiple tests, and thus some of the observed significant associations and interactions could be due to chance and need to be replicated. it is generally accepted that genetic and environmental factors influence the blood lipid and lipoprotein concentrations, but very little is known about such interactions. our findings suggest that the dietary intakes of different pufas may modify the associated effect of the genetic variation in fads on ldl and hdl concentration. our results emphasize the importance of dietary fat composition in modifying the effect of genetic susceptibility on blood lipid and lipoprotein concentrations and highlight the potential of developing individualized prevention strategies for dyslipidemia and cardiovascular disease in the future. however, for such strategies to become possible, a major challenge now is to learn more about the importance and mechanisms of interactions between genetic variants, dietary intakes, and blood lipid concentrations. it is therefore important to replicate our results in well - powered studies with good - quality dietary data. | polymorphisms of the fa desaturase (fads) gene cluster have been associated with ldl, hdl, and triglyceride concentrations. because fads converts -linolenic acid (ala) and linoleic acid into pufas, we investigated the interaction between different pufa intakes and the fads polymorphism rs174547 (t > c) on fasting blood lipid and lipoprotein concentrations. we included 4,635 individuals (60% females, 4568 years) from the swedish population - based malm diet and cancer cohort. dietary intakes were assessed by a modified diet history method including 7-day registration of cooked meals. the c - allele of rs174547 was associated with lower ldl concentration (p = 0.03). we observed significant interaction between rs174547 and long - chain -3 pufa intakes on ldl (p = 0.01) ; the c - allele was only associated with lower ldl among individuals in the lowest tertile of long - chain -3 pufa intakes (p < 0.001). in addition, significant interaction was observed between rs174547 and the ratio of ala and linoleic fa intakes on hdl (p = 0.03). however, no significant associations between the c - allele and hdl were detected within the intake tertiles of the ratio. our findings suggest that dietary intake levels of different pufas modify the associated effect of genetic variation in fads on ldl and hdl |
we report a 10-year - old girl with multiple vasculitic skin lesions and foot drop without the usual history of asthma or sinusitis which later proved to be css on biopsy. a previously healthy 10-year - old arab girl presented with bilateral asymmetrically distributed multiple tender and nonblanching erythematous skin nodules in the lower extremities, ranging between 0.5 and 1 cm in size [figure 1 ]. she was well - looking and her vital signs were as follows : temperature 36.5 c, pulse rate 90/min, blood pressure 90/60 mmhg, respiratory rate 14/min, and o2 saturation 98% in room air. physical examination of the head, eye, ear, nose throat, lungs, heart, abdomen, central nervous system, and lymph nodes did not reveal any abnormal findings. joint examination showed a mildly swollen and tender left ankle, with some restriction of dorsiflexion. the admitting diagnosis was cellulitis possibly secondary to insect bite and she was started on parenteral cloxacillin therapy with analgesics. her full blood count results were as follows : hb 13.8 g / dl, white cell count 27.6 10/l, 31% neutrophils, 10% lymphocytes, 59% eosinophils (absolute eosinophil count 14.9 10/l), and platelets 343 10/l. erythrocyte sedimentation rate was 68 mm / h and serum c reactive protein 24 mg / l (normal < 6 mg / l). there was a further rise of the white cell count to 30.5 10/l and eosinophils to 67% (20.3 10/l) within 48 h. blood and stool cultures were sterile. multiple nodular erythematous skin lesions of the lower extremities with a tender, oedematous, and erythematous surrounding skin with foot drop of the left leg. a left foot drop with sensory loss over the lateral aspect of the left leg was noted on the third day of admission. chest x - ray showed multiple fluffy bilateral perihilar infiltrates and computed tomography of the para nasal sinuses was normal. serum immunoglobulin 's levels were within normal range except for marginal elevation of ige levels. enzyme - linked immunosorbent assay for serum pr3-anti - neutrophil cytoplasmic antibodies (ancas) and mpo - anca were negative. a bone marrow aspirate done to rule out hypereosinophilic syndrome showed eosinophilic hyperplasia, with no dysplasia or neoplasia. a biopsy of one of the cutaneous lesions showed hyperplastic epidermis with intense dermis infiltration by eosinophils. these cells were also observed in subcutaneous medium - sized vessels, confirming the diagnosis of css [figure 2 ]. nerve conduction studies revealed axonal motor neuropathy of the common peroneal nerve and absent sensory potential of the medial plantar and sural nerves. hematoxylin - eosin skin biopsy specimen showing intense infiltration by eosinophils involving the subcutaneous tissue and medium - sized artery (h&e stain, 400x) intravenous methylprednisolone therapy was administered which resulted in a dramatic drop of her peripheral white cell count to 14.6 10/l and eosinophils to 0.08%. she was discharged on day 7 on daily oral prednisolone (1 mg /kg) and physiotherapy. when she was reviewed 2 weeks later, there was a significant improvement in her skin lesions and sensation with some improvement in her ankle dorsiflexion. churg strauss syndrome, originally called allergic granulomatosis and angiitis, is a primary systemic vasculitis characterised by an increase in the number of eosinophils in the peripheral blood and tissues, and is almost invariably preceded by asthma or allergic rhinitis. reports of css disease occurring in children are very rare, with most having respiratory tract involvement and a history of asthma. glucocorticoid - sparing agents used in the treatment of asthma, such as leukotriene receptor antagonists, may unmask css when oral glucocorticoids are withdrawn suggesting that the clinical picture of css is suppressed by glucocorticoid therapy. the most frequent cutaneous findings are macular - papular, nodular, or erythematous lesions on the extensors of the extremities. the characteristic pathologic changes include tissue infiltration with eosinophils, necrotizing or nonnecrotizing granulomas as well as vasculitis involving small- or medium - sized vessels. the american college of rheumatology (acr) has proposed six criteria for the diagnosis of css in adults. these are : (1) asthma (wheezing, expiratory rhonchi), (2) eosinophilia of more than 10% in peripheral blood, (3) paranasal sinus abnormality, (4) pulmonary infiltrates (may be transient), (5) histological proof of vasculitis with extravascular eosinophils, and (6) mononeuritis multiplex or polyneuropathy. the presence of four or more criteria yields a sensitivity of 85% and a specificity of 99.7%. anca positivity is rarely found in children, unlike in affected adults where they are directed against myeloperoxidase in most of the cases. she presented with vasculitic skin lesions (with biopsy - proven medium - vessel vasculitis and eosinophilic infiltrates), marked peripheral eosinophilia, peripheral mononeuropathy, pulmonary radiological infiltrates but without respiratory symptoms or a history of asthma. major organ involvement may require aggressive approach with pulse doses of intravenous corticosteroids combined with other immunosuppressive agents, such as cyclophosphamide, azathioprine, and methotrexate. the 5-year survival rate is about 25% without treatment, while with treatment, the 1-year survival rate is 90% and the 5-year survival rate is 62%, with often the need to continue low - dose corticosteroids to control residual asthma. causes of death include cardiac failure, myocardial infarction, renal failure, cerebral haemorrhage, gastrointestinal bleeding, or status asthmaticus. a high index of suspicion is needed to diagnosis css in childhood, especially when respiratory clinical manifestations are lacking. even when the clinical presentation and laboratory parameters are suggestive, histological confirmation is still indicated to differentiate it from other types of vasculitis especially in cases where eosinophilia is absent such as in patients with bronchial asthma on chronic glucocorticoid therapy. evaluation for multiorgan involvement is essential, in particular the cardiac, gastrointestinal, renal and central nervous systems, as their involvement has a negative impact on the prognosis. | churg - strauss syndrome (css) is rare in children. it consists of a small- and medium - sized vessel vasculitis, with skin and peripheral nerve involvement. it is characterized by eosinophilia, extravascular necrotizing granuloma, and eosinophilic infiltration of multiple organs particularly the lungs, but may also involve the gastrointestinal tract, the heart, and the kidneys. the condition is usually associated with a preceding history of asthma or allergic sinusitis. it has rarely been reported in children, where most of the cases had pre - existing asthma, allergic rhinitis, or atopic disease. we report a 10-year - old arab girl proven to have css, with no history of asthma or allergic rhinitis, who presented with tender cutaneous nodules of lower extremities, foot drop, and peripheral eosinophilia, without any clinical respiratory symptoms or signs. |
diagnostic laparoscopy is often used to identify specific intraabdominal pathology as the cause for chronic abdominal and pelvic pain. however, few studies have determined that laparoscopic adhesiolysis, as the only operative intervention, ameliorates a significant amount of chronic abdominal pain. furthermore, only anecdotal reports of the role of laparoscopic adhesiolysis in the setting of acute or chronic bowel obstruction have been published. our study was designed to investigate the role of laparoscopic adhesiolysis in the treatment of patients with chronic abdominal pain or recurrent bowel obstruction, not attributed to other obvious pathology. between september 1996 and april 1999, hospital records of 35 patients who underwent laparoscopic adhesiolysis were retrospectively reviewed. fifteen patients, who had other major abdominal procedures in conjunction with adhesiolysis, were excluded from the study. these procedures included cholecystectomy (7), repair of incisional hernia (2), appendectomy (2), gastrostomy (2), colon resection (1), and nissen fundoplication (1). the extensive adhesiolysis in these 15 patients who were operated on for other specific pathologies was considered incidental. therefore, these patients were excluded from our study. after obtaining approval from the institutional review board assessment of symptomatic relief was determined by the questionnaire, and peri - operative morbidity and mortality were assessed by reviewing the inpatient and outpatient charts as well as through the review of the responses to the questionnaires. of the 20 patients who underwent adhesiolysis only, 2 patients were lost to follow - up. patients comprised 17 females and 1 male, with a mean age of 52 years (range 35 to 78). all operations were completed by 1 surgeon experienced in laparoscopy, at a major medical institution under general anesthesia. thirteen operations were undertaken for chronic abdominal / pelvic pain, and 5 for bowel obstruction. of the 5 patients with bowel obstruction, 3 were hospitalized with acute bowel obstruction ; and 2 were operated on electively for treatment of chronic, intermittent bowel obstruction. the average number of previous abdominal procedures was 2.6 (range 1 - 4 ; table 1). preoperatively, patients were worked up extensively with a combination of computerized tomography of the abdomen and pelvis, upper and lower gastrointestinal contrast studies, and upper and lower gastrointestinal endoscopies. these studies were obtained to rule out obvious intraabdominal / visceral pathology that would explain the patient 's chronic abdominal pain, intestinal obstruction, or both. only when all studies were considered negative for such processes were the patients considered for laparoscopy and adhesiolysis. prior abdominal / pelvic surgeries preoperative evaluation postoperatively, 14 of the 18 patients had complete resolution of their symptoms, and an additional 2 patients had partial resolution of their symptoms (overall 88.9% response to adhesiolysis). seven of these 16 patients reported recurrence of some of their symptoms from 1 week to 6 months following their adhesiolysis. after a mean follow - up period of 11 months (range 1 32 months), overall, 14 patients (77.8%) reported an improvement in their quality of life and responded that they would have the adhesiolysis again. three patients continue to require intermittent use of medications for pain control. of the 18 operations performed, 3 resulted in enterotomies, 2 of which required conversion to laparotomy for resection of devitalized bowel. the 3 enterotomies were encountered in the 3 patients hospitalized preoperatively with acute bowel obstruction. the 15 patients who did not suffer enterotomies were all discharged within 24 hours after their operations. the remaining 3 patients were discharged from 10 days to 21 days following their operations. another patient who required laparotomy for bowel resection underwent a subsequent laparotomy for recurrent bowel obstruction. six other patients have been hospitalized for unrelated medical and surgical conditions (table 3). intestinal obstruction is commonly attributed to intraabdominal scar tissue, a claim that is frequently substantiated by operative findings in patients requiring surgical intervention. abdominal and pelvic pain in association with intraabdominal scar tissue are not as well understood. mueller and kresch have suggested that adhesions can be the cause of pain if they limit the movement or distensibility of peritoneum or bowel. stretching pain secondary to adhesions attached to the liver, intestine, or other organs may also contribute to chronic abdominal pain ; and the adhesions can partially or intermittently cause intestinal obstruction. one study noted that small adhesions appear to cause recurrent pain without other symptoms, whereas large adhesions produce pain in combination with symptoms indicative of intermittent bowel obstruction. enthusiasm for elective adhesiolysis is often limited by the concern about subsequent scar tissue formation following major laparotomy. although the etiology for intraabdominal scar tissue formation is likely to be multifactorial, the inflammatory response, which is decreased in laparoscopy versus laparotomy, has been considered a cause for subsequent scar tissue formation. therefore, it is possible that laparoscopic adhesiolysis would result in immediate resolution of symptoms attributed to intraabdominal adhesions, with less likelihood of subsequent recurrence of adhesions and symptoms. our operative technique includes complete lysis of all adhesions that have resulted in fixation of the small and large intestine to the abdominal wall. except in those patients with operative findings of an obvious transition from dilated to decompressed bowel, we do not routinely inspect the entire length of the small intestine, searching for interloop adhesions. we believe excessive manipulation of the small intestine may increase the risk of enterotomies. so far, with a mean follow - up of 11 months, the only patient who has required repeat abdominal surgery for recurrent symptoms is 1 of the 3 patients who required major bowel resection at the time of her initial adhesiolysis, supporting the adequacy of our technique of adhesiolysis. identification of other intraabdominal pathology through the extensive use of less - invasive preoperative testing should result in a low incidence of nontherapeutic adhesiolysis. fifteen patients excluded from our study had other major abdominal procedures performed at the time of their adhesiolysis. all of these patients were extensively evaluated preoperatively and found to have other possible sources for their chronic abdominal pain. during their operations, however, all 15 patients underwent similar extensive adhesiolysis to prevent a future operative procedure. we believe that an extensive pre - operative workup should be used before attributing symptoms of chronic abdominal pain to intraabdominal scar tissue. additionally, we believe that patients with unusual preoperative abdominal pain should undergo adhesiolysis at the time of their laparoscopy for other preoperatively detected intraabdominal pathology. as early as 1992, several authors suggested that laparoscopy in the setting of bowel obstruction may yield inadequate enterolysis, and is likely to be dangerous. more recently, other authors have demonstrated acceptable results with laparoscopic lysis of adhesions in the setting of acute intestinal obstruction. in our study, all 3 patients who underwent adhesiolysis after hospitalization for acute bowel obstruction sustained enterotomies. one was repaired laparoscopically, but 2 required conversion to laparotomy for resection of devitalized bowel, and 1 of the 2 patients has since required another operative procedure for recurrent bowel obstruction. in contrast, the 2 patients who underwent adhesiolysis for management of chronic bowel obstruction had no operative morbidities. based on this experience, we conclude that laparoscopic adhesiolysis performed for management of acute bowel obstruction may result in a high incidence of operative complication. an overall 77.8% improvement in symptoms following laparoscopic adhesiolysis supports liberal use of diagnostic laparoscopy and lysis of adhesions in patients with chronic abdominal pain, bowel obstruction, or both, who have previously under - gone major abdominal surgery. to improve the yield of such an approach, in conclusion, laparoscopic adhesiolysis for chronic abdominal pain, recurrent bowel obstruction, or both, is safe and effective and results in minimal peri - operative morbidity. in the setting of acute bowel obstruction, | background : major abdominal operations result in random and unpredictable scar tissue formation. intraabdominal scar tissue may contribute to recurrent episodes of bowel obstruction, chronic abdominal pain, or both. laparoscopic adhesiolysis may provide relief of symptoms in patients with prior abdominal surgery with chronic abdominal pain or recurrent bowel obstruction.methods:between september 1996 and april 1999, 35 patients underwent laparoscopic adhesiolysis. fifteen of the patients had adhesiolysis in conjunction with other major laparoscopic procedures and were excluded from the study. twenty of the patients who underwent adhesiolysis only were retrospectively assessed for symptomatic relief as well as peri - operative morbidity and mortality.results:two of 20 patients were not available for long - term follow - up. in the 18 remaining patients, laparoscopic adhesiolysis was performed on 13 patients with abdominal pain and 5 patients with recurrent bowel obstruction. the follow - up period ranged from 1 to 32 (mean 11) months. sixteen of the 18 (88.9%) operations were completed laparoscopically. two operations were converted to open for partial enterectomy. an additional enterotomy was repaired laparoscopically. all 3 operative complications were encountered in patients operated on during hospitalization for active bowel obstruction. no mortalities or blood transfusions occurred. one patient required rehospitalization for nonoperative management of an intraabdominal hematoma. fourteen of the 18 (77.8%) had subjective improvement in their quality of life after operation. only 1 patient has required repeat adhesiolysis.conclusions:laparoscopic adhesiolysis is a safe and effective management option for patients with prior abdominal surgery with chronic abdominal pain or recurrent bowel obstruction not attributed to other intraabdominal pathology. laparoscopic intervention in patients with active bowel obstruction may increase the risk of operative complications. |
the specific targeting of nanoparticles to target cells in an organ of interest has been a long - term goal of many laboratories interested in using nanoparticles to mediate the functional delivery of biopharmaceutical agents (including nucleic acids) to defined target cells. one of the main problems in using targeting ligands in conjunction with nanoparticles is the competition between specific - binding mediated cell entry and non - specific enhanced cell uptake mechanisms (andreu, 2008). previously, we have observed that non - specific effects can be completely overwhelming (waterhouse, 2005), an observation that has led to a series of further studies (wang, 2009) intended to determine and characterize those factors that might influence non - specific enhanced cell uptake mechanisms such as nanoparticle size and heterogeneity, surface hydrophilicity and charge, not to forget other factors such as ligand conformation, points of attachment and surface density. recently, we described how ph - triggered, pegylated sirna nanoparticles (also known as ph - triggered sirna - abc nanoparticles according to our recently described abcd nanoparticle structural paradigm (kostarelos and miller, 2005 ; thanou, 2007 ; miller, 2008a ; miller, 2008b). these nanoparticles with a surface - covering of 5mol% polyethylene glycol 2000 (peg) were able to mediate the passive targeting of sirna to liver cells in vivo, thereby providing proof of concept for an effective rnai therapeutic approach to the treatment of hepatitis b virus infections (sifectplus nanoparticles) (carmona, 2009). in addition, pegylated sirna nanoparticles enabled for long - term circulation (ltc sirna - abc nanoparticles ; lesirna nanoparticles) were shown to do the same to tumour cells in vivo, thereby providing proof of concept for an effective rnai therapeutic approach to cancer treatment (kenny, 2011). in the case of the ph - triggered, pegylated sirna nanoparticles, the peg coating required for the biological stabilization of these nanoparticles was introduced by a purpose - designed post - coupling methodology. here, we report how this methodology has now been adapted to introduce integrin - targeting peptide motifs onto liposome and nanoparticle surfaces and so enable integrin receptor - mediated cellular uptake of agents to cells. there have been some spectacular data obtained in recent years using v6 integrin - receptor binding motifs for receptor - mediated delivery of different imaging agents to cells in vivo (dicara, 2007 ; hausner, 2007 ; dicara, 2008 ; hausner, 2009). we have described previously the use of integrin - receptor targeting peptides to promote functional gene delivery to cells (harbottle, 1998 ; cooper, 1999). here, we now report on the use of an alternative cyclic pentapeptide construct that makes use of a single d - tyrosyl amino acid residue to present the arginine - glycine - aspartate (rgd) amino acid residue triad in an " active " conformation capable of mediating intracellular delivery through v3/5 receptor binding and internalization (chen, 2004). arginine - glycine - glutamate (rge) controls are also reported in order to provide the means to demonstrate pure integrin - receptor mediated cellular uptake by rgd presenting imaging nanoparticles. dioleoyl l--phosphatidylethanolamine (dope) 1, dimyristoyl l--phosphatidylcholine (dmpc) 2 (sigma), dope - lissamine - rhodamine b (dope - rhoda) 3 were obtained from avanti polar lipids (usa) (figure 1). general synthetic procedures were performed as described previously (carmona, 2009 ; mvel, 2010). syntheses of n - cholesteryloxycarbonyl-3,7-diazanonane-1,9-diamine (cdan) 4 and cholesteryl - aminoxy (ca) lipid 5 were performed as described previously (keller, 2003 ; oliver, 2004 ; carmona, 2009) (figure 1). the synthesis of other compounds necessary for our experiments were performed as described (scheme 1). hplc purification of final products required the use of vydac c-4 reversed phase preparative column with 1ml / min flow rate : mobile phases as follows used trifluoroacetic acid (tfa) ; a : h2o (0.1%, v / v, tfa) ; b : mecn (0.1%, v / v, tfa) ; c : meoh (0.1%, v / v, tfa). program set at : 0 - 15.0min (100%, a), 15.1 - 25.0min (0 - 100%, b), 25.1 - 45min (100%, c), 45.1 - 55min (100%, a). elution parameters : rgd - peg - cho 13a rt 15.2min, rge - peg - cho 13b rt 14.8min, prnhco - peg - cho 14 rt 15.8min, ca lipid 5 rt 23.8min, rgd - peg - ca 15a product rt 24.1min, rge - peg - ca 15b product rt 23.8min, and prnhco - peg - ca 16 product rt 26.3min ; mass spectrometry : m / z (esi) 1878 (m - h, rgd - peg - cho 13a), 1917 (mna, rge - peg - cho 13b), 1326 (mh, prnhco - peg - cho 14), 2406 (mh, rgd - peg - ca 15a), 2443 (mna, rge - peg - ca 15b), 1869 (mna, prnhco - peg - ca 16) (figure 1). main lipids were used to prepare liposomes cl1 and cl2 and hence 10mol% pegylated bcd1 and 1 - 5mol% pegylated bcd2 nanoparticles. syntheses of -terminally modified - peg - chos : i) a) side chain protected, fluorenylmethyloxycarbonyl (fmoc) amino acid residue n - terminal coupling of l - arg(npbf), l - lys(boc), d - tyr(otbu) and finally l - asp(otbu) [or l - glu(otbu) ] to glycyl-2-chlorotrityl resin 6 using 2-(1h - benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (hbtu) with di - isopropylethylamine (dipea) to mediate residue coupling and 2% (v / v) each of 1,8-diazabicyclo[5.4.0]undec-7-ene (dbu) and piperidine in dimethylformamide (dmf) for fmoc deprotection between rounds of peptide bond formation : b) resin release with 20% (v / v) each of acetic acid and trifluoroethanol (tfe) in ch2cl2, 31 - 78% ; ii) a) 3eqv diphenylphosphoryl azide (dppa) in dry dmf, 5 eqv nahco3, 0c ambient temperature, ph 8.5, 74 - 80% : b) 2.5% (v / v) each h2o and triisopropylsilane (tis) in trifluoroacetic acid (tfa), 80 - 100% ; iii) a) ho - c2h4-oh, al2o3, ccl4, reflux, 27% : b) pentafluorophenol (pfpoh), n, n'-dicyclohexylcarbodiimide (dcc), etoac, 71% ; iv) a) amino - peg - propionic acid, 4 eqv triethylamine (tea), chcl3, reflux, 84% : b) 1.2 eqv pfpoh, 1.2 eqv dcc, etoac, 76% ; v) 0.2 m na2hpo4, 0.1 m naoh, 69 - 100% ; vi) 10% (v / v) tfa in h2o, 100% ; vii) a) propylamine (prnh2), 4 eqv tea, chcl3, reflux, 100% : b) 10% (v / v) tfa in h2o, 51% (v / v). designated lipids were dissolved in chloroform at 5mg / ml and then appropriate aliquots were combined in a presilanized round - bottom flask (5ml). in each case, the organic solvent was evaporated to dryness to form an even thin lipid film that was further purged with a stream of argon gas to remove residual traces of the organic solvent. the lipid film was hydrated using double distilled h2o (ddh2o) to give a multilamellar liposome suspension that was subsequently subjected to sonication in a water bath at 40c for 30min (sonomatic water bath, langford ultrasonics, 33 khz ultrasound frequency). post - sonication, small unilamellar vesicles (50 - 80 nm) (b - component, cl1 or cl2) were diluted to 1.5mg / ml and incubated at room temperature for 15min. thereafter, appropriate aliquots of -terminally modified - peg - cho 13a, 13b or 14 (1mg / ml in water, cd molecules) were introduced for post - coupling such that their final composition was between 1 and 10mol% of total lipid. the ph of solution was monitored by ph boy (camlab ltd, cambridgeshire, uk) and adjusted to ph 4 if required by addition of small aliquots of aqueous solutions of naoh (0.99 m) or hcl (0.99 m). on completion of the reaction (approximately 16 - 24hr) (as judged by hplc analysis), the solution ph was accurately readjusted to ph 7 resulting in 10mol% pegylated bcd1 and 1 - 5mol% pegylated bcd2 nanoparticles, respectively, ready for use. 1 - 5mol% pegylated abcd2 nanoparticles were prepared by combining 1 - 5mol% pegylated bcd2 nanoparticles with various volumes of aqueous 4 mm hepes, ph 7.0 - 7.4, sirna solution (50m a - component) under heavy vortex conditions (final [sirna ] typically 100g / ml ; 7m) using a lipid : sirna ratio of 12:1 (w / w) that correlates with a lipid - nucleic acid n / p charge ratio of approximately 2. lipid compositions of two liposome formulations prepared in double distilled water with final diameters of 50 - 80 nm. for lipid structures see figure 1. these experiments were performed on a facs calibur instrument (bd biosciences). in each set of indicated experiments 1, 2 or 5mol% pegylated abcd2 nano - particles (14060 nm) were prepared with sirna ([sirna ] 80pmoles / well) and uptake studies performed with huvec cells previously grown to 80% confluent at 37c, 10% co2. dioleoyl l--phosphatidylethanolamine (dope) 1, dimyristoyl l--phosphatidylcholine (dmpc) 2 (sigma), dope - lissamine - rhodamine b (dope - rhoda) 3 were obtained from avanti polar lipids (usa) (figure 1). general synthetic procedures were performed as described previously (carmona, 2009 ; mvel, 2010). syntheses of n - cholesteryloxycarbonyl-3,7-diazanonane-1,9-diamine (cdan) 4 and cholesteryl - aminoxy (ca) lipid 5 were performed as described previously (keller, 2003 ; oliver, 2004 ; carmona, 2009) (figure 1). the synthesis of other compounds necessary for our experiments were performed as described (scheme 1). hplc purification of final products required the use of vydac c-4 reversed phase preparative column with 1ml / min flow rate : mobile phases as follows used trifluoroacetic acid (tfa) ; a : h2o (0.1%, v / v, tfa) ; b : mecn (0.1%, v / v, tfa) ; c : meoh (0.1%, v / v, tfa). program set at : 0 - 15.0min (100%, a), 15.1 - 25.0min (0 - 100%, b), 25.1 - 45min (100%, c), 45.1 - 55min (100%, a). elution parameters : rgd - peg - cho 13a rt 15.2min, rge - peg - cho 13b rt 14.8min, prnhco - peg - cho 14 rt 15.8min, ca lipid 5 rt 23.8min, rgd - peg - ca 15a product rt 24.1min, rge - peg - ca 15b product rt 23.8min, and prnhco - peg - ca 16 product rt 26.3min ; mass spectrometry : m / z (esi) 1878 (m - h, rgd - peg - cho 13a), 1917 (mna, rge - peg - cho 13b), 1326 (mh, prnhco - peg - cho 14), 2406 (mh, rgd - peg - ca 15a), 2443 (mna, rge - peg - ca 15b), 1869 (mna, prnhco - peg - ca 16) (figure 1). main lipids were used to prepare liposomes cl1 and cl2 and hence 10mol% pegylated bcd1 and 1 - 5mol% pegylated bcd2 nanoparticles. syntheses of -terminally modified - peg - chos : i) a) side chain protected, fluorenylmethyloxycarbonyl (fmoc) amino acid residue n - terminal coupling of l - arg(npbf), l - lys(boc), d - tyr(otbu) and finally l - asp(otbu) [or l - glu(otbu) ] to glycyl-2-chlorotrityl resin 6 using 2-(1h - benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (hbtu) with di - isopropylethylamine (dipea) to mediate residue coupling and 2% (v / v) each of 1,8-diazabicyclo[5.4.0]undec-7-ene (dbu) and piperidine in dimethylformamide (dmf) for fmoc deprotection between rounds of peptide bond formation : b) resin release with 20% (v / v) each of acetic acid and trifluoroethanol (tfe) in ch2cl2, 31 - 78% ; ii) a) 3eqv diphenylphosphoryl azide (dppa) in dry dmf, 5 eqv nahco3, 0c ambient temperature, ph 8.5, 74 - 80% : b) 2.5% (v / v) each h2o and triisopropylsilane (tis) in trifluoroacetic acid (tfa), 80 - 100% ; iii) a) ho - c2h4-oh, al2o3, ccl4, reflux, 27% : b) pentafluorophenol (pfpoh), n, n'-dicyclohexylcarbodiimide (dcc), etoac, 71% ; iv) a) amino - peg - propionic acid, 4 eqv triethylamine (tea), chcl3, reflux, 84% : b) 1.2 eqv pfpoh, 1.2 eqv dcc, etoac, 76% ; v) 0.2 m na2hpo4, 0.1 m naoh, 69 - 100% ; vi) 10% (v / v) tfa in h2o, 100% ; vii) a) propylamine (prnh2), 4 eqv tea, chcl3, reflux, 100% : b) 10% (v / v) tfa in h2o, 51% (v / v). designated lipids were dissolved in chloroform at 5mg / ml and then appropriate aliquots were combined in a presilanized round - bottom flask (5ml). in each case, the organic solvent was evaporated to dryness to form an even thin lipid film that was further purged with a stream of argon gas to remove residual traces of the organic solvent. the lipid film was hydrated using double distilled h2o (ddh2o) to give a multilamellar liposome suspension that was subsequently subjected to sonication in a water bath at 40c for 30min (sonomatic water bath, langford ultrasonics, 33 khz ultrasound frequency). post - sonication, small unilamellar vesicles (50 - 80 nm) (b - component, cl1 or cl2) were diluted to 1.5mg / ml and incubated at room temperature for 15min. thereafter, appropriate aliquots of -terminally modified - peg - cho 13a, 13b or 14 (1mg / ml in water, cd molecules) were introduced for post - coupling such that their final composition was between 1 and 10mol% of total lipid. the ph of solution was monitored by ph boy (camlab ltd, cambridgeshire, uk) and adjusted to ph 4 if required by addition of small aliquots of aqueous solutions of naoh (0.99 m) or hcl (0.99 m). on completion of the reaction (approximately 16 - 24hr) (as judged by hplc analysis), the solution ph was accurately readjusted to ph 7 resulting in 10mol% pegylated bcd1 and 1 - 5mol% pegylated bcd2 nanoparticles, respectively, ready for use. 1 - 5mol% pegylated abcd2 nanoparticles were prepared by combining 1 - 5mol% pegylated bcd2 nanoparticles with various volumes of aqueous 4 mm hepes, ph 7.0 - 7.4, sirna solution (50m a - component) under heavy vortex conditions (final [sirna ] typically 100g / ml ; 7m) using a lipid : sirna ratio of 12:1 (w / w) that correlates with a lipid - nucleic acid n / p charge ratio of approximately 2. lipid compositions of two liposome formulations prepared in double distilled water with final diameters of 50 - 80 nm. for lipid structures see figure 1. these experiments were performed on a facs calibur instrument (bd biosciences). in each set of indicated experiments 1, 2 or 5mol% pegylated abcd2 nano - particles (14060 nm) were prepared with sirna ([sirna ] 80pmoles / well) and uptake studies performed with huvec cells previously grown to 80% confluent at 37c, 10% co2. post - coupling represents an optimal way to attach a biological targeting ligand of interest to a nanoparticle surface with controlled mol% attachment and ligand orientation (scheme 2). our approach to the development of a post - coupling methodology has been to learn from the chemistry employed to create ph - triggered, pegylated sirna nanoparticles (as mentioned in the introduction) in order to design - hybrid targeting ligands (l) that comprise a peptide targeting moiety conjugated to a peg extension chain enabled for postcoupling to a nanoparticle surface (scheme 2). rgd peptides and corresponding rge control peptides were prepared as follows (scheme 1). starting with glycyl-2-chlorotrityl solid - phase peptide resin 6, the desired pentapeptide rgd - targeting moiety was prepared in an open chain, protected form 7 by solid - peptide synthesis, then subject to cyclization and deprotection to give cyclic pentapeptide 8a. a control cyclic - rge pentapeptide 8b (with aspartate [d ] replaced by glutamate [e ]) was prepared in an equivalent manner. thereafter, the aldehyde functionality of 4-carboxybenzaldehyde 9 was acetal protected and the carboxy function was activated for coupling by means of pentafluorophenol (pfpoh) esterification giving 10. reagent 10 was then coupled to amino - peg - propionic acid and the free carboxylate activated once again for coupling by means of pfpoh esterification giving heterofunctional peg moiety 11. the coupling of 11 to 8a or 8b resulted in rgd acetal 12a and rge acetal 12b respectively. routine acetal deprotection yielded the desired products rgd - peg - cho 13a and control rge - peg - cho 13b (scheme 1). an alternative control aldehyde was obtained through combination of heterofunctional peg moiety 11 with propylamine followed by acetal deprotection resulting in the non - peptide control product prnhco - peg - cho 14 (scheme 1). all three -terminally modified - peg - cho compounds prepared as described above were used in subsequent experiments described below. using all three -terminally modified - peg - cho compounds in turn, coupling reactions were performed individually with neutral cholesteryl - aminoxy lipid (ca) 5 (figure 1) and then with neutral liposomes (equivalent in composition to cl1, table 1). reactions were monitored by hplc and mass spectrometry in water at ph4 and shown to reach completion after 16 - 24h stirring at ambient temperature giving 15a, 15b, or 16 as appropriate. liposome - coupling experiments were then performed under similar conditions in order to couple rgd - peg - cho 13a (or control aldehydes rge - peg - cho 13b and prnhco - peg - cho 14) to alternative liposome formulations cl1 or cl2 (table 1, figure 1), making use of ca lipid 5 once again in order to present the aminoxy functional groups required for conjugation of liposome surfaces with each -terminally modified - peg - cho compound in turn (scheme 2). upon conclusion of these liposome - coupling reactions, ph was raised to 7 in order to stabilize the resulting oxime bonds and the resulting pegylated liposomes. resulting ligand - pegylated liposomes (approx 100 nm in diameter) were described as belonging to either bcd1 or bcd2 nanoparticle families depending upon whether the parent liposome formulation used in preparation was cl1 or cl2, respectively. initially bcd1 family members rgd - bcd1, rge - bcd1 and prnhco - bcd1 were prepared with 1mol% of dope - rhoda 3 for fluorescence microscopy cell uptake studies. the results demonstrate that only 10mol% pegylated rgd - bcd1 nanoparticles were capable of delivering measurable and observable levels of fluorescent - label to integrin - rich human umbilical vein endothelial cells (huvec) (figure 2a). this is very interesting in view of the physical properties of bcd1 nanoparticles, namely approx 100 nm in diameter, 10mol% pegylation with peg, and a -potential of essentially zero mv. the lack of charge originates from the fact that the majority lipid components needed to prepare cl1 liposomes were neutral while the minority ca lipid 5 component possesses an aminoxy functional group unprotonated at ph 7 due to its pka value of about 4.5 (compared with the pka values of more typical amine functional groups that are found around 10) (castro, 1990). we would argue that such nanoparticle physical properties could be an ideal starting point from which to optimize receptor specific cellular uptake of nanoparticles relative to non - specific enhanced cell uptake. subsequently, bcd2 family members rgd - bcd2, rge - bcd2 and prnhco - bcd2 were prepared with 1.0 mol% of dope - rhoda 3 and up to 5mol% of each -terminally modified - peg - cho for fluorescence - activated cell sorting (facs) studies. in addition these cationic liposome systems were further combined with a non - functional nucleic acid (small interfering rna [sirna ]) (a component) in order to neutralize the positive charge and convert the bcd2 family members in to a varieties of corresponding low charge abcd2 nanoparticles with lipid : sirna the physical properties of these nanoparticles were diameters of approximately 140 nm, up to 5mol% pegylation with peg, and a -potential of less than 10mv. in these cases, facs studies were performed with low charge 1-, 2- and 5mol% pegylated rgd - abcd2, rge - abcd2 and prnhco - abcd2 nanoparticles. in each case, the discrimination between rgd presenting and control nanoparticles was still clearly present and was found to increase as a function of the mol% pegylation coverage with peg (figure 2b). the fact that the discrimination was not as substantial as observed above (figure 2a) appears to be due to the lower mol% levels of pegylation coverage and the presence of residual nanoparticle charge. therefore, control of both pegylation levels (plus other peg - structure related criteria) and residual charges are essential in order to optimize receptor mediated cell uptake in preference to non - specific enhanced cell uptake mechanisms. schematic illustration of nanoparticle formulations involving the inclusion of an aminoxy coupling lipid by a formulation procedure known as premodification followed by the post coupling of -terminally ligand (l)-modified peg aldehydes (l - peg - cho). biological receptor mediated functional delivery of imaging agents is well established (dicara, 2007 ; dicara, 2008 ; hausner. 2007 ; hausner, 2009), even of genes (harbottle, 1998 ; cooper, 1999). however, receptor - mediated delivery can not be taken as a given following the covalent attachment of a biological targeting ligand to a nanoparticle (waterhouse, 2005 ; andreu, 2008). therefore, in our view, receptor - mediated delivery phenomena should never be treated with the casual assumption that receptor - mediation is inevitable once an appropriate receptor - specific ligand has been mounted on a nanoparticle surface. any given receptor - specific ligand should in fact be subject to a standard set of trial experiments to ensure that the biophysical properties of a nanoparticle platform and the presentation of the receptor - specific ligand are optimized for bonefide receptor - mediated delivery of the corresponding nanoparticle. in this paper we describe just such a set of trial experiments (figure 2) with sufficient controls to demonstrate clearly that specific receptor - mediated uptake by cells has been enabled over background. accordingly, we would like to propose that both studies involving first the delivery of an imaging agent and then second a potential agent of pharmaceutical interest (api), should be standard assays to perform and demonstrate before all else that a nanoparticle - attached ligand is truly a receptor - specific ligand in the context of the nanoparticle platform to which the ligand is covalently attached. if this is not shown, then nanoparticle biophysical properties, ligand attachment and orientation, and mol% ligand presentation should all be systematically altered until specific receptor - mediated cell uptake can be observed substantially over and above any non - specific enhanced cell uptake background (kamaly, 2009 ; kamaly, 2010). fluorescence microscopy of huvec cells, previously grown to 80% confluent at 37c with 10% (v / v) co2, treated with 10mol% pegylated bcd1 nanoparticles prepared from rgd - peg - cho 13a or rge - peg - cho 13b. cells were in contact with nanoparticles for at least 4hr prior to mounting and observation of rhodamine fluorescence under a nikon 600 fluorescence microscope. cell uptake was determined after 4hr using rhodamine fluorescence and detected using the fl-2 channel. ab corresponds with simple sirna - lipoplex control nanoparticles, formulated from cl2 cationic liposomes and an equivalent final [sirna ]. in our case here, we can say with confidence that our post - coupling chemistry and methodology for the attachment of integrin - targeting rgd ligands has resulted in the successful formation of integrin - targeted imaging nanoparticles that can also mediate integrin - specific delivery of an api such as sirna to v3/5 integrin - receptor presenting cells. therefore, this post - coupling chemistry and premodification - postcoupling methodology could be applicable to other nanoparticle platforms with equal success provided that the biophysical properties of the nanoparticle platform also conform to the following biophysical parameters : nanoparticle dimensions of approx 100 nm in diameter nanoparticle -potential values that converge on neutral (0 mv) nanoparticle ligand surface coverage of approx 2 mol% (or higher) further research with this and other nanoparticle systems will now be needed to demonstrate if these three nanoparticle " rules " for receptor - mediated cell entry are indeed general rules or simply guidelines for receptor - mediated cellular uptake of nanoparticle systems by corresponding receptor expressing cells in vitro, ex vivo and/or in vivo. the data described here represent the completion of a first study involving the preparation of ligand - mounted pegylated nanoparticles constructed by a bespoke pre - modification postcoupling methodology. data suggest for the first time in our hands that this methodology may be used to ensure that receptor mediated cell uptake of attached nanoparticles can be " engineered " to dominate non - specific enhanced cell uptake mechanisms. | a key goal of our research is the targeted delivery of functional biopharmaceutical agents of interest, such as small interfering rna (sirna), to selected cells by means of receptor - mediated nanoparticle technologies. recently, we described how ph - triggered, pegylated sirna - nanoparticles (ph triggered sirna - abc nanoparticles) were able to mediate the passive targeting of sirna to liver cells in vivo. in addition, pegylated sirna nanoparticles enabled for long - term circulation (ltc sirna - abc nanoparticles, lesirna nanoparticles) were shown to do the same to tumour cells in vivo. further gains in the efficiency of sirna delivery are expected to require active targeting with nanoparticles targeted for delivery and cellular uptake by means of attached biological ligands. here we report on the development of a new synthetic chemistry and a bioconjugation methodology that allows for the controlled formulation of pegylated nanoparticles which surface - present integrin - targeting peptides unambiguously and so enable integrin receptor - mediated cellular uptake. furthermore, we present delivery data that provide a clear preliminary demonstration of physical principles that we propose should underpin successful, bonefide receptor - mediated targeted delivery of therapeutic and/or imaging agents to cells. |
conventional x - ray diagnosis is a significant source of radiation exposure among the population. therefore, there is the need for x - ray examinations to be conducted using techniques that keep the patients exposure as low as possible, but does not compromise on image quality. to be able to keep doses as low as reasonable achievable, the basic safety standard (bss) recommends optimization procedures in radiology for guidance. in the optimization of x - ray examination techniques, the patient dose should be characterized by a quantity that better accounts for radiation risk. knowledge of organ and effective dose are necessary if the risk of radiation exposure is of concern. patient dose is often described by the patient s entrance surface dose, which is measured on the patient s skin at the center of the x - ray beam. an alternative is to take free - in - air measurements without the contribution of backscatter radiation from the patient and express in terms of incident air kerma (iak). radiation dose to the various organs or tissues in the body can not be measured directly in patients undergoing x - ray examinations, but they can be estimated with reasonable accuracy if sufficient data on the x - ray examination parameters are available. it is important to provide information on patient doses to all relevant stakeholders for continuous optimization. this study estimated radiation dose to organs of patients during ls radiography by using pcxmc, a pc based monte carlo program that calculates organ doses in medical x - ray examinations. this study was carried out at a university hospital using a philips x - ray machine with serial number 982527 and model number 989000085271 with total inherent filtration of 2.5 mm al. the radiographic image was processed using a cassette (18 43 cm) with a screen - film combination. in all, 100 adult patients undergoing ls radiography were randomly selected for the study. the examinations were performed by a qualified radiographer and the image quality of the exposures was passed by a consultant radiologist at the university hospital. in order to check for consistent performance of the x - ray machine, quality control assessments were performed. the output reproducibility of the x - ray machine was checked with a rad - check plus (ce inovision, nuclear associates div. of victoreen, inc., usa). the accuracy, reproducibility of the x - ray tube voltage, and timer were measured with a rmi model 240a (gammex rmi, inc., middleton, wi) using the standard procedure as given in the physics of medical imaging. the x - ray field and light beam alignment was checked by exposing a light demarcated area on a film for agreement with the radiation. free in air measurements with rad - check plus (ce inovision, nuclear associates div. of victoreen, inc., usa) placed at 100 cm from the focal spot of the x - ray tube were made, an alternative to thermoluminescence dosimeter (tld) or any other direct measuring device. free - in - air measurements of radiation were recorded varying tube voltage (kv) and current - time product (mas) combinations to cover a meaningful range available on the philips x - ray machine. the recorded output measurements in millirontgen (mr) were converted to milligray (mgy) (1 mr is equivalent to 8.73 gy in air for x - ray or gamma ray) and divided by the mas to obtain the output ratio (mgy / mas). the output ratio (mgy / mas) incident air kerma curve for the phillip x - ray machine at university hospital from the curve, the output ratio (mgy / mas) could be extrapolated with a known kv. the iak on top of the patient was estimated using equation (1) with a known focus to skin distance (fsd) and mas per examination. entrance surface air kerma (esak) was estimated using microsoft excel spread sheet by multiplying iak with a selected backscatter factor (bsf) depending on the kv (patient thickness related), filtration of the radiation, and the beam field size (patient thickness related : pcxmc calculates the field size on top of the patient based on the cassette size, focus to film distance (ffd), patients weight and height) as given by petoussi - henss. introducing different thickness of aluminum sheets in the direction of the radiation beam, and using rad - check plus (ce inovision, nuclear associates div. of victoreen, inc., usa) to measure the penetrated radiation, half value layer (hvl) of 70 - 120 kvp were estimated as shown in figure 2. using figure 2, the hvl of any operating voltage of the x - ray tube at the university hospital could be estimated. further, using the obtained hvl of the tube voltage on a reference table for high frequency direct current, 3 phase, 12 and 6 pulse generator, total filtration of the x - ray tube was extrapolated. tube voltage of the x - ray machine at university hospital with its corresponding half value layer the pcxmc, monte carlo base computer software developed by the finish radiation and nuclear safety authority was used to estimate organ and effective dose to patients undergoing ls radiography. the program uses computational hermaphrodite phantom defined by mathematical expressions to compute organ and effective doses of patients of different ages and sizes in freely adjustable x - ray projections and other examination conditions used in radiology. organ and effective doses to patients were estimated by inputting iak, operating kv that is used clinically, image size, patients height and weight, projection of the x - ray beam, and part of the body being examined into the pcxmc. the pcxmc version 1.5 calculates organ and effective doses using icrp, 1991 publication 60 recommendations. pcxmc requires the input of iak because it provides the bsf taking into consideration the tube voltage (patient thickness related), hvl, beam filtration, and the field size (patient thickness related : pcxmc calculates the field size on top of the patient based on the cassette size, ffd, patients weight and height) as given in petoussi - henss. in order to generate the required x - ray spectrum for the simulation, pcxmc requires the user to input the filter material and thickness, tube voltage, and anode angle. in order to check for consistent performance of the x - ray machine, quality control assessments were performed. the output reproducibility of the x - ray machine was checked with a rad - check plus (ce inovision, nuclear associates div. the accuracy, reproducibility of the x - ray tube voltage, and timer were measured with a rmi model 240a (gammex rmi, inc., middleton, wi) using the standard procedure as given in the physics of medical imaging. the x - ray field and light beam alignment was checked by exposing a light demarcated area on a film for agreement with the radiation. free in air measurements with rad - check plus (ce inovision, nuclear associates div. of victoreen, inc., usa) placed at 100 cm from the focal spot of the x - ray tube were made, an alternative to thermoluminescence dosimeter (tld) or any other direct measuring device. free - in - air measurements of radiation were recorded varying tube voltage (kv) and current - time product (mas) combinations to cover a meaningful range available on the philips x - ray machine. the recorded output measurements in millirontgen (mr) were converted to milligray (mgy) (1 mr is equivalent to 8.73 gy in air for x - ray or gamma ray) and divided by the mas to obtain the output ratio (mgy / mas). the output ratio (mgy / mas) incident air kerma curve for the phillip x - ray machine at university hospital from the curve, the output ratio (mgy / mas) could be extrapolated with a known kv. the iak on top of the patient was estimated using equation (1) with a known focus to skin distance (fsd) and mas per examination. entrance surface air kerma (esak) was estimated using microsoft excel spread sheet by multiplying iak with a selected backscatter factor (bsf) depending on the kv (patient thickness related), filtration of the radiation, and the beam field size (patient thickness related : pcxmc calculates the field size on top of the patient based on the cassette size, focus to film distance (ffd), patients weight and height) as given by petoussi - henss. introducing different thickness of aluminum sheets in the direction of the radiation beam, and using rad - check plus (ce inovision, nuclear associates div. usa) to measure the penetrated radiation, half value layer (hvl) of 70 - 120 kvp were estimated as shown in figure 2. using figure 2, the hvl of any operating voltage of the x - ray tube at the university hospital could be estimated. further, using the obtained hvl of the tube voltage on a reference table for high frequency direct current, 3 phase, 12 and 6 pulse generator, total filtration of the x - ray tube was extrapolated. tube voltage of the x - ray machine at university hospital with its corresponding half value layer the pcxmc, monte carlo base computer software developed by the finish radiation and nuclear safety authority was used to estimate organ and effective dose to patients undergoing ls radiography. the program uses computational hermaphrodite phantom defined by mathematical expressions to compute organ and effective doses of patients of different ages and sizes in freely adjustable x - ray projections and other examination conditions used in radiology. organ and effective doses to patients were estimated by inputting iak, operating kv that is used clinically, image size, patients height and weight, projection of the x - ray beam, and part of the body being examined into the pcxmc. the pcxmc version 1.5 calculates organ and effective doses using icrp, 1991 publication 60 recommendations. pcxmc requires the input of iak because it provides the bsf taking into consideration the tube voltage (patient thickness related), hvl, beam filtration, and the field size (patient thickness related : pcxmc calculates the field size on top of the patient based on the cassette size, ffd, patients weight and height) as given in petoussi - henss. in order to generate the required x - ray spectrum for the simulation, pcxmc requires the user to input the filter material and thickness, tube voltage, and anode angle. the x - ray machine at the university hospital was observed to be performing self consistently when the quality control results were compared with the radiation regulatory requirements of ghana as shown in table 1. quality control results the examinations considered for this study were procedures where both ls projections (anteroposterior (ap) and lateral (lat)) were performed on one patient. the average patient height and weight were observed to be 171.1 (159 - 181) cm and 80.9 (50 - 120) kg, respectively. it was also observed that the university hospital uses the same x - ray field size for both lsap and ls lat per patient collimation. the total filtration of the x - ray machine was found to be in the range of 1.8 - 2.8 mm al for the peak tube voltage range for both ls ap and lat. the radiation field area on top of the patients were recorded to be in the range of 580.64 (38.1 15.24)-767.74 (43.18 17.78) cm. table 2 shows the exposure parameters for both ls projections and the mean esak and effective dose. details of exposure parameters, esak, and effective dose the kvp used for ls lat examination is greater than that of ls ap by approximately a factor of 1.2 as evidenced in table 2. the mas for ls lat examination was observed to be greater than that of ls ap examination by approximately a factor of 1.1. there were variations in the kvp and mas due to the randomly selected patients with varying weight and height. this is due to the high kvp used for ls lat examinations because of increased attenuation in the lat region of the body. the difference in effective dose of both ls projections was insignificant, although a significant difference was observed in the esak. from the results of effective dose presented in table 2, indicates that the overall radiation health effect on patients due to ls radiography is independent of the projection (ap or lat) of the radiation beam. however, the same can not be said for the equivalent doses to patient organs due to the two ls radiography projections as shown in table 3. organ doses to patients due to lumbar spine radiography organs recording high doses in this study have been presented in table 3. the organ doses varied for both ls ap and lat radiography due to the position of these organs with respect to the orientation of the incident radiation beam on the patient. however, it was observed that dose to the pancreas and the liver recorded approximately equal values for both ls radiography projections. this is due to the anatomical position of these organs with respect to the patients orientation to the radiation beam. the stomach and the spleen recorded the highest organ dose for ls ap and lat radiography, respectively. it is expected that had the international commission on radiological protection (icrp) 103 recommended tissue weighting factors been used in the organ dose estimation, the dose to the adrenals, kidney, pancreas, spleen, and heart would have been higher than as reported in this study by fractions of factor 2.4. it is also expected that the dose to the liver and the galbladder will be lower than as reported in this study by a factor of 0.8. however, the dose to the lungs and the stomach is expected to remain unchanged. it can be seen that the esak recorded for both ls radiography projections in this study were lower than what was recorded in the other studies. this could be attributed to one or a combination of the following : the number of x - ray rooms considered for the study ; the mode of x - ray output estimation ; and the difference in the x - ray output of different x - ray tube manufactures. however, the esak recorded for ls lat examination was approximately twice that of ls ap examination in this study. the same observation was also found from the studies of muhogora., and hart. this in our view makes this study consistent with the findings of the other studies. organ doses due to ls radiography have been assessed using a monte carlo base program pcxmc (version 1.5). it was found that the stomach and spleen of patients undergoing ls radiography ap and lat received the highest dose, respectively. there was no significant difference between the effective dose to patients for both ls radiography projections in the hospital. this suggests that for ls radiography, the overall stochastic health effect of radiation to patients is independent of the projection of the examination (ap or lat) in the university hospital. | radiation dose to organs of 100 adult patients undergoing lumbar spine (ls) radiography at a university hospital have been assessed. free in air kerma measurement using an ionization chamber was used for the patient dosimetry. organ and effective dose to the patients were estimated using pcxmc (version 1.5) software. the organs that recorded significant dose due to ls radiography were lungs, stomach, liver, adrenals, kidney, pancreas, spleen, galbladder, and the heart. it was observed that the stomach recorded the highest dose (48.2 1.2 gy) for ls anteroposterior (ap). the spleen also recorded the highest dose (41.2 0.5 gy) for ls lateral (lat). the mean entrance surface air kerma (esak) of ls lat (122.2 gy) was approximately twice that of ls ap (76.3 gy), but the effective dose for both examinations were approximately the same (ls lat = 8.6 sv and ls ap = 10.4 sv). the overall stochastic health effect of radiation to patients due to ls radiography in the university hospital is independent of the projection of the examination (ap or lat). |
normal cells grow and divide in an ordered fashion, in accordance with the cell cycle. defective apoptosis (programmed cell death) which results in enhanced growth describes most cancer cells. several proteins control the timing of the events in the cell cycle, which is tightly regulated to ensure that cells divide only when necessary. the loss of this regulation is the hallmark of cancer [1, 2 ]. initially, somatic cell fusion and nuclear transplantation studies, together with the selective use of growth factors and inhibitors of macromolecular biosynthesis, established the fundamental parameters of cell cycle regulation [3, 4 ]. our understanding of the complexities of apoptosis and the mechanisms evolved by tumor cells to resist engagement of cell death has focused research effort into the development of strategies designed to selectively induce apoptosis in cancer cells [57 ]. several previous studies demonstrated that certain phytochemicals present in medicinal herbs exert antitumorigenic activity by inducing apoptosis in cancer cells [811 ]. the mechanism, of apoptosis are now mostly well known, involving activation of caspases (cysteinyl, aspartate - specific proteases), which cleave to inactivate or activate target substrates within a cell and bcl2 family members in response to a wide variety of physiological and injury - induced signals. caspases are synthesized in most if not all cells as inactive zymogens, which must be proteolytically cleaved at two (or three in some cases) aspartate residues to generate the active mature enzyme. apical and more susceptible to modification by endogenous regulatory proteins, in turn cleave and activate the downstream executioner caspases, such as caspase-3 also known as apopain, sca-1, yama, and cpp32 (alias), which enact the final, irreversible commitment to death. these then cleave their target substrates to orchestrate the proteolytic dismantling of the cell [14, 15 ]. this sequence of events culminating in the activation of caspases has been broadly categorized into two pathways, the extrinsic pathway characterized by the engagement of cell surface death receptors and the intrinsic pathway involving key mitochondrial events [5, 15 ]. the bcl2 proteins also represent a promising target for modulating tumor cell sensitivity to apoptosis [5, 16 ]. it was first human apoptotic protein, an inhibitor of apoptosis identified in 1984 [17, 18 ]. high amounts of bcl2 block the apoptotic death of a pro - b - lymphocyte cell line. thus, bcl2 is unique among protooncogenes, being localized to mitochondria and interfering with programmed cell death independent of promoting cell division. overexpression of antiapoptotic bcl2 proteins is observed in many tumor types, which may contribute to the drug - resistant state and help mediate the expansion of a transformed population by disrupting normal cell turnover [5, 20, 21 ]. so, these drugs can also eliminate certain adult cells that divide more rapidly, such as those that line the gastrointestinal tract, bone marrow cells, and hair follicles. this causes some side effects, including gastrointestinal distress, low white blood cell count, and hair loss [2224 ]. for several millennia, herbal preparations and natural remedies have been shown to be effective in treating many types of maladies [9, 25, 26 ]. although herbal therapies are becoming increasingly popular worldwide, we know little about the molecular mechanisms and active ingredients in many of those therapeutic herbs [9, 27 ]. chemical characterization and cytotoxicity screening studies on plant - based materials could lead to a discovery of new natural anticancer drugs. studied apoptosis - inducing properties of dichloromethanoic and methanolic extracts of scrophularia oxysepala in mcf-7 human breast cancer cells, and samavati. dorema glabrum, a medicinal plant of the family of apiaceae, which grows in transcaucasia (nakhichevan and armenia zones) and north west of iran (azerbaijan), in loamy or rocky slopes of aras river, was chosen to study its apoptotic effects on mouse fibrosarcoma cell line, wehi-164 cells in comparison with its effects on l929 mouse normal cells. the plant has extensive uses, for example, as an herbal remedy or food additive in the mentioned regions. this study was conducted according to the common folk beliefs of armenian and azeri people that dorema glabrum can cure many anomalies especially different kinds of cancer. of course it should be mentioned that in a preliminary work, the crude extract of the plant demonstrated antioxidant activity and antilipidemic effects. mey were collected during the fruiting stage from rocky slopes of aras river bank ; jolfa, eastern azerbaijan (38 309.2, 45 2736.2 ; 1590 m, 15 km from jolfa to st. air - dried and finely powdered seeds were subjected to extraction by refluxing methanol in a soxhlet in order to obtain its ooze. 20 mg of dried extract was dissolved in 100 l dmso and diluted with 3.90 ml rpmi-1640 to give a concentration of 5000 g / ml. this was used to treat the cells. wehi-164 cells, mouse fibrosarcoma cell line (ncbi code c200) and l929 cells, mouse normal adipose tissue cell line (ncbi code c161) were obtained from iran pasture institute. both cell lines were cultured in rpmi-1640 (sigma, ph = 7.2) containing 10% fcs (fetal calf serum) and antibiotic (100 u / ml penicillin, 100 g / ml streptomycin), placed in 37c and 5% co2 in an incubator overnight. mtt assay is one of the most useful tests for investigating cells viability and cytotoxic effects of drugs, cosmetics, and food additives. mtt (3-[4, 5-dimethyl-2-thiazolyl]-2, 5 diphenyl tetrazolium bromide), which is yellow and soluble in water, can be reduced by mitochondrial dehydrogenases of live cells to give a bluish purple and insoluble salt called formosan that can easily and rapidly be quantitated by an elisa plate reader at 570 nm to evaluate growth and viability of cells and cytotoxic effects of interventor agents [13, 32 ]. wehi-164 and l929 cells were separately seeded, in a triplicate manner, in 96-well microplates (5000 cells / well) as above. after 6 hours, both cells were treated with different concentrations (10, 30, 50, 100, 200, 300, and 400 g / ml) of methanolic extract of dorema glabrum seeds with different time periods (6, 24, and 36 hours). no plant extract was added to negative controls, but the same amount of dmso was added to eliminate its intervening effects, if any. positive control cells were treated with taxol as the same concentrations of plant extract in test cells. taxol which contains paclitaxel as the main active compound is used in chemotherapy of cancer. of course prior to treatment, the cells viability was determined by counting on a neubauer slide (hemocytometer) with the aid of trypan blue. trypan blue can penetrate into dead cells ' membrane and colour them purple. after desired time, the supernatants of all wells were discarded and washed with pbs ; then 100 l of rpmi and 50 l of mtt solution (2 mg / ml) were added to each well. following incubation at 37c for 4 hours, the liquid phase of wells was discarded again. after adding 200 l dmso and 25 l sorensen 's glycine buffer (0.1 m glycine, 0.1 m nacl ; ph = 10.5), the plates were incubated at 37c in the dark for another half an hour. at the end, absorbencies of wells were determined at 570 nm wavelength using a microplate reader (awareness technology, usa). to survey production of proteins involved in apoptosis electrophoresis and related applications have contributed greatly to the understanding of the molecular bases of cell structure and function. to enhance resolution and discrimination of proteins on the basis of molecular size rather than charge or shape the western blot is a widely accepted analytical technique used to detect specific proteins in the given sample. proteins separated by electrophoretic technique are electrophoretically transferred (electroblotted) onto a membrane. the membrane, which is now a replica of the polyacrylamide gel, is subsequently probed with antibodies to specific proteins. the primary antibodies can be revealed by an additional incubation with hrp conjugated, followed by enhanced electrochemiluminescent (ecl) detection. prior to electrophoresis, we need to extract cells ' proteins ; hence wehi-164 cells were cultured in 6-well plates (10 cells per well) with rpmi 1640 containing 10% fcs and antibiotic (total volume of 5 ml). six hours later, treatments with different concentrations of plant extract (0, 30, 50, and 100 g / ml) with different time periods (24 and 36 hours) were done. according to mtt results and ic50 value of the plant extract, there was no need to treat cells with concentrations more than 100 g / ml. the wells with no extract considered as negative controls, and to eliminate effects of dmso the same amount of dmso concentrations in test cells were added to them. and as mtt test, positive control cells were treated with taxol as the same concentrations of extract in test cells. twenty - four and 36 hours after treatment, the supernatants of all wells were gathered and kept in separate tubes. this time we did not discard the supernatants because we needed the dead cells that were affected by plant ooze to extract their proteins. all wells were washed with pbs and cells were detached from bottom of plates ' wells using trp - edta, and all liquids and cells from each well was added to its own supernatant. the tubes were centrifuged at 2000 rpm for 10 min and then washed two times with pbs at 2500 rpm for 5 min. the cells pellets were homogenized by gentle vortexing and removed to microtubes with 200 l of pbs and centrifuged at 1500 rpm for 5 min. 200 l of lysis buffer was added to each tube and following mixing gently, left on the rotator in 4c for half an hour. then, supernatants were removed to fresh microtubes and pellets were discarded. to obtain protein concentration of each sample, absorbance of each tube, at 280 nm wavelength, was read using nanodrop spectrophotometer against lysis buffer blank. as mentioned above, to investigate production of proteins involved in apoptosis, sds - page and western blotting should be performed. then, proteins on the gel were transferred to the pvdf membrane using a semidry electroblotting tank. then, the membrane was washed three times (every time 5 minutes) with wash buffer (0.05% tween 20 in pbs) and was soaked in the primary antibody (abcam, usa) with a dilution of 1/1000 on the shaker for two hours, followed by again three times washing with wash buffer. 0.03% of hrp conjugated secondary antibody (abcam, usa, 3 l in 10 ml wash buffer, at 1/5000 dilution) was used to incubate the membrane on the shaker, away from direct light, for 2 hours. after discarding the secondary antibody the protein bands in the membrane were developed using ecl substrate on radiography film. in order to investigate expression of bcl2 and caspase-3 genes, to confirm western blotting results, hence, pcr was performed on synthesized cdnas, using wehi-164 cells ' mrna. since mrna is the first thing to be synthesized in genes expression and also mrnas are very unstable, briefly (3 and 6 hours) treated cells were used as well. after detaching cells from the plates and doing the washing process with pbs, their precipitants were transferred to rnase - free microtubes. total rna was extracted using rnx - plus solutions according to the manufacturer 's protocol, and because rna is very unstable, work always should be done on ice. prior to cdna synthesis, rna contents of samples were determined using a nanodrop uv spectrophotometer. then keeping in mind the samples concentrations, the desired volume (for 20 l reaction a volume equivalent to 3 g rna) of each sample was added to reaction mixture, containing 100 pmole oligo dt in a sterile rnase - free tube. then, the mixtures were incubated at 70c for 5 minutes. after transferring the mixtures to accupower rt premix tubes, they filled up to 20 l with depc distilled water. the mixtures were vortexed to dissolve the pellets and briefly spun down. to avoid samples evaporation, one drop of mineral oil the strips were put in the thermal cycler apparatus under the following conditions : cdna synthesis at 42c for 60 minutes and 94c for 5 minutes to inactivate rtase and terminate the reaction. the volume of each reaction was considered to be 20 l. the ampliqon master mix that is 2x was used to make reaction mixture as follows : 2 l of each cdna sample was added to 10 l of master mix then 1 l of each forward and reverse primers (10 mm) and 6 l of distilled, deionised, sterile water were added to make the final volume of 20 l and mgcl2 concentrations of 1.5 mm. in the end, one drop of mineral oil was added to the top of each sample to avoid evaporation of samples. then microtubes were put in the thermal cycler apparatus under the following conditions : initial denaturation at 94c for 4 minutes, followed by 35 amplification cycles, each consisting of denaturation at 94c for 30 seconds, annealing at 58c for 30 seconds, and extension at 72c for 30 seconds, with an additional extension step at the end of the procedure at 72c for 5 minutes. all rt - pcr products were visualized by electrophoresis through 2% agarose gel followed by ethidium bromide staining. the biochemical hallmark of apoptosis is the fragmentation of the genomic dna, an irreversible event that commits the cell to die and occurs before changes in plasma membrane permeability (prelytic dna fragmentation). this assay involves extraction of dna from a lysed cell homogenate followed by agarose gel electrophoresis. after culturing and treating cells with different concentrations of plant extract (0, 30, 50, and 100 g / ml) for different time periods (24 and 36 hours), their genomic dna contents were extracted by firstly lysing the cells using 500 l of lysis buffer. then 10 l of proteinase k (fermentas, life sciences, 20 next day, 40 l of saturated nacl (5 m) was added and mixed completely, followed by incubation at 4c for 10 minutes. after 20-minute centrifugation in 12000 rpm, their upper liquids were transferred to a fresh microtube. 1 ml of cold ethanol 100% (stored in 20c) was added and then incubated at 20c for 10 minutes. after centrifugation for 15 minutes in 12000 rpm, the ethanol in upper phase was removed completely. following adding 1 ml of ethanol 70 (kept in 4c) and centrifugation for 10 minutes in 12000 rpm, the ethanol was removed completely again. the pellets were dissolved in 100 l distilled, deionized, and sterile water or te (tris / edta). after determining the samples concentrations using a nanodrop uv spectrophotometer, the equivalent amount of dna samples diluted with the 6x dna loading dye (supplied with the ladder) were subjected to 1.5% agarose submarine electrophoresis in company with dna ladder marker (fermentas, life sciences, 1 kb dna ladder). in the end, following ethidium bromide staining, the fragmented dnas bands were visualized by uv transilluminator. fusiform or spindle - like natural and live wehi-164 cells (figure 1(a)) undergo morphological changes after treatment with taxol or methanolic extract of dorema glabrum seed, and because of chromatin condensation and other changes they shrink and take a spherical shape (figure 1(b)), characteristics of apoptotic cells. mtt assay showed a time- and dose - dependent inhibition of the cell growth by plant extract (figure 2). as the figure shows, ic50 value, the concentration that causes 50% loss of cell viability, in wehi-164 cell line is about 50 g / ml in 36 hours. by contrast the plant extract had higher ic50 value (about 100 g / ml in 36 hours) for normal l929 cells, meaning that it is less toxic to the normal cells than wehi-164 cells. statistical analysis using independent t - test, which resulted in p < 0.0001, showed that cytotoxicity effects of 50 g / ml plant extract in 36 hours on wehi-164 and l929 cells are significantly different. in order to compare the effects of different concentrations of plant extract on the production of bcl2 and caspase-3, all samples were unified using -actin amount as an intrinsic factor. as it can be seen from figure 3, because antizymogen caspase-3 was used as primary antibody, in the samples related to treated cells, caspase-3 zymogene band is weakened due to its cleavage. as mentioned previously, most caspases are synthesized as inactive zymogens and must be cleaved at two or three aspartate residues to generate the active enzyme. so, both western blotting results confirmed that methanolic extract of dorema glabrum seed can induce apoptosis in wehi-164 cells. all pcr products of synthesised cdnas were subjected to electrophoresis through 2% agarose gel followed by ethidium bromide staining in order to be visualized. dna fragmentation can be analysed by the typical dna ladder formation, for which dna is extracted from the apoptotic cells and separated in an agarose gel. as shown in figure 5, treatment with dorema glabrum seed extract resulted in degradation of chromosomal dna into small internucleosomal fragments, a biochemical hallmark of cells undergoing apoptosis. despite a period in which pharmaceutical companies cut back their use of natural products in drug discovery, there are many promising drug candidates in the current development pipeline that are of natural origin. technical drawbacks associated with natural product research have been lessened, and there are better opportunities to explore the biological activity of previously inaccessible sources of natural products. with the increasing acceptance that the chemical diversity of natural products is well suited to provide the core scaffolds for future drugs, there will be further developments in the use of novel natural products and chemical libraries based on natural products in drug discovery campaigns. after all, traditional cytotoxic chemotherapy although kills cancer cells by indirectly inducing apoptosis unfortunately, side effects are brutal, and most tumors become resistant [17, 23, 24 ]. therefore, drugs that restore the apoptotic pathways have the potential for effectively treating tumors. herbal plants have been the basis for nearly all medicinal therapies since ancient times and constitute a common alternative for cancer prevention and treatment in many countries around the world. a number of chemotherapeutic agents, with properties including apoptosis induction and antiangiogenesis, have been isolated from natural products and characterized to inhibit the development of malignancies, such as curcumin from curcuma longa, epicatechin gallate from tea, paclitaxel from pacific yew, emodin, a natural anthraquinone derivative from rheum palmatum l. and honokiol, a biphenyl extract from magnolia obovata bark. dorema glabrum is a perennial medicinal plant growing in loamy or rocky slopes commonly in armenia, nakhichevan, and north west of iran that is currently used as a remedy for treating cancerous diseases in folk medicine and also as a green vegetable in domestic use. to evaluate the effects of dorema glabrum seed extract on cell proliferation and identify its therapeutic potential, we demonstrated, for the first time, the potent cytotoxicity activity of different concentrations of methanolic extract of dorema glabrum seed against wehi-164 mouse fibrosarcoma cell line and l929 normal cell line. a successful anticancer drug should kill or incapacitate cancer cells without causing excessive damages to normal cells, meaning minimum side effects. understanding the modes of action of these compounds should provide useful information for their possible applications in cancer prevention and perhaps in cancer therapy. cell cycle modulation by various natural and synthetic agents is gaining widespread attention in recent years. so, multiple techniques were used to assess the antiproliferative and apoptotic effects of d. glabrum seed methanolic extract on cancer cells. these include blebbing, loss of cell membarane symmetry and attachment, cell shrinkage, nuclear fragmentation, and chromatin condensation. in the present study, mtt assay was performed which showed that the methanolic extract of dorema glabrum seed caused growth inhibition in the wehi-164 cells in a dose- and time - dependent manner. but it appeared less toxic in low concentrations to normal or nonmalignant cells in vitro because ic50 value of the plant extract for wehi-164 cells is 50 g / ml and for l929 cells is 100 g / ml in 36 hours. this claim was confirmed by statistical analysis using independent t - test that resulted in p < 0.0001, meaning that the mean differences of cytotoxicity effects of 50 g / ml plant extract in 36 hours on wehi-164 and l929 cells are significant. thirty - sixh hours treatment was selected because in shorter times higher concentrations of plant extract were needed to cause 50% loss of cell viability. since concentrations more than 50 g / ml affect l929 cells viability too, it is prefered to choose 36-hour treatment with 50 g / ml plant extract in order to avoid massive damages to normal cells. also, we compared the effects of plant extract with the effects of taxol, an anticancer and apoptosis inducer drug, and it should be mentioned here that the effects of plant extract on both cell lines followed the same pattern as taxol effects on the cells (figure 2). chromatin condensation, cell shrinkage, and other alterations, characteristics of apoptotic cells, cause the morphology of treated cells with the plant extract, change from spindle like to spherical shape, and also make them lose their attachment (figure 1). in conclusion, treatment with the plant extract resulted in degradation of chromosomal dna into smaller fragments (figure 5), a biochemical hallmark of cells undergoing apoptosis. once more, induction of apoptosis, and not necrosis, by plant extract was confirmed because electrophoresis of necrotic cells ' dna results in smear not ladder. as it was referred, apoptosis is a consequence of a highly complex and sequential cascade of cellular events, and caspase-3 has been implicated in the execution phase of apoptosis cleaving over 100 substrates. due to our investigation results, the immunoblotting data, since 32kd caspase-3 precursor was decreased in time- and concentration - dependent manner, methanolic extract of dorema glabrum seed can induce caspase-3 activation via its proteolytic cleavage into active subunits which enact the final irreversible commitment to death. also from the immunoblotting results, the decrease in the amount of antiapoptotic bcl2 protein is clear in the cells exposed to plant extract (figure 3). it was mentioned that overexpression of bcl2 proteins is seen in different types of tumors, which can contribute to drug - resistant state. it is believed that prevailing a blockade induced by bcl2 or bcl - xl could restore normal cellular homeostasis, reverse the drug - resistant phenotype, and restore tumor cell sensitivity to conventional chemotherapeutics [5, 20, 21 ]. we have also performed rt - pcr technique and demonstrated that the plant extract - dependent apoptosis was accompanied with significant increase of caspase-3 mrna and hence its expression and decrease of that of bcl2 protein (figure 4). in conclusion, our data well established the antiproliferative effect of methanolic extract of dorema glabrum seed and clearly showed that the plant extract can induce apoptosis and not necrosis in vitro, but its activities remained unknown in vivo. these results demonstrated that dorema glabrum seed with antiproliferative properties, especially with ic50 value for cancerous cells lower than that of normal cells, might be a novel and attractive therapeutic candidate for tumor treatment in clinical practice. | we aimed to investigate the apoptotic effects of the methanolic extract of dorema glabrum seed on wehi-164, cancerous cells in comparison with l929, normal cells and compared them with the cytotoxic effects of taxol. so, mtt test and dna fragmentation assay were performed on cultured and treated cells. also electrophoresis which was followed by immunoblotting was done to survey the production of caspase-3 and bcl2 proteins, and to inquire into their relative genes expression, rt - pcr was used. according to our findings, the methanolic extract of dorema glabrum seed can alter cells morphology as they shrink and take a spherical shape and lose their attachment too. so, the plant extract inhibits cell growth albeit in a time- and dose - dependent manner and results in degradation of chromosomal dna. induction of apoptosis by the plant extract was proved by the reduction of pro - caspase-3 and bcl2 proteins and increase in caspase-3 gene expression and decrease in that of bcl2 too. our data well established the antiproliferative effect of methanolic extract of dorema glabrum seed and clearly showed that the plant extract can induce apoptosis and not necrosis in vitro. these results demonstrated that dorema glabrum seed might be a novel and attractive therapeutic candidate for tumor treatment. |
we encountered a woman with a preexisting large focal nodular hyperplasia (fnh) of the liver, persisting during two separate pregnancies. her elevated serum -glutamyltransferase and alkaline phosphatase levels before pregnancy were reduced during both pregnancies, but returned to prepregnancy levels after delivery. focal nodular hyperplasia (fnh) is a benign lesion of the liver that is not uncommon in adults and is frequently observed in healthy individuals. it is unclear, however, whether pregnancy in women with fnh increases the risks of fnh growth and complications or alters hepatic function in patients. we encountered a woman with a large fnh, who experienced two pregnancies and delivered healthy infants at term uneventfully. the courses of her pregnancies and the effects of pregnancy on fnh, including lesion size and hepatic function were evaluated at the same institution. the patient was a 25-year - old, gravida 0, para 0, female, who had been followed up since the age of 18 years at the internal medicine outpatient ward of our institution because of a large fnh. at the age of 13 years, a nodule measuring 8 cm was observed in the patient, at a medical institution near her residence. a biopsy was performed, and on histological analysis of the specimen, she was diagnosed with fnh. because the nodule was located near the hepatic hilus, surgical intervention was not performed. at the age of 25 years, the patient married and moved near our hospital. an mri taken before her first pregnancy showed that the nodule was 11 cm in diameter (figs.1a and 2a). a high intensity area was observed at the center of the lesion, a finding compatible with central scar tissue, with a moderately high intensity area observed at the surface of the lesion. (c) during her second pregnancy at 34 weeks of gestation. before becoming pregnant, her liver function tests showed that her serum aspartate aminotransferase (ast) and alanine aminotransferase (alt) concentrations were in the normal ranges, but her -glutamyltransferase (ggt) and alkaline phosphatase (alp) concentrations were elevated, to 651 iu / l and 407 u / l, respectively. after conceiving, she was referred by her physician to our outpatient ward at 9 weeks of gestation. during her first pregnancy, her serum concentrations of liver enzymes decreased to the normal range without any medication (fig.3). magnetic resonance imaging performed at 37 weeks of gestation showed that the size of the fnh was unchanged, with no signs of bleeding or degeneration of the lesion (figs.1b and 2b). the concentrations of aspartate aminotransferase (ast), alanine aminotransferase (alt), lactate dehydrogenase (ldh), alkaline phosphatase (alp), and -glutamyltransferase (ggt) are shown in u / l., she delivered a normal, healthy female infant, weighing 3084 g and with 1 and 5 min apgar scores of 8 and 9, respectively, by vaginal delivery. her postpartum recovery was normal, except that her serum ggt and alp concentrations became elevated, returning to their prepregnancy levels. this pregnancy was also uneventful (figs.1c, 2c, and 3) and, at 40 weeks of gestation she delivered a normal, healthy male infant, weighing 3130 g and with 1 and 5 min apgar scores of 9 and 10, respectively, by vaginal delivery. her postpartum course was again uneventful, and both mother and child were discharged after 5 days. the size of the fnh remained the same as before her first pregnancy, despite two pregnancies and deliveries. during both pregnancies, her serum concentrations of liver enzymes after conceiving, she was referred by her physician to our outpatient ward at 9 weeks of gestation. during her first pregnancy, her serum concentrations of liver enzymes decreased to the normal range without any medication (fig.3). magnetic resonance imaging performed at 37 weeks of gestation showed that the size of the fnh was unchanged, with no signs of bleeding or degeneration of the lesion (figs.1b and 2b). the concentrations of aspartate aminotransferase (ast), alanine aminotransferase (alt), lactate dehydrogenase (ldh), alkaline phosphatase (alp), and -glutamyltransferase (ggt) are shown in u / l. her first course of pregnancy was uneventful. at 39 weeks, she delivered a normal, healthy female infant, weighing 3084 g and with 1 and 5 min apgar scores of 8 and 9, respectively, by vaginal delivery. her postpartum recovery was normal, except that her serum ggt and alp concentrations became elevated, returning to their prepregnancy levels. this pregnancy was also uneventful (figs.1c, 2c, and 3) and, at 40 weeks of gestation she delivered a normal, healthy male infant, weighing 3130 g and with 1 and 5 min apgar scores of 9 and 10, respectively, by vaginal delivery. her postpartum course was again uneventful, and both mother and child were discharged after 5 days. the size of the fnh remained the same as before her first pregnancy, despite two pregnancies and deliveries. during both pregnancies, her serum concentrations of liver enzymes focal nodular hyperplasia is a common type of hepatic mass in adults, with an incidence of approximately 3%. we encountered a woman who had fnh prior to her first pregnancy and followed her throughout two courses of pregnancy. in contrast, the serum concentrations of liver enzymes were decreased during both pregnancies, and returned to their prepregnancy levels soon after the deliveries. a previous study described the effects of 41 pregnancies on fnh in 37 women, finding that the size of the fnh was not affected by pregnancy. another study evaluating the effects of pregnancy on fnh size in patients found that the mean tumor size before pregnancy was 58.5 22.7 mm. tumor size remained constant in seven of these 20 patients (35%) and declined in 10 (50%). however, fnh size in multiparous women was assessed using a questionnaire, not by actual measurement. furthermore, that study evaluated only lesion size, not changes in serum enzyme concentrations. in our patient, the fnh was > 11 cm in diameter, the largest fnh reported to date, and its size was not affected by either of her two pregnancies. there have been no reports on changes in the serum concentrations of liver enzymes during pregnancy in women with fnh. in contrast, serum alp and ggt values, which were elevated before and after her pregnancies, decreased to normal levels during her pregnancies. alp and ggt are enzymes present in the cell membranes of the bile duct, gall bladder, pancreas, and kidneys. serum concentrations of alp elevate by placental origin and ggt are not affected in pregnancies in women without fnh, but were shown to be higher in few pregnant women (< 1%) with intrahepatic cholestasis. pregnancy may alter the results of liver function tests, with placental steroids playing a role. during pregnancy, the production of placental steroids increases the serum estrogen and progesterone concentrations compared with the nonpregnant state. for example, a 9-year study of 216 women in france showed that neither the size nor the numbers of fnh lesions are influenced by oral contraceptive use. although estrogen receptors were shown to be present in the cytosol and nuclei of hepatic cells including the tissue of fnh, few estrogen receptors are present on the cell surface. estrogen and progesterone affect the mrna expression and activity of cyps (cytochrome p450) in primary human hepatic cells, as shown in a recent study. these findings potentially provide a basis to understand the mechanism of altered drug metabolism during pregnancy. however, further studies are needed to investigate the lowering of serum alp and ggt in fnh. in conclusion, we have described here a single patient with fnh of the liver complicating pregnancy and postpartum delivery twice over a 4-year observation period. fnh size was not affected by pregnancies, whereas liver function test results improved, allowing these enzymes to return to their prepregnancy levels after deliveries. lesion size and abnormally elevated serum liver enzyme concentrations are not indicated for surgery in pregnant women with asymptomatic fnh. our findings indicate that women with large fnh, even those with elevated serum liver enzymes, should not be discouraged from pregnancy. | key clinical messagewe encountered a woman with a preexisting large focal nodular hyperplasia (fnh) of the liver, persisting during two separate pregnancies. fnh size was not affected by either pregnancy. her elevated serum -glutamyltransferase and alkaline phosphatase levels before pregnancy were reduced during both pregnancies, but returned to prepregnancy levels after delivery. |
the overall prevalence of hypertension (htn) in the population has been reported between 6% and 32%. disturbed calcium metabolism may play an important role in the patho - physiology of essential hypertension. ionized calcium (ca) acts as an intracellular second messenger in excitation - contraction coupling in vascular smooth muscle (vsm) cells. the free intracellular calcium concentration determines the tension in vsm cells, thereby contributing to peripheral vascular resistance (pvr). zidek. also found an increased intracellular calcium activity in normotensive subjects with a familial hypertensive disposition in comparison with normotensives without family history of htn. three extracellular calcium fractions : ionized, protein - bound, and complexed calcium are in equilibrium with one another in the serum. ionized calcium (ca) is the physiologically active form whereas protein - bound calcium is apparently inactive. the function of complexed calcium, which is bound to small anions such as bicarbonate, citrate, phosphate, and lactate, is uncertain. treatment of htn is multi - dimensional comprising of dietary modification, weight reduction, regular exercise, and medication. the calcium channel blockers (ccbs) namely nifedipine, amlodipine, diltiazem, verapamil, etc., form one of the mainstay treatments in htn because they considerably reduce the morbidity and mortality in htn and coronary artery disease. ccbs block the voltage - gated calcium channels in vsm cells resulting in vasodilatation and decrease in peripheral resistance. since ccbs act on sarcolemma and reduce the entry of ca from extracellular fluid (ecf) to intracellular fluid (icf), it is assumed that the serum (extracellular) calcium levels may be altered in hypertensive individuals who are on ccbs. this study may give an insight into the role of deranged calcium homeostasis in htn. a total of 31 individuals including 19 hypertensive patients and 12 normotensive subjects in the age group of 3570 years and belonging to both genders attending the out - patient department of general medicine were enrolled into the study after taking written informed consent and approval from institutional ethics committee. hypertensive patients were grouped into two : those who were on ccbs (n = 9) and those who were on antihypertensive medication other than ccbs (n = 10). systolic blood pressure (sbp) more than 140 mmhg and/or diastolic blood pressure (dbp) more than 90 mmhg with or without previous diagnosis of hypertension were included in hypertensive group. subjects with sbp less than 140 mmhg, dbp less than 90 mmhg, and without previous diagnosis of hypertension were considered normotensive. individuals on supplementation with calcium and vitamins including vitamin d were excluded from the study. subjects with history of diabetes mellitus, ischemic heart disease, retinopathy, neuropathy, and nephropathy were also excluded. the blood pressure (bp) was measured in the right arm of the subject in sitting position using sphygmomanometer (diamond company, pune, india) after adequate relaxation. sbp and dbp were recorded, pulse pressure (pp) and mean arterial blood pressure (mabp) calculated. ionized calcium (ca) was measured by accucare calcium arsenazo iii kit (lab - care diagnostics pvt. ltd. at a neutral ph, ca forms a complex with arsenazo iii, the color intensity of which is directly proportional to the concentration of calcium in the sample. statistical analysis was performed using spss version 13.0 (spss inc., il, usa) and origin pro version 8.0 (origin lab, ma, usa). differences between the groups were analyzed using analysis of variance (anova) followed by tukey honest significant difference (hsd) post hoc test. the blood pressure (bp) was measured in the right arm of the subject in sitting position using sphygmomanometer (diamond company, pune, india) after adequate relaxation. sbp and dbp were recorded, pulse pressure (pp) and mean arterial blood pressure (mabp) calculated. ionized calcium (ca) was measured by accucare calcium arsenazo iii kit (lab - care diagnostics pvt. ltd., sarigam, india) according to manufacturer 's instructions. at a neutral ph, ca forms a complex with arsenazo iii, the color intensity of which is directly proportional to the concentration of calcium in the sample., il, usa) and origin pro version 8.0 (origin lab, ma, usa). levene statistic was performed to test the homogeneity of variances. differences between the groups were analyzed using analysis of variance (anova) followed by tukey honest significant difference (hsd) post hoc test. nevertheless, in patients on ccb treatment, serum ca is more compared with patients on antihypertensive medication other than ccb but these differences are statistically not significant [figure 1 ]. spearman 's correlation between htn and serum calcium levels is not significant (p = 0.18). demographic and test parameters of the participants differences in serum calcium levels between normotensives and hypertensives serum ca levels in different groups htn is a multi - factorial disorder in which various physiological mechanisms participate to elevate bp. many hypotheses were proposed about the possible mechanisms underlying essential htn including derangements in serum electrolytes and water balance. the present study shows no significant difference in serum calcium in hypertensive group compared with normotensive group which is consistent with the findings of kosch. however the results are contradictory with that of others who reported a significant decrease in serum calcium in patients with essential htn compared with normotensive subjects. serum calcium levels were significantly decreased in both males and females with essential htn and their first - degree relatives when compared with the normotensive controls. also reported reduced calcium in males with elevated dbp. in the present study the serum calcium levels did not correlate with the levels of bp. serum calcium was found to be significantly correlated with sbp only among those under the age of 40 years. abnormal cellular ion transport resulting in altered membrane control over intracellular calcium may be related to essential htn. changes in magnesium levels may contribute to altered cell membrane calcium binding in essential htn. the free intracellular calcium concentration determines the tension in vsm cells, and thus peripheral vascular resistance. increased serum calcium is observed in hyperparathyroidism, vitamin d intoxication, multiple myeloma and some neoplastic diseases of bone. decreased serum calcium is observed in hypoparathyroidism, vitamin d deficiency, steatorrhea, nephrosis, and nephritis. alternations in intracellular calcium are thought to be involved in the common pathway mediating the secretion and action of many hormones, including the pressor action of catecholamines and angiotensin ii. 25 dihydroxy vitamin d, levels of plasma renin activity, circulating ionized calcium contribute to the pathophysiology of essential htn. the effects of abnormal calcium and calcium regulating hormone levels may extend to the bp control centers of the central nervous system (cns), particularly the nucleus tractus solitarius. hypertensive patients on ccb have serum calcium levels more than those on antihypertensive medications other than ccb, although the difference is statistically not significant. | background : the alterations in extracellular calcium level may influence intracellular calcium level and possibly play a role in the pathogenesis of essential hypertension.aim:the purpose was to find out the association between serum calcium levels and hypertension ; and to compare the serum calcium levels between normotensive controls, hypertensive subjects on calcium channel blockers, and hypertensive subjects on antihypertensive medication other than calcium channel blockers.materials and methods : thirty one individuals including normotensives (n = 12) and hypertensives (n = 19) were enrolled for the study and their blood pressure recorded. hypertensive group was sub divided into two : hypertensives on calcium channel blockers and hypertensives on antihypertensive medication other than calcium channel blockers. serum calcium levels were measured by accucare calcium arsenazo iii kit. differences between the groups were analyzed using anova.results:no significant difference in serum calcium level was found between normotensive and hypertensive groups ; and no correlation was found between calcium levels and the blood pressure. also the difference in serum calcium levels in hypertensive group on calcium channel blockers and those on antihypertensive other than calcium channel blockers was insignificant.conclusions:serum calcium levels are tightly regulated. subtle changes in serum levels do not affect blood pressure. |
in recent years, there has been an increasing interest in applying multivariate pattern (mvp) analysis to neural data, especially from functional magnetic resonance imaging (fmri ; haynes and rees, 2006 ; norman., 2006 ; o'toole., 2007), more than a decade after the first application in studies employing positron emission tomography (pet ; moeller and strother, 1991 ; kippenhahn., 1992) and fmri (e.g., mcintosh., 1996). employing multivariate statistical learning methods allows refocusing research on how information is encoded, instead of exclusively looking at where it could be detected in the brain (o'toole., 2007). (2004) who revealed a combinatorial encoding of object category information in human ventral temporal cortex. shortly after these initial studies, kamitani and tong (2005) showed that the already known fine - grained columnar representation of visual orientation in primary visual cortex is represented in fmri data, despite a coarser spatial resolution. (2008) demonstrated that even more information is contained in the fmri signal, by being able to reconstruct actual visual stimuli from blood oxygenation level - dependent (bold) response patterns in early visual cortex. unlike the aforementioned studies, their model was able to generalize from unstructured basic visual features to geometric shapes and letters. in addition to the most popular mvp analysis approach (i.e., classification), analysis of the full covariance structure of a dataset can also be used to investigate similarity structures of brain response patterns. this transformation of neural responses into the domain of stimulus space (o'toole., 2007) represents a powerful tool for exploratory analysis that can, for example, help to deduce the purpose of a functional processing unit of the brain. their flexibility even allows for comparative analyses across different species. by employing such a technique, kriegeskorte. (2008) showed striking correlations between the similarity structure of object category representations in the ventral processing stream of humans and monkeys. finally, mvp analyses are suitable and have been used to validate computational models of processing streams in the human brain. kay. (2008), for example, constructed a receptive - field model of early visual processing, and trained it on bold - response patterns in early visual cortex, associated with the presentation of over a thousand natural images. they then used this model to identify more than a hundred novel images using a relatively simple nearest - neighbor classification algorithm, hence providing evidence for the plausibility of the model architecture. this recent progress in neuroscience research was made possible by the work on statistical learning methods that has been done mostly in the nips community over the past decades. despite significant differences in terminology and methods, many cognitive neuroscientists are nevertheless familiar with the concept of mvp classification, because of common roots in connectionism in psychological research in the 1980s and early 1990s. from a conceptual point of view, studying classifier performance when predicting category labels of brain response patterns is very similar to the analysis of behavioral data of humans performing a categorization task (e.g., in a typical two- alternative - forced - choice paradigm). procedures, such as those originating in signal detection theory (green and swets, 1966), are well understood and provide familiar measures (e.g., d and receiver operating characteristics curves, roc) to assess the quality of human or algorithmic classifier model performances. more recent research on kernel - based methods in machine learning (ml) shows sometimes striking similarity with categorization models in cognitive psychology, or neuron tuning curves in theoretical neuroscience (jkel., 2009). however, there are certain problems associated with the adoption of statistical learning methods to answer neuroscientific questions. this article will point out some critical aspects that significantly impact this emerging field. the examples of studies employing mvp analyses listed in the previous section offer a glimpse at the explanatory power and flexibility of these techniques. however, as with all other methods, one has to be careful to obey limitations and requirements of a particular method, since inappropriate use (e.g., double - dipping ; kriegeskorte., 2009) or interpretation therefore, a proper assessment of the value of a scientific study requires knowledge about whether employed methods were used appropriately. the use of terms like mind - reading or decoding may be taken as an indication that mvp analyses are automatically deciphering the encoding of information in the brain. however, the generalization accuracy of a model alone (even if it is perfect) does not justify concluding that it identified the true neural signal of an underlying cognitive process. for example, a classifier is simply distinguishing between abstract patterns. without an appropriate experimental design and further investigation, it remains unknown what stimulus property (if any) is reflected in the data. in the context of psychological experiments, confounds always limit the interpretability of experimental results. however, the enormous flexibility of statistical learning techniques theoretically allows them to pick up any signal in a dataset (e.g., differences in stimulation frequency). consequently, studies employing these methods have to be carefully planned to ensure their validity, but (even given an appropriate experimental design) the suitability assessment of methods remains difficult if they are not a part of the standard toolbox of scientists and reviewers in a certain field, since in this case there is no common ground to base an evaluation on. for the conventional univariate statistical parametric mapping (spm) based approach, there is a huge amount of literature that allows one to derive at least a reasonable guess of many parameters that have to be considered in each analysis. based on this literature, any scientist evaluating a particular study should be able to decide whether a reasonable spatial filtering kernel was used during processing, or whether the data was modeled with an appropriate hemodynamic response function. for mvp analysis of fmri, there is only a handful of articles concerned with the evaluation of different methods. this is, of course, not very surprising, since the total number of studies using this approach is negligible in comparison to, at least, 15 years of very productive spm - based fmri data analysis. there is an increasing number of studies that employ mvp analysis aiming to answer actual scientific questions despite the absence of a tested set of good practices. if an effect is found using similar or even different methods, by different research groups using different data acquisition equipment, its existence will generally be accepted. however, in the context of mvp analyses of fmri data, replicating a study is hindered by at least two main factors. first, in contrast to the ml community, datasets are typically not available to the general public. the second factor is that published studies generally only contain a verbal description of the applied analysis. this second factor is much less important for studies employing conventional spm - based fmri data analysis, since the majority is performed by using one of the popular fmri toolkits, like afni (cox, 1996), brainvoyager (goebel., 2006), fsl (smith., 2004), or spm (friston., 1994). the behavior of these toolkits is known, they are available to the whole research community, and the algorithms they implement are published in peer - reviewed publications. all these aspects allow one to summarize an analysis by listing relatively few parameters. for an mvp analysis, an analysis generally involves the combination of many different tools, combined with custom, usually unpublished code. for a researcher intending to replicate a study, translating a verbal potentially incomplete or too superficial description into running analysis code is a lengthy and error - prone task that turns a replication attempt into a costly project. to make a replication effort worthwhile for the majority of mvp analysis - based studies, analysis descriptions should be provided in a form that captures every detail of a procedure and is nevertheless comprehensible by scientists in the field. ideally, a publication should be accompanied by the actual source code that was used to run an analysis, in addition to a verbal description (hanke., 2009b). access to the source code can immediately facilitate validation and replication efforts, enabling the potential for timely feedback with respect to newly developed analysis strategies, while simultaneously fostering the use of successful methods. up to now, only a small fraction of the available algorithms and procedures originating from research on statistical learning has been tested regarding their applicability to neuroimaging data, and cognitive science research questions and paradigms. potential implications of a particular classification algorithm, or feature selection procedure on the interpretation of the results, are yet to be fully explored. a systematic evaluation and formulation of guidelines for mvp analyses of neural data represents a herculean task that requires the joint effort of the neuroscience community. fortunately, an initial set of articles providing an overview of various important aspects of mvp analysis for different target audiences has appeared (e.g., jkel. 2009). however, while there are versatile ml libraries and statistical learning frameworks, such as weka (hall., 2009) and shogun (sonnenburg., 2006), none of them is specifically geared toward the analysis of neuroimaging data. in addition, other neuroimaging - aware mvpa software and pipeline packages described in the literature are closed - source, covered by a restrictive license, or intentionally focused on a specific analysis technique (rex., 2003 ; mcintosh., 2004 ; 2004). to facilitate the exploration, application, and evaluation of mvp analysis in the context of neuroimaging research, we recently introduced a free and open - source, cross - platform analysis framework, called pymvpa (hanke., 2009a, b). it aims to bridge the gap between the rich set of algorithms and procedures originating from research on statistical learning, and the specific properties and requirements of neuroimaging data. the ability of this generic scripting environment to access a huge code base in various languages, combined with its syntactical simplicity, makes it an ideal tool for implementing and sharing ideas among scientists from numerous fields and with heterogeneous methodological backgrounds. the recent python in neuroscience special issue of the journal frontiers in neuroinformatics listed a number of versatile neuroscience projects and software libraries that make use of python, and extend it as a rich high - performance computing environment. the approach of pymvpa is to combine as many of the available building blocks as possible into a consistent framework that allows one to build processing pipelines of arbitrary complexity. whenever feasible, we used existing software implementations in various programming languages, instead of rewriting algorithms in python itself. this strategy strives to focus users on a single implementation of an algorithm and by that, increase the likelihood to detect and fix errors. a key feature of pymvpa is that it abstracts the peculiarities of neuroimaging datasets and exposes them as a generic data array that is compatible with most ml software packages. however, information relevant to the neuroscience context (e.g., spatial metrics of fmri voxels) is retained and accessible within the framework. this allows for transparent conversion of datasets or results from a generic representation (e.g., a numerical vector) into the native data space (e.g., a brain volume). this aspect of pymvpa offers the possibility to write and test neuroscience - aware algorithms that are suitable to address the underlying goal of cognitive neuroscience : to understand how the brain performs the information processing that results in complex behavior. consider, for example, a feature selection algorithm. in ml research, feature selection is usually performed to remove unimportant information from a dataset that does not improve, or even has a negative impact on, the generalization of a particular classifier. in the neuroscience context, however, the primary focus is not on the accuracy level, but on the structure and origin of the information that allows for correct predictions to be made the accuracy simply has to be reasonably high to allow for an interpretation of the model at all. careless removal of features that provide redundant information can have significant side - effects on the conclusions that can be drawn from an analysis. pymvpa is explicitly designed to address the demand for methodologies that acknowledge the specifics of cognitive neuroscience research. the mentioned exposure of spatial data metrics inside the framework, for example, allows to easily develop neuroscience - aware methods that aggregate information from generic feature - based algorithms to derive decisions regarding the selection of whole functional areas. all processing units in the pymvpa framework, such as classifiers or other dataset measures, are designed to have compatible interfaces that allow for modular adjustments to an analysis pipeline. for example, a particular statistical learning algorithm can be replaced for another, without having to adjust any other part of the intended analysis pipeline (e.g., preprocessing, feature selection, or cross - validation procedures). this property makes it very easy to benchmark the performance of various algorithms on a particular dataset which represents a key task when evaluating generic algorithms in the neuroscience domain. additionally, pymvpa provides extensive debugging and logging functionality, aggregation, and intermediate storage of results ; plus convenient ways to interface with generic, as well as domain - specific visualization software. altogether, the availability of these basic housekeeping features allow researchers to focus on aspects important for neuroscience research, instead of low - level software engineering tasks. despite its high - level programming interface (hanke., 2009a), pymvpa nevertheless allows one to modify its behavior in great detail. its modular architecture is easily extensible with novel algorithms and support for additional data formats. like pymvpa itself, virtually all software that it interfaces with is free and open - source. this makes it an ideal environment for in - depth verification, validation, and comparison of existing and newly developed algorithms. pymvpa acknowledges this fact and offers an abstraction layer that can represent data regardless of its original dimensionality or dataspace metrics, while nevertheless exposing these properties inside the framework. using this functionality, we have shown in hanke. (2009b) that pymvpa can be used to implement a unified analysis pipeline for a whole spectrum of neural data modalities. despite modality - specific data properties, pymvpa allowed for easy and straightforward initial preprocessing (e.g., input, detrending, normalization), and analysis with statistical learning methods (figure 1). to address the original research questions, the results could be conveniently visualized and interpreted in the original domain - specific data space. using this uniform approach, we were not only able to replicate previous findings, but also to obtain additional, sometimes thought - provoking results. to encourage other researchers to verify and extend the suggested methodology, the complete source code and a sample fmri dataset modality - independent data analysis with pymvpa. on the left side : typical preprocessing steps for data from electroencephalography (eeg), functional magnetic resonance imaging (fmri), and extra - cellular recordings (ecr) are shown. after initial modality - specific preprocessing, pymvpa transforms data into a simple array representation that is compatible with generic machine learning software implementations. at the final stage of an analysis, pymvpa allows for easy back - projection of the results into the original modality - specific data space. data modality - independent analysis opens the door to combine the power of the plethora of available invasive and non - invasive data recording techniques. while they are all measuring reflections of the same underlying neural signals, each of them is offering a unique set of properties in terms of spatio - temporal resolution, signal to noise, data acquisition cost, applicability to humans, and the corresponding neural correlates that result from the measurement process. neuroscientists often focus on only one or a smaller subset of these neural modalities, partly due to the kinds of questions investigated, and partly due to the cost of learning to analyze data from these different modalities. the availability of a generic framework, such as pymvpa, that provides a uniform processing pipeline, can encourage the exchange of available methodologies among research communities specialized in the analysis of a particular data modality. to facilitate the exploration, application, and evaluation of mvp analysis in the context of neuroimaging research, we recently introduced a free and open - source, cross - platform analysis framework, called pymvpa (hanke., 2009a, b). it aims to bridge the gap between the rich set of algorithms and procedures originating from research on statistical learning, and the specific properties and requirements of neuroimaging data. the ability of this generic scripting environment to access a huge code base in various languages, combined with its syntactical simplicity, makes it an ideal tool for implementing and sharing ideas among scientists from numerous fields and with heterogeneous methodological backgrounds. the recent python in neuroscience special issue of the journal frontiers in neuroinformatics listed a number of versatile neuroscience projects and software libraries that make use of python, and extend it as a rich high - performance computing environment. the approach of pymvpa is to combine as many of the available building blocks as possible into a consistent framework that allows one to build processing pipelines of arbitrary complexity. whenever feasible, we used existing software implementations in various programming languages, instead of rewriting algorithms in python itself. this strategy strives to focus users on a single implementation of an algorithm and by that, increase the likelihood to detect and fix errors. a key feature of pymvpa is that it abstracts the peculiarities of neuroimaging datasets and exposes them as a generic data array that is compatible with most ml software packages. however, information relevant to the neuroscience context (e.g., spatial metrics of fmri voxels) is retained and accessible within the framework. this allows for transparent conversion of datasets or results from a generic representation (e.g., a numerical vector) into the native data space (e.g., a brain volume). this aspect of pymvpa offers the possibility to write and test neuroscience - aware algorithms that are suitable to address the underlying goal of cognitive neuroscience : to understand how the brain performs the information processing that results in complex behavior. consider, for example, a feature selection algorithm. in ml research, feature selection is usually performed to remove unimportant information from a dataset that does not improve, or even has a negative impact on, the generalization of a particular classifier. in the neuroscience context, however, the primary focus is not on the accuracy level, but on the structure and origin of the information that allows for correct predictions to be made the accuracy simply has to be reasonably high to allow for an interpretation of the model at all. careless removal of features that provide redundant information can have significant side - effects on the conclusions that can be drawn from an analysis. pymvpa is explicitly designed to address the demand for methodologies that acknowledge the specifics of cognitive neuroscience research. the mentioned exposure of spatial data metrics inside the framework, for example, allows to easily develop neuroscience - aware methods that aggregate information from generic feature - based algorithms to derive decisions regarding the selection of whole functional areas. all processing units in the pymvpa framework, such as classifiers or other dataset measures, are designed to have compatible interfaces that allow for modular adjustments to an analysis pipeline. for example, a particular statistical learning algorithm can be replaced for another, without having to adjust any other part of the intended analysis pipeline (e.g., preprocessing, feature selection, or cross - validation procedures). this property makes it very easy to benchmark the performance of various algorithms on a particular dataset which represents a key task when evaluating generic algorithms in the neuroscience domain. additionally, pymvpa provides extensive debugging and logging functionality, aggregation, and intermediate storage of results ; plus convenient ways to interface with generic, as well as domain - specific visualization software. altogether, the availability of these basic housekeeping features allow researchers to focus on aspects important for neuroscience research, instead of low - level software engineering tasks. despite its high - level programming interface (hanke., 2009a), pymvpa nevertheless allows one to modify its behavior in great detail. its modular architecture is easily extensible with novel algorithms and support for additional data formats. like pymvpa itself, virtually all software that it interfaces with is free and open - source. this makes it an ideal environment for in - depth verification, validation, and comparison of existing and newly developed algorithms. pymvpa acknowledges this fact and offers an abstraction layer that can represent data regardless of its original dimensionality or dataspace metrics, while nevertheless exposing these properties inside the framework. using this functionality, we have shown in hanke. (2009b) that pymvpa can be used to implement a unified analysis pipeline for a whole spectrum of neural data modalities. despite modality - specific data properties, pymvpa allowed for easy and straightforward initial preprocessing (e.g., input, detrending, normalization), and analysis with statistical learning methods (figure 1). to address the original research questions, the results could be conveniently visualized and interpreted in the original domain - specific data space. using this uniform approach, we were not only able to replicate previous findings, but also to obtain additional, sometimes thought - provoking results. to encourage other researchers to verify and extend the suggested methodology, the complete source code and a sample fmri dataset modality - independent data analysis with pymvpa. on the left side : typical preprocessing steps for data from electroencephalography (eeg), functional magnetic resonance imaging (fmri), and extra - cellular recordings (ecr) are shown. after initial modality - specific preprocessing, pymvpa transforms data into a simple array representation that is compatible with generic machine learning software implementations. at the final stage of an analysis, pymvpa allows for easy back - projection of the results into the original modality - specific data space. data modality - independent analysis opens the door to combine the power of the plethora of available invasive and non - invasive data recording techniques. while they are all measuring reflections of the same underlying neural signals, each of them is offering a unique set of properties in terms of spatio - temporal resolution, signal to noise, data acquisition cost, applicability to humans, and the corresponding neural correlates that result from the measurement process. neuroscientists often focus on only one or a smaller subset of these neural modalities, partly due to the kinds of questions investigated, and partly due to the cost of learning to analyze data from these different modalities. the availability of a generic framework, such as pymvpa, that provides a uniform processing pipeline, can encourage the exchange of available methodologies among research communities specialized in the analysis of a particular data modality. the emerging field of mvp analysis of neural data is beginning to complement the established analysis techniques, and has great potential for novel insights into the functional architecture of the brain. even if an mvp analysis is not a mind - reader (logothetis, 2008), it undoubtedly represents a major step forward in the analysis of the brain function. its flexibility acknowledges the complexity of neural signals, and hence can help to advance the understanding of brain function. however, there are many open questions on how the wealth of statistical learning algorithms can be applied optimally in this domain. although evaluating use cases and identifying potential pitfalls in the neuroscience context is an ambitious and demanding task, it urgently needs to be done to ensure valid analysis and unbiased conclusions. pymvpa is a generic framework that is explicitly tailored toward mvp analyses of neural data. its versatility on data from various modalities has already been shown by us and others (hanke., 2009b ; sun., 2009). the offered transparency in expressing complex processing pipelines hopefully will facilitate the systematic evaluation of statistical learning methods in the neuroscience context, and will serve as a solid foundation for collaborative and derivative research efforts. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. | encouraged by a rise of reciprocal interest between the machine learning and neuroscience communities, several recent studies have demonstrated the explanatory power of statistical learning techniques for the analysis of neural data. in order to facilitate a wider adoption of these methods, neuroscientific research needs to ensure a maximum of transparency to allow for comprehensive evaluation of the employed procedures. we argue that such transparency requires neuroscience - aware technology for the performance of multivariate pattern analyses of neural data that can be documented in a comprehensive, yet comprehensible way. recently, we introduced pymvpa, a specialized python framework for machine learning based data analysis that addresses this demand. here, we review its features and applicability to various neural data modalities. |
rett syndrome (rtt) is a rare form of autism spectrum disorder (asd), which mostly affects girls with worldwide prevalence rate ranges from 1 : 10,000 to 1 : 20,000 live births [15 ]. rtt is a clinically defined condition with a large spectrum of phenotypes associated with a wide genotypic variability [6, 7 ]. classic or typical rtt, the most common form of the condition, is caused in about 9095% of cases by de novo mutations in the mecp2, a gene mapped on chromosome x and encoding methyl - cpg binding protein 2 [7, 8 ]. the clinical picture of classical form progresses through 4 stages and is characterized by normal development for the first 6 to 18 months, followed by loss of purposeful hand movements, failure of speech development, autistic - like behavior, slowed brain and head growth, and mental retardation. to date, it is not known how mecp2 mutations lead to rtt phenotypes ; therefore the identification of the pathways that are affected by mecp2 functions could bring new insight in the rtt pathogenetic mechanisms. mecp2 was originally thought to function as a transcription repressor by binding to methylated cpg dinucleotides, but recent studies have individuated more functions related to mecp2 [10, 11 ]. in fact mecp2 is now considered a multifunctional protein, since it is implicated not only in genome transcriptional silencing, but also in transcriptional activation, by regulating chromatin and nuclear architecture ; therefore, its malfunction or mutation can lead to severe cellular function alterations. hence, it is very difficult to understand the link between mecp2 mutation and the clinical feature present in rtt. one of the most common approaches used to better understand the molecular pathways involved in genetic disorders has been the determination of gene expression profiling, since it provides the opportunity to evaluate possible transcriptome alterations at both gene and gene - network levels. this approach should not be considered an end point but a magnifying lent where new aspects involved in the diseases can be discovered and then studied. so far, only a handful of studies have investigated the gene expression profiles of rtt children in tissues, that is, postmortem brain samples, or in cells, such as clones of fibroblasts isolated from skin biopsies [13, 14 ] and immortalized lymphoblastoid cell lines [1416 ]. moreover, several studies have performed microarray gene expression analysis using in vitro cellular models representing mecp2 deficiency induced by sirnas or cells and tissues from rtt mouse models, but to our knowledge there are no data on microarray analysis from the aim of this study was to evaluate the gene expression patterns in pbmc isolated from rtt patients. this approach lets us bypass some of the limitations / variables of the previous gene arrays studies on rtt, such as the use of postmortem samples, gene - modified cells and murine tissues that do not always reflect all features of the human disease. in fact, studying ex vivo samples, such as pbmc, provides some advantage that can be summarized by the fact pbmc are the only readily available cells in humans ; various studies showed disease - characteristic gene expression patterns in pbmc that can be easily obtained. our results identified a clear difference in gene expression profile between control and rtt patients, with almost 500 genes being deregulated, suggesting several new pathways involved in this disorder. the study included 12 female patients with clinical diagnosis of typical rtt (mean age : 10.9 4.9 years, range : 622) with demonstrated mecp2 gene mutation and 7 sex- and age - matched healthy controls (mean age : 15.1 9.03 years, range : 432). rtt diagnosis and inclusion / exclusion criteria were based on the recently revised rtt nomenclature consensus. all the patients were consecutively admitted to the rett syndrome national reference centre of the university hospital of the azienda ospedaliera universitaria senese (aous). table 1 presents the demographic and genetic characteristics of the enrolled patients subjected to microarray analysis. blood sampling in the control group was carried out during routine health checks, sports, or blood donations, while blood sample in patients were obtained during the periodic clinical checks. the study was conducted with the approval of the institutional review board and all informed consents were obtained from either the parents or the legal tutors of the enrolled patients. blood was collected in heparinized tubes and all manipulations were carried out within 30 minutes after sample collection. pbmc were separated from whole blood by density gradient centrifugation using ficoll - paque plus (ge healthcare europe gmbh, milan, italy). after pbmc isolation, total rna was extracted from cells using rneasy mini kit (qiagen, hilden, germany), according to the manufacturer 's instructions. the total nucleic acid concentration and purity were estimated using a nanodrop spectrophotometer (nanodrop technologies, wilmington, de). quality of rna was checked on agilent bioanalyzer (agilent technologies, santa clara, ca). for the microarray processing, rna was amplified and labeled using the affymetrix whole - transcript (wt) sense target labeling protocol. affymetrix genechip human gene 1.0 st arrays were hybridized with labeled sense dna, washed, stained, and scanned according to the protocol described in wt sense target labeling assay manual. briefly, 100 ng of total rna was reverse transcribed into double - stranded cdna with random hexamers tagged with a t7 promoter sequence. the double - stranded cdna was subsequently used as a template and amplified by t7 rna polymerase, producing many copies of antisense crna. in the second cycle of cdna synthesis, random hexamers were used to prime reverse transcription of the crna from the first cycle to produce single - stranded dna in the sense orientation. dutp was incorporated in the dna during the second - cycle, first - strand reverse transcription reaction. this single - stranded dna sample was then treated with a combination of uracil dna glycosylase (udg) and apurinic / apyrimidinic endonuclease 1 (ape 1) that specifically recognized the unnatural dutp residues and broke the dna strand. dna was labeled by terminal deoxynucleotidyl transferase (tdt) with the affymetrix proprietary dna labeling reagent that is covalently linked to biotin. 5 g of labeled cdna was hybridized to the human gene 1.0 st array at 45c for 17 hours. the arrays were washed and stained in affymetrix fluidics station 450 and scanned using the affymetrix genechip scanner 3000. the signal intensities from each chip were preliminarily normalized with rma method and filtered by iqr filter choosing as threshold a value of 0.25. this specific filter removes the probesets that do not present changes in their expression across the analyzed microarray. the threshold of 0.25 is an intermediate value that retains the probesets that show significative changes of their signal at least in the 25% of analyzed microarray. sam approach uses permutation based statistics and is a valid method to analyze data that may not follow a normal distribution, outperforming other techniques (e.g., anova and classical t - test), which assume equal variance and/or independence of genes. limma fit each gene expression to a linear regression model testing the significativity of the distance from the model with a t - test robust against nonnormality and inequality of variances. in order to characterize the biological processes enriched in the list of modulated genes, a statistical analysis of overrepresented gene ontology (go) terms (bp level 5) was performed with david (database for annotation, visualization, and integrated discovery) web server (david bioinformatics resources). only the go terms enriched with a p value < 0.05 corrected with the benjamini and hochberg method were selected and hierarchically clusterized (ward 's method) according to their semantic after clusterization the best number of clusters was identified according to the analysis of the silhouette scores and the medoid term in each cluster was selected as the representative member of the cluster. each cluster represents a group of biological processes with similar e / o related functions. for confirmation of affymetrix expression microarray results, rt - qpcr analysis was performed as previously described. for validation, six of the differentially expressed genes, validation was done in a randomly selected subset of the original samples (submitted for microarray analysis) that included 3 healthy controls and 3 rtt patients. primer pairs were obtained from the real - time pcr genbank primer to hybridise unique regions of the appropriate gene sequence : gsto1 (fw : 5-aga gtt gtt ttc taa ggt tct gac t-3) and (rw : 5-act tca ttg ctt cca gcc gt-3), product length 116 bp ; psmb10 (fw : 5-aca gac gtg aag cct agc ag-3) and (rw : 5-acc gaa tcg tta gtg gct cg-3), product length 294 bp ; cox8a (fw : 5-gcc aag atc cat tcg ttg cc-3) and (rw : 5-tct ggc ctc ctg tag gtc tc-3), product length 137 bp ; hist1h1b (fw : 5-ccc ggc taa gaa gaa ggc aa-3) and (rw : 5-aca gcc ttg gtg atc agc tc-3), product length 99 bp ; mmp9 (fw : 5-gtc cgt gag ggt gtt gag tg-3) and (rw : 5-act gct caa agc ctc cac aa-3), product length 145 bp ; arhgap11b (fw : 5-aac tgc cag agc cca ttc tc-3) and (rw : 5-gtc tgg tac acg ccc ttc tt-3), product length 295 bp. gapdh (fw : 5-tga cgc tgg ggc tgg cat tg-3 and rw : 5-ggc tgg tgg tcc agg ggt ct-3, 134 pb) was used in our experiments as internal standard. as previously described, samples were compared using the relative cycle threshold (ct) method (livak and schmittgen 2001). after normalization to more stable mrna gapdh, the fold increase or decrease was determined with respect to control, using the formula 2, where ct is (gene of interest ct)(reference gene ct) and ct is (ct experimental)(ct control). results are the means sem of three independent experiments, each analysed in triplicate. p < 0.001 versus control (one - way anova followed by bonferroni 's posttest). given that rtt results from dysfunction of the transcriptional modulator mecp2, several strategies have been developed to identify its target genes in order to gain insights into the disease pathogenesis [1218 ]. in our study, to identify gene expression changes associated with and potentially related to mecp2 mutations and to delineate alterations of pathways associated with the disease, we evaluated and compared transcriptomic profiles in pbmc from rtt patients and control subjects. this work showed for the first time, to our knowledge, altered expression of a large set of genes that may help elucidating and explaining the link between mecp2 and some of the molecular and cellular aspects observed in rtt patients. in our previous studies on rtt we were able to show increased levels of oxidative stress (os) markers, such as isoprostanes (isops) and 4-hydroxynonenal protein adducts (4-hne pas) [22, 23 ], and increased ubiquitination and degradation of oxidatively modified proteins, but how mecp2 mutation is able to affect cellular redox balance and proteins turn over still needs to be defined. using the sam and limma methods, we have defined a set of genes differentially expressed in rtt patients with respect to controls (healthy subjects) and a significant overlap was found by comparing results from two approaches. a cut - off level based on a minimum of 1-fold change in expression resulted in a list of 482 common deregulated genes, while 10 genes were suggested only by sam and only 1 gene was indicated by limma. among the shared genes, 430 showed significant upregulation, while 52 were downregulated in rtt compared to controls (figure 1 and supplemental tables 1 and 2). genes with the strongest changes in expression, both upregulated (fc 2) and downregulated (fc 1.2), are listed in tables 2 and 3, respectively. the complete list of differentially expressed genes, both upregulated and downregulated (fc 1), is shown in supplemental tables 1 and 2. it is evident from this first set of data that mutations in mecp2 influence more the genes upregulation with respect to the downregulation. this is in part in line with the first functions that have been attributed to mecp2, such as a gene expression repressor [10, 11 ]. in addition, it is worth it to underline that both screening approaches used in this study (limma and sam) almost overlap between them making the results more reliable. next, using the david databases, we performed the functional annotation of the significant genes and identification of the biochemical pathways in which they are involved. a comparison of the differentially regulated mrna transcripts in rtt pbmc compared to control group shows significant changes in cellular pathways. in particular, we identify 10 major clusters corresponding to 62 biological processes enriched by 146 genes (table 4). these clusters highlight key biological pathways related to mitochondrial function and organization (i.e., mitochondrial atp synthesis coupled to electron transport, inner mitochondrial membrane organization such as : atp5a1, cox6c, etfa, uqcrq, timm10, and tspo), cellular protein metabolic process (i.e., regulation of protein ubiquitination, regulation of ubiquitin - protein ligase activity, and proteasomal ubiquitin - dependent protein catabolic process such as psma2, psmd6, ube2e3, and ufc), rna processing (i.e., nuclear mrna splicing and spliceosome assembly and rna elongation from rna polymerase ii promoter such as rpl15), dna organization in chromatin, and cellular macromolecular complex assembly (i.e., nucleosome assembly, dna packaging, and protein complex biogenesis such as hist1h4l, h2afz, top2a, and hmgb2). among these clusters, the most significantly regulated transcripts include those encoding several subunits of mitochondrial respiratory chain complexes and thus linked directly to mitochondrial atp production and, indirectly, to potential reactive oxygen species (ros) generation. in particular, ndufa1, ndufab1, ndufa2, and ndufb6, all components of mitochondrial complex i (nadh : ubiquinone oxidoreductase), showed the greater changes with a fc of more than 2. moreover, other subunits of complex i (ndufv2, ndufs4, ndufa9, ndufs6, ndufb10, ndufb4, ndufc2, ndufb2, ndufs5, ndufc1, ndufb9, and ndufa8) were clearly upregulated in rtt group. complex i plays a vital role in cellular atp production, the primary source of energy for many crucial processes in living cells. it removes electrons from nadh and passes them by a series of different protein coupled redox centers to the electron acceptor ubiquinone. because complex i is central to energy production in the cell, it is reported that its malfunction results in a wide range of neuromuscular diseases. some of them are due to mutations in the mitochondrial genome, but others, which result from a decrease in the activity of complex i or an increase in the production of ros, are not well understood. the production of ros by complex i is linked to parkinson 's disease and to ageing [26, 27 ] and this is in line with rtt since it is now well documented as an increased os condition in this pathology [22, 23 ]. another gene involved in complex i function is ndufv2 that was also clearly upregulated in our study. mutations in this gene are implicated in parkinson 's disease, bipolar disorder, and schizophrenia and have been found in one case of early onset hypertrophic cardiomyopathy and encephalopathy ; also it has been shown for ndufa2, a subunit of the hydrophobic protein fraction of the complex i. mutations in this gene are associated with leigh syndrome, an early onset progressive neurodegenerative disorder. of note is ndufab1, which is a carrier of the growing fatty acid chain in fatty acid biosynthesis in mitochondria and alteration in fatty acid levels has been noted in asd [28, 29 ]. not only complex i subunits were upregulated in rtt, but also we have detected an upregulation of genes involved in all the five complexes of the electron transport chain. in fact, also sdhb (succinate dehydrogenase complex, subunit b, and iron sulfur (ip)) gene encoding for a subunit of mitochondrial complex ii (succinate : ubiquinone oxidoreductase) was significantly upregulated. this subunit is responsible for transferring electrons from succinate to ubiquinone (coenzyme q). complex ii of the respiratory chain, which is specifically involved in the oxidation of succinate, carries electrons from fadh to coq. of note, also 3 genes, uqcrq, uqcrfs1, and uqcrh, encoding for subunits of complex iii (ubiquinol - cytochrome c oxidoreductase), were upregulated with a mean fc = 1.60. this complex plays a critical role in biochemical generation of atp, contributing to the generation of electrochemical potential by catalyzing the electron transfer reaction from ubiquinol to cytochrome c coupled with proton translocation across the membrane. lines of evidence report that in mouse models some of the promoters of ubiquinol - cytochrome c reductase subunit are able to be targeted by mecp2 protein, contributing to the development of the pathology. furthermore, the cytochrome c gene (cycs) together with genes encoding subunits of mitochondrial complex iv (cytochrome c oxidase) (cox14, cox7a2, cox6c, cox7c, and cox8a) was upregulated with a mean fc of circa 1.5. of note the encoded protein accepts electrons from cytochrome b and transfers them to the cytochrome oxidase complex. this protein is also involved in initiation of apoptosis and this would be in line with previous studies that have shown a possible involvement of apoptosis in rtt [31, 32 ], although only few studies have investigated the role of apoptosis in rtt and the current literature is still controversial. it receives an electron from each of the four cytochrome c molecules and transfers them to one oxygen molecule, converting molecular oxygen to two molecules of water. in the process, via a protons translocation, it is able to generate atp. this data would suggest that rtt patients are in continuous new atp synthesis and this could be a consequence of new protein synthesis. our data are in line with a previous work by kriaucionis where the authors have analyzed the gene profile in the brain of rtt animal model. they have shown that there were several mitochondrial abnormalities and an upregulation of both complexes i and iii subunits. in particular they were also able to show a correlation between upregulation of complexes i and iii and the animal symptoms severity with a significant increase in mitochondrial respiratory capacity and a reduction in respiratory efficiency. the defect appears to be associated with respiratory complex iii, which is also upregulated in our study, and that containing the uqcrc1 protein. in addition it has been shown that mecp2 binds to the promoter of the uqcr gene in vivo and that uqcr mrna expression is elevated in brains of mecp2-null mice that have acquired neurological symptoms and this is in line with our results. finally, we also observed in rtt pbmc an upregulation of mitochondrial complex v (atp synthase) subunits (atp5a1, atp5ep2, atp5j2, and atp5o) together with atpase inhibitory factor 1 gene (atpif1). mitochondrial membrane atp synthase is a master regulator of energy metabolism and cell fate ; therefore, a misregulation of this gene can be associated with altered atp production and cell metabolism. it is interesting to note that also the atpase inhibitory factor 1 (atpif1) that inhibits the activity of the mitochondrial h - atp synthase was upregulated, and this lets us speculate the existence of an aberrant loop between making new atp and inhibiting its production. in addition, recent findings indicate that atpif1 has additional functions by promoting adaptive responses of cell to ros, a condition (os) that has been well documented to be present in rtt [22, 23 ]. similarly, other genes related to the atp synthesis showed significant expression changes (i.e., cyb5a, cyb561d2, etfa, ldhb, pdhb, and surf1). for instance, etfa (electron transfer flavoprotein, alpha polypeptide) serves as a specific electron acceptor for several dehydrogenases and in mitochondria it shuttles electrons between primary flavoprotein dehydrogenases and the membrane - bound electron transfer flavoprotein ubiquinone oxidoreductase. in addition, ldhb encodes lactate dehydrogenase b, an enzyme that catalyzes the reversible conversion of lactate and pyruvate and nad and nadh, in the glycolytic pathway, being therefore correlated with atp generation. of note is the upregulation of surf1 (surfeit 1) gene that encodes a protein localized to the inner mitochondrial membrane and thought to be involved in the biogenesis of the cytochrome c oxidase complex. related to mitochondrial structure / organization, we found upregulation of 7 translocase genes (timm10, tspo, timm9, timm17a, tomm7, timm13, and timm8b) involved in proteins import into mitochondrion (mean fc of 1.34) and of several mitochondrial ribosomal proteins (mrpl13, mrpl20, mrpl21, mrpl33, mrpl51, mrpl52, mrps25, mrps30, mrps33, mrps36, rpl10a, rpl13, rpl15, rpl22, rpl22l1, rpl26, rpl31, rpl32, rpl39l, rps10, rps25, rps26, rps26p11, rps29, rps5, and rps7) with a mean fc of 1.5. all together these lines of evidence seem to suggest an increased mitochondrial activity that might be linked to the observation of the pathologic phenotype. anyways at this stage of the study, it is not possible to determine whether or not there is an increase in atp levels. it is possible to speculate that increased genes related to mitochondrial subunits could be a consequence of increased cells request of energy (atp). this hypothesis is in line with recent study where the authors have shown decreased levels of atp in brain mouse rtt. recent discussions regarding a possible connection between rtt and mitochondrial dysfunction have generated significant interest. the basis for these discussions is related in part to the common features of rtt on the one hand and disorders of mitochondrial function on the other. of interest is the fact that a patient with symptoms normally associated with mitochondrial disorders harbored mutations in the mecp2 gene. this overlap between symptoms of rtt and mitochondrial disorders recalls early reports of structural abnormalities [36, 37 ] and defects in the electron transport chain [37, 38 ] in mitochondria from skin and muscle biopsies of rtt patients. moreover, about half of rtt patients were reported to have elevated levels of circulatory lactic or pyruvic acid, which might be caused by defects in the efficiency of the respiratory chain and urea cycle complexes, both of which are mitochondrial [3941 ]. several disorders related to the brain are a consequence of mitochondrial alteration with the resultant increase of os and in certain cases the cell apoptosis. as rtt is not a neurodegenerative disorder, any contribution of mitochondrial dysfunction to rtt symptoms may take the form of chronic mitochondrial underperformance, rather than catastrophic failure leading to neuronal death. to date, no systematic study of mitochondrial function in individuals with rtt has presented whether these findings represent a primary or secondary effect ; that is, are they involved directly in the clinical features of rtt or do they reflect effects of these clinical features on mitochondrial function ? prior to identification of mutations in mecp2 in 1999, several reports appeared to be related to mitochondrial structure and function [3654 ]. however, since 2001, publications on a possible role of mitochondria in the pathogenesis of rtt have been very few [36, 54 ]. in a recent work, investigators in australia reported gene expression results from postmortem brain tissue of individuals with rtt and normal controls. one gene related to a mitochondrial enzyme, cytochrome c oxidase subunit 1, had reduced expression in rtt tissue raising the possibility that loss of mecp2 function could be responsible. however, whether this is a primary or secondary finding remains to be established and provides an important target for further investigation. in summary, while mitochondrial abnormalities related to structure and functions have been reported, sufficient information is lacking as to the precise role of such abnormalities in rtt. as mentioned, alteration of mitochondrial functions is often correlated with os and, in particular, the mitochondrial sites that are often invoked as the most important mitochondrial superoxide producers are in respiratory complexes i and iii [56, 57 ] and this can explain the increased os levels found in rtt patients. the presence of a redox unbalance in rtt is confirmed by the upregulations of several genes involved in redox homeostasis such as superoxide dismutase 1, catalase, and peroxiredoxin 1 (sod1, cat, and prdx1) with a 1.6, 1.14, and 1.12 fc, respectively. moreover, glutathione s - transferase omega 1, microsomal glutathione s - transferase 2, and microsomal glutathione s - transferase 3 (gsto1, mgst2, and mgst3) are also overexpressed in rtt with a fc = 2.1. for instance, sod1 upregulation could be a consequence of increased superoxide production by aberrant activation of complex i and iii and the dismutation of superoxide in h2o2 can explain the increased expression of cat and prdx1. it is likely to believe that the compensatory antioxidant system is not quite sufficient to quench ros production and this could explain the high os level present in rtt [22, 23 ]. for this reason the induction of glutathione s - transferase omega-1 (gsto1) is not surprising. this enzyme is involved in the detoxification mechanisms via conjugation of reduced glutathione (gsh) to oxidativelly modified proteins (carbonyls and 4-hne pas). in addition, our results showed the upregulation of mgst2 and mgst3 which are the microsomal glutathione s - transferases ; this data correlates very well with our previous findings where rtt patients showed an increased level of 4-hne pas, as mgst2 is able to also conjugate 4-hne with gsh. we also observed an increased expression of mrna for alcohol dehydrogenase 5 (adh5), aldo - keto reductase family 1, member a1 (akr1a1) and aldehyde dehydrogenase 1 family, member a1 (aldh1a1), all enzymes involved in lipid peroxidation products detoxification. in fact, rtt pbmc microarray data revealed increased expression levels of genes associated with protein turnover, such as genes encoding proteasome subunits (psma2, psma3, psma5, psma7, psmb1, psmb10, psmc5, psmc6, psmd6, and psmd9) ; furthermore, proteasome maturation protein (pomp), involved in proteasome assembly, and genes regulating the activity of the ubiquity ligases (rbx1, ufc1, ccnb1ip1, and daxx) are also up regulated (table 2), suggesting an increase in cell and protein degradation processes. this could also be a consequence of oxidized proteins and the presence of 4-hne pas. this evidence is supported by the observed upregulation of the ubiquitin - conjugating enzyme e2e3 (ube2e3, fc = 1.19) that accepts ubiquitin from the e1 complex and catalyzes its covalent attachment to other proteins. however, this is in contrast with some lines of evidence that link rtt to the downregulation of ubiquitin conjugating enzymes (ube3a) by mecp2. overall the effect of mecp2 on ube3a regulation is still controversial. in fact, there is even a recent work that did not find any difference in ube3a expression between wild type and a rtt mouse with the mutation r168x. in general, the levels of protein ubiquitination, that is one of the steps to degrade modified proteins, are increased in rtt. in addition several histone related genes (hist1h1b, hist1h2ab, hist1h2ai, hist1h2aj, hist1h2al, hist1h2bb, hist1h2bh, hist1h2bm, hist1h3b, hist1h3f, hist1h3i, hist1h3j, hist1h4d, hist1h4f, and hist1h4) were downregulated with a mean fc of 1.28 suggesting a reduced production of proteins necessary to the dna chromatin assembly. in general, histone modifications are very dynamic and include acetylation, methylation, isomerization, phosphorylation, and ubiquitination. it is easy to understand that modifications such as histone methylation can display additional complexity since the degree of methylation is very variable (mono-, di-, or trimethylation). furthermore, combinations or sequential additions of different histone marks can affect the chromatin organization and subsequently alter the expression of the corresponding target genes. in our case, several genes related to histone expression were downregulated and this can dramatically affect gene expression. for instance, histone h1 protein binds to linker dna between nucleosomes forming the macromolecular structure known as the chromatin fiber. histones h1 are necessary for the condensation of nucleosome chains into higher - order structured fibers. acts also as a regulator of individual gene transcription through chromatin remodeling, nucleosome spacing, and dna methylation. we have detected a down - regulation of several h1 subunits ranging from 2- to 3-fold in rtt patients. next, we wanted to confirm the differential expression observed for selected mrnas, on an individual basis, by rt - qpcr. six genes were selected based on their different patterns of expression (3 up- and 3 downregulated). assessment of their mrna expression levels by rt - qpcr accurately reflected those obtained by microarray profiling (figure 2), thereby confirming the validity of our microarray results. the levels of three mrnas encoding for glutathione s - transferase omega 1 (gsto1), proteasome (prosome, macropain) subunit, beta type-10 (psmb10), and cytochrome c oxidase subunit viiia (cox8a) were upregulated in rtt patients by 2.5-, 2- and 2.2-fold, respectively (figure 2), very similarly to the levels measured by gene array. in contrast, histone cluster 1, h1b (hist1h1b), matrix metallopeptidase 9 (mmp9), and rho gtpase activating protein 11b (arhgap11b) were downregulated by 50% in rtt patients, as compared to healthy subjects (figure 2) and also in this case similar expression was detected by gene array analysis. our microarray data reveal an altered gene expression profile in rtt lymphomonocytes with the upregulation of genes related to mitochondrial biology and ubiquitin - proteasome proteolytic pathway. in particular, the overexpression of the genes involved in atp synthesis processes means the tendency of cells to show an altered energy requirement, perhaps to cope with the increased activities of protein degradation. on the other hand, it should be noted that mitochondrion plays essential roles in mediating the production of ros and these in turn cause damage to proteins, as well as lipids and nucleic acids. to remove damaged molecules, in a kind of vicious circle, increased cellular proteolytic activity requires an extra mitochondrial atp production with a further ros generation. this picture is consistent with our previous reports, indicating the alteration of redox status in rtt patients, coupled with the increased ubiquitination and degradation of oxidatively modified proteins (scheme 1). in conclusion, these findings on transcriptional profiling in rtt patients reveal new molecular mechanisms underlying rtt phenotype, suggesting that mitochondrial - atp - proteosome are likely to have direct actions on redox balance in rtt syndrome. furthermore, it confirmed a possible indirect role of os in pathogenesis and progression of disorder. | rett syndrome (rtt) is mainly caused by mutations in the x - linked methyl - cpg binding protein (mecp2) gene. by binding to methylated promoters on cpg islands, mecp2 protein is able to modulate several genes and important cellular pathways. therefore, mutations in mecp2 can seriously affect the cellular phenotype. today, the pathways that mecp2 mutations are able to affect in rtt are not clear yet. the aim of our study was to investigate the gene expression profiles in peripheral blood lymphomonocytes (pbmc) isolated from rtt patients to try to evidence new genes and new pathways that are involved in rtt pathophysiology. limma (linear models for microarray) and sam (significance analysis of microarrays) analyses on microarray data from 12 rtt patients and 7 control subjects identified 482 genes modulated in rtt, of which 430 were upregulated and 52 were downregulated. functional clustering of a total of 146 genes in rtt identified key biological pathways related to mitochondrial function and organization, cellular ubiquitination and proteosome degradation, rna processing, and chromatin folding. our microarray data reveal an overexpression of genes involved in atp synthesis suggesting altered energy requirement that parallels with increased activities of protein degradation. in conclusion, these findings suggest that mitochondrial - atp - proteasome functions are likely to be involved in rtt clinical features. |
fanconi syndrome (fs) is a rare disease characterized by defects in proximal tubular function, including impairment of reabsorption of solutes such as glucose, uric acid, phosphate, amino acid, and bicarbonate. patients with fs may present normoglycemic glycosuria, low molecular weight proteinuria, hypophosphatemia, and normal anion gap metabolic acidosis. multiple myeloma is a neoplastic bone marrow disease characterized by clonal proliferation of plasma cells and overproduction of monoclonal protein. free light chain overproduction is associated with toxic effects to proximal tubular cells in the kidneys, which may induce fs. in this case, the patient who had presented proteinuria initially was diagnosed with fs and multiple myeloma, after reviewing her results from blood laboratory work, urine analysis, and bone marrow examination. in addition, kidney pathology confirmed the presence of rod - shaped casts in proximal tubules. a 48-year - old woman visited the nephrology clinic for proteinuria, which was detected at a local hospital. she had been producing foamy urine and experiencing nocturia for 2 months, and she was suffering from bilateral flank pain for 6 months. she had no specific underlying disease or related family history. however, she had been taking a course of chinese medicine for the past 6 months. at presentation, her vital signs were stable (blood pressure : 128/80 mmhg, heart rate : 62 beats / minute, respiration rate : 18 breaths / minute, body temperature : 36.4c), and her general physical examination was unremarkable. results from the blood testing, which included complete blood count, coagulation test, total bilirubin, aspartate transaminase, alanine transaminase, cholesterol, glucose, erythrocyte sedimentation rate, c - reactive protein, and thyroid function test were in the normal range. the patient s protein level was 6.6 g / dl and her albumin level was 4.9 g / dl, so her globulin was low (1.6 g / dl). her creatinine was 1.02 mg / dl, with a mildly decreased estimated glomerular filtration rate of 58 ml / minute/1.73 m. hypouricemia (0.9 mg / dl) and hypophosphatemia (2.3 mg / dl) were observed. serum sodium / potassium / chloride (139/3.5/109 mmol / l) and calcium (8.8 mg / dl) were in normal ranges. arterial blood gas analysis showed normal anion gap metabolic acidosis (ph 7.324, pco2 31.2 mmhg, po2 108.9 mmhg, hco3 15.9 mmol / l). urine dipstick testing showed the following characteristics : specific gravity (1.036), ph (6.5), blood (+), albumin (+ +), and glucose (+ +). a urine electrolyte test showed 51 mmol / l of sodium and 23.5 mmol / l of potassium. a spot urine test showed a urine protein / creatinine ratio of 10.61 mg / mgcr and a urine albumin creatinine ratio of 401.69 g / mgcr. based on the above information, we concluded that the patient had generalized proximal tubular dysfunction and overflow proteinuria. the patient had an elevated serum kappa / lambda ratio of 5,113.1. through urine protein electrophoresis, the m peak was observed behind the beta globulin region (2,911.6 mg / day). bone marrow examination showed normocellular marrow with 24% cd138 + plasma cell staining with kappa restriction. a renal biopsy was performed for accurate diagnosis of fs and to exclude renal amyloidosis or monoclonal immunoglobulin (ig) deposition disease. immunofluorescence staining for iga, igg, igm, c3, kappa, and lambda was negative. under electron microscopy, the glomerular basement membrane was slightly irregular in contour with mild effacement of epithelial foot processes. numerous rod- or rhomboid - shaped crystalline inclusions were present in the cytoplasm of proximal tubular epithelial cells (fig. most of the crystalline inclusions were electron dense and floating in the cytoplasm (fig. however, they were not found in the glomerular cells including podocytes. there were no amyloid fibrils, granular deposits, or immune type electron densities. finally, she was diagnosed as having multiple myeloma (kappa type) with fs. kidney pathology confirmed the presence of rod - shaped crystalline inclusions in proximal tubular cells. plasma cells were observed in<5% of aspirated bone marrow, although cd138 + cells were counted in up to 5% of bone marrow with kappa restriction. currently, the patient has survived for 1 year after autologous stem cell transplantation and she has received thalidomide maintenance ; however, her proximal tubular dysfunction has not improved (table 1). hypouricemia (1.3 mg / dl) and glycosuria were still observed in her last laboratory tests. phosphate level was normal (3.0 mg / dl) under intake of 2,040 mg of phosphate and 0.75 g of calcitriol daily. to maintain a potassium level of 4 mmol / l and prevent metabolic acidosis, the patient was taking 3,600 mg of potassium chloride, 100 mg of spironolactone, and 3,000 mg of sodium bicarbonate daily. urine albumin to creatinine ratio showed a similar level with baseline (440 g / mgcr) and urinary protein to creatinine ratio was much decreased to 1.69 mg / mgcr, which reflected reduced overflow proteinuria and persistent tubular proteinuria. this patient presented with proteinuria and upon evaluation was found to have normal anion gap metabolic acidosis, normoglycemic glycosuria, hypophosphatemia, and hypouricemia. therefore, we suspected that she had fs. bone marrow examination showed normocellular marrow with 24% cd138 + plasma cells in kappa restriction. therefore, she was diagnosed with multiple myeloma and light chain proximal tubulopathy. in multiple myeloma, renal insufficiency is a common complication. up to 50% of patients with multiple myeloma initially present with kidney injury. acute renal failure in myeloma is often caused by excessive production and filtration of free light chains. overproduced light chains can exert direct toxic effects onto kidney cells and generate myeloma casts by binding to tamm - horsfall proteins. cast nephropathy is the most common pattern of renal parenchymal disease associated with multiple myeloma. amyloidosis and monoclonal immunoglobulin deposition disease are the other types of deposition diseases that occur less frequently. hypercalcemia, drug toxicity, and volume depletion also contribute to acute kidney injury in multiple myeloma cases. excessively filtrated monoclonal light chains, usually composed of kappa light chains restricted to the v i subgroup, generate crystals within the cytoplasm of proximal tubular epithelial cells. these monoclonal kappa light chains have variable domains that are resistant to degradation by proteases in lysosomes. mutations of variable domains of kappa light chain, which is changing serine 30 to alanine, isoleucine, or leucine residue, increase the hydrophobicity of the cdr - l1 loop. fragments of the variable domain that accumulate in the cytoplasm are responsible for intracellular crystal formation and tubular dysfunction. the crystalline inclusions generated from light chains can induce cytotoxicity in the proximal tubular epithelial cells. they are associated with proximal tubular dysfunction including hypophosphatemia, hypouricemia, aminoaciduria, glycosuria, and metabolic acidosis. also, disruption of phagocytes that contain crystals may induce tissue injury, subsequently leading to tubular atrophy and interstitial fibrosis. it is unclear whether achieving a hematologic response by treating multiple myeloma can resolve renal proximal tubular dysfunction. gailani reported a hematologic response in a myeloma case by irradiation, followed by melphalan, prednisone, and vincristine. after myeloma treatment, the patient s hypophosphatemia, hypouricemia, and glycosuria were normalized. uchida found that after treatment for multiple myeloma, urinary bence - jones protein disappeared and glycosuria, aminoaciduria, phosphaturia, and metabolic acidosis all improved. it was reported by von scheele that glycosuria improved in a patient with fs with myeloma after 6 weeks of melphalan therapy ; however, despite achieving a partial hematologic response after myeloma therapy, renal tubular acidosis, glycosuria, and hypophosphatemia persisted in the patient. it is important to prescribe both phosphate and vitamin d to correct hypophosphatemia and to prevent metabolic bone disease, because impairment of vitamin d activation mechanisms also contributes to hypophosphatemia in fs. for normalization of metabolic acidosis, 1015 meq / kg / day of sodium bicarbonate we report a case of fs without renal failure, where the patient was ultimately diagnosed with multiple myeloma, demonstrating crystalline inclusion in proximal tubular cells on kidney biopsy. adult patients with fs should be evaluated for the presence of bence - jones proteinuria and plasma cell dyscrasia should be excluded as an underlying factor. | fanconi syndrome (fs) is a rare condition that is characterized by defects in the proximal tubular function. a 48-year - old woman was admitted for evaluation of proteinuria. the patient showed normal anion gap acidosis, normoglycemic glycosuria, hypophosphatemia, and hypouricemia. thus, her condition was compatible with fs. the m peak was found behind the beta globulin region in urine protein electrophoresis. upon bone marrow examination, we found that 24% of cells were cd138 + plasma cells with kappa restriction. from a kidney biopsy, we found crystalline inclusions within proximal tubular epithelial cells. thereafter, she was diagnosed with fs accompanied by multiple myeloma. the patient received chemotherapy and autologous stem cell transplantation, and obtained very good partial hematologic response. however, proximal tubular dysfunction was persistent until 1 year after autologous stem cell transplantation. in short, we report a case of fs accompanied by multiple myeloma, demonstrating crystalline inclusion in proximal tubular cells on kidney biopsy. |
in orthodontic treatment, tooth movement is related to the response to applied orthodontic forces that cause remodelling of the periodontal tissues, especially alveolar bone. the typical 2- to 3-year treatment period is burdensome for patients, so it is very important to accelerate alveolar bone remodelling during treatment in order to abbreviate the required time. recently, various biostimulatory effects of low energy laser irradiation that involves wound healing and fibroblast proliferation, collagen synthesis, and nerve regeneration have been reported. in particular, the acceleration of bone regeneration by laser treatment has been a focus of contemporary researches. therefore, if laser irradiation can cause the acceleration of bone remodelling, it may also have great benefit in abbreviating the orthodontic treatment period. the present study was designed to examine the effects of low - energy laser irradiation on alveolar bone remodelling during orthodontic tooth movement and on formation of new keratinized gingiva. low level laser therapy (lllt) is also known as soft laser therapy and bio - stimulation. the use of lllt in health care has been documented in the literature for more than 30 years and many research studies have demonstrated that lllt is effective for some specific applications in dentistry. recently, various effects of low - energy laser irradiation have been reported, including wound healing, fibroblast proliferation, collagen synthesis and nerve regeneration. in particular, the focus of recent studies has been the acceleration of bone regeneration by laser treatment. in orthodontics, low - energy laser irradiation can be useful for several different treatments, such as : reduction of post - adjustment pain, treatment of traumatic ulcers in the oral mucosa induced by an orthodontic appliance, increasing keratinized gingiva. it is also been demonstrated that lllt stimulates the velocity of tooth movement and the osteoclastogenesisin the pressure site via stimulation of the activator receptor of nuclear factor kappa b (rank)/rank ligand (rankl) system and by the expression of the macrophage colony - stimulating factor (m - csf)/its receptor (c - fms) expressions. the mechanisms of low level laser therapy are complex, but essentially rely upon the absorption of particular visible red and near - infrared wave - lengths in photoreceptors within sub - cellular components, particularly the electron transport (respiratory) chain within the membranes of mitochondria. the absorption of light by the respiratory chain components causes a short - term activation of the respiratory chain, and oxidation of the nadh pool. this stimulation of oxidative phosphorylation leads to changes in the redox status of both the mitochondria and the cytoplasm of the cell. the electron transport chain is able to provide increased levels of promotive force to the cell, through increased supply of atp, as well as an increased in the electrical potential of the mitochondria membrane, alkalization of the cytoplasm, and activation of nucleic acid synthesis. because atp is the energy currency for a cell, lllt has a potent action that results in stimulation of the normal functions of the cell. by increasing the respiratory metabolism of the cell this has relevance in terms of mast cells, which are triggered to respond by ionic gradients. in addiction it has been demonstrated that laser irradiation stimulates cellular proliferation and differentiation of osteoblast lineage nodule - forming cells, especially in committed precursors, resulting in an increase in the number of differentiated osteoblastic cells as well as in bone formation. this vasoactive effect is of relevance to the treatment of joint inflammation such as those may occur in the tmj. there is direct evidence that 660, 820, and 940 nm light can trigger mast cell degranulation. these types of cell are distributed preferentially about the microvascular endothelium in skin, oral mucosa and dental pulp. mast cells in these locations contain the pro - inflammatory cytokine tnf (tumor necrosis factor) in their granules. release of tnf promotes leukocyte infiltration of tissues by enhancing expression of endothelial - leukocyte adhesion molecules. in addition, must cell proteases, such as chymase, facilitate entry of leukocytes into tissues. because mast cells play a fundamental role in controlling leukocyte traffic, modulation of mast cell functions by lllt can be of considerable importance in the treatment of sites of inflammation in the oral cavity. a final aspect of the effect of lllt on cells is related to the effects of laser light on the cytoskeleton. several studies have suggested that lllt can modulate cell behaviour by causing re - arrangements of the cytoskeleton. myofibroblasts are responsible for the contraction force during wound healing. these cells are observed in normal tissue, granulation one, and some pathological conditions. because lllt is an effective stimulator of differentiation to myo - fibroblasts lllt has also been proven to reduce synthesis of inflammatory mediators in neural tissue, as well as more rapid maturation and regeneration, particularly axonal growth. it also reduces pain in patients suffering from post - herpetic neuralgia, from cervical dentinal hypersensitivity, and from periodontal pain during orthodontic tooth movement. in conclusion low level laser therapy accelerates wound healing and reduces pain, by stimulating oxidative phosphorylation in mitochondria and modulating inflammatory responses. by influencing the biological function of a variety of cell types, it is able to exert a range of several beneficial effects upon inflammation and healing. the low friction philosophy is based on the principle of using just enough force to initiate tooth movement - the threshold force. the underlying principle behind the threshold force is that it must be low enough to prevent occluding the blood vessels in the periodontal ligament. this allows the cells and the necessary biochemical messengers to be transported to the site where bone resorption and apposition will occur, thus permitting tooth movement. a passive self - ligation mechanism has the lowest frictional resistance of any ligation system. the forces generated by the archwire are transmitted directly to the teeth and the supporting structures without absorption or transformation by the ligature system. the forces generated by elastomeric ligatures can have unwanted effects on treatment progress. compared with conventional edgewise appliances, the use of passive self - ligation results in a significant reduction in the : use of anchorage devices, because the frictional resistance generated by ligatures is not present. it was demonstrated that passive self - ligating appliances use less anchorage than conventional appliances. this supports the reduction in the use of anchorage devices experienced by users of passive self-ligation.use of intraoral expansion auxiliaries because the force of the archwire is not transformed or absorbed by the ligatures and the necessary expansion can be achieved by the force of the archwires.need for extractions to facilitate orthodontic mechanics because alignment is not hindered by ligature frictional resistance and can therefore largely be achieved with small diameter copper nickel titanium archwires. tooth alignment therefore places minimal stress on the periodontium and the possibility of iatrogenic damage to the periodontium is reduced. use of anchorage devices, because the frictional resistance generated by ligatures is not present. it was demonstrated that passive self - ligating appliances use less anchorage than conventional appliances. this supports the reduction in the use of anchorage devices experienced by users of passive self - ligation. use of intraoral expansion auxiliaries because the force of the archwire is not transformed or absorbed by the ligatures and the necessary expansion can be achieved by the force of the archwires. need for extractions to facilitate orthodontic mechanics because alignment is not hindered by ligature frictional resistance and can therefore largely be achieved with small diameter copper nickel titanium archwires. tooth alignment therefore places minimal stress on the periodontium and the possibility of iatrogenic damage to the periodontium is reduced. in addition, a passive self - ligation system provides three key features : very low levels of static and dynamic friction.rigid ligation due to the positive closure of the slot by the gate or slide.control of tooth position because there is a slot of adequate width and depth. rigid ligation due to the positive closure of the slot by the gate or slide. this allows extended intervals between treatment visits, particularly in the early stages of treatment, a reduced number of visits during a course of treatment, and shortened treatment times. the static friction developed by passive self - ligating brackets is almost negligible. as angulation or inclination is applied to the bracket, binding occurs although the force generated by this binding is less for self - ligating brackets than for conventional ligation. if alignment and space closure can be achieved more quickly with self - ligating brackets due to reduced friction combination between lllt and low friction techniques during the orthodontic treatment of teeth erupted in oral mucosa (without keratinized gum) makes possible the formation of new keratinized gingiva. low level laser therapy (lllt) is also known as soft laser therapy and bio - stimulation. the use of lllt in health care has been documented in the literature for more than 30 years and many research studies have demonstrated that lllt is effective for some specific applications in dentistry. recently, various effects of low - energy laser irradiation have been reported, including wound healing, fibroblast proliferation, collagen synthesis and nerve regeneration. in particular, the focus of recent studies has been the acceleration of bone regeneration by laser treatment. in orthodontics, low - energy laser irradiation can be useful for several different treatments, such as : reduction of post - adjustment pain, treatment of traumatic ulcers in the oral mucosa induced by an orthodontic appliance, increasing keratinized gingiva. it is also been demonstrated that lllt stimulates the velocity of tooth movement and the osteoclastogenesisin the pressure site via stimulation of the activator receptor of nuclear factor kappa b (rank)/rank ligand (rankl) system and by the expression of the macrophage colony - stimulating factor (m - csf)/its receptor (c - fms) expressions. the mechanisms of low level laser therapy are complex, but essentially rely upon the absorption of particular visible red and near - infrared wave - lengths in photoreceptors within sub - cellular components, particularly the electron transport (respiratory) chain within the membranes of mitochondria. the absorption of light by the respiratory chain components causes a short - term activation of the respiratory chain, and oxidation of the nadh pool. this stimulation of oxidative phosphorylation leads to changes in the redox status of both the mitochondria and the cytoplasm of the cell. the electron transport chain is able to provide increased levels of promotive force to the cell, through increased supply of atp, as well as an increased in the electrical potential of the mitochondria membrane, alkalization of the cytoplasm, and activation of nucleic acid synthesis. because atp is the energy currency for a cell, lllt has a potent action that results in stimulation of the normal functions of the cell. by increasing the respiratory metabolism of the cell this has relevance in terms of mast cells, which are triggered to respond by ionic gradients. in addiction it has been demonstrated that laser irradiation stimulates cellular proliferation and differentiation of osteoblast lineage nodule - forming cells, especially in committed precursors, resulting in an increase in the number of differentiated osteoblastic cells as well as in bone formation. this vasoactive effect is of relevance to the treatment of joint inflammation such as those may occur in the tmj. there is direct evidence that 660, 820, and 940 nm light can trigger mast cell degranulation. these types of cell are distributed preferentially about the microvascular endothelium in skin, oral mucosa and dental pulp. mast cells in these locations contain the pro - inflammatory cytokine tnf (tumor necrosis factor) in their granules. release of tnf promotes leukocyte infiltration of tissues by enhancing expression of endothelial - leukocyte adhesion molecules. in addition, must cell proteases, such as chymase, facilitate entry of leukocytes into tissues. because mast cells play a fundamental role in controlling leukocyte traffic, modulation of mast cell functions by lllt can be of considerable importance in the treatment of sites of inflammation in the oral cavity. a final aspect of the effect of lllt on cells is related to the effects of laser light on the cytoskeleton. several studies have suggested that lllt can modulate cell behaviour by causing re - arrangements of the cytoskeleton. myofibroblasts are responsible for the contraction force during wound healing. these cells are observed in normal tissue, granulation one, and some pathological conditions. because lllt is an effective stimulator of differentiation to myo - fibroblasts, the process of wound healing should be accelerated. lllt has also been proven to reduce synthesis of inflammatory mediators in neural tissue, as well as more rapid maturation and regeneration, particularly axonal growth. it also reduces pain in patients suffering from post - herpetic neuralgia, from cervical dentinal hypersensitivity, and from periodontal pain during orthodontic tooth movement. in conclusion low level laser therapy accelerates wound healing and reduces pain, by stimulating oxidative phosphorylation in mitochondria and modulating inflammatory responses. by influencing the biological function of a variety of cell types, it is able to exert a range of several beneficial effects upon inflammation and healing. the low friction philosophy is based on the principle of using just enough force to initiate tooth movement - the threshold force. the underlying principle behind the threshold force is that it must be low enough to prevent occluding the blood vessels in the periodontal ligament. this allows the cells and the necessary biochemical messengers to be transported to the site where bone resorption and apposition will occur, thus permitting tooth movement. a passive self - ligation mechanism has the lowest frictional resistance of any ligation system. the forces generated by the archwire are transmitted directly to the teeth and the supporting structures without absorption or transformation by the ligature system. the forces generated by elastomeric ligatures can have unwanted effects on treatment progress. compared with conventional edgewise appliances, the use of passive self - ligation results in a significant reduction in the : use of anchorage devices, because the frictional resistance generated by ligatures is not present. it was demonstrated that passive self - ligating appliances use less anchorage than conventional appliances. this supports the reduction in the use of anchorage devices experienced by users of passive self-ligation.use of intraoral expansion auxiliaries because the force of the archwire is not transformed or absorbed by the ligatures and the necessary expansion can be achieved by the force of the archwires.need for extractions to facilitate orthodontic mechanics because alignment is not hindered by ligature frictional resistance and can therefore largely be achieved with small diameter copper nickel titanium archwires. tooth alignment therefore places minimal stress on the periodontium and the possibility of iatrogenic damage to the periodontium is reduced. use of anchorage devices, because the frictional resistance generated by ligatures is not present. it was demonstrated that passive self - ligating appliances use less anchorage than conventional appliances. this supports the reduction in the use of anchorage devices experienced by users of passive self - ligation. use of intraoral expansion auxiliaries because the force of the archwire is not transformed or absorbed by the ligatures and the necessary expansion can be achieved by the force of the archwires. need for extractions to facilitate orthodontic mechanics because alignment is not hindered by ligature frictional resistance and can therefore largely be achieved with small diameter copper nickel titanium archwires. tooth alignment therefore places minimal stress on the periodontium and the possibility of iatrogenic damage to the periodontium is reduced. in addition, a passive self - ligation system provides three key features : very low levels of static and dynamic friction.rigid ligation due to the positive closure of the slot by the gate or slide.control of tooth position because there is a slot of adequate width and depth. rigid ligation due to the positive closure of the slot by the gate or slide. this allows extended intervals between treatment visits, particularly in the early stages of treatment, a reduced number of visits during a course of treatment, and shortened treatment times. the static friction developed by passive self - ligating brackets is almost negligible. as angulation or inclination is applied to the bracket, binding occurs although the force generated by this binding is less for self - ligating brackets than for conventional ligation. if alignment and space closure can be achieved more quickly with self - ligating brackets due to reduced friction combination between lllt and low friction techniques during the orthodontic treatment of teeth erupted in oral mucosa (without keratinized gum) makes possible the formation of new keratinized gingiva. the sample of this clinical trial comprised a total of 22 patients and 27 teeth (10 upper cuspids, 8 lower cuspids, 3 upper first bicuspids, 2 upper second bicuspids, 3 lower first bicuspids and 1 lower second bicuspid) erupted in oral mucosa completely without keratinized gingiva. vestibular infra - bone position of cuspids was detected in 4 of these patients [figure 1 ]. all the patients were treated with passive self - ligating orthodontic appliance system using termicarchwire 0 - 13 am orth and these teeth were bio - stimulated with laser (lllt). we used different brackets, in particular : time (am orth) in 70% of treated subjects (time2 for 40% and time3 for 30%), smartclip (3 m unitek) in 15%, and empower (am orth) in 15%. a diode laser with a wavelength of 980 nm and continuous wave at 1 watt output power was used in the study. 25 : surgical approach with diode laser. time 2 appliance (am orth) the average age of the sample was 13,5-years - old and the female patients were 13 instead the male ones were 11. the laser used is a diode laser 980 nm (wiser - doctor smile). the beam was delivered by a plane wave optical fiber (doctor smilemanipoloondapiana) and irradiation was administered by placing the end of the optical fiber tip direct where missing keratinized gingiva, on cement - enamel junction (cej) of upper and lower cuspids and bicuspids undergoing orthodontic tooth movement (1,5 cm as minimum on defocalization, as prescribed by the producer, doctor smilemanipoloondapiana). irradiation was performed for 50 seconds at each point 3 times, every 2 minutes on relaxing time, (a total of 150 seconds) once a month on months 0 - 6 (a total of seven times). we utilized 1 watt, continuous wave, as indicated by the producer (doctor smile - manipoloondapiana) the energy density corresponding to an exposure time of 150 seconds was150j / cm, (every second the fluence is 1j / cm). to assess the amount of increasing keratinized gingiva every months, measurement were made using a periodontal probe before (t0) and after laser irradiation (months 1, 2, 3, 4, 5, and 6) ; each measurement was taken twice and it was used the average value. the experimental periods for observation were set at 1, 2, 3, 4, 5 and 6 months after irradiation. at the time t0 the measurement of keratinized gingiva was 0 mm because the teeth analyzed were erupted in ectopic site in oral mucosa [table 1 ]. measurement of increasing keratinized gingiva using a periodontal probe before (t0) and after laser irradiation (months 1, 2, 3, 4, 5, and 6) descriptive statistics, including the mean and standard deviation (sd) were calculated for each patient and for each monthly measurement. statistical analysis of data was performed using the analysis of variance (anova) with software for windows (ver. 5.0). descriptive statistics, including the mean and standard deviation (sd) were calculated for each patient and for each monthly measurement. statistical analysis of data was performed using the analysis of variance (anova) with software for windows (ver. 5.0). the average keratinized gingiva at the end of the study is 3.1 mm and the mean increasing at each month is 0,49 mm [figure 3 ]. 3,45 mm of attached gingiva around the crown of 25, after 6 month of self- ligating orthodontic therapy and llt biostimulation as discussed in the paper passive self - ligation offers the most direct transmission of force from archwire to tooth with very low friction, secure ligation, and excellent control of tooth position. all contemporary modalities of orthodontic treatment can achieve tooth alignment ; passive self - ligation, however, does achieve results effectively, and in a way that corresponds with patient wishes. in addition, practitioners experienced with the technique perceive that additional, unexpected patient benefits occur that are not traditionally associated with conventional orthodontic treatment. teeth can moved with or through bone depending on the strain generated in the surrounding periodontium. the force system applied attempted to generate a maximum distribution of low forces, thus avoiding local necrosis and ischemia of the periodontal ligament. in addition it can be assumed that the patient 's oral hygiene was maintained during treatment. in order to improve the effects of low friction in bone remodeling, we have tested the combined use of laser, in particular the low energy laser therapy. while much of the initial work with lllt used the helium - neon gas laser (= 632.8 nm), nowadays most lllt clinical procedures are undertaken using semiconductor diode lasers operating (at = 830 nm or = 980 nm wavelengths), as the one we have used in this study. since wavelength is the most important factor in any type of phototherapy, the clinician must consider which wavelength are capable of producing the desired effects within living tissues. the typical power output for a low level laser device used for this therapy is of the order of 10 - 50 mw, and total irradiances at any point are of the order of several joules. thermal effects of lllt on dental tissues are not significant, and do not contribute to the therapeutic effects seen. the wavelengths used for lllt have a low absorption coefficient in hemoglobin and water, and consequently, a high penetration depth in the irradiated soft and hard tissue from 3 mm to up to 15 mm. the extensive penetration of red and near - infrared light into tissues has been documented by several investigators. as the energy penetrates tissues, there is multiple scattering by both erythrocytes and microvessels, and so both blood rheology and the distribution of microvessels in the tissue influence the final distribution pattern. laboratory studies of low level laser effects have demonstrated a range of bio - stimulation effects : for fibroblasts, increased proliferation, maturation and locomotion have been noted, as well as transformation to myofibroblasts, reduced production of pro - inflammatory prostaglandin e2, and increased production of basic fibroblast growth factor. these effects have been reported for fibroblasts from the skin, buccal mucosa and gingival, all of which show increased proliferation at low doses (e.d., high dose lllt suppresses both fibroblast proliferation and autocrine production of basic fibroblast growth factor. several studies have been conducted to understand how different approaches on implant surface can influence survival of the same. histological studies in oral mucosal wound healing have demonstrated that laser irradiation improves wound epithelialization, cellular content, granulation tissue formation, and collagen deposition in laser - treated wounds, compared to untreated sites. in conclusion patients with teeth erupted in ectopic position get benefit from combined use of lllt and self ligating orthodontic appliance in order to improve the formation of new area of keratinized gingival in particular in this study we obtained about 3,1 mm of this one. in the field of orthodontics, low energy laser irradiation is utilized for several different types of orthodontic treatment, such as reduction of post adjustment pain or treatment of traumatic ulcers in the oral mucosa caused by the appliance. however, scant information is available concerning the effects of lllt on bone remodelling during orthodontic tooth movement. low friction in addition to lllt can stimulate keratinized gingiva under maintenance of good oral hygiene in patients with teeth erupted in oral mucosa completely without keratinized gingival. low forces can stimulate the periodontal remodelling instead low energy laser irradiation can enhance the differentiation of periodontal undifferentiated cells into fibroblastic lineage in order to form keratinized gingiva. | background : recently, various biostimulation 's effects of low energy laser irradiation have been reported. the present study was designed to examine the effects of low - energy laser irradiation on alveolar bone remodelling during orthodontic tooth movement and finally on formation of new keratinized gingiva.materials and methods:22 patients and 27 teeth in vestibular mucosal without keratinized gingiva were selected. every patient was treated with self ligating appliances. in every orthodontic session the patient was treated with diode laser biostimulation. at the moment of debonding, 27 teeth involved in the research were evaluated in terms of quality and quantity of attached gingiva. bop and cal loss were investigated.results:every tooth considered at the end of orthodontic treatment showed an attached gingiva around the crown : the average of keratinized gingiva at the end of the study was 3.10 mm and the mean increasing at each month was 0,49 mm.conclusions:the combination between self ligating appliances and laser 's biostimulation could improve the differentiation of periodontal ligaments stem cells in fibroblasts, able to promote attached gingiva around the crown of the teeth erupted in oral vestibular mucosa. |
breast cancer is an increasingly visible disease, and a rapidly growing cause of mortality, in developing countries. in africa, where breast cancer may often present at an earlier age and can progress more aggressively [2, 3 ], little is known about pathways and triggers for women to take action (e.g., seek medical advice) based on their recognition of symptoms. in ethiopia, breast cancer is typically a fatal disease with high mortality [4, 5 ], unlike the experience of the western world where breast cancer is frequently treatable and with lower mortality. ethiopia has an increasingly comprehensive set of breast cancer prevention, diagnosis, and treatment interventions available for women though stigma toward cancer, poor knowledge and awareness of cancer signs and treatability, and system overload continue to account for delays in reaching care. an important component of the knowledge - action chain is understanding ethiopian women 's recognition of symptoms of breast cancer and their motivations for taking action. ethiopian women typically present for care at a late stage in the disease, where treatment is most ineffective, and while system - related barriers to care account for a portion of that delay in access, women 's attitudes and lack of awareness of breast cancer symptoms also account for a stalled initiation of action. as the health system and treatments available for breast cancer in ethiopia continually expand and are accessible to the population, more women can potentially access care at an earlier time when treatment may be more useful, provided women recognize and take action when they experience a symptom that could potentially signal breast cancer. this evaluation includes a qualitative and quantitative assessment of the experience of participants with breast cancer who, ultimately, successfully accessed the services of the ethiopia breast cancer project (ebcp) and reports on their recognition of symptoms, their attitudes upon noticing those symptoms, and their motivations for and experiences with taking action. this project focuses upon women who accessed care through the ethiopia breast cancer project (ebcp). ebcp aims to strengthen human resource capacity, technical competency, and advocacy and improve access to treatment for breast cancer in ethiopia, working closely with all related departments and services of tikur anbessa hospital (tah), the ministry of health, and the ethiopian cancer association. patients, their families, and health practitioners were interviewed as part of the larger impact assessment using semistructured interview protocols developed following open - ended ethnographic interviews and observations. interviewers were fluent in written and spoken amharic and english and sequentially identified ebcp program participants to interview over a one - month span. interviewers participated in a one - day training session that included : study overview, ethical conduct of research, role play, and pilot test interviews and review. the teams debriefed each day with investigators for major points and discussion items for the group. qualitative data was analyzed through theme analysis predominantly using atlas.ti, version 5.5 (atlas.ti scientific software development gmbh, berlin, germany), and quantitative data was analyzed using jmp, version 8 (sas institute, inc., where necessary, translated english grammar in direct quotes is corrected from transcriptions to ease readability and where necessary, placed in the first person context, though content remains unchanged. in total, 55 patients directly participated in the study, in addition to 14 proxies (children, spouses, others) representing other patients, for a total of 69 patients represented. most participants were married and female, and more than half were age 50 years or younger. about two - thirds of participants lived in addis ababa, and almost three - quarters of the population was ethiopian orthodox. two - thirds of the participants were diagnosed with breast cancer in the immediate two years prior to the assessment. when compared to the clinic treatment population for the first half of 2008 from ebcp programmatic data (unpublished), the study sample did not differ significantly from the clinic treatment population on any of the following parameters : age, gender, or residence of patient. participants were asked in an open - ended way to tell their story of how they came to learn they had breast cancer. if necessary, participants were prompted for relative dates when they first noticed symptoms, when they first accessed care, and their navigation of the care system (see dye.). these narratives of participants ' experiences were subsequently coded to identify symptoms, triggers for action, timing of action, and contextual factors occurring in their stories. this project was reviewed and approved by the addis ababa university faculty of medicine irb. additionally, project team members were trained in research ethics using materials from the citiprogram (https://www.citiprogram.org/). participant names were not collected as part of this project, and indirect identifying information was grouped and presented in general categories or in aggregate. as shown in table 1, most participants in the assessment (82.6 percent) indicated that a lump was the first symptom (of what was to become breast cancer) that they experienced. the only other significantly mentioned symptom noted in participants ' narratives of their cancer experience was a sensation of itching or burning, either on the breast or at a lymph node site. most participants indicated that they also, ultimately, experienced subsequent symptoms, most commonly described as pain (often near or around the site of the original lump), which was experienced by 36 percent of participants. also some participants noted that they found additional lumps (14.5 percent) subsequent to their first symptom. nearly all participants (89.9 percent) indicated that they noticed a lump at some point prior to seeking advice for their symptoms. most participants did not expect that the lump they noticed was of any concern, at least initially : about 2 years back, i found a small hard lump over my left breast, but since it was small and i had no pain, i was not that much concerned about it. (case 56) three years back, i started feeling sharp, tingling kind of pain in my right breast. i went to a nearby clinic and was diagnosed to have a cold, and i started oral medication. then after some time, i developed a pea - sized hard nodule over the same breast, which was getting bigger at time passed on by. but, starting last year, the lump become painful and we took her to [a clinic ]. similarly, as shown in table 2, most participants (69.6 percent) ignored their symptoms, at least initially, for an average of more than one and a half years until they took some form of action. while many participants were ambiguous or uncertain for how long they ignored their symptoms, more than one - third indicated that they waited at least a year or more, some waiting as long as five or six years. this period represents the time participants waited to take action regarding their symptoms ; it does not reflect typically additional delays throughout the care system once the participant made a decision to access it. after a year, i went to [eastern ethiopia ] where my biggest child lives. it was painless at that time and i thought it will resolve by its own. finally the lump became harder it be come [sic ] very painful within two years. i went to a health center the physician examined me and told me that it is breast cancer, and i will not live more than 2 months (case 41) finally, participants indicated that the most common reason for initiating some form of action to address their symptoms (table 3)including seeking a traditional or contemporary healer was that they experienced symptoms in addition to their primary one (42.0 percent). further, 21.7 percent of participants indicated that they took action because of a change in their original symptom. taken together, nearly three - fourths of all participants in the assessment were motivated to take action because of changes in or additions to their symptoms. an additional 5.8 percent indicated that family pressure motivated them to take action, and also 5.8 percent indicated that they did not deliberately take action but rather their breast cancer was detected secondarily at another health care visit. before 2 years, i started feeling sharp pain over my left breast with a little swelling. but i did not do anything about it or told anybody because i was under the consideration that it will go away by itself. but after about 4 months, i had to go to a nearby health center because the mass was getting bigger and the pain was getting worse. (case 49) three years back, i started nursing my last child. i went to a physician [who ] told me that it is cold and gave me an antibiotic. after a month of this medication i noticed a small lump on the same breast. i showed it to my husband, and my sisters they told me that it simple lump and not to worry about it. after 2 and 1/2 years, i met my old friend who works in addis ababa as a nurse and i showed her too. she told me to go to the health center and get treated. after doing some investigation (case 27) two years back, i noticed a small nodule over my left breast but i was not that much concerned about it. then when i got pregnant in that year, the mass started becoming very painful which forced to seek medical attention. then i was told that it could be cancer and that i would get the treatment after giving birth. clearly, in ethiopia lumps are the first noticeable signs of breast cancer typically recognized by participants. almost all of the participants in this study noticed a lump at some point and most participants also dismissed the lump, at first, as nothing to be concerned with., participants noticed more lumps or changes in symptoms (pain, itching), and that triggered them to seek advice, from a traditional healer, clinic, or other health care source. a few women were motivated to seek advice from their friends or family, and for some their cancer was discovered in the course of obtaining medical care for something else. several other studies have similarly found that lumps are the dominant symptom noticed by women with breast cancer and that most women find lumps as their primary symptom [1215 ]. further, breast pain was identified as a trigger for seeking care in ghana, and montazeri. showed that painless mass was the most commonly recognized symptom in iran, though still only 44% identified it as potentially cancer. studies also indicate that women in low - resource areas delay seeking care longer than women in other parts of the world, with delays of a year or more from detection of symptom to seeking advice [12, 14, 15, 17 ]. this delay could reflect the relatively recent (and yet incomplete, in many areas) inclusion of cancer in public health programs and awareness campaigns. african women already face considerable delays in accessing care through overburdened health care systems and with limited resources ; adding more than a year of delay from noticing a symptom to action increases the chances that their disease will progress significantly before care initiates. notably, this study included participants who, at least eventually, obtained care at the national cancer hospital in ethiopia. even among these women who obtained care, delay from noticing to acting on symptoms was considerable. this study is limited in that delay, symptom recognition, and the dynamics of triggers for action are unknown among women not entering the care system in ethiopia. given the importance of lumps in initiating the cascade of events that could lead to breast cancer diagnosis, increasing women 's knowledge of lumps as a trigger for advice may help reduce the lag time between initial notice of the symptom and eventual action in ethiopia. focused public campaigns and other strategies to increase awareness may be effective in promoting action when confronted with a lump (e.g., see remennick). strengthening the capacity of the health system to respond to women 's queries about lumps is also necessary, especially moving local primary care sites the most common initial point of entry for breast cancer patients in ethiopia to the basic level of capacity recommended by the breast health global initiative. though there is debate about the effectiveness of breast self - examination (bse) in lowering breast cancer mortality (see reeler.), perhaps promoting breast lump awareness to encourage women to seek advice on lumps promptly, in a resource - poor country such as ethiopia, may trigger action and entry into care sooner, while more comprehensive population - based and -accessible screening and diagnostic programs are developed and implemented over time. | objective. this study assessed the initial experiences, symptoms, and actions of patients in ethiopia ultimately determined to have breast cancer. methods. 69 participants in a comprehensive breast cancer treatment program at the main national cancer hospital in ethiopia were interviewed using mixed qualitative and quantitative approaches. participants ' narratives of their initial cancer experience were coded and analyzed for themes around their symptoms, time to seeking advice, triggers for action, and contextual factors. the assessment was approved by the addis ababa university faculty of medicine institutional review board. results. nearly all women first noticed lumps, though few sought medical advice within the first year (average time to action : 1.5 years). eventually, changes in their symptoms motivated most participants to seek advice. most participants did not think the initial lump would be cancer, nor was a lump of any particular concern until symptoms changed. conclusion. given the frequency with which lumps are the first symptom noticed, raising awareness among participants that lumps should trigger medical consultation could contribute significantly to more rapid medical advice - seeking among women in ethiopia. primary care sites should be trained and equipped to offer evaluation of lumps so that women can be referred appropriately for assessment if needed. |
participants consisted of 4 students who attended two special education schools at the elementary level. all students were diagnosed as having adhd, and their age range was between 9 to 11 years. three of the four students were diagnosed with a combined type of adhd, and one person was diagnosed to have the hyperactive impulsive type by a child psychiatrist. the researcher directly observed the inattentive behaviors (such as inattention to teacher and materials, not following through instructions, leaving seat) of the participants in the classroom. inattentive behaviors of participants (target behaviors) were observed during two sessions each week (each session lasted 30 minutes). all target behaviors were recorded using duration per occurrence recording procedure, and percentages of the behaviors were calculated. self - monitoring of attention was used as a cognitive - behavioral technique for increasing self - control in the participants. the programs included the following components : self - monitoring cues tape : an audiotape including tones or beeps at irregular intervals. when a student first begins to use self - monitoring, the pre - recorded tones are essential for his / her success (5). a self - monitoring card : it includes a self - assessment question (was i paying attention ?) and spaces in which participants indicate being off - task or on - task when the tones sound. percentages of inattentive behaviors in experimental phases during self - monitoring, students were engaged to work in classroom assignments or follow their teacher 's instructions (5). the desired goal of self - monitoring was to achieve self - control in classroom situation and to work without external cues features (tape and card). thus, during self - monitoring sessions, external cues were phased out, and students were expected to control their off - task behaviors internally. the results indicated that the percentages of off - task or inattentive behaviors in the classroom situation reduced as a result of the self - monitoring program. this decreasing trend from baseline to the intervention phase is displayed for all the four participants. levels of inattentive behaviors in the follow - up are approximately similar to the intervention, and are lower than the baseline phase (particularly for the first and fourth participants). during self - monitoring, students were engaged to work in classroom assignments or follow their teacher 's instructions (5). the desired goal of self - monitoring was to achieve self - control in classroom situation and to work without external cues features (tape and card). thus, during self - monitoring sessions, external cues were phased out, and students were expected to control their off - task behaviors internally. the results indicated that the percentages of off - task or inattentive behaviors in the classroom situation reduced as a result of the self - monitoring program. this decreasing trend from baseline to the intervention phase is displayed for all the four participants. levels of inattentive behaviors in the follow - up are approximately similar to the intervention, and are lower than the baseline phase (particularly for the first and fourth participants). direct observation of inattentive behaviors in a school- based special education situation showed that the behaviors reduced in the intervention phase of a single - subject experimental design. however, maintaining the treatment effects was difficult for the participants. based on the results of this study, two important points should be considered : firstly, increased effectiveness of self - monitoring technique was only observed in the context of the intervention phases and not in the follow - up phases. in other words, secondly, individual differences are important in accepting treatment. from a practical perspective, the findings of this study indicate that self - monitoring techniques can be applied for students with adhd in school - based special education settings. with respect to the short - term effectiveness of self - monitoring technique, continued treatment for adhd is needed so that parents and teachers could understand the outcomes of each approach. lastly, it should also be noted that parent management training (6, 7) is an important factor to be considered in cbt techniques. | beneficial effects of stimulants on core symptoms of attention deficit hyperactivity disorder (adhd) have been reported in several studies. behavioral interventions have also been proposed as empirically supported interventions for adhd. although cognitive - behavioral therapies (cbt) have been criticized for the lack of evidence - based data, some studies have indicated the positive effects of cbt techniques on children with attention deficit hyperactivity disorder (adhd). this article reports the effects of self - monitoring technique, as a cbt technique, on inattentive behaviors of children with adhd. |
a prospective case series of 10 patients was conducted by the national university hospital, diabetic foot team, in singapore. the study population was made up of six males and four females with a mean age of 54 (range : 3766 years). in this study, the indications for usage of renasys go included ray (toe and metatarsal) amputation wounds, post - drainage wounds of abscess and post - debridement wounds for necrotizing fasciitis and ulcers of the heel, dorsum of foot and of the sole. four ulcers were ray amputation wounds, two were wounds post - drainage of abscess, one was on the dorsum of the foot, one was on the left shin, one was on the left foot and one was on the right calf. each ulcer was classified as either a grade 2 ulcer or a grade 3 ulcer, according to wagner 's classification (10). documentation in the study protocol included the patient 's demographics, diabetic history, presence of complications and infection markers. all wounds were monitored and photographed as they progressed and wound dimensions were measured with a ruler (fig. the location and diagnosis of the ulcer was recorded as well as the presence of any granulation tissue. clinical investigations performed prior to therapy included markers of infection (leukocyte count, c - reactive protein and erythrocyte sedimentation rate). a swab from the wound was sent for culture and sensitivity tests before application of the npwt system. the npwt settings, mode of application, date of initiation and end of npwt, number and date of npwt dressing changes were recorded. prior to renasys - go npwt treatment, adequate surgical debridement was performed on all wounds to remove any necrotic and infected tissue. the npwt dressing was cut to fit the size and shape of the wound and the number of pieces used recorded to ensure all pieces were removed at each dressing change. the wounds were sealed using semi - permeable film drape and a hole was cut in the center of the film. the soft port was placed directly over the hole in the film and its tubing was connected to the canister tubing. for all patients, the npwt device was set at continuous or mode with negative pressure of 100 or 120 mmhg. the pressure settings were dependent on the amount of exudate from the wound and patient 's skin sensitivity since pressures may need to be lower to reduce pain on the skin. npwt dressing changes were performed every 4872 hours by a trained medical officer or nurse. the number of npwt dressing changes ranged from two to nine dressings, while the duration of npwt treatment was 726 days. in this study, the indications for usage of renasys go included ray (toe and metatarsal) amputation wounds, post - drainage wounds of abscess and post - debridement wounds for necrotizing fasciitis and ulcers of the heel, dorsum of foot and of the sole. four ulcers were ray amputation wounds, two were wounds post - drainage of abscess, one was on the dorsum of the foot, one was on the left shin, one was on the left foot and one was on the right calf. each ulcer was classified as either a grade 2 ulcer or a grade 3 ulcer, according to wagner 's classification (10). documentation in the study protocol included the patient 's demographics, diabetic history, presence of complications and infection markers. all wounds were monitored and photographed as they progressed and wound dimensions were measured with a ruler (fig. the location and diagnosis of the ulcer was recorded as well as the presence of any granulation tissue. clinical investigations performed prior to therapy included markers of infection (leukocyte count, c - reactive protein and erythrocyte sedimentation rate). a swab from the wound was sent for culture and sensitivity tests before application of the npwt system. the npwt settings, mode of application, date of initiation and end of npwt, number and date of npwt dressing changes were recorded. prior to renasys - go npwt treatment, adequate surgical debridement was performed on all wounds to remove any necrotic and infected tissue. the npwt dressing was cut to fit the size and shape of the wound and the number of pieces used recorded to ensure all pieces were removed at each dressing change. the wounds were sealed using semi - permeable film drape and a hole was cut in the center of the film. the soft port was placed directly over the hole in the film and its tubing was connected to the canister tubing. for all patients, the npwt device was set at continuous or mode with negative pressure of 100 or 120 mmhg. the pressure settings were dependent on the amount of exudate from the wound and patient 's skin sensitivity since pressures may need to be lower to reduce pain on the skin. npwt dressing changes were performed every 4872 hours by a trained medical officer or nurse. the number of npwt dressing changes ranged from two to nine dressings, while the duration of npwt treatment was 726 days. the age of patients in this study ranged from 37 to 66 (median : 54). five patients were malays, three indians, and two chinese. complications of dm seen in the study patients included peripheral neuropathy, vasculopathy, nephropathy and diabetic retinopathy. comorbidities which affected the study group included hypertension, hyperlipidemia and peripheral vascular disease. patient characteristics table 2 shows the renasys - go npwt settings for the patients. a pressure of 100 mmhg was applied to four of the wounds, while a pressure of 120 mmhg was applied to the remaining six wounds. wounds were administered npwt for an average of 15.9 days (range 726) while an average of 5.2 npwt dressings (range 29) was used per patient in the study group. table 3 illustrates the changes in wound size and final outcome of wounds for all patients. the remaining two patients demonstrated an increase in size of the wound due to repeated debridements. seven of the 10 wounds healed by a split - thickness skin graft (stsg) and three wounds healed by secondary closure. treatment details and machine settings the average size of the wounds at the beginning of the study was 39.95 cm (range : 5.00156.00 cm), and by the end of the study, the average wound size had reduced to 37.339 cm (range : 6.00149.80 cm). a 42-year - old indian male presented with a dorsal foot ulcer wagner grade 3 and exposed tendons. risk factors associated with healing consisted of an 8-year history of type 2 dm and hyperlipidemia. the patient had previously undergone three surgical wound debridements and ray amputation of the big toe in january 2013 in a different facility where he was offered a below - the - knee amputation. patient 's dorsalis pedis and posterior tibial pulses were palpable, with ankle brachial index and toe brachial index readings of 1.23 and 0.87, respectively and a capillary refill timing of 16.0% for hba1c, 12.0 g / dl for hb and 35 function indicated a level of 105 mol / l for creatinine and 6.6 mmol / l for blood urea nitrogen. hydrosurgical debridement of the infected ulcer was carried out before application of the renasys - go npwt dressing. a final debridement along with a stsg was then performed, followed by the npwt application. the wound was inspected 5 days after the stsg and healed completely after 2 months. the wound area was decreased by 31.00% (fig. (d, e) post - ssg day 5 and week 8, respectively. a 42-year - old indian male presented with a dorsal foot ulcer wagner grade 3 and exposed tendons. risk factors associated with healing consisted of an 8-year history of type 2 dm and hyperlipidemia. the patient had previously undergone three surgical wound debridements and ray amputation of the big toe in january 2013 in a different facility where he was offered a below - the - knee amputation. patient 's dorsalis pedis and posterior tibial pulses were palpable, with ankle brachial index and toe brachial index readings of 1.23 and 0.87, respectively and a capillary refill timing of 16.0% for hba1c, 12.0 g / dl for hb and 35 renal function indicated a level of 105 mol / l for creatinine and 6.6 mmol / l for blood urea nitrogen. hydrosurgical debridement of the infected ulcer was carried out before application of the renasys - go npwt dressing. a final debridement along with a stsg was then performed, followed by the npwt application. the wound was inspected 5 days after the stsg and healed completely after 2 months. (d, e) post - ssg day 5 and week 8, respectively. the effectiveness of npwt for diabetic foot wounds is well supported by previous research (58). has shown to achieve comparable results for both chronic and acute wounds (9). the present study is the first one in singapore to explore the effectiveness of this newer system for diabetic lower limb ulcers. compared to conventional dressings, npwt provides a controlled wound environment that is separate from the external surroundings. this enables wound healing to occur under clean conditions, with moisture level controlled by altering pressure settings. hence, the healing of chronic wounds such as diabetic foot ulcers is enhanced by the usage of npwt dressings., showing a 49% compared to 7.7% reduction in wound depth and 59% compared to 0.1% reduction in wound volume when diabetic foot ulcers were treated with vac therapy as compared to moist gauze dressings (11). our study showed an average reduction in wound area of 22.4%, with eight out of 10 wounds having a decreased wound area after treatment. a freshly granulating wound bed indicates that the wound has entered the proliferative stage of wound healing and permits either secondary closure or the usage of other wound closure techniques.. showed that wounds treated with npwt dressing, either continuous or intermittent, granulated better than those treated using conventional dressing (12). in this study, the time required to achieve wound bed preparation for surgical intervention was taken as the time from initiation of renasys - go therapy to the achievement of a continuous and fresh bed of granulation in the wound. all wounds were able to achieve adequate granulation tissue before closure by secondary healing (three out of 10 patients) or by a successful stsg (seven out of 10 patients). the renasys - go therapy was observed to be beneficial in reducing bacterial infection in the wounds studied. this potential benefit of npwt was suggested in a swine model study by morykwas. a significant reduction in the bacterial load of inflicted chronic wounds was achieved by the fifth day for those treated with npwt, but required an additional 6 days to reach the same reduced level in wounds untreated with npwt. in our study, culture and sensitivity tests found colonization of all 10 wounds at the start of npwt (table 1). however, after npwt treatment, all 10 wounds showed clearance of bacterial infection, allowing surgical intervention to be undertaken or secondary closure to occur. in this study, the average duration required to complete the npwt was 15.9 days, with a range from 7 to 26 days. however, in singapore, the cost of renasys - go therapy is relatively high and adds on to patients hospitalization costs. as such, to make this npwt more affordable for our patients, stsg was performed once the wound bed preparation was achieved with sufficient granulation tissue and wound cultures being negative for bacterial growth. it is also interesting to note that the average length of time taken for our npwt treatment (15.9 days) was shorter than the average length of time taken for other similar conventional treatments (23.3 days) (15). along with the international expert panel on negative pressure wound therapy have recommended a range of npwt settings between 50 and 150 mmhg (16). lower negative pressures may be considered for pain reduction, and higher negative pressures may be considered for high volume of wound exudates (16). in our study, npwt settings were between 100 mmhg (four patients) to 120 mmhg (six patients). lower negative pressures (100 mmhg) were used for pain reduction and higher negative pressures (120 mmhg) were used for a high volume of wound exudates. limitations of this study includes a low patient number, short term follow - up and lack of comparison of the renasys - go therapy versus other forms of npwt or conventional dressings. with the positive results seen in our case series, we recommend future studies such as a prospective randomized controlled clinical trial comparing the efficacy of renasys - go therapy versus other treatment modalities compared to conventional dressings, npwt provides a controlled wound environment that is separate from the external surroundings. this enables wound healing to occur under clean conditions, with moisture level controlled by altering pressure settings. hence, the healing of chronic wounds such as diabetic foot ulcers is enhanced by the usage of npwt dressings., showing a 49% compared to 7.7% reduction in wound depth and 59% compared to 0.1% reduction in wound volume when diabetic foot ulcers were treated with vac therapy as compared to moist gauze dressings (11). our study showed an average reduction in wound area of 22.4%, with eight out of 10 wounds having a decreased wound area after treatment. a freshly granulating wound bed indicates that the wound has entered the proliferative stage of wound healing and permits either secondary closure or the usage of other wound closure techniques. morykwas. showed that wounds treated with npwt dressing, either continuous or intermittent, granulated better than those treated using conventional dressing (12). in this study, the time required to achieve wound bed preparation for surgical intervention was taken as the time from initiation of renasys - go therapy to the achievement of a continuous and fresh bed of granulation in the wound. all wounds were able to achieve adequate granulation tissue before closure by secondary healing (three out of 10 patients) or by a successful stsg (seven out of 10 patients). the renasys - go therapy was observed to be beneficial in reducing bacterial infection in the wounds studied. this potential benefit of npwt was suggested in a swine model study by morykwas. a significant reduction in the bacterial load of inflicted chronic wounds was achieved by the fifth day for those treated with npwt, but required an additional 6 days to reach the same reduced level in wounds untreated with npwt. in our study, culture and sensitivity tests found colonization of all 10 wounds at the start of npwt (table 1). however, after npwt treatment, all 10 wounds showed clearance of bacterial infection, allowing surgical intervention to be undertaken or secondary closure to occur. in this study, the average duration required to complete the npwt was 15.9 days, with a range from 7 to 26 days. however, in singapore, the cost of renasys - go therapy is relatively high and adds on to patients hospitalization costs. as such, to make this npwt more affordable for our patients, stsg was performed once the wound bed preparation was achieved with sufficient granulation tissue and wound cultures being negative for bacterial growth. it is also interesting to note that the average length of time taken for our npwt treatment (15.9 days) was shorter than the average length of time taken for other similar conventional treatments (23.3 days) (15). along with the international expert panel on negative pressure wound therapy have recommended a range of npwt settings between 50 and 150 mmhg (16). lower negative pressures may be considered for pain reduction, and higher negative pressures may be considered for high volume of wound exudates (16). in our study, npwt settings were between 100 mmhg (four patients) to 120 mmhg (six patients). lower negative pressures (100 mmhg) were used for pain reduction and higher negative pressures (120 mmhg) were used for a high volume of wound exudates. limitations of this study includes a low patient number, short term follow - up and lack of comparison of the renasys - go therapy versus other forms of npwt or conventional dressings. with the positive results seen in our case series, we recommend future studies such as a prospective randomized controlled clinical trial comparing the efficacy of renasys - go therapy versus other treatment modalities. in this study, the renasys - go npwt system has been shown as beneficial in the treatment of diabetic lower limb ulcers and wounds, which may include wounds with exposed tendons, fascia or bone after surgical debridement. in addition, this npwt system was able to prepare all wounds for closure via stsg or secondary healing by promoting sufficient granulation tissue and by reducing bacterial infection of the wounds in a reasonable amount of time (average of 15.9 days). the authors declare that they have no conflict of interest and have not received any funding or benefits from industry to conduct this study. | introductionthis case series aims to study the effectiveness of renasys - go negative pressure wound therapy system in the healing of diabetic lower limb ulcers.materials and methodsan electronic vacuum pump (renasys - go, smith & nephew gmbh) was used to apply negative pressure wound therapy on wounds, with pressure settings determined according to clinical indication. changes in wound dimension, infection status and duration of treatment were recorded over the course of renasys - go therapy in 10 patients with diabetic lower limb ulcers.resultshealing was achieved in all wounds, three by secondary closure and seven by split - thickness skin grafting. eight wounds showed a reduction in wound size. the average duration of treatment with renasys - go therapy was 15.9 days, and all wounds showed sufficient granulation and were cleared of bacterial infection at the end of therapy.conclusionsrenasys - go therapy may be beneficial in the treatment of diabetic lower limb ulcers and wounds. in this study, which included wounds presenting as post - surgery ray amputation, metatarsal excision wounds, post - debridement abscesses and ulcers, the renasys - go therapy prepared all wounds for closure via split - thickness skin grafting or secondary healing by promoting granulation tissue and reducing bacterial infection in approximately 2 weeks. |
while public health and biodefense often have overlapping elements, the capabilities needed to support each mission are not identical. thus, the dhs scientific response to ebola required initial investments of time and resources to develop additional capabilities and protocols, contributing to the time lag in obtaining actionable results. we are currently working to expand capabilities to hasten our next response, which should generate results weeks to months faster in the next crisis. this includes identifying and establishing protocols for working with a wide range of disease transmission matrices (ie, blood, sweat, tears, etc) and surfaces that may harbor microbes (eg, wood, carpet, furniture, clothing). by developing agent - agnostic capabilities, dhs s&t will be able to respond quickly and efficiently to a broad array of public health events, generating complete, informative, actionable data no matter what the future threats might be. generating and using a master question list for ebola was central to s&t 's communication, research prioritization, and response. it allowed us to work closely across the federal government including hhs, dod, and the white house via the ebola task force through informal communication, regular meetings, scientific information sharing, and formal coordination to maximize unique resources and mission space while collectively supporting a synergistic response. it is by no means revolutionary that organizing known information and using it to make informed decisions is helpful, but a crisis response is less about breakthroughs and more about systematic application of tried and true principles. we are currently working to develop master question lists for a number of diseases representing high - impact human health risks. these master question lists follow the same general structure as the ebola list, with careful attention to ensure a broad applicability. for example, one category is decontamination : what are effective methods to neutralize the virus in the environment ? rather than ebola - specific questions, such as how are west africa ebola treatment units decontaminated ? these categories, as delineated for ebola in figure 1, allow a guided flexibility to adapt the master question list framework to a variety of pathogens. master question lists will aid in research prioritization by identifying where current knowledge gaps reside, matching gaps to existing roles and responsibilities across the us government, and preventing duplication of effort. developing these knowledge resources in advance will facilitate better communication and a faster, more organized response, both within dhs and across the federal government. this success was recently demonstrated with the zika virus outbreak, where dhs developed a master question list, which was requested by and shared with departments across the interagency. in addition, we have collected 76 scientific and technical questions asked of us in the frenetic days following the initial ebola diagnosis in the united states, including questions from dhs components, academics, private companies, and us and international government groups. while some of these requests were specific to one group, many more were broadly applicable. often, questions were relevant to multiple groups despite being asked by a single group. the majority of the requests could be answered from existing information, highlighting the need for scientific expertise to be available for crisis response. the questions affecting the broadest stakeholder base were regarding screening and surveillance (likely due to the fear of the virus entering the united states and spreading domestically). analysis also showed that s&t had the most information to share, and the office of health affairs was the biggest customer of this information (this group also had the largest overlap with cdc mission areas). working together with the dhs offices of policy, health affairs, and fema, s&t is helping our department define the requirements to codify and implement policy changes to improve future responses. this includes identifying the needs and responsibilities of each group and establishing best practices for information sharing between groups. we have identified what information was requested by each group to support their ebola response, and we are asking them to expand that input by viewing all 76 requests to see where the needs and resources of others might be helpful. we will then attempt to extrapolate the needs and responsibilities to a broader, disease - agnostic set of requirements. these exercises will strengthen the communication infrastructure and highlight where information is available, leading to faster future responses for dhs and the federal government. we never want a crisis, but one good thing that comes out of critical situations is that they bring people together in a unity of effort. dhs employees from all the components and scattered across the country were reaching out to each other to ensure a safe and effective response. by nature we always turn to those we know and trust, but an ad hoc communication network is not ideal, as it results in pockets of information sharing, filtering of requests and results, and exclusion of those who may not be connected but very much need to know. for example, the safety of reusable equipment was discussed extensively to ensure healthcare facilities would not inadvertently infect nave patients and also to ensure that diagnostic samples were not contaminated by previously used equipment. preexisting laboratory expertise was leveraged to alleviate concerns, and this information was broadly shared within ad hoc networks. however, there were clear failures, such as when a new group was referred to dhs almost a year later asking the same questions. a faster, more effective, and broader network can be created by identifying positions rather than individuals. at dhs s&t, we have approached this problem through parallel top - down and bottom - up approaches, identifying the positions we think are relevant in each dhs component as well as asking components to identify relevant experts. in addition, future crises will be addressed through the strong executive secretariat system that exists for coordinating decision making and managing information flow. these communication mechanisms and experts will be part of the information - sharing exercises described above. all dhs components and elements will have the opportunity to promote the needs and capabilities of their organization, establishing formal relationships for future responses. in a crisis situation, each party has a limited time - frame in which to contribute and have an impact. while no response will ever be perfect, we should all strive to use the best available data to make decisions. : (1) clearly define research in the context of the larger problem. why should policymakers (and through them, the public) care about your research ? this can be set up in advance by developing research directions and project plans where applications (ie, guiding policy) are an end - goal. (2) ensure results are actionable and clear ; note risks, but do not obscure conclusions with an excess of footnotes and nuances. (3) present results in a way that will maximize their impact on nontechnical communities. some scientific journals have begun to include a short author summary in addition to the abstract. this could be an ideal place to convey actionable results to decision makers in clear, nontechnical terms. the article will still contain all the relevant technical details, and those will be available to anyone reading the article, but this executive summary can concisely guide nontechnical audiences. after all, do n't we all want to see our science improve the world ? while all of this may be challenging, there are many great resources available to improve science communication skills, such as the alan alda center for communicating science at stony brook university or the american association for the advancement of science, among others. for government decision makers, we recommend actively seeking out and leveraging scientific information and experts when making decisions and determining policy. increasingly, science and technology are inextricably dual - use, meaning that they drive both new threats and the needed solutions to our nation 's challenges. cultivation and use of a technical core is inextricably linked to meeting the demands of the future, and experts are necessary to help chart a successful course. we at dhs s&t are developing a new initiative called the science advisory group of experts (sage) to help us incorporate science early and often in decision - making processes. this effort pre - identifies scientists in academia or private industry with recognized expertise pertaining to dhs mission areas who are willing to be on stand - by to provide technical advice and support practical decision making based on actionable scientific data, particularly in the event of a crisis. to our colleagues in government, we are willing to share this rolodex of expertise. and to our fellow scientists, if you are interested in joining the sage team, please contact dhs s&t. at the time of this writing, there are a number of diseases threatening both the united states and international communities. the zika virus outbreak has been declared a public health emergency, and mers, chikungunya, avian influenza, and measles are also of concern. we want to prepare before an emergency to ensure that our response is better, faster, and cheaper, no matter what or where the disease might be. we need to develop protocols, methods, information resources, and communication plans as part of our response infrastructure and have that infrastructure and expertise embedded in our response plans before the next outbreak occurs. we need to work with the policy and technical communities to ensure that, where possible, actionable scientific data are used to shape decisions and guide operations. it is our hope that melding science, policy, and decision making will result in faster and more effective responses to future crises. | the department of homeland security 's (dhs) science and technology (s&t) directorate plays a role in public health that extends beyond biodefense. these responsibilities were exercised as part of the 2014 - 16 ebola outbreak, leading to productive and beneficial contributions to the international public health response and improved operations in the united states. however, we and others have identified numerous areas for improvement. based on our successes and lessons learned, we propose a number of ways that dhs, the interagency, and academia can act now to ensure improved responses to future public health crises. these include pre - developing scientific capabilities to respond agnostically to threats, and disease - specific master question lists to organize and inform initial efforts. we are generating dhs - specific playbooks and tools for anticipating future needs and capturing requests from dhs components and our national and international partners, where efforts will also be used to refine and exercise communication and information - sharing practices. these experiences and improvement efforts have encouraged discussions on the role of science in developing government policy, specifically responding to public health crises. we propose specific considerations for both scientists and government decision makers to ensure that the best available science is incorporated into policy and operational decisions to facilitate highly effective responses to future health crises. |
a 58-year - old woman presented to the emergency department with a stuporous mental state, ophthalmoplegia, and severe dysarthria. she had a prior history of cardiac valve replacement surgery for both mitral and aortic regurgitations 10 years earlier. her national institutes of health stroke scale (nihss) score was 14 and modified rankin scale (mrs) score was 5. a ct scan of the head five hours after the onset of symptoms showed a hyperdense basilar artery on two consecutive 5-mm cut images, indicating an acute basilar artery embolism. after obtaining written informed consent for the endovascular therapy from the patient 's family, the angiographic procedure began 5.5 hours after the onset of stroke. at this point, a loading dose of abciximab (0.25 mg / kg) was administered intravenously. rapid recanalization was achieved using a minimum dose of thrombolytic agents by performing direct angioplasty first followed by intra - arterial thrombolysis. the balloon catheter was advanced into the occlusion site and inflated to 4 atm for 30 seconds. immediately after the angioplasty, the basilar artery was partially recanalized with a restoration of the blood flow to the both superior cerebellar arteries. a microcatheter was then placed just proximal to the occlusion site, and 400,000 units of urokinase were infused at a constant rate for one hour. the completion angiograms demonstrated a partial recanalization of the basilar artery with embolus remaining in the basilar tip and the restoration of blood flow to both the posterior and superior cerebellar arteries. in addition, angiograms demonstrated a single perforating thalamic artery that was divided into two branches (figs. 1b, c). after the endovascular procedure, the patient showed progressive clinical improvement and regained complete consciousness within a few hours. a non - enhanced ct scan obtained immediately after the endovascular therapy showed bilateral symmetrical high attenuations in the paramedian thalamic regions (fig. the maximum hounsfield unit of hyperdense lesions was 67. on the following day, her nihss score was 6 and the patient developed hypersomnolence, severe memory impairments, and vertical gaze palsy. the follow - up nonenhanced ct scan obtained 24 hours after completing the intra - arterial thrombolytic therapy showed the disappearance of the hyperdense lesions and the development of hypodensities in the bilateral paramedian thalamic regions without a hemorrhagic transformation (fig. after three months, the patient improved to functional independence (nihss score from 14 to 4 ; mrs score from 5 to 1) but continued to have both short and long - term memory dysfunction as well as vertical gaze palsy. a hyperdense lesion observed on the post - therapeutic, nonenhanced ct scans obtained immediately after intraarterial thrombolysis has been described as either a hemorrhage or an extravasation of the contrast medium given during the angiographic procedures performed during the thrombolysis procedures (2 - 4). yoon. (1) reported that a hyperdense lesion that has different characteristics from a hemorrhage in terms of both the ct findings and the clinical outcome could be categorized as two entities : contrast enhancement and contrast extravasation. in their study, contrast enhancement was defined as a hyperdense lesion that disappeared on the 24-hour follow - up ct scan, without leaving a hematoma cavity or a mass effect, and contrast extravasation as a hyperdense lesion with a maximum hu > 90, which persisted on the 24-hour follow - up ct scan. they also concluded that both the contrast enhancement and contrast extravasation develop primarily in the region of the basal ganglia in accordance with the specific vascular territory (1). the ct findings in the present case are consistent with contrast enhancement, which occurred in the thalamus after an intra - arterial thrombolysis procedure, according to the definition by yoon. it has been reported that contrast enhancement and contrast extravasation occur primarily in the basal ganglia after intra - arterial thrombolysis in those patients with an occlusion of the middle cerebral artery (1 - 4). to the best of our knowledge, there are no reports regarding thalamic enhancement after thrombolysis in patients with an acute occlusion of the basilar artery. the prolonged infusion of thrombolytic agents and contrast materials in the vicinity of the perforating thalamic branches may have resulted in the leakage of the contrast medium through the injured blood brain barrier (bbb) of the perforating microvessels supplying the thalamus. this can lead to the gradual accumulation of contrast medium, which manifests as contrast enhancement on the nonenhanced ct scans obtained immediately after the thrombolytic procedure. in this case, the contrast enhancement occurred in the symmetrical bilateral paramedian regions of both thalami. the suggested mechanism for this finding is a disruption of the bbb of a single perforating thalamic artery (paramedian artery), which supplies the paramedian part of the upper midbrain and thalamus, including the intralaminar nuclear group and most of the dorsomedial nucleus (5). in this case, angiography clearly demonstrated a single paramedian artery. it was reported that a single perforating thalamic artery can supply the paramedian territory on both sides, and an occlusion of this artery can result in corresponding bilateral infarctions in the paramedian aspects of the thalami (6). a cadaver dissection study reported that the origin of the perforating thalamic arteries was unilateral in eight of 12 brains (7). in conclusion, contrast enhancement can occur in the thalamus after an intra - arterial thrombolysis procedure in patients with an acute basilar artery occlusion. in addition, a pathological process involving a single perforating thalamic artery can result in symmetrical bilateral disease in the paramedian regions of both thalami. | a 58-year - old woman presented with an acute embolic occlusion of the distal basilar artery. she underwent angioplasty and intra - arterial thrombolysis. angiography performed after recanalization revealed a single perforating thalamic artery. a nonenhanced ct scan carried out immediately after the procedure revealed hyperdense lesions in the bilateral paramedian portions of the thalami, which disappeared on the 24-hour follow - up ct scan. three months later, the patient improved to functional independence, but had some memory dysfunction and vertical gaze palsy. this case suggests that contrast enhancement or extravasation can occur in the thalamus after intra - arterial thrombolysis performed to recanalize a basilar artery occlusion. |
zoon 's balanitis or balanitis circumscripta plasmacellularis is a benign chronic disease of unknown origin. zoon 's balanitis, unlike some other inflammatory penile dermatoses, is generally not thought to be a precursor for neoplasia. we herein report the case of an uncircumcised man, who developed clinically and histopathologically evident squamous cell carcinoma of the penis in an area affected by zoon 's balanitis. a 71-year - old uncircumcised man presented with a 3-month history of extremely painful genital sores associated with swollen foreskin and dysuria, not preceded by any flu - like symptoms. the patient reported that he has suffered from zoon 's balanitis for 12 years, confirmed by a histopathological examination. he treated the condition with topical corticosteroids with relative improvement and paid periodic visits for 7 years ; after that he stopped performing any follow - ups. clinical examination revealed a well - demarcated, highly indurated, glistening erythematous plaque (about 3 cm in diameter) on the glans and adjacent preputial mucosa. a 3-mm punch biopsy of the glans was performed showing typical features of a moderately differentiated squamous cell carcinoma (nests of atypical cells with abundant eosinophilic cytoplasm and large vesicular nucleus) (fig. zoon 's balanitis is characterized by a solitary red - orange plaque of the glans and prepuce. although the etiology is unknown, different factors have been reported to be involved in its genesis (local infections, poor hygiene, heat, friction, and constant rubbing). histopathologic examination demonstrate an inflammatory infiltrate of predominantly plasma cells, atrophy of epidermis, spongiosis, tortuous dermal blood vessels, and exocytosis of inflammatory cells [2, 3 ]. it is generally considered to be a benign condition, and its association with malignancies has been rarely reported. in 1999, a case of penis carcinoma arising in a patient with zoon 's balanitis was described by joshi, implying that this inflammatory lesion could be a premalignant condition or could contribute to the development of squamous cell carcinoma. after this report, the hypothesis that zoon 's balanitis may be followed by penile carcinoma or premalignant lesions started to arise. in 2001, bunker claimed that there were zoonoid changes in clinical and histological features in some cases of lichen sclerosus, lichen planus, bowenoid papulosis and penile cancer. these zoonoid changes could suggest that zoon 's balanitis per se is a premalignant condition. recently, starritt and lee reported a case of erythroplasia of queyrat of the glans on a background of zoon 's plasma cell balanitis. the hypothesis of the possible association between zoon 's balanitis and penile cancer could also be linked to the concept of inflammation as a cofactor in carcinogenesis. in 1863, virchow noted a connection between inflammation and cancer and sustained that this link may have implications for cancer prevention and treatment. therefore, prevention of penis carcinoma is possible, including early detection and relative treatment of risk factors (phimosis, inflammatory conditions, multiple sexual partners, and hpv infections) and precancerous lesions [9, 10 ]. this case highlights the importance of frequent follow - ups for zoon 's balanitis ; topical treatment, such as corticosteroids and calcineurin inhibitors [4, 5, 7 ], may be important to prevent the development of a secondary carcinoma. in case of resistance to topical treatments, circumcision should be recommended because it could be curative and also protective against penile carcinoma. | zoon 's balanitis or balanitis circumscripta plasmacellularis is a chronic disease of unknown origin. this condition usually manifests in middle - aged or elderly uncircumcised men. although of unknown etiology, different factors have been reported to be involved in its genesis (local infections, poor hygiene, heat, friction, and constant rubbing). it is generally considered to be a benign condition, and its association with malignancies has been rarely reported. we report the case of an uncircumcised man, who developed clinically and histopathologically evident squamous cell carcinoma of the penis in an area affected by zoon 's balanitis. |
the genomic revolution raised expectations of unprecedented advances in health care,1,2 but actual scientific progress remains constrained by the double bind of limited available empirical data and concerns over human safety in research. failure to address this issue could result in general disappointment over inadequate translation of scientific knowledge into actual improvements in the quality of human life, leading to loss of confidence in science - based initiatives and continued trends toward pseudoscientific and antiscientific alternatives epitomized by the antivaccination movement.3 on a more positive note, timely resolution of the issue could empower the global health system. this commentary thus aims to outline possible future directions of health research with emphasis on antibody - mediated immunity as a concept central to pharmaceutical product development that prioritizes human safety. global health may be defined as collaborative transnational research and action for promoting health for all, wherein health for all refers back to the 1978 declaration of alma ata as proclaimed by the world health organization (who).4 in the preamble to the who constitution, health itself is defined as a state of complete physical, mental and social well - being and not merely the absence of disease or infirmity, which has been criticized as practically meaningless.5 a more pragmatic view of health is that of just health, which entails meeting health needs fairly via consensus - building among stakeholders, subject to real - world constraints on available resources.6 to inform the negotiation of just health, health outcomes must be predicted for resource - allocation options. modern science, in the sense of empirically validated predictive mathematical models,7,8 provides means for predicting outcomes relevant to health and other human needs, thereby enabling translation of scientific knowledge into practical applications.9 this motivates the pursuit of biomedical research, often with the expectation that reductionist approaches will continue to drive the generation of new knowledge.10 undeniably, reductionism has enabled science - driven technological revolutions, but negative unintended consequences of technological change caution against overly simplistic reductionist approaches.11 global health is thus conditioned by the bioethical principle of nonmaleficence (i.e., avoidance of causing harm), which is subsumed under the precautionary principle (i.e., assigning the burden of proof to proponents of activities that may threaten health and the environment).12 such risk aversion is reflected in prevailing regulatory regimes, which remain committed to using animal models for evaluating safety under the assumption that such models are valid for human biomedical applications despite interspecies biological differences.13 meanwhile, animal - welfare concerns present a growing barrier to animal use, especially with the validity of animal models being called into question.14 hence, translational science must meet demands for evidence of safety while being denied conventional means for producing such evidence. animal - based safety studies might be supplanted by phase 0 clinical trials, whereby pharmacokinetic and pharmacodynamic properties of investigational new drugs are initially explored using subtherapeutic doses administered to healthy human volunteers.15,16 still, even seemingly low doses may produce adverse effects in ways that are difficult to predict for new drugs by virtue of their novelty. biological complexity limits the applicability of reductionism framed with deterministic linear mathematical models,8 which historically have dominated science as they are more computationally tractable and humanly comprehensible than stochastic nonlinear mathematical models more appropriate for describing and predicting biological systems behavior. such stochastic models must be developed and validated on the basis of empirical data if they are to be useful for health - care decision support.7 unfortunately, the required data are still critically lacking ; and even if this paucity of data were overcome, uncertainty would still be inherent in the predictions generated by any stochastic models developed. reluctance to permit initial trials of novel interventions on the grounds that they entail unacceptably uncertain risk levels itself limits the generation of new empirical data that might yield better estimates of the said risk levels, thereby perpetuating a vicious circle of risk aversion and limited capacity for risk assessment. to break the aforementioned vicious circle, one possibility is to develop new interventions in conjunction with specific countermeasures from the outset, in the interest of safety. where the interventions would entail the administration of pharmaceutical agents, the countermeasures could include the administration of specific antidotes, such that new pharmaceutical agents would be concomitantly developed in tandem with their antidotes. given a novel candidate pharmaceutical agent, a corresponding prospective antidote might be developed on the basis of specific ligand - receptor interactions. if the agent is regarded as a ligand that binds to a particular target receptor, the antidote might be an alternative ligand that competitively binds to the same receptor ; otherwise, the antidote might be an alternative receptor that competes with the target receptor for binding by the agent. developing antidotes as alternative ligands necessitates sufficiently detailed knowledge of the target receptors, which nonetheless fails to guarantee production of alternative ligands that actually function as antidotes (e.g., as pharmacologic antagonists rather than pharmacologic agonists). in contrast, developing antidotes as alternative receptors is amenable to implementation by means of a generally applicable strategy, namely the production of specific antibodies against virtually any agent, such that antidote effects are realized as antibody - mediated immunity. the production of antibodies against pharmaceutical agents is well - known for biologicals, which tend to be immunogenic. this is problematic where biologicals elicit antibody responses that compromise efficacy and result in hypersensitivity exemplified by serum sickness, wherein a systemic hypersensitivity reaction develops against heterologous serum proteins, as may occur with serum therapy (i.e., passive immunization using serum antibodies). serum therapy was extensively employed in the treatment of infectious diseases prior to the widespread availability of antibiotics17 and is a classic example of antibody - based prophylactic and therapeutic approaches, for which antibodies of human origin or having humanized sequences tend to be preferred over unmodified nonhuman antibodies in contemporary clinical practice.18 a more recent example of antibody - mediated immunity to biologicals is that of neutralizing autoantibodies against therapeutic erythropoietin, which tend to be produced as a result of pharmaceutical product adjuvanticity due suboptimal manufacturing processes.19 such problems can occur where proteins in biologicals contain potentially immunogenic t - cell epitopes as is typical even among autologous proteins, which has motivated efforts to minimize the immunogenicity of biologicals via t - cell epitope deletion.20 yet, the immunogenicity of even autologous proteins suggests the possibility of producing functionally neutralizing antibodies against virtually any self - protein. neutralizing anticytokine antibodies have thus been developed for the treatment of inflammatory conditions wherein excessive cytokine production contributes to immunopathology;21 and such antibodies may also serve as antidotes to therapeutic cytokines, for mitigating cytokine - mediated toxicity. although such drugs are typically haptens, antibodies may nonetheless be produced against them (e.g., by effectively rendering them immunogenic via conjugation to strongly immunogenic carrier molecules). this enables, for example, the production of antibodies against the cardiac glycoside digoxin, which yield fab fragments useful as an antidote for toxicity due to digoxin and even other structurally similar cardiac glycosides.22,23 to place the role of antibodies as antidotes into proper perspective, this is but only one aspect of antibody - mediated immunity, which encompasses a rich variety of immune effector mechanisms that can potentially contribute to the control and prevention of clinical conditions in general.24 whereas such immunity was previously thought of as being confined to extracellular spaces, effector mechanisms have been identified that operate within intracellular spaces as well, for example, within endosomes (e.g., of epithelial cells wherein internalized dimeric iga complexed with the polymeric - immunoglobulin receptor pigr mediates intracellular neutralization of invading virions25) and even cytoplasm (e.g., of cells expressing the ubiquitin ligase trim21, which catalyzes the ubiquitinylation of igg - bound viral capsids to facilitate their proteasomal degradation and thereby interrupt the viral replication cycle26). antibody - mediated immunity can contribute to the control of both infectious and noninfectious diseases, in the latter case possibly by selective targeting of self molecules. however, antibody - mediated immunity itself may contribute to adverse effects, incuding antibody - mediated enhancement of infection (as observed among various viral and cellular pathogens,27 especially enveloped viruses in settings of low - affinity antibody binding and low antibody concentration) and excessive loss of self - molecule function. from the standpoint of safety, passive immunization by administering exogenous antibodies is preferable to active immunization that induces endogenous antibody production, considering the potential complications of active immunization in the event of adverse antibody - mediated effects. although such effects could be mitigated in either passive or active immunization schemes by administering an antidote (e.g., an antibody that binds and thereby neutralizes the antibody mediating adverse effects), passively acquired antibodies eventually would be eliminated from the recipient, in which case administration of the antidote might be unnecessary if antibody elimination kinetics are sufficiently rapid ; but active immunization may induce protracted endogenous production of antibodies, such that the antidote might be administered repeatedly over a prolonged and possibly indefinite period. furthermore, in the absence of any available antidote, the adverse effects of active immunization might be essentially permanent, which argues for the development of antidotes for such effects where active immunization is contemplated. the preceding considerations, particularly the potential benefits of developing antidotes to prophylactic and therapeutic agents, suggest a general strategy for pharmaceutical product development based on monoclonal antibodies. structurally distinct monoclonal antibodies may be generated against a pharmaceutical agent, such that each monoclonal antibody may serve as a distinct alternative antidote to the agent, thereby providing a range of possible antidotes from which may be selected one that is most appropriate for administration to a particular patient (e.g., to avoid idiosyncratic hypersensitivity reactions). nevertheless, the historical cost - effectiveness of antibody - mediated immunity in public - health terms has been overwhelmingly due to active immunization, via mass vaccination, rather than passive immunization. although novel prophylactic and therapeutic approaches based on antibody - mediated immunity are likely to be more acceptable in terms of safety if initially realized via passive rather than active immunization, economic considerations would motivate subsequent attempts to induce similar immunity via active immunization (e.g., by vaccination), which would entail polyclonal rather than monoclonal antibody responses. this poses the problem of developing vaccines that are efficacious yet safe, particularly in the sense of also developing antidotes that mitigate antibody - mediated adverse effects that might occur consequent to active immunization with the vaccines. b - cell epitope specificities of monoclonal antibodies produced against the same immunogen may vary widely even among clones from the same immunized individual. although investigators may selectively retain clones with desirable properties (e.g., secretion of protective antibodies) while discarding other clones with undesirable properties (e.g., secretion of non - protective antibodies that actually enhance infection), such artificial selection is much less feasible in vivo. this presents a major challenge when attempting to develop active - immunization schemes (e.g., vaccination) that recapitulate antibody - mediated immunity achieved via passive immunization with monoclonal antibodies. if, for example, a protein contains multiple structurally distinct b - cell epitopes, in - vivo polyclonal antibody responses to the protein are likely to yield antibodies that target the different epitopes, albeit to varying extents as a function of multiple factors including immunogen structure (which may be at least partially denatured in the course of immunization28), host genetic background (e.g., favoring tolerance toward epitopes similar to those of self antigens) and history of prior immunization (e.g., resulting in original antigenic sin,29 wherein subsequent immune responses are biased toward epitopes resembling previously encountered ones) ; at worst, the immune responses can be completely non - protective (e.g., by failing to produce protective antibodies against key target epitopes) and also harmful (e.g., by producing antibodies that mediate enhancement of infection). to address the problem of developing active - immunization schemes for optimal targeting of polyclonal antibody responses against polyepitopic targets, artificial means may be employed to avoid counterproductive immunodominance (i.e., to artificially bias immune responses toward protective epitopes and away from other epitopes that may be non - protective and even disease - enhancing). one proposed approach is immune refocusing,30 whereby immune responses are selectively focused on intended target epitopes (e.g., of pathogen - neutralizing antibodies) on engineered variants of naturally - occurring macromolecular targets from which other potentially immunodominant epitopes (e.g., immunological decoys evolved by pathogens to evade host immune systems) have been deleted ; however, this is technically challenging insofar as the selective epitope deletion should maintain the intended target epitopes intact. an alternative approach is the use of peptide - based immunogens to elicit antipeptide antibodies that cross - react with functionally critical epitopes on the intended macromolecular targets.31 the use of antipeptide antibodies to produce intended biological effects is straightforward where the actual target is itself a peptide, especially where it is so short as to comprise only one or very few b - cell epitopes, for example, in the case of the octapeptide angiotensin ii.32 in such cases, the immunizing peptide may structurally mimic the target peptide such that the elicited antipeptide antibodies cross - react with the target peptide. a major challenge is posed by the problem of ensuring that antipeptide antibodies actually cross - react with protein targets ; even where the immunizing - peptide sequence is chosen to exactly match a segment of a target protein with which the antipeptide antibodies thus obtained are intended to cross - react, cross - reaction may fail to occur (e.g., because the corresponding protein sequence is structurally inaccessible or in a conformation different from that of the immunizing peptide). moreover, where cross - reaction does actually occur, this fails to ensure that intended biological effects (e.g., protection against rather than enhancement of disease) are produced. these challenges historically have hindered progress toward developing peptide - based vaccines, in spite of extensive computationally - guided work focused mainly on b - cell epitope prediction.33 nevertheless, the development of peptide - based immunogens as vaccines has become the subject of renewed interest in recent years, especially with improvements in computational support for vaccine design and in vaccine technology overall.34,35 with regard to b - cell epitope prediction for peptide - based immunogen design, the computational problem is now better understood in terms of physicochemical and biological correlates,36,37 and many new epitope - prediction tools are now available.38 furthermore, the realizations that antibody - mediated immunity may pose unacceptable risk of harm in certain cases (e.g., due to antibody - dependent enhancement of disease) and that purely cell - mediated immunity (i.e., based on adaptive t - cell immunity) may be effective in disease prevention suggest the prospect of peptide - based vaccines designed to include primarily (if not exclusively) t - cell epitopes.39 within the conceptual framework of pharmaceutical agents and antidotes that has been outlined thus far, risk aversion toward peptide - based vaccines could be mitigated by developing antibodies and peptides as antidotes. beyond in - silico preliminary analyses to avoid introducing potentially harmful peptide - vaccine sequences (e.g., those similar to epitopes of self, food or common environmental components), candidate vaccine peptides could be incorporated into immunogens for producing antipeptide monoclonal antibodies as candidate therapeutic or prophylactic agents. before administering these antibodies to humans, prospective antidotes to the antibodies could be developed for use in the event of any adverse antibody - mediated effects that might be observed among human subjects. the antidotes could be anti - idiotypic monoclonal antibodies (e.g., generated via immunization with the antipeptides antibodies or variable - region fragments thereof) or the peptides themselves (e.g., as unconjugated haptens in monomeric form to avoid proinflammatory immune - complex formation via antibody cross - linking), such that the anti - idiotypic antibodies would inhibit antigen binding by the antipeptide antibodies while the peptides would directly block the antipeptide - antibody paratopes. insofar as proteins and peptides are typically metabolized in vivo via main - chain peptide - bond hydrolyis,40 human - safety concerns over the prospective antidotes would be less problematic than for more exotic xenobiotics whose in - vivo toxicity is much more difficult to predict (e.g., in view of potentially complex biotransformation pathways41). the prospective antidotes might be less costly to manufacture as peptides rather than anti - idiotypic antibodies considering that chemical synthesis of the peptides would circumvent resource - intensive antibody production, although regulators may favor novel antibodies over small - molecule new chemical entities.42 demonstrating the safety of antipeptide antibodies in humans may provide justification for further studies on active immunization with peptide - based immunogens, toward vaccine development. efficacy of the antipeptide antibodies for therapeutic or prophylactic immunization could motivate further vaccine - development efforts, although such efforts might nonetheless be justified on the basis of cell - mediated immunity. in the event of any adverse effects due to endogenous antipeptide antibodies elicited by immunization with the peptide - based immunogens, the effects might be mitigated by administering peptides previously developed as antidotes to similar antibodies used for passive immunization. thus developing peptide - based vaccines as synthetic chemical products would conceivably entail lower manufacturing costs and fewer biosafety concerns relative to conventional vaccine - production technologies that utilize biological materials. in contrast to biologically derived proteins and more complex materials (e.g., containing viable pathogens or attenuated variants thereof), synthetic peptides are more amenable to nondestructive lyophilization for extension of product shelf life by avoiding solvent - mediated hydrolysis, such that a shift from conventional to peptide - based vaccines could obsolete the cold chain, which is an extremely crucial yet technically and economically demanding aspect of conventional vaccine logistics.43 peptide - based vaccines and, more generally, peptide - based immunogens could also play an instrumental role in the production of polyclonal antipeptide antibodies as therapeutic and prophylactic agents, considering that such antibodies could be recovered from human sources (e.g., via processing of expired human - donor plasma from medical blood - product banking facilities) or even immunized animals (as has been practiced since the advent of serum therapy, noting that this still remains a cost - effective measure, e.g., for treating tetanus using equine antitetanus antiserum,44 in the absence of contraindications such as known allergies or potential hypersensitivity reactions due to repeated dosing). the development of new therapeutic and prophylactic pharmaceutical agents in tandem with their corresponding antidotes is a plausible generally applicable strategy to resolve the current impasse over human - safety concerns as regards pharmaceutical products. in this context, antibodies can potentially fulfill dual roles as both antidotes and pharmaceutical agents in their own right, possibly for passive - immunization approaches that may provide proof of concept for subsequent active - immunization approaches such as vaccination. pharmaceutical antipeptide antibodies in particular can be targeted against individual epitopes, thereby facilitating evaluation of these epitopes from the perspectives of safety and efficacy, as a prelude to selective combination of epitopes in the design of peptide - based immunogens as vaccines. ultimately, the proliferation of geographically dispersed low - cost manufacturing of peptide - based vaccines and of pharmaceutical antipeptide antibodies could better enable the global health system for high - capacity yet locally responsive health - product development and distribution, proceeding along paths that explicitly prioritize human safety in scientifically well - grounded and practically meaningful ways toward the goal of just health. | if new scientific knowledge is to be more efficiently generated and applied toward the advancement of health, human safety must be more effectively addressed in the conduct of research. given the present difficulties of accurately predicting biological outcomes of novel interventions in vivo, the imperative of human safety suggests the development of novel pharmaceutical products in tandem with their prospective antidotes in anticipation of possible adverse events, to render the risks of initial clinical trials more acceptable from a regulatory standpoint. antibody - mediated immunity provides a generally applicable mechanistic basis for developing antidotes to both biologicals and small - molecule drugs (such that antibodies may serve as antidotes to pharmaceutical agents as a class including other antibodies) and also for the control and prevention of both infectious and noninfectious diseases via passive or active immunization. accordingly, the development of prophylactic or therapeutic passive - immunization strategies using antipeptide antibodies is a plausible prelude to the development of corresponding active - immunization strategies using peptide - based vaccines. in line with this scheme, global proliferation of antibody- and vaccine - production technologies, especially those that obviate dependence on the cold chain for storage and transport of finished products, could provide geographically distributed breakout capability against emerging and future health challenges. |
purified herba epimedii extract (chuan - ke - zhi, ckz), a chinese medicine preparation, has already attracted more and more attention in recent years for its curative effects on respiratory system diseases, such as bronchial asthma, chronic bronchitis, and chronic obstructive pulmonary emphysema [14 ]. and this purified extract was also reported to be able to regulate human humoral and cellular immunity [59 ] ; therefore, it has been gradually extended to the treatment of various cancers and immunological diseases [1013 ]. although ckz has been clinically applied for many years, there are still some difficulties and challenges on its in vivo process and mechanism of action. nowadays the pharmacokinetic research of traditional chinese medicine (tcm) are usually carried out in accordance with the research method of western medicine ; however, not like western medicine tcm always contains numerous compounds, which generally display different pharmacokinetic behaviour and may interact with each other in organism. therefore in order to evaluate the rationality and compatibility of chinese herbal compound formula, it is obviously important and necessary to investigate the pharmacokinetics of marker component as well as the possible interaction of other ingredients contained in tcm. according to the existing phytochemical and pharmacological studies [1420 ], herba epimedii contains a large number of flavonoid glycosides. most of them are characterized by an isopentenyl group substitution at the c8 position of flavonoid skeleton. these 8-isopentenyl flavonoid glycosides, of which epimedins a, b, and c and icariin are the principal and representative, are thought to be main bioactive components of herba epimedii and its extract preparation. as a purified extract, ckz contains more abundant 8-isopentenyl flavonoid glycosides than this crude drug, and epimedin c is the most dominant compound, which is visually exhibited in figure 1. in the present study, therefore, the pharmacokinetic research of ckz firstly focused on the pharmacokinetic behaviour of epimedin c in rat, and then on the base of established and validated quantitative method for epimedin c in rat plasma, we compared the pharmacokinetic differences of epimedin c after intramuscular administration of individual epimedin c, combination solution of epimedins a, b, and c and icariin, and ckz to rats. finally, considering the poor oral absorption of epimedin c [22, 23 ], the absolute bioavailability of epimedin c administrated intramuscularly was investigated for the first time in this study. overall, our study could facilitate the understanding of absorption mechanism of flavonoid glycosides in herbal formulations and provide reference for the clinical application of this purified herba epimedii extract. epimedin a (purity 98%) and epimedin b (purity 98%) were purchased from shanghai ronghe pharmaceutical technology development co., ltd. epimedin c (purity 98%), icariin (purity 98%), and naringin (purity 98%) used as internal standard (is) were provided by the national institute for the control of pharmaceutical and biological products (beijing, china). (lot number 11072502, guangzhou, china), and the concentration of epimedins a, b, and c and icariin was determined to be 81.38, 68.49, 667.5, and 132.5 g / ml, respectively. hplc - grade methanol and acetonitrile were obtained from fisher company (fisher scientific, fairlawn, nj). the deionized water was prepared by a millipore purification system (millipore, milford, ma, usa) and filtered with 0.2 m membranes. hplc was performed on an agilent eclipse xdb - c18 (150 2.1 mm i.d. 5 m) column using an agilent 1100 series hplc system (agilent technologies, inc., usa) equipped with a g1311a binary pump, a g1379a vacuum degasser unit and a g1313a autosampler, and a g1316a sl thermostated column compartment. the temperature of column chamber was set at 40c. the mobile phase consisted of acetonitrile and 0.1% formic acid (35 : 65, v / v). an isocratic elution mode was adopted with a flow rate of 220 l / min. the hplc system was coupled to a triple - quadruple tandem mass spectrometer (api-3000 pulsar, applied biosystems, foster city, usa) equipped with an electrospray ionization (esi) source. analyst 1.4 workstation (applied biosystems, foster city, usa) was used for the control of equipment, data acquisition, and analysis. for the optimization of ms / ms parameters, the standard solutions of epimedin c and is prepared with 50% acetonitrile were infused at a flow rate of 20 l / min using a syringe pump (harvard apparatus, holliston, ma, usa). finally, the instrument was operated with the ionspray voltage, deflector voltage (df) and multiplier voltage (cem) at 5000, 200, and 2200 v, respectively. nitrogen was used as nebulizer gas (8 l / min), as auxiliary gas (7.5 l / min at 350c), and as curtain gas (8 l / min). the optimized mrm fragmentation transitions were m / z 867.3 659.3 for epimedin c and m / z 579.2 271.1 for is. the experimentation on rats obtained the approval from an independent ethics committee at guangdong provincial hospital of chinese medicine (approval number 2012038). the experiment was performed at an spf level laboratory, authorized by guangdong provincial government. sprague - dawley rats (300 20 g) were purchased from guangdong medicinal laboratory animal center. the animals fasted for 12 h with free access to water prior to administration. the four groups were administrated as follows : two groups dosed with epimedin c aqueous solution by intramuscular and intravenous administration, respectively, one group dosed with ckz by intramuscular administration, and one group intramuscularly administrated with the combination solution of epimedins a, b, and c and icariin whose contents are the same as those in ckz. the four aqueous solutions were administered to rat at the same epimedin c dosage (approximately 0.33 mg / kg). about 350 l of blood sample was collected from the suborbital vein into heparinized tubes before drug administration (0 h) and at 3, 6, 10, 15, 25, 35, and 45 min and 1, 2, 3, 4, 6, and 8 h after intramuscular and intravenous administration. and then each blood sample was centrifuged at 3000 rcf for 15 min at 4c, and the plasma was transferred into 1.5 ml centrifuge tube and stored at 80c until analysis. during routine analysis, each analytical run included blank rat plasma, a set of calibration samples, and a set of qc samples. a simple solid - phase extraction (spe) method was used for extracting epimedin c from rat plasma. the agilent ods - c18 spe cartridge was preconditioned with 1 ml methanol followed with 1 ml water. a 100 l aliquot of plasma sample was spiked with 10 l of is working solution (1 g / ml). the combination was vortexed for 60 s and then added to a pretreated cartridge. after the sample was drawn through the spe bed, the cartridge was washed with 1.0 ml water, followed by vacuumizing for 1 min to remove the aqueous effluent. the methanol eluate was transferred to a clean test tube and evaporated to dryness under a gentle stream of nitrogen at 40c. the residue was redissolved in 150 l of mobile phase (35% acetonitrile containing 0.1% formic acid) and centrifuged by a microspin filter tube (0.22 m nylon, alltech) at 10,800 g for 1 min. a 10 l aliquot of the solution was injected into lc - ms / ms for analysis. blank rat plasma samples were spiked with epimedin c standard solutions to determine the final concentration ranges (1, 3, 10, 30, 100, 300, and 1000 ng / ml). the curves were fitted by a weighted (1/x) least - squares linear regression method through the measurement of the peak - area ratio of epimedin c to is. the acceptance criterion for a calibration curve was a correlation coefficient (r) of 0.99 or better, and each back - calculated standard concentration must be within 15% deviation from the nominal value except at the lower limit of quantitation (lloq), for which the maximum acceptable deviation was set at 20%. the lloq was defined as the lowest concentration on the standard curve at which the standard deviation was within 20% and accuracy was within 100 20%, and it was established using five samples independent of standards. the analyte response at this concentration level should be > 5 times the baseline noise. to evaluate the accuracy and precision of established method, qc samples at four concentration levels (3, 30, 300, and 800 ng / ml) were analyzed in five replicates on three validation days. the assay accuracy was expressed as (observed concentration / nominal concentration) 100%. the accuracy was required to be within 85115%, and the intra- and interday precision are not to exceed 15%. to investigate the effect of the sample preparation, the extraction recovery and matrix effect were determined by comparing the peak areas between three types of samples : (1) plasma spiked with known amount of epimedin c before sample preparation ; (2) plasma spiked with known amount of epimedin c after sample preparation ; (3) the standard solution of epimedin c. the difference between peak areas of sample 2 and sample 3 reflects the extent of ion suppression. the accuracy (trueness) of the method was calculated by comparing theoretical and experimentally measured analyte levels. the stability of epimedin c in rat plasma was evaluated using 20 qc samples (five samples at each concentration). the stability was tested under the following conditions : (1) freeze - thaw stability of epimedin c in rat plasma through three freeze - thaw cycles ; (2) short - term stability of epimedin c in rat plasma at room temperature for 6 h ; (3) long - term stability of epimedin c in rat plasma stored at 80c for 30 days ; (4) postpreparative stability of epimedin c in rat plasma stored in the autosampler at 25c for 24 h. the validated method was applied to the pharmacokinetic and bioavailability studies of epimedin c in rats. the maximum plasma concentration (cmax) and time to cmax (tmax) were obtained directly from the concentration - time curves. the area under the concentration - time curve (auc0) was calculated using linear trapezoidal rule. the mean residence time (mrt) was calculated as aumc0/auc0, where aumc0 represents the area under the first moment plasma concentration - time curve. the mass spectrometric behaviour of epimedin c and is was studied using both positive and negative ion esi. it was found that epimedin c and is had good responses in negative ion detection mode with a low background noise level. the ms / ms spectra of epimedin c and is were shown in figure 3. the former was discarded because of its high noise level and the interference of endogenous substances. the latter offered a very clean sample which made the quantitative method robust and scalable. consequently, the spe method was used for sample preparation and further optimized by testing and comparing the influence of different solid - phase extraction cartridges (c8, c18, and hlb) on the recovery of analyte and is. agilent c18 spe cartridge was the best owing to its low background noise, ease of sample preparation, and relatively high extraction recovery. the optimal conditions were presented above. in order to improve the peak shape and enhance the signal response of analytes, and to reduce the run time, different analytical columns and mobile phase compositions were tried to achieve good resolution and symmetric peak shapes for epimedin c and is. by comparison with shiseido capcell pak c18 (150 2.0 mm i.d. 5 m) column, agilent xdb c18 (150 2.1 mm i.d. 5 m) column could obtain better chromatographic behaviour and higher signal response for the analytes. at last, the agilent xdb c18 (150 2.1 mm i.d. 5 m) column was selected for analysis. the modifier of mobile phase of acetonitrile - water binary solvent system was screened from ammonium acetate, acetate acid, and formic acid. as a result, the mobile phase consisting of acetonitrile - water containing 0.1% formic acid with an isocratic elution could improve the symmetry of peak shape and enhance the signal response. the flow rate was also optimized and finally set at 0.22 ml / min. under the above - mentioned hplc conditions, the retention time of epimedin c and is was within 3 min, and no interfering substance was detected at the retention time of analytes in blank rat plasma samples (shown in figure 4). the specificity of the analytical method was evaluated by analyzing individual blank plasma samples from six different sources. all samples were found to have no interference with endogenous substances and is at the respective retention position of epimedin c. typical mrm chromatograms of blank plasma spiked with is, plasma spiked with epimedin c, and the rat plasma sample after intramuscular administration of epimedin c, flavone combination, and ckz were shown in figure 4. the regression equation, linear range, and correlation coefficient of epimedin c were y = 0.00461x + 0.002 (1.001000 ng / ml) and r = 0.9981. the llod with a signal - to - noise (s / n) ratio of > 5 of epimedin c was 0.04 ng / ml, while the lloq with an s / n ratio of > 10 was 0.05 ng / ml, which was sensitive enough for pharmacokinetic study of epimedin c in rat plasma. intra- and interday precision and accuracy were calculated by analysis of variances, based on replicate analyses (three days, four concentrations, each n = 5) of qc samples. the mean extraction recovery rates of epimedin c from rat plasma were 86.6 3.1, 80.7 4.3, 80.1 2.6, and 78.4 1.1 at low, medium, and high concentrations, and the matrix effects were 90.71 5.3, 95.95 2.49, 94.57 0.82, and 100.9 2.21, respectively. the result of stability experiment showed that epimedin c was stable in autosampler (24 h) at 25c, bench - top (6 h) at room temperature, and under repeated three freeze / thaw cycles and frozen condition at 80c within 30 days (table 2), as rsds were within 15% for both the low and high concentrations. the method showed good precision, repeatability, and stability and was suitable for determination of epimedin c in biological samples. after administration of epimedin c, ckz, and combination of epimedins a, b, and c and icariin, the plasma epimedin c concentrations were successfully determined by lc - ms / ms method described above. the mean plasma concentration - time profiles of epimedin c were illustrated in figure 5. the result indicated that epimedin c exhibited rapid absorption with the plasma peak concentrations occurring at around 10 min and were quickly eliminated thereafter. in addition, the plasma concentration - time curves of epimedin c after intramuscular administration showed a second peak or shoulder in most rats. as shown in table 3, the data indicated that many pharmacokinetic parameters were significantly different among three intramuscular administration groups. values of the area under the plasma concentration versus the time curve (auc0) were 271.03 44.39, 311.41 128.11, and 448.45 137.74 for intramuscular administration of epimedin c, combination of four flavones, and ckz, respectively. compared with administration of individual epimedin c, auc of epimedin c in ckz group was statistically different (p < 0.05), while after administration of combination of four flavones, the auc value was also higher than that of individual epimedin c but the difference was not significant. the mrt0 and t1/2 values of epimedin c after administration of ckz and combination of four flavones were longer than corresponding values after administration of epimedin c, and the mrt0 value was significantly different (p < 0.01) among three groups. drug clearance (clz) is defined as the volume of plasma that would contain the amount of drug excreted per minute. in the present study, drug clearance (clz) of epimedin c was 1.26 0.21, 1.20 0.41, and 0.79 0.19 for epimedin c, combination, and ckz group, respectively. the data demonstrated that the systemic clearance of epimedin c is significantly different (p < 0.01) between epimedin c group and ckz group. in summary, longer mrt0 and t1/2, higher auc, and lower clz values of epimedin c in ckz group than epimedin c group implied that the purified herbal preparation may be superior to single compound in prolonging the duration of action of epimedin c. the bioavailability of flavonoids has received extensive attention for many years due to its poor oral absorption characteristic. information on the comparative kinetic and bioavailability studies of flavonoid glycosides from herba epimedii is important for understanding the biological effects of the herbal drug and its preparation. it has been reported that the oral bioavailability of individual epimedin c was higher than that of herba epimedii extracts. our results, on the contrary, turned out that, after intramuscular administration of the purified extract to rats, the absorption and bioavailability of epimedin c enhanced significantly in comparison with those of individual epimedin c administered intramuscularly, which suggests other components in cki could improve the bioavailability of epimedin c. because of the synergistic effects of other herbs / components, chinese herbal formula is usually more potent than individual herb / component, which has been confirmed by more and more researches on chinese medicine formula such as huangqin decoction [2427 ]. the purified herbal preparation might be more effective than individual epimedin c via intramuscular administration in clinical application. moreover, the above - mentioned report also demonstrated that epimedin c is poorly absorbed following oral administration of both individual compound and herbal extract and the absolute bioavailability of epimedin c is not more than 0.58% in rat. in the present study, we observed, compared to the oral administration, that epimedin c was quickly absorbed with an extremely high bioavailability after intramuscular administration ; the absolute bioavailability of this flavonoid glycoside was about 100%. clearly, an injectable form of epimedin c may be more effective and reasonable for the clinical application of the purified herba epimedii extract preparation. our study was designed to evaluate the pharmacokinetic parameters of epimedin c after a single dose of epimedin c, combination of four flavonoid glycosides, and ckz administered, respectively, to rats via intramuscular and intravenous injection and to compare the relative and absolute bioavailability of epimedin c. although there have been investigations on the pharmacokinetics of epimedin c administrated orally [22, 23 ], to the best of our knowledge, this is the first study to reveal the comparative pharmacokinetic and absolute bioavailability of epimedin c administrated intramuscularly. the results showed that among three intramuscular administration groups the differences of pharmacokinetic parameters such as clz, mrt0, and auc0 were statistically significant (p < 0.05). with the obtained pharmacokinetic results, it can be concluded that (1) after intramuscular administration of the purified herba epimedii extract to rats, the absorption and bioavailability of epimedin c enhanced significantly in comparison with those of epimedin c administered individually. in brief, other ingredients in the purified extract can affect the pharmacokinetic behaviour of epimedin c in rats ; (2) as for epimedin c, intramuscular administration is superior to oral administration due to higher absolute bioavailability ; (3) the results presented in this study implicated that the purified herbal preparation might be more effective and safer than individual compound. in conclusion, results from this work could provide some guidance for the clinical applications of this herbal drug extract. and in terms of the bioavailability of epimedin c | chuan - ke - zhi (ckz), a purified herba epimedii extract, is a potent chinese medicine preparation whose main bioactive components are a class of flavonoid glycosides such as epimedins a, b, and c and icariin. and epimedin c is far more abundant than other flavones in this extract. this study aims to investigate the pharmacokinetic and bioavailability of epimedin c and what effects, if any, other ingredients in ckz have on its pharmacokinetics. epimedin c, ckz, and a combination of epimedins a, b, and c and icariin were, respectively, administrated to rats, and then the pharmacokinetic parameters of epimedin c in the three groups were calculated and compared. the result indicated that clz, mrt0, and auc0 of epimedin c were significantly different among the three groups (p < 0.05), and compared with the epimedin c group, the absorption of epimedin c significantly increased in the ckz group. furthermore, in this study the absolute bioavailability of epimedin c was also investigated by comparing intramuscular and intravenous administration of epimedin c. as a result, epimedin c could be quickly absorbed with extremely high absolute bioavailability after intramuscular administration. |
in 2006, an unidentified respiratory disease occurred in rhesus monkeys (macaca mulatta) at a breeding farm in the guangxi zhuang autonomous region in southern china. the farm, the largest in china, comprised 31,260 monkeys, of which 1 in 5 was unweaned. the morbidity rate in young monkeys was 60%, with an 30% death rate (25% and 5%, respectively, for adults). in 2007, surviving monkeys were vaccinated with an inactivated suspension made from the livers and lungs of dead animals. after vaccination, the number of cases decreased during 2007 and 2008 to 100200 per year. cases occurred throughout 2006. because most authorized suppliers of monkeys to research laboratories in china obtain their breeding stock from this farm, the disease spread throughout china, particularly to experimental animal facilities in wuhan, kunming, and beijing (14). the disease also was introduced into a few wildlife parks in china ; however, perhaps because of the low population density of susceptible animals in these locations, further spread has not been reported. initially, cdv was not suspected as the causative agent of the monkeys illness. in late 2008, however, tissue specimens from infected animals that had been stored in a freezer for 2 years were analyzed and found to contain cdv. four serum samples from adult monkeys whose illnesses had naturally resolved had titers of 432 virus neutralizing antibodies against cdv, whereas virus neutralizing antibodies could not be detected in 3 serum samples from uninfected monkeys. after this identification, all experimental monkeys were vaccinated with attenuated cdv vaccine starting in early 2009. whether the vaccines actually boosted immunity or whether they were simply given coincident with waning of the outbreak from increasing immunity, the number of cases has since remained low (130 in 2009 and 2030 in 2010 [not all confirmed ]). additionally, anti - cdv serum seemed to help infected animals recover more rapidly from the infection. infected monkeys initially displayed measles - like signs, including respiratory signs ; anorexia ; fever ; and red rashes over the entire body, with reddening and swelling of the footpads ; conjunctivitis ; and thick mucoid nasal discharge. postmortem examination demonstrated discrete purple or rosy rashes on the body, with macules of 24 mm (figure 1, panel a), which in some cases were confluent and had a dark rosy color. rashes on the face were vesicular and ulcerated after suppuration, forming a scab. other signs included congestion, redness and swelling of the pars oralis pharyngis, diffuse hemorrhagic spots on the papillae of the tongue, suppurative conjunctivitis (figure 1, panel b), and rhinitis with copious thick mucous exudates. blood stasis patches in the lungs and interstitial fibrosis were observed in most affected lungs. infected monkeys also had blood stasis in parts of the liver, with tiny khaki - colored hemorrhagic spots on the surface. a) rash ; b) suppurative conjunctivitis. because of the signs of a measles - like infection, identification of the etiologic agent first focused on measles virus and later on cdv. reverse transcription pcr amplification of lung specimens by using virus - specific primers was negative for measles virus but positive for cdv. the supernatant of ground liver samples also was positive for cdv by immunochromatographic analysis (bit rapid color cdv cassette, bioindist co. ltd. lung specimens also infected tree shrews (12 months old) after subcutaneous injection, resulting in excitement and death from encephalitis and enterohemorrhage. the full - length viral genome was amplified and sequenced (genbank accession no. we constructed phylogenetic trees by the neighbor - joining method, using the full sequence of this virus and others available in genbank. the full genome of this isolate shared the highest homology with a ferret isolate (accession no. ay649446) from the united states, and a dog isolate (accession no. the l gene showed the highest identity (95.6%96.5%) with that of us ferret and raccoon isolates (accession nos. ay386316 and ay466011) ; phylogenetic analysis of the h gene indicated an eastern asian source of this isolate, which shared high homology with isolates from different species of animals in china, taiwan, and japan (figure 2, panel b). overall, the amino acid homology of the guangxi isolate with the others was 96.0%97.3%, clustering it in a large clade that includes cdv isolates from asia. nevertheless, the isolate is unique in that it contains multiple amino acid changes in its viral structural proteins, none of which have been found in other isolates. phylogenetic analysis of the canine distemper virus by comparison of the genome or gene of the monkey isolate with other canine distemper virus isolates. fx, fox ; cn, people s republic of china ; rd, raccoon dog ; dg, dog ; tw, taiwan ; mky, monkey ; cnkm, kunming, people s republic of china ; cnbj, beijing, people s republic of china ; jp, japan ; dm, denmark ; pdn, lynx pardinus ; sp, spain ; ft, ferret ; us, united states ; gm, germany ; bg, badger ; lp, lesser panda ; mk, mink ; rc, raccoon ; lpd, leopard ; nl, the netherlands. reasons for the epidemic remain unclear. the first monkey to contract the infection was in the farm in guangxi ; however, the source of the infection is unknown because there were no dogs or other fur - bearing animals at the farm. breeding facilities were self - contained, with no introduction of external animals and no other farms nearby. one possible source of infection is contact by the monkeys at the farm with local wild monkeys. another possibility is spillover of the virus from a stray dog carrying cdv that became adapted to the new host. large - scale breeding and caging of the monkeys might have contributed to increasing susceptibility to canine distemper infection. reared in groups of 2030 within fenced - off areas or in pens of several hundred, cdv appears to have been transmitted by droplets derived from feces or other body discharges, all of which contained cdv. this canine distemper outbreak poses a threat to monkey populations. because the guangxi farm routinely supplies monkeys for animal facilities throughout china, monitoring for canine distemper in its monkeys, including those shipped to other animal facilities, as well as human handlers, is advisable to prevent possible secondary spread and interspecies transmission. although cdv spread has been largely controlled by inactivated and live canine distemper vaccines, sporadic cases still occur and the high number of mutations in the virus makes future transmission unpredictable. therefore, surveillance for canine distemper should be considered among monkey populations and among humans who have close contact with them. | since 2006, canine distemper outbreaks have occurred in rhesus monkeys at a breeding farm in guangxi, people s republic of china. approximately 10,000 animals were infected (25%60% disease incidence) ; 5%30% of infected animals died. the epidemic was controlled by vaccination. amino acid sequence analysis of the virus indicated a unique strain. |
warts are benign epithelial neoplasms of the skin and mucosa resulting from human papillomavirus (hpv) infection. they are a common dermatologic complaint, with an estimated incidence of 10% in children and young adults (1). even if disease progression is naturally self - limited, the course of the disease is unpredictable, and treatment may be necessary. several treatment methods are available for treatment of warts ; however, most methods have specific disadvantages and side effects. topical management requires the application of drugs for long durations and treatment success is, therefore, highly dependent on patient compliance (2). laser treatment is based on the principle of photodermal or photomechanical destruction of the target tissue. depending on the pulse duration and energy density, this may result in the coagulation (photodermal effect) or blasting (photomechanical effect) of these structures (2). many studies have used 585-nm pulsed dye lasers for the treatment of warts, with the wart blood vessels as the target tissue (1 - 4). this is because hemoglobin in blood has strong absorption peaks at wavelengths ranging from 585 to 595 nm (5). moreover, hemoglobin has a significant, albeit more modest, absorption peak between 800 and 1,100 nm. therefore, it has been postulated that 1,064-nm nd : yag lasers could be used in the treatment of telangiectases (5). in addition, since there is decreased light absorption by melanin at this wavelength, there is reduced risk of pigmentary side effects (4). previous studies have shown that 1,064-nm nd : yag lasers used at longer widths can be used to treat telangiectasias of the lower extremities (5), face (6), and venous lake (7). we used a long - pulsed nd : yag laser in the treatment of warts, with the wart blood vessels as the target, and evaluated clinical outcomes. over the course of 1 yr, 369 patients (mean age, 21 yr ; range, 3 - 67 yr) (table 1) with recalcitrant or untreated warts were treated with a long - pulsed nd : yag laser. informed written consent was obtained from all participants before inclusion in the study. in all, 21 patients were lost during follow up, and therefore, the data of 348 patients were evaluated. warts were classified into three types - verruca vulgaris (212 patients), deep palmoplantar warts (68 patients), and periungual warts (68 patients). prior to the study, photographs of all the lesions were taken and the size of each lesion was recorded. the study employed a 1,064-nm long - pulsed nd : yag laser (cutera, inc., the following parameters were used : spot size, 5 mm ; pulse duration, 20 msec ; and fluence, 200 j / cm. prior to laser treatment, warts were treated with emla cream or a local lidocaine injection and were peeled with a razor blade. one or two courses of slightly overlapping laser pulses were applied to each wart, covering the wart itself and a 1-mm margin on the surrounding skin. around the third treatment date, crusts were found to be formed on the treated parts. at 1 to 2 weeks after treatment, the crusts were removed, and the warts were cleared, leaving only a small scar (figs. 5 - 7). clearance was defined as the complete absence of a clinically apparent wart, and treatment failure was defined as a persistent lesion after four treatments. over the course of 1 yr, 369 patients (mean age, 21 yr ; range, 3 - 67 yr) (table 1) with recalcitrant or untreated warts were treated with a long - pulsed nd : yag laser. informed written consent was obtained from all participants before inclusion in the study. in all, 21 patients were lost during follow up, and therefore, the data of 348 patients were evaluated. warts were classified into three types - verruca vulgaris (212 patients), deep palmoplantar warts (68 patients), and periungual warts (68 patients). prior to the study, photographs of all the lesions were taken and the size of each lesion was recorded. the study employed a 1,064-nm long - pulsed nd : yag laser (cutera, inc., the following parameters were used : spot size, 5 mm ; pulse duration, 20 msec ; and fluence, 200 j / cm. prior to laser treatment, warts were treated with emla cream or a local lidocaine injection and were peeled with a razor blade. one or two courses of slightly overlapping laser pulses were applied to each wart, covering the wart itself and a 1-mm margin on the surrounding skin. around the third treatment date, crusts were found to be formed on the treated parts. at 1 to 2 weeks after treatment, the crusts were removed, and the warts were cleared, leaving only a small scar (figs. 5 - 7). clearance was defined as the complete absence of a clinically apparent wart, and treatment failure was defined as a persistent lesion after four treatments. the average number of treatment sessions required for clearance was 1.49 (range, 1 - 4 sessions) (fig. 64% of warts were cleared, while 96% (336/348) of warts were cleared after the fourth treatment (fig. 2). verruca vulgaris responded better than the other types of warts and required fewer treatments for clearance (mean, 1.35 sessions). 1). the clearance rate after the first treatment was also higher in the verruca vulgaris group (72.6%) than 64.7% in the periungual warts group and 44.1% in the deep palmoplantar warts group (fig. the size of the warts was reduced by an average of 10% based on the diameter. this was observed in 69.1% of the verruca vulgaris warts, 52.5% of the periungual warts, and 27.4% of the deep palmoplantar warts. histopathologic examination after treatment showed separation of the dermoepidermal junction, epidermal necrosis, and red blood cell (rbc) extravasation. destroyed blood vessels surrounded by a dense inflammatory infiltrate were also shown in the dermis (fig. 4). since most (82%) patients who were anesthetized with emla cream complained of serious pain during treatment, local lidocaine injections were administered during the second treatment. other side effects included transient numbness (15%), hemorrhagic bullae (7%), hyperpigmentation (5%), and hypopigmentation (4%). in addition, 2% of patients with periungual warts experienced nail dystrophy. during a median follow - up period of 2.24 months (range, 2 - 10 months), warts are very common, with an incidence of approximately 10% in children and young adults. approximately 70% of these patients were between 10 and 39 yr of age, and 50% presented to a dermatologist. these complications, along with cosmetic embarrassment and risk of translocation to other areas of the skin, are indications for treatment, which can be challenging. many different types of therapy have been used in the treatment of warts (9 - 13). commonly used methods of physical destruction include surgical excision, electrodesiccation, cryosurgery, and pulsed dye or carbon dioxide laser therapy. chemical destruction can be induced with salicylic acid, cantharidin, formaldehyde, or glutaraldehyde, among other agents. more recently, immunomodulators such as interferon, systemic retinoids, cimetidine, and topical imiquimod have been used (14 - 16). this prospective study evaluated the efficacy of long - pulsed nd : yag lasers in the treatment of warts. the results show that long - pulsed nd : yag lasers are a safe and effective approach to wart treatment, with clearance rates higher than those achieved with other common therapies. previous studies of cryotherapy in the treatment of warts report clearance rates ranging from 63% to 69% (10, 17, 18). in a study using imiquimod, 56% of the patients reported total clearance (19). the pooled data from six randomized controlled studies demonstrated a cure rate of 75% in those treated with salicylic acid compared with 48% in the control group (21). one study performed using bleomycin prick method demonstrated complete resolution of warts in 92% of participants (22). long - pulsed nd : yag lasers emit a visible light spectrum at 1,064 nm. when a 1,064-nm long - pulsed nd : yag laser is used on the skin, a short, strong laser pulse is absorbed by red structures such as cutaneous blood vessels. as a result, blood vessels heat up rapidly and burst. upon blood vessel rupture, the mechanism of action of a long - pulsed nd : yag laser in the treatment of warts is not fully understood. dilated vessels in the papillary dermis are a characteristic feature of warts (23). light microscopic evaluation of treated areas at 7 days after treatment in this study showed separation of the dermoepidermal junction, epidermal necrosis, and rbc extravasation. in addition to these findings, destroyed blood vessels in the dermis were found to be surrounded by a dense inflammatory infiltrate. this destruction may obliterate the nutrient supply to the wart or destroy the rapidly dividing epidermal cells that contain hpv. minimal destruction of the surrounding tissue is anticipated with long - pulsed nd : yag lasers. in our study, the overall clearance rate was 96% (336 of the 348 treated warts were eradicated). the clearance rate of verruca vulgaris after the first treatment was very high (72.6%), whereas the clearance rate of deep palmopantar warts after the first treatment was low (44.1%). the average number of treatments required for clearance was 1.49. during a median follow - up period of 2.24 months (range, 2 - 10 months), 11 relapses were seen (recurrence rate 3.3%). this low recurrence rate seems to be due to the short follow - up period in this study ; therefore, a study with a longer follow - up period and examination of recurrence is needed. the clearance rate after pulsed dye laser treatment, which employs the same mechanism of action as the nd : yag laser, has been reported to be 48% to 92% (1 - 4), while the laser used in the current study showed a higher clearance rate (96%). therefore, a comparative study between these two types of lasers is required. side effects noted in our study included transient pain during treatment (82%), posttreatment numbness (15%), hemorrhagic bullae (7%), hyperpigmentation (5%), and hypopigmentation (4%). side effects were generally mild and did not prevent normal activity. in conclusion, long - pulsed nd : yag lasers are a safe and effective treatment for warts, with response rates higher than those obtained with conventional therapies. no single optimal treatment has been inclicated for warts ; therefore, long - pulsed nd : yag lasers should be considered a reasonable addition to the therapeutic options available. future studies examining optimal laser parameters and treatment intervals would expand our knowledge on how best to use long - pulsed nd : yag laser therapy in managing warts. | various treatment methods have been adopted in the management of warts ; however, there is still no consensus on first - line treatment. this study was designed to evaluate the efficacy of long - pulsed nd : yag laser in the treatment of warts. over the course of 1 yr, 369 patients with recalcitrant or untreated warts were exposed to a long - pulsed nd : yag laser. the following parameters were used : spot size, 5 mm ; pulse duration, 20 msec ; and fluence, 200 j / cm2. no concomitant topical treatment was used. in all, 21 patients were lost during follow up ; hence, the data for 348 patients were evaluated. the clearance rate was 96% (336 of the 348 treated warts were eradicated). the clearance rate of verruca vulgaris after the first treatment was very high (72.6%), whereas the clearance rate of deep palmopantar warts after the first treatment was low (44.1%). during a median follow - up period of 2.24 months (range, 2 - 10 months), 11 relapses were seen (recurrence rate, 3.27%). in conclusion, long - pulsed nd : yag laser is safe and effective for the removal or reduction of warts and is less dependent on patient compliance than are other treatment options. |
during our continuing search for structurally unique secondary metabolites from marine dinoflagellates, we have isolated a series of cytotoxic macrolides, amphidinolides, as well as long chain polyhydroxyl metabolites from dinoflagellates amphidinium sp.. (strain number y-52), which was isolated from the inside cells of the okinawan marine acoel flatworm pseudaphanostoma luteocoloris, and isolated polyhydroxyl metabolites, luteophanols a ~ c [23 ]. further investigation of extracts of the cultured dinoflagellate (y-52) led to the isolation of a new polyhydroxyl metabolite, luteophanol d (1), possessing two tetrahydropyran rings and twenty three hydroxy groups on c63-linear aliphatic chain. in this paper the dinoflagellate was unialgally cultured at 25 c for two weeks in seawater medium enriched with 1% es supplement. the extract was partitioned between hexane and 1 m nacl aq, and the aqueous phase was successively extracted with chcl3 and then n - buoh. the n - buoh soluble materials were subjected to gel filtration on sephadex lh-20 (meoh and then meoh / h2o, 1:1) followed by purification with reversed - phase hplc (meoh / h2o) to afford luteophanol d (1, 0.00023 %, wet weight). esims of luteophanol d { 1, []d + 4.7 (c 0.25, meoh) } showed the pseudomolecular ion peak at m / z 1329 (m+na), and its molecular formula, c66h114o25, was established by hresims [m / z 1329.7522 (m+na), -2.5 mmu ]. the uv and ir spectra indicated the presence of conjugated diene chromophore (max 232 nm) and hydroxy group (max 3430 cm), respectively. the h and c nmr data revealed that 1 contained two sp quaternary carbons, twelve sp methines, two sp methylenes, twenty - seven sp methines, of which twenty - six were oxymethines, twenty - one sp methylenes including one oxymethylene, and two methyl groups. since eight out of ten elements of unsaturation implied by the molecular formula were accounted for, 1 was inferred to possess two rings. this suggested that luteophanol d (1) was a congener of luteophanol b or c. the structure of luteophanol d (1) was elucidated by extensive 2d nmr experiments including h - h cosy, hohaha, roesy, hsqc, and hmbc. detailed analyses of h - h cosy, hohaha, and hsqc spectra revealed the proton - proton connectivities from h2 - 1 to h2 - 27, from h3 - 65 to h2 - 39, and from h-41 to h2 - 63. the hmbc spectrum of 1 showed cross - peaks for h-29/c-27, h2 - 66/c-39, h2 - 66/c-41, and h2 - 39/c-40, indicating connecitivities of c-27 to c-28, c-39 to c-40, and c-40 to c-41. two tetrahydropyran rings (c-32 to c-36 and c-43 to c-47) were assigned by hmbc cross - peaks for h-32/c-36 and h-47/c-43. five di- and one tri - substituted double bonds were indicated to have all e geometries by roesy data (h-30/h3 - 65, h-54/h-56, h-55/h-57, h-56/h-58, and h-57/h-59) and h - h coupling constants (j12,13 = 16 hz, j16,17 = 15 hz, and j51,52 = 15 hz). relative stereochemistry of the two tetrahydropyran rings (c-32 to c-36 and c-43 to c-47) was elucidated on the basis of roesy data and h - h coupling constants (j32/33 = ~0 hz, j33/34 = ~0 hz, j34/35a = 10 hz, j34/35b = 2 hz, j35a/36 = 10 hz, j35b/36 = 2 hz, j43/44a = 10 hz, j43/44b = 2 hz, j44a/45 = 9 hz, j44b/45 = 2 hz, j45/46 = ~0 hz, and j46/47 = ~0 hz) of 1 (figure. thus, luteophanol d (1) was assigned as a luteophanol b congener lacking one methylene between c-52 and c-54 of luteophanol b. luteophanol d (1) possesses two tetrahydropyran rings and twenty - three hydroxyl groups on a c63 - 1inear carbon chain with one exo - methylene and two methyl branches. luteophanol d (1) contains a hydrophilic diene portion at c-54 ~ c-59, whereas known polyhydroxyl metabolites, amphidinols [48 ] and lingshuiols [910 ], previously isolated from dinoflagellates amphidinium sp. since the polyketide chain of 1 was shorter than those of luteophanols b and c by only one carbon, this truncation of the carbon chain might be occurred by unusual dinoflagellate polyketide biosynthesis. luteophanol d (1) exhibited antibacterial activity against micrococcus luteus (mic, 33 g / ml). ir and uv spectra were recorded on jasco ft / ir-5300 and jasco ubest-35 spectrophotometers, respectively. the 3.35 and 49.8 ppm resonances of residual cd3od were used as internal references for h and c nmr spectra, respectively. (strain number y-52) was unialgally cultured at 25 c for two weeks in seawater medium enriched with 1% es supplement. the harvested cells of the cultured dinoflagellate (385 g wet weight, from 725 l of culture) were extracted with meoh (1 l x 3). the meoh extract (15.19 g) was partitioned between hexane (500 ml x 3) and 1 m nacl aq., and the aqueous phase was successively extracted with chcl3 (500 ml x 3) and then n - buoh (500 ml x 3). the n - buoh soluble fraction (4.47 g) was subjected to a sephadex lh-20 column [meoh and then meoh / h2o, (1:1) ] followed by reversed - phase hplc (develosil ods-5, 10 x 250 mm ; eluent : 55% meoh / h2o then 45% meoh / h2o ; flow rate : 2.5 ml / min ; uv detection at 226 nm) to afford luteophanol d (1, 0.9 mg, 0.00023 % wet weight). colorless amorphous solid ; []d + 4.7 (c 0.25, meoh) ; uv (meoh) max 232 nm (22800) ; ir max 3430, 2920, 1640, and 1050 cm ; esims m / z 1329 (m + na) ; hresims m / z 1329.7522 (m + na)+. calcd. for c66h114o25na, 1329.7547 ; h nmr (cd3od / c5d5n 2:1) 3.62 (2h, h2 - 1) ; 3.75 (m, 1h, h-2) ; 1.50 (1h, h-3a) ; 1.65 (1h, h-3b) ; 1.47 (1h, h-4a) ; 1.75 (1h, h-4b) ; 1.50 (2h, h2 - 5) ; 3.63 (1h, h-6) ; 1.50 (2h, h2 - 7) ; 1.47 (1h, h-8a) ; 1.75 (1h, h-8b) ; 1.47 (1h, h-9a) ; 1.58 (1h, h-9b) ; 3.68 (1h, h-10) ; 2.25 (2h, h2 - 11) ; 5.80 (1h, h-12) ; 5.63 (1h, h-13) ; 4.17 (1h, h-14) ; 2.35 (2h, h2 - 15) ; 5.67 (1h, h-16) ; 5.76 (1h, h-17) ; 2.30 (1h, h-18a) ; 2.70 (1h, h-18b) ; 3.75 (1h, h-19) ; 3.77 (1h, h-20) ; 2.67 (1h, h-21) ; 3.77 (1h, h-22) ; 3.95 (1h, h-23) ; 1.70 (1h, h-24a) ; 2.13 (1h, h-24b) ; 4.02 (1h, h-25) ; 1.70 (2h, h2 - 26) ; 2.10 (1h, h-27a) ; 2.32 (1h, h-27b) ; 5.70 (1h, h-29) ; 4.80 (1h, h-30) ; 3.90 (1h, h-31) ; 4.33 (1h, h-32) ; 4.40 (1h, h-33) ; 4.20 (1h, h-34) ; 2.02 (1h, h-35a) ; 2.11 (1h, h-35b) ; 3.68 (1h, h-36) ; 3.77 (1h, h-37) ; 1.75 (1h, h-38a) ; 2.19 (1h, h-38b) ; 2.32 (1h, h-39a) ; 2.69 (1h, h-39b) ; 4.45 (1h, h-41) ; 3.58 (1h, h-42) ; 4.30 (1h, h-43) ; 1.70 (1h, h-44a) ; 2.40 (1h, h-44b) ; 4.26 (1h, h-45) ; 4.39 (1h, h-46) ; 4.08 (1h, h-47) ; 4.28 (1h, h-48) ; 4.70 (1h, h-49) ; 5.90 (1h, h-50) ; 5.86 (1h, h-51) ; 2.18 (2h, h2 - 52) ; 1.65 (1h, h-53a) ; 1.70 (1h, h-53b) ; 4.18 (1h, h-54), 5.73 (1h, h-55) ; 6.30 (1h, h-56), 6.30 (1h, h-57), 5.78 (1h, h-58), 4.18 (1h, h-59), 1.60 (1h, h-60a) ; 1.65 (1h, h-60b) ; 2.15 (2h, h2 - 61) ; 5.83 (1h, h-62) ; 4.93 (1h, h-63a) ; 5.01 (1h, h-63b) ; 1.18 (d3h, h3 - 64) ; 1.75 (3h, h3 - 65) ; 5.03 (1h, h-66a) ; 5.18 (1h, h-66b) ; c nmr (cd3od / c5d5n 2:1) c-1, 68.3 ; c-2, 74.0 ; c-3, 35.5 ; c-4, 23.8 ; c-5, 39.4 ; c-6, 72.8 ; c-7, 39.4 ; c-8, 23.8 ; c-9, 38.8 ; c-10, 72.8 ; c-11, 42.3 ; c-12, 129.4 ; c-13, 137.7 ; c-14, 74.0 ; c-15, 43.0 ; c-16, 131.2 ; c-17, 131.7 ; c-18, 39.2 ; c-19, 74.0 ; c-20, 80.4 ; c-21, 35.9 ; c-22, 81.1;c-23, 73.2 ; c-24, 42.3 ; c-25, 72.5 ; c-26, 37.7 ; c-27, 37.5;c-28, 139.4 ; c-29, 127.7 ; c-30, 68.6 ; c-31, 73.3 ; c-32, 80.2 ; c-33, 69.8 ; c-34, 68.2 ; c-35, 31.6 ; c-36, 76.6 ; c-37, 75.3 ; c-38, 33.7 ; c-39, 28.9 ; c-40, 152.9 ; c-41, 77.4 ; c-42, 76.2 ; c-43, 71.5 ; c-44, 32.7 ; c-45, 68.1 ; c-46, 69.8 ; c-47, 81.4 ; c-48, 73.0 ; c-49, 75.0 ; c-50, 130.5 ; c-51, 135.4 ; c-52, 30.4 ; c-53, 38.8 ; c-54, 73.0 ; c-55, 132.3 ; c-56, 131.5 ; c-57, 131.5 ; c-58, 138.5 ; c59, 73.0 ; c-60, 38.5 ; c-61, 31.4 ; c-62, 140.4 ; c-63, 115.9 ; c-64, 7.8 ; c-65, 18.2;c-66, 113.5. ir and uv spectra were recorded on jasco ft / ir-5300 and jasco ubest-35 spectrophotometers, respectively. the 3.35 and 49.8 ppm resonances of residual cd3od were used as internal references for h and c nmr spectra, respectively. the dinoflagellate amphidinium sp. (strain number y-52) was unialgally cultured at 25 c for two weeks in seawater medium enriched with 1% es supplement. the harvested cells of the cultured dinoflagellate (385 g wet weight, from 725 l of culture) were extracted with meoh (1 l x 3). the meoh extract (15.19 g) was partitioned between hexane (500 ml x 3) and 1 m nacl aq., and the aqueous phase was successively extracted with chcl3 (500 ml x 3) and then n - buoh (500 ml x 3). the n - buoh soluble fraction (4.47 g) was subjected to a sephadex lh-20 column [meoh and then meoh / h2o, (1:1) ] followed by reversed - phase hplc (develosil ods-5, 10 x 250 mm ; eluent : 55% meoh / h2o then 45% meoh / h2o ; flow rate : 2.5 ml / min ; uv detection at 226 nm) to afford luteophanol d (1, 0.9 mg, 0.00023 % wet weight). colorless amorphous solid ; []d + 4.7 (c 0.25, meoh) ; uv (meoh) max 232 nm (22800) ; ir max 3430, 2920, 1640, and 1050 cm ; esims m / z 1329 (m + na) ; hresims m / z 1329.7522 (m + na)+. calcd. for c66h114o25na, 1329.7547 ; h nmr (cd3od / c5d5n 2:1) 3.62 (2h, h2 - 1) ; 3.75 (m, 1h, h-2) ; 1.50 (1h, h-3a) ; 1.65 (1h, h-3b) ; 1.47 (1h, h-4a) ; 1.75 (1h, h-4b) ; 1.50 (2h, h2 - 5) ; 3.63 (1h, h-6) ; 1.50 (2h, h2 - 7) ; 1.47 (1h, h-8a) ; 1.75 (1h, h-8b) ; 1.47 (1h, h-9a) ; 1.58 (1h, h-9b) ; 3.68 (1h, h-10) ; 2.25 (2h, h2 - 11) ; 5.80 (1h, h-12) ; 5.63 (1h, h-13) ; 4.17 (1h, h-14) ; 2.35 (2h, h2 - 15) ; 5.67 (1h, h-16) ; 5.76 (1h, h-17) ; 2.30 (1h, h-18a) ; 2.70 (1h, h-18b) ; 3.75 (1h, h-19) ; 3.77 (1h, h-20) ; 2.67 (1h, h-21) ; 3.77 (1h, h-22) ; 3.95 (1h, h-23) ; 1.70 (1h, h-24a) ; 2.13 (1h, h-24b) ; 4.02 (1h, h-25) ; 1.70 (2h, h2 - 26) ; 2.10 (1h, h-27a) ; 2.32 (1h, h-27b) ; 5.70 (1h, h-29) ; 4.80 (1h, h-30) ; 3.90 (1h, h-31) ; 4.33 (1h, h-32) ; 4.40 (1h, h-33) ; 4.20 (1h, h-34) ; 2.02 (1h, h-35a) ; 2.11 (1h, h-35b) ; 3.68 (1h, h-36) ; 3.77 (1h, h-37) ; 1.75 (1h, h-38a) ; 2.19 (1h, h-38b) ; 2.32 (1h, h-39a) ; 2.69 (1h, h-39b) ; 4.45 (1h, h-41) ; 3.58 (1h, h-42) ; 4.30 (1h, h-43) ; 1.70 (1h, h-44a) ; 2.40 (1h, h-44b) ; 4.26 (1h, h-45) ; 4.39 (1h, h-46) ; 4.08 (1h, h-47) ; 4.28 (1h, h-48) ; 4.70 (1h, h-49) ; 5.90 (1h, h-50) ; 5.86 (1h, h-51) ; 2.18 (2h, h2 - 52) ; 1.65 (1h, h-53a) ; 1.70 (1h, h-53b) ; 4.18 (1h, h-54), 5.73 (1h, h-55) ; 6.30 (1h, h-56), 6.30 (1h, h-57), 5.78 (1h, h-58), 4.18 (1h, h-59), 1.60 (1h, h-60a) ; 1.65 (1h, h-60b) ; 2.15 (2h, h2 - 61) ; 5.83 (1h, h-62) ; 4.93 (1h, h-63a) ; 5.01 (1h, h-63b) ; 1.18 (d3h, h3 - 64) ; 1.75 (3h, h3 - 65) ; 5.03 (1h, h-66a) ; 5.18 (1h, h-66b) ; c nmr (cd3od / c5d5n 2:1) c-1, 68.3 ; c-2, 74.0 ; c-3, 35.5 ; c-4, 23.8 ; c-5, 39.4 ; c-6, 72.8 ; c-7, 39.4 ; c-8, 23.8 ; c-9, 38.8 ; c-10, 72.8 ; c-11, 42.3 ; c-12, 129.4 ; c-13, 137.7 ; c-14, 74.0 ; c-15, 43.0 ; c-16, 131.2 ; c-17, 131.7 ; c-18, 39.2 ; c-19, 74.0 ; c-20, 80.4 ; c-21, 35.9 ; c-22, 81.1;c-23, 73.2 ; c-24, 42.3 ; c-25, 72.5 ; c-26, 37.7 ; c-27, 37.5;c-28, 139.4 ; c-29, 127.7 ; c-30, 68.6 ; c-31, 73.3 ; c-32, 80.2 ; c-33, 69.8 ; c-34, 68.2 ; c-35, 31.6 ; c-36, 76.6 ; c-37, 75.3 ; c-38, 33.7 ; c-39, 28.9 ; c-40, 152.9 ; c-41, 77.4 ; c-42, 76.2 ; c-43, 71.5 ; c-44, 32.7 ; c-45, 68.1 ; c-46, 69.8 ; c-47, 81.4 ; c-48, 73.0 ; c-49, 75.0 ; c-50, 130.5 ; c-51, 135.4 ; c-52, 30.4 ; c-53, 38.8 ; c-54, 73.0 ; c-55, 132.3 ; c-56, 131.5 ; c-57, 131.5 ; c-58, 138.5 ; c59, 73.0 ; c-60, 38.5 ; c-61, 31.4 ; c-62, 140.4 ; c-63, 115.9 ; c-64, 7.8 ; c-65, 18.2;c-66, 113.5. | luteophanol d (1), a new polyhydroxyl linear carbon - chain metabolite, has been isolated from the cultured marine dinoflagellate amphidinium sp., which was isolated from okinawan marine acoel flatworm pseudaphanostoma luteocoloris. the structure of 1 was elucidated by detailed analyses of 2d nmr spectra. luteophanol d (1) possesses two tetrahydropyran rings and twenty - three hydroxyl groups on c63-linear aliphatic chain with one exo - methylene and two methyl branches. |
acute aortic dissection is defined as the rapid development of a false, blood - filled channel within the tunica media of the aorta. three acute aortic dissections are ultimately diagnosed out of every 1000 emergency department patients presenting with acute back, chest, or abdominal pain. mortality in untreated acute aortic dissection is estimated at more than 1% per hour after the onset of symptoms, whereas 30-day survival for appropriately treated patients is greater than 80%. therefore, the timely diagnosis and rapid management of acute aortic dissection are of paramount importance to the emergency physician. diagnosis is delayed more than 24 hours after the initial presentation in almost half of all cases, highlighting the need for emergency physicians to maintain appropriate clinical suspicion for acute aortic dissection in patients with chest, back, or abdominal pain. failure to diagnose acute aortic dissection carries a significant risk for poor outcomes because of the consequences of progressive disease (e.g., aortic rupture) and the possibility of treating a falsely diagnosed myocardial infarction (mi) or pulmonary embolism (pe) with anticoagulation, a potentially catastrophic error. surgery on the aortic arch requires cardiopulmonary bypass, profound hypothermia, and a period of circulatory arrest. focal and diffuse neurologic deficits are the major complications associated with the resection of the aortic arch, occurring in 3% to 18% of patients. a 38-year - old woman was admitted to the emergency room with chest pain and dyspnea. the patient s past surgical history was the bental operation, performed 7 years earlier, and the stenting of the descending aorta 6 months previously due to aortic dissection. the patient s medical history was significant for hyperthyroidism and asthma, and her physical examination revealed blood pressure of 160/90 mmhg, heart rate of 90 beats per minute, metallic sound on heart auscultation, reduced breathing sounds in the left lung on lung auscultation, soft abdomen without tenderness, and symmetric and present peripheral pulses in the extremities. additionally, the electrocardiogram (ecg) showed normal sinus rhythm and 1-mm st depression in leads avl, v5, and v6, and chest x - ray demonstrated a white left lung. (figure 1) chest x - ray showing extreme dissection of the aorta (arrows) echocardiography demonstrated normal left ventricular chamber size, no left ventricular hypertrophy, moderate reduction in systolic function, global wall motion abnormality, ejection fraction of 35%, smoky pattern in the left ventricle, normal left atrium, normal right ventricle size and no right ventricular hypertrophy, normal mitral valve and no mitral stenosis or mitral regurgitation, normal hemodynamic of the aortic valve prosthesis (aortic valve replacement was performed 7 years earlier), normal prosthetic ascending aorta, aneurismal descending aorta, normal flow pattern of the descending thoracic aorta, and a large aneurysm (8 5 cm) filled with clot at the proximal portion of the descending aorta with reduced blood flow in the true lumen and deviation of the flow in the abdominal aorta - suggestive of aortic dissection. echocardiography also revealed normal pulmonary valve with no pulmonary stenosis or pulmonary insufficiency, normal tricuspid valve with trivial tricuspid regurgitation but no tricuspid stenosis, normal drainage of the pulmonary vein, greater systolic pressure than diastolic pressure, no ventricular septal defect, no atrial septal defect, normal inferior vena cava with 50% respiratory variation, and no pericardial effusion. ct angiography of the aorta with contrasted media revealed remarkable dilatation of the descending thoracic aorta (100-mm total transverse diameter) due to aortic aneurysm without active dye flow - containing thrombosis, a stent containing flow inserted in the aorta (figure 2), normal aorta at the root and the ascending portion, mild pleural reaction in the left pleural space, evidence of dissection with flow in the true and false lumens of the abdominal aorta, flow in the renal arteries, enlarged left ventricular chamber, and normal main pulmonary artery and pulmonary artery branches. coronal reformatted image, demonstrating the dissection of the aortic arch, involving the innominate and left common carotid arteries. (complete arrows show false lumens and head arrows show true lumens) aortography showed a significant endoleak (type i) from the proximal part of the graft into the false lumen. the patient had sinus tachycardia (140150/min), which was treated with metoral (100 mg / tds). two arterial lines were inserted in the right radial and right femoral arteries to monitor the blood pressure above and below the aortic arch. the involvement of the aortic and carotid arteries, given the fact that the patient had dissection and aneurysm as well as tachycardia and high blood pressure, rendered perianesthetic cerebral oxygenation extremely crucial. it is absolutely vital that all the above - mentioned factors be meticulously controlled in order to prevent the aggravation of the patient s conditions. overall, our anesthetic plan - including techniques, drugs, and monitoring for this complicated case - was individualized to enhance the outcome of the procedure. indeed, a rationale existed for choosing the right radial and right femoral artery for invasive pressure monitoring. furthermore, right radial arterial pressure monitoring was important because the procedure required clamping of the left subclavian artery and femoral arterial pressure monitoring allowed us the assessment of distal aortic perfusion. anesthesia was induced by using sufentanil in incremental and titrated doses up to 20 cc, propofol in incremental and titrated doses up to 20 mg, and cisatracurium (0.2 mg / kg). additionally, neuromuscular block was monitored with a nerve stimulator with train of forth (t.o.f) mode to ensures adequate neuromuscular block. while in deep anesthetic state and deep neuromuscular block, the patient was intubated without any bucking and hemodynamic changes. for hemodynamic management during instrumentation, anesthesia maintenance was achieved using the balanced technique, utilizing propofol infusion (75100 mcg / kg / min), narcotic (sufentanil [12 mcg / kg / h ]), and cisatracurium (2 mcg / kg / min). hemodynamic management was accomplished by maintaining the central venous pressure between 12 and 16 cmh2o in tandem with the infusion of tng (1050 mcg / min) and sodium nitroprusside at an infusion rate of 1.55 mcg / min. bolus doses of esmolol (0.51 mg / kg) were delivered via the central venous pressure line to control unexpected tachycardia. in addition to invasive blood pressure, central venous pressure, temperature, heart rate, ecg, and end tidal co2 monitoring, the patient was monitored during the entire time of anesthesia and procedure with bilateral regional cerebral tissue oximetry (rso2) with the invos in which regional saturation system measures changes in oxygen saturation levels beneath the sensor, allowing doctors and nurses to measure site - specific oxygen saturation levels. troy mi) ensures adequate bilateral hemispheral oxygenation. during anesthesia and procedure, especially during carotid clamping, rso2 was above 60%, which is higher than the acceptable lower limit of rso2 (55%) (67% in the right side and 66% in the left side). right clamp time was 25 minutes and left common carotid clamp time was 20 minutes. basal activated clotting time (act), measured for coagulation monitoring, was 100 seconds. heparin (100 mg) was administered, and second act was measured to be 345 seconds. after mid - sternotomy, the innominate and left common carotid arteries were found dissected ; accordingly, proximal and distal control was done. first, a 16 gel - sealed tube was anastomosed to the side of the ascending graft and then an end - to - side anastomosis to the innominate artery was performed. for the left common carotid artery, a 12 gel - sealed tube was anastomosed end to side to the ascending aorta graft and thereafter end to end to the left common carotid artery without cardiopulmonary bypass (figure 3). she had only a moderate rise in creatinine (up to 3.84 mg / dl), which later returned to the normal value of 1.61.9 mg / dl during hospitalization. she was followed up for 4 weeks and then monthly for one year, during which time no problems were detected. end - to - side anastomosis of the innominate (black arrow) and left common carotid arteries (white arrow) to the ascending aorta in aortic arch dissection, monitoring of vulnerable organs and careful management are crucial. in the case of the involvement of arteries which supply the brain blood flow, monitoring of end - organ oxygenation near - infrared photons are injected into the skin over the forehead. after being scattered about inside the scalp, skull, and brain, some fractions of the injected photons survive to return and exit the skin (reflectance). by measuring the quantity of the returning photons and their wavelength, one can infer the spectral absorption of the underlying tissue and make some conclusion about its average oxygenation. to reduce the interference of extra - cerebral oxygenation, invos utilizes two source detectors in spacing : a near and a far source detector. theoretically, subtraction of some of the near signals from the far signals should leave a signal origination, predominantly in the brain cortex. the potential for neurological injury is the achilles heel of aortic and heart surgery, but the technological advancement and innovations in anesthetic techniques have allowed us to offer surgical treatment to patients at the highest risk for neurological injury. near - infrared spectroscopy devices demonstrate a significant correlation with cerebral venous oxygen saturation monitoring (sjo2) and mixed venous oxygen saturation. yamamoto - k. reported that near - infrared spectroscopy is suitable for monitoring immediate change in oxygenation during carotid surgery. monitoring the cerebral rso2 in coronary artery bypass graft surgery patients prevents profound cerebral desaturation and it is associated with significantly lower incidence rates of major organ dysfunction. as was mentioned in the previous paragraphs, in the case of the dissection of the aortic arch and its branches, we can handle the aortic arch branches without cardiopulmonary bypass but by using rso2, which prevents cerebral ischemia. in this complicated case study, patient had previously undergone aortic valve replacement and had a large number of coexisting medical conditions such as replacement of the ascending aorta and intra - arterial proximal descending aortic stenting. therefore, debranching of the aortic arch branches was performed via meticulous control of hemodynamic and brain oxygenation as well as rso2 instead of cardiopulmonary bypass or deep hypothermic circulatory arrest. to our knowledge, this technique has not been previously reported in literature and may extend to a larger subset of patients who might otherwise have been excluded. in addition, regional oxygen saturation correlates well with the mean flow velocity during carotid clamping. it appears that cerebral oximetry is a satisfactory and possibly superior device for monitoring the adequacy of cerebral perfusion and oxygenation during carotid endarterectomy (cea) in comparison with transcranial doppler (tcd). the validity of rso2 also has been assessed by comparison with direct microprobe measurement of brain tissue oxygen partial pressure and reasonable agreement between these two measures has been found. the pre - induction baseline brain rso2 for this case was right 80% and left 70%. previous studies have stated that a brain rso2 50 scale units appears to represent an increased risk for hypoxic injury and that a drop by 20% below baseline is clinically important. in the case of our patient - at any given time during anesthesia and procedure, the rso2 values were not below 50% scale units or 20% below baseline. this case presented a rare challenge of handling aortic dissection without cardiopulmonary bypass, only by using rso2. this method prevents cerebral ischemia without any profound neurologic complications due to the use of cardiopulmonary bypass and deep hypothermic cardiac arrest. further debranching of the aortic arch, especially vertebral arteries debranching with this method, requires further investigations. | abstractaortic dissection begins with the formation of a tear in the aortic intima, and it directly exposes an underlying diseased medial layer to the driving force of the intraluminal blood. this blood penetrates the diseased medial layer and cleaves the media longitudinally, thereby dissecting the aortic wall. herein, we report the case of a 38-year - old woman, who presented with chest pain and dyspnea. after physical examination, laboratory evaluation, echocardiography, and ct angiography, extensive aortic dissection was diagnosed involving the innominate and left common carotid arteries. accordingly, the debranching of the aortic arch arteries was performed. during the procedure, the patient was monitored with bilateral regional cerebral tissue oximetry. the patient did not show any signs of complications either in the postoperative period or at postoperative three - month weekly follow - up or at subsequent monthly follow - up for the past year. |
recent advances in nanotechnology have enabled the development and use of carbon nanotubes as field - effect transistors (cntfets). these devices have advantages over traditional metal oxide semiconductor transistors, including higher circuit integration,1 increased gain, and higher mobility.2 the device consists of a single - wall (swcnt) or multi - wall (mwcnt) carbon nanotube acting as the channel of a field - effect transistor, connected to two metal source and drain electrodes, and has its drain current controlled by a gate electrode. in general, a positive gate voltage shifts the fermi level of the carbon nanotube toward the conduction band while a negative gate voltage shifts the fermi level toward the valence band.3 hysteresis is a peculiar effect in cntfets and is observed when the gate voltage is swept in the forward and reverse directions, leading to a hysteretic effect that is difficult to predictably analyze due to the different nonlinear effects that are present. hysteresis is typically related to charge injection from the carbon nanotube into the gate oxide dielectric, particularly at large gate bias, where the charges then become confined until the gate polarity reverses as a part of the voltage sweep.4 mathematical models have been developed to characterize hysteresis behavior, such as the limiting - loop proximity (lp),5 preisach,6 and jiles has not generated accurate approximations for cntfet drain current versus gate voltage or isd vg electrical characteristics primarily because they do not take into account the specific properties of the nanotube. one theoretical model often used to describe cntfets was proposed,8 based on the physical effects of ballistic conduction, wherein electrons in the nanotube are not affected by scattering.9 ballistic transport has nearly been achieved in cntfet modeling by the use of proper contact materials such as palladium and gold, thereby reducing transport barriers and approaching the theoretical conductance limit of the device (gon 4q2/h).10 the main objective of this work is to develop a reliable mathematical model by which to determine drain current by considering the physical basis of cntfet conduction, which includes the hysteresis effect. positive results were obtained, considering that some fitting parameters required estimation while instrument accuracy affected some of the obtained values. the results demonstrate that hysteretic effects can indeed be modeled and can potentially be used in a controlled manner for a variety of applications. in this paper we describe the ballistic theory pertaining to carbon nanotube transistor behavior, including the relationship between vg, vsd, isd, and the transfer characteristics of the cntfet. adaptations to the lp model to account for hysteresis, based on ballistic theory, are presented. we will also show the experimental procedures, methods, and materials used in order to obtain the device and data ; explain the methodology used for the acquisition of the model parameters ; present the results for different experiments and compare the data with simulations from the adapted model ; and express conclusions about these comparisons. due to its dimensions, a carbon nanotube can be considered a quasi one - dimensional element, whose diameter and chirality directly affect its properties. there is no boundary scattering in pristine nanotubes due to the lack of surface bonds and a cylindrical structure. thus, for long - length nanotubes, quasi - ballistic transport can be observed.11,12 for a cntfet, when contact is made between the carbon nanotube and the metal electrodes, a potential, or schottky barrier is formed at the metal since gold was used as the contact material in this study, having a large work function, the schottky barrier height became small. hence, in this paper, we only consider the ballistic transport within the carbon nanotube channel and assume that the gate voltage only modulates the energy bands of the carbon nanotube channel between the source and drain.8 the channel potential can be described as : s = vg, for vg01,fordvgdt, 500550 m thickness, 0.0010.005 cm resistivity) produced by nova electronic materials ltd (flower mound, tx, usa) were used in this work. thin silicon dioxide (sio2) layers were grown on the wafer by a typical dry wet dry oxidation process at a temperature of 1,100c for 10 minutes, 70 minutes, and 10 minutes, respectively. prior to the catalyst spinning procedure, the silicon wafer was cut into samples measuring 1010 mm, which were ultrasonically degreased in trichloroethylene (c2hcl3), acetone ((ch3)2co), and isopropyl alcohol (c3h8o), rinsed in deionized water, and dried in nitrogen. a solution of ferric nitrate nonahydrate (1.6 mg) + precursor moo2(acetylacetonate ligand)2 complex (0.5 mg) + aluminum oxide (15 mg) dissolved in methanol (20 ml) was used as the catalyst for carbon nanotube growth. catalyst islands are patterned, using electron beam lithography and lift - off on boron - doped silicon substrates with 300 nm sio2 grown by thermal dry oxidation. swcnts are grown from these catalyst islands using chemical vapor deposition with a methane and hydrogen gas mixture (total flow 60 sccm) in a 750c900c750c three - zone furnace set. the swcnt samples were grown with methane flow (32 sccm) and hydrogen flow (28 sccm). after imaging as - grown nanotubes with a field - emission scanning electron microscope, source / drain electrodes are patterned by electron beam lithography. the width of the source and drain metal electrodes are 1 m with a 1.5 m separation. the metals chromium and gold (thicknesses of 30 /1,000) are deposited by sputtering and followed by lift - off in acetone. surface morphology of the grown layers was examined by field - emission scanning electron microscopy using a zeiss supra 55-vp fesem microscope (carl zeiss meditec ag, jena, germany). micro - raman spectroscopy was carried out at room temperature using a renishaw raman microscope (invia ; renishaw, wotton - under - edge, uk) employing the output of an ar+ laser (20 mw power) for excitation at =514.5 nm. a labview 2013 sp1 software program (national instruments corporation, austin, tx, usa) was implemented in an ieee-488 environment, using a computer to control the model 4140b pa meter / dc voltage source (hewlett - packard inc., palo alto, ca, usa), and connected to a four - probe micromanipulator system for characterization of device parameters. all devices were measured at room temperature (300 k) in a controlled pressure (1 atm + 0.12 kpa in h2o) and humidity (30%) environment. for the determination of the lp model parameters w and vc, we used graphic analysis based on the simulation results. figure 2 describes the link between these parameters and their visual representation in the theoretical model. w is the horizontal distance between the two curves, obtained by tracing two lines in parallel with the inclination of the curves in their transition regions. thereafter, vc is obtained by determining the horizontal midpoint of w at the vertical point located in the off - state saturation current value. although 0, 1, and 2 are inherent to the physical characteristics of the cntfet, as mentioned in the model description section, there is no known mathematical relation that could allow the prior acquisition of these parameters. thus, we considered each transistor as a stand - alone device, obtaining the slope of the curve in the transition region for each device and drain - source voltage by the minimization of deviations between the data and the model curves in this region. in this way, with at least three measurements of for different values of vsd, it is possible to determine 0, 1, and 2 for a device by a linear system. figure 3a shows a long and straight single semiconducting nanotube contacted by two gold electrodes to form a cntfet, and figure 3b illustrates the typical hysteresis behavior of the nanotube transistor for drain current versus gate voltage (with vsd=0.02 v). data were taken at room temperature and the device is operated with gate voltages between 10 v < vg < + 10 v, in the asymmetric hysteretic transition region. this result corroborates well with the data from a previous work,19 in which the showed that the device conductance was increased for negative gate voltages. we used the ballistic transport model8 and lp model5 for hysteresis modeling of cntfets. due to the effect complexity, this modeling is susceptible to nonlinearities, so we have incorporated effects in our model to include energy bands, memory phenomena, minor loop accommodation, and stabilization. figure 4a and b, respectively, show typical raman spectrum and typical radial breathing mode peaks ranging from 100 to 300 cm that were used to estimate the diameter of the swcnt.20 we observed two typical swcnt peaks located at 1,350 cm (d - band) and 1,590 cm (g - band). the diameter distribution of the carbon nanotubes ranged between 1.0 nm and 2.2 nm depending on the different argon concentrations. in order to determine the carbon nanotube diameter, the raman shift intensities were utilized and, based on the values in the table, we determined the diameter to be 1.0 nm, so that 1 was calculated as 1~0.45/d (nm) ev.21 contact resistance was measured in saturation current, and we obtained approximately 100 k in this device. simulation was performed at room temperature, using matlab (mathworks, natick, ma, usa), on the device for different values of vsd and different vg variances. for the device, tox=300 nm and we used fitting parameters 0=0.992, 1=0.05 v and 2=0.024 v. figure 5a c display curve modeling for vg between 2.5 and + 2.5 v, 5.0 and + 5.0 v, and 10.0 and + 10.0 v, respectively, and for vsd equal to 0.03 v, 0.02 v, and 0.01 v. the results highlight how the gate - source voltage sweep affects hysteresis parameters vc and w. a larger sweep enlarges w and shifts vc toward negative values of vg, while changing vsd does not affect these parameters. it is noted that the bottom curves of the model do not reach the same value of the upper curves on negative voltage limit, and the reason we suggest is that the devices did not reach their maximum saturation current, and, for this, the negative voltage limit should be lower. despite this observation and some inaccuracies due to measurement and parameter deviation, a good fitting this work makes some contributions regarding cntfet modeling : first, simulations with higher biases of vg were performed, while several articles modeling cntfet devices used a smaller gate voltage sweep (eg, 0 to + 0.05 volts) in simulations, in comparison to our work, and second, the description of hysteresis effect by using lp parameters showed that hysteretic loop width and shift is sensitive to gate voltage bias. in order to evaluate the accuracy of the proposed model, three other devices were submitted to measures, for vg between 10 v and + 10 v, with the same procedures and conditions described earlier in the experimental procedure section. subsequently, the experimental results were employed in simulations for the modeling of the devices. table 2 describes the parameters used for the devices and the relative error of the comparison between the experimental and model curves. it is noted that, with the results shown in table 2, the model is applicable to other devices with different characteristics and behavior. in spite of the expected relative errors, since the measurement accuracy and model approximations might interfere in the modeling, these differences do not attenuate the effectiveness of the proposed work. in this work, we successfully modeled isd vg curves including hysteresis effects, for different voltage values and parameters, as a result of mathematical adaptations over the main expression, respecting the physical concepts of ballistic theory. the lp model contributed to the characterization, as we could use its parameters to distinguish the two curves caused by hysteresis. one improvement that we intend to demonstrate in a future work is the prior identification of the hysteretic parameters without using graphic methods. nonetheless, the significant contribution presented in this article is that, given a device with its parameters, it is possible to mathematically localize a bias point over the experimental curves within a desired region of operation, thereby permitting accurate control of the cntfet for specific applications, such as nonvolatile memory devices. | theoretical models are adapted to describe the hysteresis effects seen in the electrical characteristics of carbon nanotube field - effect transistors. the ballistic transport model describes the contributions of conduction energy sub - bands over carbon nanotube field - effect transistor drain current as a function of drain - source and gate - source voltages as well as other physical parameters of the device. the limiting - loop proximity model, originally developed to understand magnetic hysteresis, is also utilized in this work. the curves obtained from our developed model corroborate well with the experimentally derived hysteretic behavior of the transistors. modeling the hysteresis behavior will enable designers to reliably use these effects in both analog and memory applications. |
although four major models are presently being debated, only the out - of - africa suite has received strong support from interpretations of both the fossil record and dna [16 ]. yet, the evolutionary origin of aboriginal australians remains controversial owing to the presence of considerable variability in cranial morphology during the pleistocene and interpretations of its possible phylogenetic importance [1, 2, 718 ]. moreover, there is sharp disagreement about the possible alternative causes of this variation and its significance to a global understanding of the evolutionary history of modern homo sapiens [1, 2, 11, 12, 17, 18 ]. long before a human fossil record was known for australia, various speculative evolutionary sequences were devised linking nonmodern hominins to aboriginal australians. in particular, it was proposed that pithecanthropus (homo erectus) or the late - surviving ngandong population of this species (sometimes referred to h. soloensis) played a role in their origins. this idea has been an enduring theme of palaeoanthropology for more than 100 years : from klaatsch through to westaway and groves. a recent major review of the question of modern human origins identified three major issues for australian palaeoanthropology to be resolved : (1) the relationship of the first australians to later inhabitants of the continent, (2) whether late pleistocene morphological diversity may have been accentuated by the severity of the last glacial maximum, leading to isolation and the forcing of morphological change in some australian populations, and (3) if archaic populations such as those known from ngandong did survive into the late pleistocene, an analogous situation to that in europe might have existed, raising the possibility of gene flow with dispersing h. sapiens., the very earliest fossil remains from australia (e.g., willandra lakes human 3) do seem to fit metrically and morphologically within the range of living aborigines. stringer 's points 2 and 3 relate to the possible cause(s) of cranial robusticity in some pleistocene / early holocene australians. that is, whether such features arose as a result of natural selection acting on populations within australia or were brought here by people who evolved from, or hybridised / admixed with, a nonmodern population in southeast asia (i.e., the solo / ngandong hominins). australian palaeoanthropological theory and method continues to be dominated by adaptationist accounts of robusticity and population history. the assumption that the cranium is optimised part by part and that atomising its form into traits assumed to be heritable units, functionally discrete, to have been shaped by natural selection and, therefore, positively associated with reproductive success, remains the core proposition of the field. in the present contribution, it is argued that the failure to fully consider alternative (nonadaptationist) approaches is a major reason why the interrelated issues of firstly, the cause(s) of cranial robusticity and, secondly, its relevance to reconstructing the origins of aboriginal australians remain unresolved. this paper commences with a review of the history of ideas regarding cranial robusticity and the origins of aboriginal australians. then, an alternative to atomisation, herein called the ontogenetic approach, is described and some key concepts underpinning it are introduced. finally, this approach is applied to some characters used to support the ngandong ancestry model for aboriginal australians. ever since blumenbach 's de generis humani varietate nativa of 1795, european scientists have been attempting to understand the affinities and hence origins of aboriginal australians. blumenbach had at hand the skulls of several australians provided to him by joseph banks. however, only with the wider exploration of the continent by europeans and the beginnings of a global trade in human cranial remains during the 19th century was a systematic effort made to understand their skeletal morphology. during this period, the deep antiquity of the earth was beginning to be established, including an ancient origin for humankind as presented in lyell 's antiquity of man. in lyell 's volume, huxley 's (1863) first comparisons of aboriginal skulls to neanderthal remains were noted, indicating various morphological resemblances (not exclusive though, or implying ancestry). as the first anthropologist to study human origins from comparative anatomical and fossil sources, and placing his ideas within the darwinian evolutionary framework, huxley can reasonably be considered the founder of the discipline of palaeoanthropology. moreover, as aboriginal australians were central to his ideas, as well as to the early development of this scientific field, huxley ensured their place at the centre of debate surrounding human origins, a position they have held for close to 150 years. during the early 20th century, many researchers continued to focus on documenting similarities in cranial form between australians and the neanderthals [2426 ], inspired by huxley 's earlier and highly influential work (see figure 1). however, following the discovery of fossil human - like remains in indonesia by dubois, the view eventually emerged that australians may actually have descended from a local population within southeast asia, either pithecanthropus or homo soloensis [28, 29 ] (page 162) as proposed by huxley (see other criticisms in [30, 31 ]). however, the idea of a regional evolutionary sequence between aboriginal australians and pithecanthropus in southeast asia seems not to have been in the minds of these workers. instead, they saw aboriginal australians as representatives of the most primitive type of living h. sapiens and had in mind a global evolutionary sequence in which solo man (ngandong) and rhodesian man (kabwe) were examples of proto - australians, belonging together to living humans in h. sapiens. also, dubois [28, 29 ] thought that the wadjak remains he recovered from indonesia were australoid although they now seem to be terminal pleistocene in age and are probably not related to aboriginal australians. with the ideas of weidenreich [33, 34 ] and coon, the pithecanthropoid - australoid both these workers believed that modern humans around the world had evolved from regional populations of pithecanthropus (today h. erectus). thus, the regional lineage from this taxon through to aboriginal australians was part of a global process, which howells dubbed the candelabra hypothesis. parenthetically, it should be noted that this idea was not universally supported ; for example, hrdlika preferred a neanderthal stage in human evolution and considered aborigines to belong to an old race of whites, borrowing heavily from huxley, while birdsell 's views are strikingly similar to the contemporary out - of - africa theory. the increase in the number of fossilised human remains recovered during controlled excavations from the 1960s onwards [3943 ], and demonstration of a pleistocene occupation of the australian continent [44, 45 ] encouraged renewed interest in the pithecanthropoid - australoid lineage hypothesis [3943 ]. again, it must be stressed that not all researchers accepted this hypothesis, macintosh in particular reversing his earlier endorsement. during the 1980s and 1990s, this idea was reformulated, initially as the regional continuity hypothesis and later as the global multiregional model of modern human origins [48, 49 ]. at its core was the notion of a deep - time southeast asian - australian clade supported by evidence of morphological continuity in the skull and dentition between lower and upper pleistocene nonmodern hominins in indonesia through to recent aboriginal australians. as thorne and wolpoff stated, in no other region can a specimen (i.e., sangiran 17) be found that combines so many features that seem unique or at least of high frequency in pleistocene australians it is important to note that many proponents of the multiregional model have since the early 1990s regarded h. erectus to be a junior synonym of h. sapiens ; thus, the emergence of modern humans is seen by them as a process occurring within a single, long lasting, and widely distributed evolutionary lineage, or species. moreover, thorne [5153 ] proposed that two populations had colonised australia at different times during the pleistocene in his dihybrid model, the merging of the two giving rise to modern aborigines. group descended from h. erectus and the second a later arriving gracile population originating in pleistocene china and, like modern east asians, evolving from east asian h. erectus. although the order of arrivals had to be revised (reversed) once it was established that gracile crania like wlh3 were actually geologically much older than any robust remains recovered from various localities [15, 53 ]. the first emerged from a test of multiregionalism in southeast asia - australia using the wlh50 calvaria (see figure 2) [54, 55 ]. it was found that six of the seven ngandong calvaria examined were phenetically closer to wlh50 than to any other specimens considered, including early modern humans from skhul and qafzeh. in this work, it was concluded that the results implied a dual ancestry for aboriginal australians because there is no evidence suggesting wlh-50 can be grouped with either late pleistocene africans or levantines to the exclusion of the ngandong sample that is, it was argued that australians are descended from both an ancient regional population (e.g., solo / ngandong) and recent modern humans from africa through a process of reticulation (admixture / hybridisation). the present author has made similar conclusions employing different fossils and an alternative, multivariate, methodology. however, his findings were argued to be consistent with the assimilation model of modern human origins [56, 57 ], a hypothesis receiving stronger support from genetic studies. weidenreich in a personal communication to birdsell changed his mind late in life and also thought australians had a dual ancestry. the present author has, however, changed his views and no longer considers interbreeding to provide a parsimonious explanation for aboriginal australian morphology or the origins of these people. the final recent variant was proposed by webb and borrows heavily from birdsell 's trihybrid hypothesis [60, 61 ], as have thorne 's later ideas. webb speculated that the first population to colonise australia was the species h. soloensis, an upper pleistocene descendent of javan h. erectus. he argued that it migrated to the continent as early as 130150 ka and ultimately adapted to local conditions, founding the robust australian pleistocene / early holocene population. further, he contended that modern humans entered australia sometime between 50 ka and 75 ka, tracing their origins back to africa. these people are argued to have been negrito or negrito - like, being of small stature, a feature developed the two species (h. soloensis and h. sapiens) are argued to have gradually formed a single population through genetic mixing, but the process was dominated by the modern group, which had the larger population and constantly, albeit slowly, receives fresh genes through a series of migrations arriving throughout the glacial maximum and after, into the early holocene [14, page 269 ]. many objections to southeast asian - australian regional continuity and the related dihybrid origins model have been offered over the last 30 years or so. some opponents have argued that perceived similarities between robust individuals and h. erectus may simply represent the retention of plesiomorphies of later homo by aboriginal australians [11, 63 ]. indeed, larnach found that aborigines are closest in cranial morphology among living people to h. erectus but stressed their resemblance is never close he also noted that the solo / ngandong population exhibits a number of autapomorphies not found among aboriginal australians (see also [65, 66 ]), and if australians are descended from solo man, then sometime during that descent these unique traits were lost and their sites reverted to a similar state to that obtaining in homo erectus [64, page 170 ]. thus, if regional continuity is correct, pithecanthropus (h. erectus) must have given rise to the robust pleistocene australian group in a separate event, the ngandong population (h. soloensis) being a late surviving and autapomorphic descendent, and unrelated to australians. durband has pointed out that the available data for javan h. erectus especially from the facial skeleton (i.e., n = 1 or sangiran 17) is too inadequate to make reliable phylogenetic inferences. moreover, the dihybrid model and by implication regional continuity has been rejected by most australian workers on the basis of its failure to take adequate account of spatiotemporal variability in cranial and dental morphology, including size, within a single continental population [810, 12, 18, 6875 ], or at the least its failure to reject this as a null hypothesis. moreover, some individual features underpinning regional continuity such as vault thickness have been proposed as selected for in response to violence and cranial trauma (see below for a discussion of epigenetic explanations for some robust traits). finally, the conceptual underpinnings of the dihybrid / migrationist model have been deemed to be founded in a palaeontological (i.e., typological) rather than population - based approach, as would be demanded by the synthetic evolutionary paradigm. as noted above, most studies of australian robusticity have assumed that the morphological characters under investigation are genetic (or heritable). this concept is used in its broader sense to mean that the phenotype of an individual is a good predictor of the genotype. however, it has become a truism of developmental biology that the old and compelling idea that there exist specific genes for virtually every structural detail throughout the craniofacial complex is simply not true [77, page 230 ]. the more that is learned about the genes and developmental processes forming the cranium, the less tenable the atomisation of complex organs like the cranium has become. some concepts described in this section indicate powerfully why this is the case and underpin the need for an alternative paradigm in australian palaeoanthropology. estimates of the heritability for many commonly employed cranial measurements and traits are available (e.g., [7982 ]). although there are some uncertainties surrounding the application of sample - specific heritability estimates to other populations, they nonetheless provide insight into the potential reliability of particular traits / variables as well as a broader understanding of the heritability and integrated nature of cranial ontogeny. heritabilities vary widely for standard craniometric variables : h = 0.000 0.000 to 0.867 0.156. moreover, many variables have been found to exhibit heritability estimates that do not differ from zero (i.e., h = 0) [79, 81 ]. in a recent investigation, only two commonly employed measurements have been found to exhibit high heritability values : palate breadth (mab) and nasal height (nlh). moreover, major length (gol), breadth (xcb), and height (bbh) dimensions exhibited low to moderate, but statistically significant heritability estimates. however, many important regional measurements such as frontal chord (frc), bifrontal breadth (fmb), biasterionic breadth (asb), bizygomatic breadth (zyb), and nasal breadth (nlb) are characterised by low and nonsignificant heritability estimates. additionally, these estimates show a spatial pattern whereby lower estimates tend to be associated with the face. in many cases, they are associated with measurements for areas involving attachment sites for the muscles of mastication suggesting an important role for epigenesis in their ontogeny. endocranial dimensions show moderate to high heritability estimates including important measures of human basicranial size and angulation. these results highlight the reasonably high heritability and considerable ontogenetic integration (correlation among dimensions) of human endocranial form, but contrast with low - moderate heritability for measurements on the ectocranial surface. in a recent study of the heritability of cranial dimensions employing a 3d approach, martnez - abadas. found a broadly similar pattern to previous (2d) studies [7981 ]. overall, dimensions showed low - moderate heritability with about 72% being significant (i.e., h > 0). interestingly, they found the face to be the region with the highest number of significantly heritable traits and highest mean heritability, followed by the cranial base and the neurocranium. within regions they found the orbit, nasal part, neurocranial vault, and basicranium to be characterised by low to moderate heritability, while the masticatory apparatus exhibited low heritability, a finding consistent with carson 's results. between cranial regions, they also found low - moderate heritability confirming the concept of ontogenetic integration of the cranium. show that the cranial base, neurocranium, and face are characterised by similar levels of heritability and also strongly point to an important role for epigenesis in ontogeny (see also [80, 81, 8386 ]). epigenesis is defined as the developmental interactions among cells, tissues, and their environments. it can translate localised developmental alterations into integrated and widespread morphological changes and provide a fundamental mechanism for introducing flexibility into developmental programs including phenotypic plasticity [8386 ]. lieberman. have discussed three types of epigenetic interactions during ontogeny : (1) primary interactions, occurring at the cellular level, (2) secondary interactions, involving those between adjacent tissues during growth, and (3) tertiary interactions, in which interactions occur throughout ontogeny between cells within a unit (e.g., via hormones) and the rest of the organism as well as the environment. one important and widely discussed concept underpinning epigenesis in the ontogeny of the cranium is the functional matrix hypothesis (fmh) of moss. he described the fmh in the following way : the developmental origin of all cranial skeletal elements (e.g., skeletal units) and all their subsequent changes in size and shape (e.g., form) and location, as well as their maintenance in being, are always, without exception, secondary, compensatory, and mechanically obligatory responses to the temporally and operationally prior demands of their related cephalic nonskeletal cells, tissues, organs, and operational volumes (e.g., the functional matrices) [88, page 9 ]. the fmh explains many aspects of cranial development and is widely regarded to be an important concept surrounding secondary epigenetic interactions during cranial ontogeny (e.g., [77, 89 ]). this law is actually a poorly defined and frequently criticised term usually applied as a catch - all concept to refer to the adaptation of bone to mechanical stimuli [77, 78, 91, 92 ]. put simply, wolff 's law is, an extension of the old and trusted idea that form is interrelated with and inseparable from function that bone grows and develops in such a manner that the composite of physiologic forces exerted on it are accommodated by the bone 's developmental processes [77, page 233 ]. historically, one problem with this concept 's use has been that it was frequently invoked to argue for uniform responses such as bone deposition in the face of biomechanical stress particular from muscle, when very often the opposite response, resorption, had occurred [77, 91 ]. thus, while in its general form this law remains valid and useful, it is now widely acknowledged that just how bone responds to mechanical stress is complex and varies according to its location and whether stress is direct or is mediated by other tissue. the reader is refereed to some recent and more detailed reviews of this concept [77, 78, 92 ]. the external environment through tertiary epigenetic interactions has long been argued to be an important determinant of cranial form. for example, the shapes of the neurocranium and nose have long been linked to climatic adaptation [93100 ] and facial form to diet or masticatory practices (see [101104 ] : see below for a more detailed discussion of this idea). a 3d study of cranial morphology compared to neutral genetic population distances found that the human cranium does preserve a signal of population history. however, historical signatures are not equal across cranial regions and seem to be largely concentrated in the temporal bone and neurocranium. modularity is a general property of biological systems, from molecular to ecosystemic levels of interaction. in ontogeny, it represents the observation that morphological features do not vary independently but are integrated with each other, reflecting coordination in development, function, and evolution. complexes that are highly integrated internally but are relatively independent of each other [86, page 628 ]. the morphological characters within modules are characterised by three major properties : (1) they collectively serve a common functional role, (2) they are tightly integrated by pleiotropic effects of genetic variation, and (3) they are relatively independent of other modules. this independence among structures (modules) allows unrelated components to vary and evolve separately, but the integration within the units maintains functionally necessary relationships among traits [8587, 106113 ]. morphological integration assumes that developmentally / functionally related traits are coinherited and will produce coordinated responses in evolution [8587, 106113 ]. the cranium is divided along ontogenetic lines into three regions : the cranial base, neurocranium, and face. as the cranial base is in an evolutionary sense the oldest structure, it is phylogenetically highly conserved and believed to be subject to stronger genetic influence in ontogeny than the neurocranium and face [77, 113116 ]. moreover, it widely is assumed that the face is the most sensitive region to epigenetic factors as it stops growing later in ontogeny than either the cranial base or neurocranium [77, 114, 115 ]. thus, it is subject to greater influence from mechanical loading during mastication and from various environmental factors during ontogeny [116119 ]. there is, however, considerable evidence that the face and neurocranium are characterised by both integration between regions and considerable modularity, or region - specific integration [77, 8083, 85, 87, 115, 120123 ]. moreover, this pattern seems to characterise humans, chimpanzees, and gorillas [106, 120, 124, 125 ] and is known to be phylogenetically conserved in mammals [107, 126 ]. cranial shape patterns appear early in ontogeny and remain from early childhood until adulthood, including in depository and resorptive cranial growth fields [115, 128 ]. although this is somewhat simplistic as endocranial shape continues to change in humans through adolescence, well after brain growth it has ceased. a range of studies aiming to test and develop enlow 's ideas about facial and cranial growth [77, 115, 130 ] have been published over the past two decades and employed a range of techniques including 3d morphometrics [119, 122, 123, 130144 ]. many studies have focussed on interactions between the cranial base, neurocranium, and facial form, and some major findings include that larger relative brain size is associated with a larger basicranial angle, while large faces may produce the opposite situation, although the influence of facial size seems to be weaker than for brain size. lieberman. [122, 123 ] also found that cranial proportions (neurocranial shape, or degree of brachycephaly versus dolichocephaly) depend on interactions between cranial base width and brain volume. this has been confirmed by 3d studies showing that changes in cranial base width play an important role in cranial shape variation, including facial width. moreover, studies of the heritability of cranial regions confirm strong covariation between the breadth measures of major developmental regions of the skull. additionally, independent of age and size, an important proportion of cranial shape variability seems to be traceable to differences in the position and orientation of the face and masticatory system relative to the braincase. experimental research using mice suggests that integration in the mammalian skull is highly structured following a hierarchical scheme dominated by covariation between the widths of the neurocranium and the basicranium and, to a lesser extent, also the face. thus, the cranial base can be thought of as the skull 's central integrator [122, 123 ]. it strongly influences overall cranial shape, constraining facial breadth, height, and length, and neurocranial breadth and length, helping prevent the different regions from evolving independently and would preserve the functional and architectural requirements of the skull [82, page 29 ]. the above review of some ontogenetic concepts and recent research findings provide the context for a reconsideration of the possible causes of cranial robusticity among australians. there have been very few synthetic attempts to explain it using nonphylogenetic or nonadaptationist approaches. one important, but overlooked, example is that of howells who proposed that robusticity may have been a phenotypic plastic response to some regional and transient environmental stimulus. that is, the phenomenon would be, not adaptation and selection, resulting in genetic change in the post - mungo population, but a reversible phenotypic shift, on an unchanged genetic basis, toward larger size and related allometric effects on the face and mandible particularly, producing in some relatively smaller - brained individuals the special flattened and narrow frontal which is so striking in cohuna and some of the kow swamp skulls (pages 646 - 647). this explanation is broadly consistent with the ontogenetic approach outlined above, being in large part a putative example of tertiary epigenetic interactions. the cultural practice of artificial deformation, another example of tertiary interactions, has been argued to have produced or exaggerated the acute angle of the frontal squama, angulation of the parietals, and aspects of occipital squama morphology in a small number of robust australian crania such as kow swamp 5, cohuna, some coobool creek crania and nacurrie 1 [9, 146151 ]. other epigenetic factors have been proposed to explain vault thickness, including, increased levels of growth hormone, nutritional phosphorous deficiency, and inherited anaemia [13, 14, 152, 153 ]. the latter (pathology) hypothesis has been examined to some extent by stuart - macadam, curnoe and thorne, westaway, and curnoe but requires further scrutiny (see below). lahr and wright using multivariate statistics considered spatial (or architectural) aspects of cranial form and proposed that the superstructures (see below) characteristic of cranial robusticity may be integrated and covary allometrically. for australian aborigines, they concluded that like other human populations the expression of robust characters was likely a response to some functional complex, possibly mastication. they also suggested that the distinctive anatomical combination present in australian crania of a very narrow vault and pronounced robusticity, does not represent a plesiomorphic state inherited from either early modern humans or the solo / ngandong population [156, page 184 ]. moreover, they proposed that allometry in combination with this regional morphology might explain extreme robusticity seen in the pleistocene of australia. however, in attempting to explain the origins of this cranial morphology, they turned again to an explanation involving natural selection during the settlement of australia. in recent research by the present author, the ontogenetic framework was adopted in an attempt to explore nonphylogenetic explanations for cranial robusticity among pleistocene / early holocene australians. it was concluded that robusticity among these individuals may have been the result of the complex developmental and functional interplay between (1) a large neurocranium, (2) narrow cranial base, (3) large viscerocranium with considerable high midfacial projection, and (4) large dentition, especially the posterior teeth, with their resultant large jaws (mandible and maxilla) and high volume of masticatory muscles. while it was suggested that these features were probably heritable to some extent, other factors such as body size, advanced physiological age, environmental effects from the physical demands of a hunter - gatherer lifestyle in an arid zone, dietary factors including food abrasiveness and limited preparation of food, and the use of teeth as tools may all have been factors affecting (exaggerating) the expression of robusticity in the australian context. as noted above, pathological processes are suggested to have greatly enlarged the already thick vault bones of wlh50 (figure 2). webb [13, 14, 152, 153 ] has listed three indicators of pathology in this individual : (1) uniform vault thickness, (2) identification of the hair - on - end sign on lateral radiographs of the calvaria, and (3) a thin vault cortex. figure 3 compares vault thickness at eight locations in wlh50 with the median and range for the remaining willandra lakes series, as measured by webb. the large absolute thickness of the wlh50 vault is striking although thickness at one landmark (inion) does lie well within the range of the remaining willandra lakes sample. it is also very clear that vault thickness in wlh50 is far from uniform, as it varies between locations in a similar magnitude to the medians for the willandra lakes series (figure 3). the magnitude of change (difference) between each of eight locations was calculated for wlh50 and the rest of the willandra lakes series, and the median value is actually larger in this individual (2 mm versus 1 mm). moreover, a nonparametric mann - whitney u - test showed that the sample medians are statistical indistinguishable (n = 7/54, u = 187.5, p =.98 ; monte carlo p =.98). thus, wlh50 does not possess a vault of uniform thickness when compared to the rest of the willandra lakes human sample. figure 4 is a lateral radiograph of wlh50 taken in 2006 by the author and a. thorne. after careful inspection, no indication of the hair - on - end sign can be seen. figure 5 is a lateral slice of a ct - scan of wlh50 also taken by the author and a. thorne in 2006. again, in this and every slice inspected by the present author (245 slices in three planes, or 735 images), no evidence of the hair - on - end sign can be found. there is, however, evidence for abrasion on the external cortex around bregma (figure 5), which may have been mistaken by webb for the vertical spiculations sometimes associated with porotic hyperostosis. it is also clear in figures 4 and 5 that the external table of wlh50 is far from thin. to assess this quantitatively, the ratio of diploe to external table thickness was calculated from thickness measurements made on ct - scans just lateral to the median sagittal plane by the author. external table thickness was chosen because along with the diplo this vault component is altered in cases of porotic hyperostosis. the median of this ratio for wlh50 measured at nine locations shows the diplo to be 1.4 times as thick as the external table, while in wlh3 the median shows it to be 0.9 times as thick. while wlh50 certainly does exhibit relatively thinner external table, it is far from thin. finally, the results of a mann - whitney u - test showed the median difference between wlh3 and wlh50 to be statistically nonsignificant (n = 9/23, u = 74, p =.22 ; monte carlo p =.23). table 1 lists 16 morphological traits for wlh50 used to support a role for the solo / ngandong population in the evolution of aboriginal australians (i.e., a reticulate or dual ancestry model) [54, 55 ]. these nonmetric traits were originally selected in order to avoid duplicating features that seemed to reflect the consequences of the same anatomical variation [55, page 294 ]. while somewhat ambiguous, this statement is understood by the present author to imply that traits were employed that were believed to be developmentally and functionally nonintegrated and, therefore, to be characterised by weak or absent covariation. this assumption is crucial to an evaluation of the phyletic valency of cranial robusticity in the australian context and the implicit assumption that characters shared by australian and solo / ngandong crania are homologous. (the reader is referred also to bruer. who provided a critical evaluation of the character coding and statistical methods employed by hawks. and wolpoff..) all of the characters shown in table 1 belong to a single cranial (developmental) unit, namely, the neurocranium. a small part of the lateral cranial base is preserved while the facial skeleton is missing save a fragment of zygomatic bone. moreover, these traits represent two subunits of the neurocranium most of them belong to the cranial vault (14), with the remaining (2) belonging to the orbit subunit (table 1). given the now well - established finding of ontogenetic modularity and integration of the neurocranium (e.g., [77, 8083, 8589, 106144 ]), there are good reasons to be suspicious about the use of the atomisation in this instance. according to the functional matrix hypothesis, the cranium comprises various functional cranial components (fcc), which are ontogenetically integrated. one fcc contains the brain, cerebellum, and ocular globe, sharing a common embryological origin in the neural tube. this fcc develops in an integrated way being subjected to common heritable and epigenetic factors. the neurocranium largely develops as a result of the passive displacement of the vault bones, occurring within a connective tissue stroma, which is enlarged by the growing brain. as a result, all three parts of this fcc follow a common growth trajectory, reaching 9096% of their adult size by about age 6 - 7 [77, 89, 115, 129 ]. however, the neurocranium also forms an integrated unit with the cranial base, the neurobasicranial complex, and its form is strongly influenced by factors such as basicranial flexion, while the developing face also epigenetically fine - tunes basicranial morphology, indirectly altering the form of the cranial vault [122, 123, 131, 132, 136, 138144 ]. additionally, he suggested that superstructures develop in response to tension (pull) from muscles, a hypothesis subsequently verified experimentally with respect to some cranial crests [77, 78, 91, 92, 115, 160, 161 ], implying that they belong to, or sit only upon, the outer bony table [91, page 208 ]. however, this situation is not universal for superstructures and their development is complex. as enlow has shown [77, 91, 115, 161 ], the external surface of a bone is frequently shifted from an endosteal position. this means that developmental changes within the endocranium as well as epigenetic factors acting on the external table determine the morphology of the vault. moreover, different processes may be involved in remodelling the neurocranium in spatially adjacent regions, and different regions of the cranium may exert influence over the growth of a single structure at different ontogenetic stages. for example, about midway up the frontal squama is a reversal line from resorptive (inferiorly) to depositional (superiorly) remodelling. once the frontal lobes have largely completed their enlargement, the internal table stops growing, while the outer table continues to remodel, progressively separating the inner and outer tables, and replacing through resorption cancellous bone to form the frontal sinus. however, this continued growth now results directly from the anterior remodelling of the upper face or nasomaxillary complex. further, the orbit (subunit) is contained within this region, and the inner table of its roof (endocranial side) is resorptive while the external (orbital plate) is depository. as the orbital roof continues to remodel inferiorly along with the growth of the frontal lobes of the brain, the orbit must grow anteriorly to provide sufficient room for the growing eye ; it is displaced outwards with other bones of the orbit which form part of the face according to the v principle. in the case of the supraorbital torus, also considered to be a superstructure by weidenreich, there is now abundant evidence that this feature develops epigenetically as a result of the spatial relationships among the face, neurocranium, and cranial base rather than biomechanical stresses during mastication [77, 115, 162167 ], contra. for example, figure 6 is a holocene san skull indicating stress gradients from masticatory stress as analysed with the finite element method. the supraorbital region is unaffected by strain in this homogenous 3d model (but see similar results using a heterogeneous model). thus, while 14 characters might be regarded as superstructures of the cranial vault, developing to provide muscle attachments and remodelling in response to mechanical stress, research into the supraorbital torus makes clear that the two orbital roof features lack epigenetic influences from the muscles of mastication. the supraorbital develops as a result of a complex interplay between the shape of the neurocranium and breadth of the basicranium, growth of the frontal lobes of the brain, development of the frontal sinus, remodelling of the orbit to accommodate the growing eye, and growth of the nasal region, which develops in response to lung enlargement and in particular affects the superior orbital rim, and the anteroposterior length of the face seems also to be a determinant of its position relative to the frontal squama and, therefore, the degree of frontal recession seen [77, 115, 122, 123, 137, 162171 ]. moreover, crania with high midfacial projection exhibit a long anterior sphenoid resulting in a viscerocranium positioned anterior to the neurocranium [133, 134, 170, 171 ] and in a prominent supraorbital. finally, the development of the supraorbital region may even be epigenetically influenced by exercise through the effects of hormones seemingly independently from (but commonly with) other structures such as thickened vault bones (see [172, 173 ] ; see also). as noted above, mitteroecker and bookstein have found four common cranial factors or trajectories across a range of primates including humans, which are integrated through genetic effects such as pleiotropy and linkage, as well as epigenetic processes. factor 1an enlarged and prognathic maxilla, relatively small cranial capsule, cranial crests, and enlarged zygomatic arches are features epigenetically associated in primates mainly as a result of masticatory muscle action. these are all characteristics of cranial robusticity and while they can be observed as a package in better - preserved crania (e.g., cohuna, kow swamp 1), some of them are observable in incomplete remains such as wlh50 (table 1, figures 2 and 7) [2, 814, 1618, 4143, 4652, 54, 55, 64, 72, 151 ]. an enlarged and prognathic maxilla, relatively small cranial capsule, cranial crests, and enlarged zygomatic arches are features epigenetically associated in primates mainly as a result of masticatory muscle action. these are all characteristics of cranial robusticity and while they can be observed as a package in better - preserved crania (e.g., cohuna, kow swamp 1), some of them are observable in incomplete remains such as wlh50 (table 1, figures 2 and 7) [2, 814, 1618, 4143, 4652, 54, 55, 64, 72, 151 ]. factor 2this pattern contrasts broad and short crania with narrow and long crania (brachycephalic versus dolichocephalic crania), including both the face and the neurocranium, and also involves changes in the overall size of the face relative to the neurocranium. all robust australians (e.g., cohuna, ks1, wlh50) exhibit narrow and long crania (figures 2 and 7) [2, 814, 1618, 4143, 4652, 64, 72, 151 ]. overall neurocranial length, breadth, and height are characterised by moderate but significant levels of heritability, suggesting a large amount of variation in these features determined by epigenetic forces [79, 81, 82 ]. moreover, while their faces are absolutely long and consistent with this factor (e.g., ks1, ks5, cohuna), they differ in exhibiting absolutely and relatively broad faces [2, 8, 9, 1114, 17, 18, 4143, 4749, 51, 52, 151 ]. however, facial shape has been found to exhibit weaker correlations with neurocranial and basicranial shape than these regions have with each other, perhaps explaining why the face is the most variable part of the skull in humans. additionally, the dimensions of facial length, breadth, and height are characterised by moderate heritability indicating that additive genetic variation accounts for approximately 30% of the phenotypic variation [79, 81, 82 ]. further, facial breadth is well known to be under strong ontogenetic influence from the muscles of mastication [171, 174178 ]. a positive correlation between total masticatory muscle size (cross - sectional area) and body size (stature and weight) has also been found [171, 174178 ]. the body size of the wlh50 individual, like other pleistocene aboriginal australians, would have been large by later holocene and contemporary standards [8, 17 ]. in fact, metrical dimensions of size and endocranial volume reveal it to be one of the largest pleistocene modern humans found anywhere in the world and comparable in size to early african crania such as from herto, and larger than many nonmodern individuals (compare with data in white.), including h. erectus calvarii. this pattern contrasts broad and short crania with narrow and long crania (brachycephalic versus dolichocephalic crania), including both the face and the neurocranium, and also involves changes in the overall size of the face relative to the neurocranium. all robust australians (e.g., cohuna, ks1, wlh50) exhibit narrow and long crania (figures 2 and 7) [2, 814, 1618, 4143, 4652, 64, 72, 151 ]. overall neurocranial length, breadth, and height are characterised by moderate but significant levels of heritability, suggesting a large amount of variation in these features determined by epigenetic forces [79, 81, 82 ]. moreover, while their faces are absolutely long and consistent with this factor (e.g., ks1, ks5, cohuna), they differ in exhibiting absolutely and relatively broad faces [2, 8, 9, 1114, 17, 18, 4143, 4749, 51, 52, 151 ]. however, facial shape has been found to exhibit weaker correlations with neurocranial and basicranial shape than these regions have with each other, perhaps explaining why the face is the most variable part of the skull in humans. additionally, the dimensions of facial length, breadth, and height are characterised by moderate heritability indicating that additive genetic variation accounts for approximately 30% of the phenotypic variation [79, 81, 82 ]. further, facial breadth is well known to be under strong ontogenetic influence from the muscles of mastication [171, 174178 ]. a positive correlation between total masticatory muscle size (cross - sectional area) and body size (stature and weight) has also been found [171, 174178 ]. the body size of the wlh50 individual, like other pleistocene aboriginal australians, would have been large by later holocene and contemporary standards [8, 17 ]. in fact, metrical dimensions of size and endocranial volume reveal it to be one of the largest pleistocene modern humans found anywhere in the world and comparable in size to early african crania such as from herto, and larger than many nonmodern individuals (compare with data in white.), including h. erectus calvarii. factor 3encompassing relative size of the midface and neurocranial globularity, two characteristics that are tightly associated during postnatal ontogeny. while sample size disallows a proper comparison, robust australian individuals such as ks1 are certainly characterised by a relatively narrow midface (zmb / nph = c66%) compared with the gracile keilor cranium (c76%) (data from). moreover, these and other crania are strongly contrasted in their degree of neurocranial globularity ; robust crania exhibiting highly angulated vaults, with receding frontal squamae (table 1, figure 2) [2, 8, 9, 1114, 17, 18, 4143, 4749, 51, 52, 151 ]. in contrast, individuals such as wlh1, wlh3, and keilor possess more globular crania (see figure 7) [2, 9, 13, 14, 17, 41, 42, 5153, 71, 72, 151 ]. encompassing relative size of the midface and neurocranial globularity, two characteristics that are tightly associated during postnatal ontogeny. while sample size disallows a proper comparison, robust australian individuals such as ks1 are certainly characterised by a relatively narrow midface (zmb / nph = c66%) compared with the gracile keilor cranium (c76%) (data from). moreover, these and other crania are strongly contrasted in their degree of neurocranial globularity ; robust crania exhibiting highly angulated vaults, with receding frontal squamae (table 1, figure 2) [2, 8, 9, 1114, 17, 18, 4143, 4749, 51, 52, 151 ]. in contrast, individuals such as wlh1, wlh3, and keilor possess more globular crania (see figure 7) [2, 9, 13, 14, 17, 41, 42, 5153, 71, 72, 151 ]. factor 4a roundish and relatively short and high neurocranium to elongated, ellipsoidal crania.. robust australian crania all exhibit ellipsoidal crania (superior outline) ; many have an occipital bun and lambdoid flattening, and all exhibit prominent browridges by modern human standards (see figures 2 and 7) [2, 8, 9, 1114, 17, 18, 4143, 4749, 51, 52, 151 ]. although the wlh50 calvaria is actually relatively tall in addition to being very long, this probably results from the combination of large endocranial volume and very narrow cranial base (see [17, 156 ] ; see also [180, 181 ]). a range of other factors interacting in a complex manner are also known to influence the development of the supraorbital part, cranial shape being one of them (see above). a roundish and relatively short and high neurocranium to elongated, ellipsoidal crania. exaggeration of the latter generates an occipital bun and lambdoid flattening along with large browridges. robust australian crania all exhibit ellipsoidal crania (superior outline) ; many have an occipital bun and lambdoid flattening, and all exhibit prominent browridges by modern human standards (see figures 2 and 7) [2, 8, 9, 1114, 17, 18, 4143, 4749, 51, 52, 151 ]. although the wlh50 calvaria is actually relatively tall in addition to being very long, this probably results from the combination of large endocranial volume and very narrow cranial base (see [17, 156 ] ; see also [180, 181 ]). a range of other factors interacting in a complex manner are also known to influence the development of the supraorbital part, cranial shape being one of them (see above). it is interesting also to note that mitteroecker and bookstein found that their factors 1 and 2 were the most highly conserved among humans, chimpanzees, and gorillas, with all three sharing a single ontogenetic trajectory owing to the presence of tight integration during development. many of the traits listed in table 1 can be explained by variation within these two highly constrained modules. trajectories for factors 3 and 4 were found to deviate considerably in humans compared to chimpanzees and gorillas suggesting that certain human cranial features have decoupled during evolution. they opined that this difference was best explained by local developmental factors unique to humans such as relative brain size, posture, and locomotion (factor 3), and facial shape, mainly involving the browridge (factor 4). in the end, this research indicates that selection does not act on individual features, but instead cranial traits form part of an integrated and heritable set of factors or trajectories. australian cranial robusticity lies on the trajectory for all four factors but is never as extreme in its expression as seen among nonmodern hominins. figure 7 presents 3d models (scaled to the same length) constructed by the author from ct - scans of wlh3 and wlh50, a holocene african hunter - gatherer and various pleistocene (nonmodern) hominins. compared to wlh3 and the holocene san individual, wlh50 does exhibit a receding frontal squama, prominent browridge, and some projection of the occipital. however, compared to all nonmodern, middle pleistocene calvarii, wlh50 is tall, has a more upright frontal squama, which bulges anteriorly (prebregmatic eminence), and has relatively rounded parietal and occipital profiles and mild lambdoidal flattening. moreover, it lacks the strongly projecting browridge and a posttoral shelf so evident in all middle pleistocene remains, whether belonging to h. erectus, h. heidlebergensis / rhodesiensis, or archaic h. sapiens / h. helmei. its profile including the frontal squama is quite rounded (globular) compared to nonmodern crania. thus, the purported similarities between wlh50 and h. erectus seem to have been greatly exaggerated, particularly when a comparison involving middle pleistocene african crania, the putative ancestors of all modern humans, is made. instead, wlh50 simply presents as a more rugged version of the wlh3 morphology, the two resembling each other in their angle of the posterior part of the frontal squama and profile of the parietals and occipital ; when compared with the san cranium, and indicating regional - specific features for australian crania. moreover, the comparatively modest differences in shape between wlh3 and wlh50 seem to be explicable in terms of the ontogenetic trajectories described by mitteroecker and bookstein. another important recent study of cranial robusticity with strong bearing on the morphology of wlh50 and other pleistocene / early holocene australians is that of baab.. they tested for integration / correlation among various robusticity traits in a global sample of humans including recent aboriginal australians. overall, they found that crania with more prognathic faces, expanded glabellar and occipital regions, and longer skulls were more robust than those with rounder vaults and more orthognathic faces. this supports the findings of lahr and wright and, more broadly, the results of mitteroecker and bookstein. baab. also found evidence for significant positive but weak coexpression among all robust traits (frontal trigone, sagittal keel, infraglabellar notch, supraorbital torus, zygomaxillary tubercle, and prebregmatic eminence) with the exception of the occipital torus. they did, however, find that the supraorbital torus is strongly and significantly correlated with an infraglabellar notch and moderately and significantly correlated with a zygomaxillary tubercle and a zygomatic trigone. moreover, moderate and significant correlations were found between the zygomaxillary tubercle and bregmatic eminence, inferolateral rounding of the orbit, infraglabellar notch, and zygomatic trigone ; between a bregmatic eminence and inferolateral rounding of the orbit ; infraglabellar notch and zygomatic trigone. they concluded that epigenesis through masticatory function was the most likely cause of the development of these traits.. failed to show correlation with the other robust traits they examined, namely, the occipital torus. for example, perez. have described a positive association between occipital muscle attachment areas and head shape (dolichocephaly) among males in a sample of south american hunter - gatherer crania. thus, it may simply be an epigenetic consequence of an elongated cranium, in - line with other changes such as a protruding occipital and formation of an occipital bun [106, 122, 123, 180, 181 ]. functionally, this feature is suggested to provide attachment for nuchal ligament or to represent an area of resorptive bone developing in response to bending forces along the nuchal torus. irrespective of its cause, this feature is actually found in both wlh3 and wlh50 (personal observation) and can not, therefore, be considered a defining trait of australian robusticity nor evidence bearing on their phylogenetic history. the issue of vault ridges (or keeling) has not been explicitly addressed in most ontogenetic studies of the human cranium. although baab. did find statistically significant moderate correlations between a sagittal keel and bregmatic eminence, both features are associated with cranial vault sutures. their development may, therefore, be part of the integrated process of ontogeny and result from epigenetic factors relating to cranial shape and mechanical forces. the research of baab. also highlights that while integration and modularity are fundamental to understanding cranial ontogeny, certain features sometimes show lower than expected correlation and covariation than expected. a number of researchers have specifically addressed the issue of the mechanical effects of mastication on cranial sutures experimentally. in vivo studies found that strain (tension or compression) along sutures was not the result of torsion but rather the localised effects of masticatory actions. strain was found to be significant along all of the major vault sutures, and its effects varied depending upon the muscle involved. for example, while masseter contraction tensed the coronal suture, the temporal caused compression at this region. moreover, an earlier study found that even a relatively small bite force was sufficient to cause separation of the sagittal suture in juvenile monkeys, from which they concluded that such separation might act as a major factor in the local control of osteogenesis of the sutures [189, page 907 ]. additionally, sun. confirmed earlier findings of significant masticatory strain along vault sutures and further found that synostosis (normal fusion) resulted in increased suture strain and enhanced bone growth on the ectocranial surface leading to thicker bones in adults. finally, the sutures have also been suggested to play a role in dissipating masticatory strain, while the diplo may also play a similar role ; although these hypotheses require further testing. clinical interest in cranial ridges along the major vault sutures stems from understanding the causes and treatment of abnormal head shape as result of congenital or acquired conditions (such as birth injuries) or pathophysiological processes leading to clinical craniosynostosis (premature suture closure). however, in the absence of changes to vault or facial shape, they are considered normal variants. the metopic ridge can arise in an infant (before two years of age) from premature closure of the metopic suture, while a midline bony ridge over vertex from front to back may develop following premature fusion of the sagittal suture in adults prior to 40 years of age. coronal bony ridges can also form in cases of premature craniosynostosis, again arising any time before normal closure of the coronal suture (typically around 40 years of age). premature synostosis of one or more sutures is accompanied by compensatory growth, both in other sutures, and by remodelling (appositional growth) of other parts of the skull. premature closure of sutures prevents separation of the bones and affects skull growth in a direction perpendicular to the affected suture, leading to skull deformations [194, 195 ]. much later in life (i.e., around 40 years of age), premature craniosynostosis of the sagittal and coronal sutures can occur and leads in mild cases to the development of sagittal and coronal ridges. among robust australian crania, the parietals are relatively long and the lower occipital scale (nuchal plane) is unusually relatively short. moreover, premature craniosynostosis of the sagittal suture at around 40 years of age can result in dolicocephaly, accompanied by a narrow head with bitemporal widening, sometimes frontal and occipital bossing, and a midline bony ridge over the vertex from front to back. the current clinical incidence of synostosis is 1 in 2,0003,000 newborns although syndromic craniofacial malformations involving more than one suture occur in about 15% of cases. the aetiology of craniosynostosis is heterogeneous : hereditary, mechanical, teratogenic, and idiopathic. multiple genetic and environmental causes have been identified. among the latter are rickets caused by vitamin d deficiency or resistance, chronic renal failure, hypothyroidism and hypophosphatasia, and multiple causes of abnormal fetal positioning in utero leading to constraint of the fetal skull. for example, white found an incidence of 3% in a sample of normal mayan skulls, rising to 31% among those from the precontact period ; and in 41% of them premature synostosis was apparently explained by artificial deformation. this lends support to suggestions that artificial deformation may be an exaggerating influence in the robusticity of pleistocene / holocene australians [9, 146150 ]. the idea that aboriginal australians posses an unusual and phylogenetically informative pattern of cranial robusticity has been important in palaeoanthropology for about 150 years. there has, however, been a wide range of views about specific aspects of their evolutionary history. particularly, whether or not pleistocene nonmodern populations in southeast asia (i.e., pithecanthropus, h. soloensis, or h. erectus) played a role in their evolutionary history. irrespective of whether the multiregional, assimilation, or out - of - africa model has been supported, most investigations have taken the human cranium to be optimised part by part, and atomised its morphology into traits assumed to be heritable units, functionally discrete, shaped by natural selection and, therefore, to be positively associated with reproductive success in order to make phylogenetic inferences. yet, a large body of research from the morphological sciences, including anatomy and embryology, pathology, genetics, and evolutionary biology makes it clear that these assumptions may be unrealistic and that atomisation should be used with great care in phylogenetic reconstruction. the human (vertebrate) cranium is characterised by ontogenetic and functional modularity, with much integration / covariation among the elements within each module, as well as between them. moreover, epigenesis is a powerful, pervasive, and very complex force, its influences being felt in all levels of organisation during ontogeny, from cellular interactions to those among adjacent tissues to interactions with the external environment of an organism. additionally, while many aspects of cranial size and shape are heritable, epigenesis may determine 70% or more of variation seen within populations with well - established pedigrees [7982 ]. finally, many of the traits designated robust are also characterised by some ontogenetic integration and are likely to derive from a set of common epigenetic factors, which includes the spatial effects / constraints of size and shape, for example, central integrating features such as the cranial base and mechanical effects from the muscles of mastication. while there is clearly a strong message in this research for palaeoanthropology in general, its implications for studies of australian aboriginal robusticity are particularly acute. phylogenetic reconstruction across all organisms and employing all methods involves a set of important assumptions about the traits used. for example, lieberman lists seven such assumptions, the first four being relatively uncontroversial, with the remaining three particularly relevant here although hardest to satisfy ; (5) characters should reflect independent units of information about phylogenetic relationships, (6) they must be heritable if they are to provide reliable information on ancestry and descent, and (7) they should be equivalent units or homologous structures. the ontogenetic approach outlined here suggests that these assumptions probably can not be met in the case of phylogenetic reconstructions involving robust australian crania when using atomised traits. most characters are unlikely to be independent units, being integrated in their development and influenced by a complicated set of epigenetic interactions [77, 8389, 106144, 160167, 169178, 180183, 188197 ]. they also show mostly low to moderate heritability [7982 ] and are in some instances clearly not homologous in modern humans and nonmodern hominins. for example, a character used widely in studies of the evolutionary origins of aboriginal australians is the possession of a flat frontal squama among robust individuals [2, 8, 9, 1114, 1618, 3943, 4749, 51, 52, 63, 72, 151 ]. putting aside epigenetic explanations involving pseudopathology [17, 146150 ], this trait fails to satisfy all three assumptions of phylogenetic reconstruction. the size and shape of the growing brain in accordance with the functional matrix hypothesis [77, 88, 114, 115 ] and its epigenetic interactions with the developing cranial base moreover, there are also influences from the growing face although, they are more subtle than those of the basicranium [122, 123, 131, 132, 136, 138144 ]. the anteroposterior length of the face seems also to be a determinant of its position relative to the frontal squama and, therefore, the degree of frontal recession present ; crania with high midfacial projection exhibit a long anterior sphenoid, resulting in a viscerocranium positioned anterior to the neurocranium [122, 123, 131134 ]. in nonmodern hominins like h. erectus, a flat frontal squama results from a complex combination of a small frontal brain lobe, broad cranial base, combined high midfacial projection and long anterior sphenoid. however, in anatomically modern humans with our large brains, including relatively broad and steep frontal lobe, angulation of the frontal squama results from the brain being epigenetically forced to grow excessively posteriorly by a narrow cranial base resulting in a long and tall cranium [17, 156, 180, 181 ]. moreover, a flat frontal squama is associated with a browridge, the orbital roof being forced to grow forward to accommodate the growing eye, and an occipital bun, the result of the posterior growth of the brain within a narrow vault. additionally, because the brain and its frontal lobe are relatively broader and more globular (taller) in modern humans and our face characterised by significant retraction [137, 170 ], the brow is never as prominent in modern humans as it is in nonmodern hominins including in wlh50 (see figure 7) [122, 123, 181, 199 ]. as noted above, wlh50 is among the largest modern humans found in the fossil record globally and is comparable in size to the earliest modern remains such as from herto (see data in), and larger than many nonmodern crania including h. erectus. while large cranial size and a narrow cranial base, as seen in wlh50, are certainly plesiomorphic traits, they are not shared with h. erectus or other nonmodern hominins but are plesiomorphies of anatomically modern h. sapiens [17, 122, 123 ]. moreover, they seem to have been important influences on the form of this calvaria and other australian remains. to conclude, there is now ample evidence that the atomisation of cranial robusticity has provided a misleading picture of the evolutionary history of aboriginal australians. moreover, models suggesting that living australians as demonstrated by their robust cranial morphology can trace their origins to the nonmodern hominins of pleistocene southeast asia should be reconsidered in light of major developments in the morphological sciences. in 1976, howells issued a challenge to australian palaeoanthropology to reject a null hypothesis that differences between gracile and robust crania can be explained by phenotypic plasticity alone. it is time to reconsider the adaptationist program and to take a more parsimonious approach to explaining aboriginal australian origins, one that takes account of the complex processes involved in the ontogeny of the human cranium rather than just assuming that natural selection explains every subtle variation seen in past populations. | the origin of aboriginal australians has been a central question of palaeoanthropology since its inception during the 19th century. moreover, the idea that australians could trace their ancestry to a non - modern pleistocene population such as homo erectus in southeast asia have existed for more than 100 years, being explicitly linked to cranial robusticity. it is argued here that in order to resolve this issue a new program of research should be embraced, one aiming to test the full range of alternative explanations for robust morphology. recent developments in the morphological sciences, especially relating to the ontogeny of the cranium indicate that character atomisation, an approach underpinning phylogenetic reconstruction, is fraught with difficulties. this leads to the conclusion that phylogenetic - based explanations for robusticity should be reconsidered and a more parsimonious approach to explaining aboriginal australian origins taken. one that takes proper account of the complex processes involved in the growth of the human cranium rather than just assuming natural selection to explain every subtle variation seen in past populations. in doing so, the null hypothesis that robusticity might result from phenotypic plasticity alone can not be rejected, a position at odds with both reticulate and deep - time continuity models of australian origins. |
all the samples were collected from patients undergoing surgical ablation of larynx squamous cell carcinoma at the head and neck surgery division of the department of ophthalmology, otorhinolaryngology and head & neck of ribeirao preto medical school, usp (brazil). inclusion criteria were : histopathological diagnosis of lscc, elective surgeries for lscc in patients without previous treatments and patients ' allowance to donate part of their tumor for genetic studies. exclusion criteria were : doubtful diagnosis of lscc, unavailable post - surgical follow - up, patients without complete clinical data or signed agreement for collection of samples. a total of twenty nine lscc tumor samples and thirteen adjacent non - neoplastic tissues proceed for the transcriptome analysis. clinical information and tnm staging classification of the lscc patients most of the patients were male (96.8%), all of them were smokers and alcoholic, including both current and former. thirty five percent of the tumors were originated from glottis, followed by larynx (29.0%) and supraglottis (22.6%). patients were further classified according to the tnm system : 48.4% of the patients were in early staging and 51.6% in late staging. this study was approved by the ethics committee of ribeirao preto medical school, university of sao paulo (usp) (proc. after lscc histopathological confirmation and microdissection of the tumors from their non - neoplastic adjacent tissue, the samples were store in liquid nitrogen. after extraction, the rna was purified with rneasy kit (qiagen, usa) and quantified with nanodrop spectrophotometer (thermo scientific, usa). its quality was evaluated by 1.5% agarose gel electrophoresis (28s and 18s ribosomal rna detection). for the microarray analysis it was used the whole human genome oligo microarray kit 4 44k (agilent, g4112f, usa). cdna was hybridized to the microarray chip in the fluidics station 450 system (affymetrix, usa), using the quick amp labeling one - color kit (agilent, usa). as an internal mrna control, the one color rna spike - in kit was used (agilent, usa). agilent feature extraction software (version 9.5.3.1) was used to analyze acquired array images. plain text files were loaded and processed into r with the bioconductor package agi4 44pre - process. the cv.rep.probes function was used to estimate the percent of coefficient of variation (% cv) for replicated non - control probes, inferring the reproducibility of the arrays (fig. the gprocessedsignal and the gbgused values of each array were loaded as foreground and background signals, respectively into an rglist object. the following flags were used to filter probes : control, well above bg, is found, well above neg ctrls, is saturated, population outlier, and non uniform outlier. additionally probe signals with at least 90% of good features in an experimental condition were selected. it was performed by looking for arrays (a and b), which the sum (s) of the distances (d) to all other arrays, sa = b dab was exceptionally large ; calculating the kolmogorov smirnov statistic (ka) between each array and the pooled data distribution ; and computing hoeffding 's statistic (da) on the joint distribution of a and m for each array (m = log2(i1) log2(i2) ; a = 1/2 (log2(i1) + log2(i2)), where i1 is the intensity of the array studied, and i2 is the intensity of a pseudo-array that consists of the median across arrays). stagewise normalization is recommended when data include technical and biological replicates producing a better median chip. then, quantile normalization methods were applied, within groups and subsequently between all arrays. each set of replicated non - control probes has been collapsed into a single value computed as the median of the probes intensities belonging to the same set. principal component analysis (pca) was applied to classify samples by tumor and adjacent non - neoplastic tissue as demonstrated on fig. we compared the gene expression between tumor and adjacent non - neoplastic tissue using a non - paired - t test analysis. false discovery rate (fdr) adjusted p - value 10 were accepted to consider genes to be differentially expressed, identifying a total of 81 probes above this cut - off (fig. 3). then we investigated the biological process of upregulated and downregulated genes in tumor compared to normal samples with web - based gene set analysis toolkit (webgestalt). p - value was calculated using hypergeometric distribution and fdr as multiple hypothesis test correction method. tissue morphogenesis (go : 0009888) and differentiation (go : 0032332) are the main biological process in upregulated genes while cytokines and growth factor genes are highly expressed in adjacent non - neoplastic patients, suggesting a role on lscc tumorigenesis. all the samples were collected from patients undergoing surgical ablation of larynx squamous cell carcinoma at the head and neck surgery division of the department of ophthalmology, otorhinolaryngology and head & neck of ribeirao preto medical school, usp (brazil). inclusion criteria were : histopathological diagnosis of lscc, elective surgeries for lscc in patients without previous treatments and patients ' allowance to donate part of their tumor for genetic studies. exclusion criteria were : doubtful diagnosis of lscc, unavailable post - surgical follow - up, patients without complete clinical data or signed agreement for collection of samples. a total of twenty nine lscc tumor samples and thirteen adjacent non - neoplastic tissues proceed for the transcriptome analysis. clinical information and tnm staging classification of the lscc patients most of the patients were male (96.8%), all of them were smokers and alcoholic, including both current and former. thirty five percent of the tumors were originated from glottis, followed by larynx (29.0%) and supraglottis (22.6%). patients were further classified according to the tnm system : 48.4% of the patients were in early staging and 51.6% in late staging. this study was approved by the ethics committee of ribeirao preto medical school, university of sao paulo (usp) (proc. after lscc histopathological confirmation and microdissection of the tumors from their non - neoplastic adjacent tissue, the samples were store in liquid nitrogen. after extraction, the rna was purified with rneasy kit (qiagen, usa) and quantified with nanodrop spectrophotometer (thermo scientific, usa). its quality was evaluated by 1.5% agarose gel electrophoresis (28s and 18s ribosomal rna detection). for the microarray analysis it was used the whole human genome oligo microarray kit 4 44k (agilent, g4112f, usa). cdna was hybridized to the microarray chip in the fluidics station 450 system (affymetrix, usa), using the quick amp labeling one - color kit (agilent, usa). as an internal mrna control, the one color rna spike - in kit was used (agilent, usa). agilent feature extraction software (version 9.5.3.1) was used to analyze acquired array images. plain text files were loaded and processed into r with the bioconductor package agi4 44pre - process. the cv.rep.probes function was used to estimate the percent of coefficient of variation (% cv) for replicated non - control probes, inferring the reproducibility of the arrays (fig. the gprocessedsignal and the gbgused values of each array were loaded as foreground and background signals, respectively into an rglist object. the following flags were used to filter probes : control, well above bg, is found, well above neg ctrls, is saturated, population outlier, and non uniform outlier. additionally probe signals with at least 90% of good features in an experimental condition were selected. it was performed by looking for arrays (a and b), which the sum (s) of the distances (d) to all other arrays, sa = b dab was exceptionally large ; calculating the kolmogorov smirnov statistic (ka) between each array and the pooled data distribution ; and computing hoeffding 's statistic (da) on the joint distribution of a and m for each array (m = log2(i1) log2(i2) ; a = 1/2 (log2(i1) + log2(i2)), where i1 is the intensity of the array studied, and i2 is the intensity of a stagewise normalization is recommended when data include technical and biological replicates producing a better median chip. then, quantile normalization methods were applied, within groups and subsequently between all arrays. each set of replicated non - control probes has been collapsed into a single value computed as the median of the probes intensities belonging to the same set. principal component analysis (pca) was applied to classify samples by tumor and adjacent non - neoplastic tissue as demonstrated on fig. 2. we compared the gene expression between tumor and adjacent non - neoplastic tissue using a non - paired - t test analysis. false discovery rate (fdr) adjusted p - value 10 were accepted to consider genes to be differentially expressed, identifying a total of 81 probes above this cut - off (fig. then we investigated the biological process of upregulated and downregulated genes in tumor compared to normal samples with web - based gene set analysis toolkit (webgestalt). p - value was calculated using hypergeometric distribution and fdr as multiple hypothesis test correction method. tissue morphogenesis (go : 0009888) and differentiation (go : 0032332) are the main biological process in upregulated genes while cytokines and growth factor genes are highly expressed in adjacent non - neoplastic patients, suggesting a role on lscc tumorigenesis. we describe detailed technical methods to reproduce the analysis of whole human genome oligo microarray kit (agilent, g4112f, usa) from twenty - nine lscc tumor samples and thirteen adjacent non - neoplastic tissues. lscc is the second most common malignancies of the head and neck tumors, and this high frequency may be associated with the lifestyle of patients,,. the high quality gene expression data are concordant with important clinical parameters, demonstrating the relevant expression profile signature of differentially expressed genes. the following is the supplementary data related to this article.supplemental table 1tumor sample description. tumor sample description | laryngeal squamous cell carcinoma (lscc) is one of the most common malignancies of the head and neck tumors zhang., 2013 [1 ]). previous studies have associated its occurrence with social activities, such as tobacco and alcohol consumption (hashibe., 2007a [2 ] ; hashibe., 2007b [3 ] ; shangina., 2006 [4 ]). here, we performed a genome - wide gene expression profiling in thirty - one patients positively diagnosed for lscc, in order to investigate new targets involved in tumorigenesis. |
the current recommendation for treatment of bilateral inguinal hernia is to repair both sides during the same surgery and anaesthetic procedure and to use the mesh on the transverse or pre - peritoneal fascia. laparoscopic hernia repair became feasible after the incorporation of the principles of the technique described by stoppa. two alternative techniques were improved and have been used routinely : the transabdominal preperitoneal (tapp) and the total extraperitoneal (tep). both approaches use meshes larger than those used with an open anterior approach to reinforce the abdominal wall in the inguinal region and perform parietalisation of the spermatic cord structures. studies have shown evidence favouring the techniques with a tension - free mesh because they cause less pain and a lower post - operative recurrence. laparoscopic inguinal hernia repair has existed for almost 20 years, but its acceptance is not unanimous. some factors such as the need for extensive training, the tenuous benefit to the patient, and the higher cost explain the hesitation to adopt the laparoscopic approach as a standard procedure for all patients. the present clinical trial was designed to compare the results of bilateral inguinal hernia repair between patients who underwent the conventional stoppa technique and laparoscopic total extraperitoneal repair (lte) with a single mesh and without staple fixation. this controlled, randomised clinical trial was approved by the ethics committee for the research projects analysis of the clinics hospital / university of so paulo medical school (hcfmusp), and was conducted as part of the general surgery and trauma subject at hcfmusp between september 2010 and february 2011. prior to being included, the patients were informed about the study and then filled out the informed consent term that is standardised in the institution. totally, 50 patients were considered eligible for the study : male patients with bilateral inguinal hernia (nyhus classification), older than 25 years, with a body mass index (bmi) below 29 and with american society of anaesthesiologists (asa) class i and ii. patients with scrotal inguinal hernia, younger than 25 years, with a bmi above 29, asa class iii and iv, as well as patients with liver disease and anaemia who were immunocompromised or who previously underwent infraumbilical surgery were excluded from the study. randomisation was performed to offer the 50 patients the same opportunity to belong to either the ' stoppa ' group or the ' lte ' group. for that purpose, computer software determined the sequence of the surgeries (stoppa or lte). blood samples were collected from the patients 24 h before and 24 h and 48 h after the procedure for c - reactive protein (crp), haemoglobin level, haematocrit level, and leucocytes / mm measurements. patients responded to a questionnaire to assess their pain intensity from the 1 to 7 post - operative days, their physical functioning, the impact of pain on physical activities, and daily activities (three domains of the short form-36 (sf-36)) and post - operative comfort (carolinas comfort scale). pain intensity was assessed using the pain visual analogue scale (vas) with values ranging from 1 (no pain) to 10 (worst possible pain). the carolinas comfort scale assesses the comfort associated with eight types of usual physical movements : bending over, standing up, sitting down, getting out of bed, coughing, walking, climbing or descending stairs and exercising. a monofilament, macroporous polypropylene mesh (26 cm 36 cm) was used. the mesh was cut so that its size and shape would fit the anatomical characteristics of the patient. the width of the prosthesis was determined by the distance between the two anterior superior iliac spines minus 2 cm (w). the height of the prosthesis was defined by the distance between the upper edge of the pubis to 1.5 cm above the imaginary line that joins the anterior superior iliac spines, plus 4 cm (h). the mesh was cut and configured according to the lower recesses of the retropubic and retroinguinal regions to ensure that the mesh will be well positioned and stretched without folds [figure 1 ]. l (mesh length) = distance between anterior superior iliac spine-2 cm ; h (mesh height) = distance between the base of the penis and the imaginary line of the two anterior superior iliac spines for stoppa group, a pfannenstiel incision was performed 3 cm above the pubis. both retropubic space of the retzius and the right and left the retroinguinal space of bogros were dissected through this incision. the vas deferens and spermatic vessels were dissected out and separated from the peritoneum, allowing the bilateral parietalisation of these structures. the mesh was placed between the components of the spermatic cord and the peritoneum, with no need to split the mesh. a polypropylene mesh with an appropriate size and shape was used according to the anatomical characteristics of the patient. finally, the mesh did not display wrinkles or folds between the muscle and the peritoneum. for lte group, the surgery was initiated with a 3-cm transverse incision in the midline, 1.5 cm above the imaginary line that joins the two anterior superior iliac spines. a blunt dissection was performed between the transverse fascia and parietal peritoneum using a sponge clamp with gauze towards the pubis under direct vision, separating the bladder and parietal peritoneum from the anterior abdominal wall and exposing the retropubic space of retzius. laterally, the retroinguinal space of bogros was dissected between the anterior wall and peritoneum until the level of the previous iliac spines. after the dissection of the pre - peritoneal space, a 10-mm permanent trocar was introduced through the incision, which was narrowed around the trocar with continuous vicryl 00 sutures. carbon dioxide was then insufflated into the pre - peritoneal space until a pressure of 12 mmhg was reached. next, a 30 laparoscope (10 mm) was introduced, and the prevesical and retroinguinal space were examined. under the laparoscopic vision, a 5-mm trocar was then sequentially introduced in the right and left iliac fossa. the identification, dissection of the spermatic cord structures and parietalisation were similar to that performed for open surgery. after the dissection, the mesh was introduced into the pre - peritoneal space by pulling it with the thread placed in the suprapubic region that also helped to fix the mesh. at the end of the intervention, operation duration was recorded (in minutes) and defined as the time interval between the beginning of the incision and final stitch in the skin. the quantitative variables were expressed as the mean standard deviation and were analysed using student 's t - test. to compare the biochemical, haematological, pain and sf-36 domain parameters between the two groups and over time, two - way analysis of variance for repeated measures was used followed by the bonferroni and tukey 's post - hoc tests. graphpad software version 5.0 for windows (graphpad software, san diego, ca, usa) and spss version 18.0 6 edition (spss inc., chicago, il, usa) were used for statistical analysis and to create the plots. a p 0.05). regarding the mean operation time (min), the lte procedure was longer than the stoppa procedure (134.6 38.3 vs. 90.6 41.3 ; p 0.05) [table 2 ]. patients characteristics and quality of life in pre - operative period (mean sd), as groups values of crp, leucocytes, haemoglobin and haematocrit on pre - operative period, 1 and 2 post - operative day, as groups pain intensity [table 3 ], physical functioning, physical limitation, the impact of pain on daily activities, and the carolinas comfort scale [table 4 ] were similar among groups (p > 0.05). pain intensity value from 1 to 7 post - operative day, according to vas assessments of physical functioning, physical limitation, the impact of pain on daily activities and the carolinas comfort scale assessed on pre - operative period, 7 and 15 post - operative day, as groups complications occurred in 88% of stoppa group (22 patients) and 64% in lte group (16 patients) (p < 0.05). in stoppa group, there were 11 haematomas, 11 seromas, and five bruises. in three patients, complications of lte group were seven haematomas, seven bruises and seven seromas, and no intervention was necessary. the hernia repair approach was and still is another topic for discussion. in the open approach, there are debates concerning what would be the best alternative for hernia repair : conventional bilateral hernia repair using an anterior approach or an incision either longitudinally or transversely with a posterior approach of the preperitoneal space. with the improvement of laparoscopic techniques for inguinal hernias repairs, using both the tapp and tep approaches, the best results of laparoscopic techniques were obtained with bilateral inguinal hernias. in the present study, stoppa and lte approaches were similar regarding the extension of the dissected area in the preperitoneal space, mesh size, and procedure used to fix the meshes. the main difference between the two techniques was access : the pfannenstiel incision was used in the stoppa group, and the laparoscopic incision was used in the lte group. in the present study, the preperitoneal space was dissected under direct vision without using the dissection balloon. a randomised, multicentre study compared the dissection of the preperitoneal space with and without a dissection balloon. the results showed no difference in the time spent to create the preperitoneal space, and the number of conversions was similar. the authors concluded that a dissection balloon was unnecessary, and the cost of operation was lower in the group without the balloon. the laparoscopic insertion and manipulation of two smaller meshes in the preperitoneal space is easier than that using a larger mesh. some studies have reported that a larger mesh that covers the entire myopectineal orifice has the advantage of reducing recurrence near the pubis. another study showed no difference in the early and late outcomes when one or two meshes were used for the laparoscopic repair of bilateral inguinal hernias ; however, the cost was lower when a single mesh was used. the intensity of the inflammatory response is directly proportional to the mesh size. in the present study, a single mesh was used so that inflammatory responses to the mesh during the acute phase would be similar in the two groups. the fixation of the mesh to the pectineal ligament, pubis, and transverse fascia followed the standard procedures used since the introduction of laparoscopic hernia repair. because of the frequent incidence of persistent pain resulting from nerve damage by the staples, some researchers have supported non - fixation of the mesh. this idea was based on the stoppa approach that used large meshes in the pre - peritoneal space without fixing them, obtaining good results. the intra - abdominal pressure can hold the mesh in place, when a large piece of mesh is used (at least 10 cm 15 cm) and placed without any wrinkles between the tissue layers and when the mesh covers all the hernia defect. one study showed no movement of an unfixed mesh up to the 3 month after surgery as assessed by chest x - ray. two randomised controlled studies have shown that the non - fixation of the mesh did not increase the recurrence rate, and there was a trend toward a reduction in the incidence of neuralgia compared with the group where staples were used to hold the mesh in place. in the current study, the mesh was fixed in both the tep and stoppa groups, with only one retropubic stitch. in the present study, one study revealed that the operation time was similar between the stoppa and laparoscopic tapp approaches. studies comparing the stoppa and lte approaches also found that the laparoscopic procedure was longer. in bilateral inguinal hernia repair via lte, the amplitude of the pre - peritoneal space is smaller than when using other approaches, requiring the surgeon to perform a gradual dissection, thus explaining the longer time required for this procedure. on the 1 and 2 post - operative days, the crp levels were statistically significantly higher in the stoppa group. significant differences were observed when analysing the progression of the crp levels during the post - operative period, confirming the progressive increase in the crp levels during the analysed periods. during the post - operative period without complications, crp levels rise gradually until they reach a peak on the 2 day after surgery. the crp measurement is quantitative, and changes in crp levels are directly proportional to the intensity of surgical trauma. in the current study, the crp levels indicated that the intensity of the surgical trauma was higher in the stoppa group, confirming data from previous studies. on the 1 post - operative day, the surgical trauma increased the leucocytes, similarly in both the stoppa and tep groups. despite a reduction, the leucocytes remained elevated during the 2 post - operative day, with no difference between the groups. the two surgical approaches analysed similarly increased the leucocytes, a finding that is in agreement with the literature. the concentration of haemoglobin and the haematocrit value during the pre - operative period were similar in both groups. on the 1 post - operative day, the surgery caused a decrease in the levels of haemoglobin and haematocrit in both groups and remained stable on the 2 post - operative day, with no differences between the groups. the haemoglobin concentration and the haematocrit value were determined to measure the occurrence of bleeding during the operation. a decrease of approximately 1 g / dl in the haemoglobin levels and a haematocrit decrease by 2% on the 1 post - operative day were not exclusively due to blood loss. the saline infusion during surgery contributed to reduced haemoglobin and haematocrit levels during the post - operative period. most of the studies that compare the different techniques for inguinal hernia repair use the vas to assess pain intensity. in the literature, the vas was applied on the 1, 2 and 3 post - operative days, and all of the studies revealed that the group of patients submitted to laparoscopic surgeries had less pain than the group submitted to the stoppa procedure. a study revealed that not only the pain was more intense in the stoppa group but also the analgesic consumption was higher. another study that used on - demand analgesia reported vas values of 5.7 and 1.7 in the stoppa group and 4.7 and 0.7 in the tapp group on the 1 and 2 post - operative days, respectively. in the present study, where standardised regular analgesia was used, the pain intensity assessed from the 1 to the 7 post - operative days was similar in both groups. the maximum value reached in the vas was 3.4 on the 2 post - operative day in the lte group and on the 3 post - operative day in the stoppa group. therefore, with regular post - operative analgesia for bilateral inguinal hernia repair, pain intensity is reduced to minimum values, regardless of the approach used. the present study did not provide evidence of faster patient recovery in the lte group, although there was a trend towards this outcome in this group. the values of physical functioning and physical limitation on the 7 post - operative day in both groups were smaller than those observed before surgery, showing the negative influence of the surgery in these perceptions by the patient. on the 15 post - operative day, the scores of these perceptions exceeded the value obtained before surgery in the lte group, but the values in the stoppa group were lower than those observed before surgery despite being greater than those on the 7 day. considering the values obtained, a minimum of 300 subjects in each group would be required to ensure that the physical functioning and physical limitation during the post - operative period are influenced by the type of approach under the conditions used in this study. considering the daily activities domain, the effect of pain was similar between the groups. the pre - operative scores were similar in both groups and smaller than those obtained on the 7 and 15 post - operative days. therefore, in the patient 's perception, the pain caused by the surgery exceeded the pain caused by the hernia. this study demonstrated that the pain assessed by the vas and its impact on routine physical activity have very close absolute values and were not a good parameter for differentiation. the carolinas comfort scale, a specific instrument to assess quality of life in patients after mesh hernia repair, analyses the feeling of pain and of the mesh and limitations when bending over, standing up, sitting down, sitting during daily activities, coughing, walking, climbing or descending stairs, and exercising. in the present study, the obtained score indicated a small discomfort before surgery that was similar between the groups. on the 7 post - operative day, discomfort was higher in both groups, confirming the influence of the surgery on discomfort ; no statistically significant difference was observed between the groups. on the 15 post - operative day, the scores of both groups returned to the pre - operative levels in both groups. according to the study that validated the carolinas comfort scale, vascular, vesical or intra - abdominal visceral injuries did not occur during the surgery ; there was no bleeding or infections in surgical wounds during the post - operative period, as described in some studies. minor complications were observed, such as the presence of bruises, haematomas, and seromas, which were present in 22 (88%) patients from the stoppa group and 16 (64%) patients from the lte group. haematomas from three of 11 patients from the stoppa group were punctured to drain the collection. the present study indicates that laparoscopic bilateral inguinal hernia repair using the lte approach results in less surgical trauma, as assessed by the crp levels. the pain intensity during the post - operative period was low when analgesia was administered and did not differ between the two groups, suggesting that the analgesic medication controls the pain and maintains pain at low levels of intensity, regardless of the approach used. although the results show a trend favouring the laparoscopic approach, the present work could not conclude that the recovery was faster. a study with regular analgesia and a greater number of participants the comparative study between the stoppa and lte approaches for the bilateral inguinal hernia repair demonstrated the following : the lte approach showed less surgical trauma despite the longer operation time;quality of life during the early post - operative period were similar ; andcomplication rates were higher in the stoppa group. the lte approach showed less surgical trauma despite the longer operation time ; quality of life during the early post - operative period were similar ; and complication rates were higher in the stoppa group. | background : the present clinical trial was designed to compare the results of bilateral inguinal hernia repair between patients who underwent the conventional stoppa technique and laparoscopic total extraperitoneal repair (lte) with a single mesh and without staple fixation.patients and methods : this controlled, randomised clinical trial was conducted at general surgery and trauma of the clinics hospital, medical school, the university of so paulo between september 2010 and february 2011. totally, 50 male patients, with a bilateral inguinal hernia, older than 25 years were considered eligible for the study. the following parameters were analysed during the early post - operative period : (1) the intensity of surgical trauma, operation time, c - reactive protein (crp) levels, white blood cell count, bleeding and pain intensity ; (2) quality of life assessment ; and (3) post - operative complications.results:lte procedure was longer than the stoppa procedure (134.6 min 38.3 vs. 90.6 min 41.3 ; p 0.05). there was no difference in pain during the 1st and 7th post - operative, physical functioning, physical limitation, the impact of pain on daily activities, and the carolinas comfort scale during the 7th and 15th post - operative (p > 0.05). complications occurred in 88% of stoppa group (22 patients) and 64% in lte group (16 patients) (p < 0.05).conclusion : the comparative study between the stoppa and lte approaches for the bilateral inguinal hernia repair demonstrated that : (1) the lte approach showed less surgical trauma despite the longer operation time ; (2) quality of life during the early post - operative period were similar ; and (3) complication rates were higher in the stoppa group. |
the advent of minimally invasive surgery has provided surgeons new techniques for treating clinical disease. within the field of spinal surgery, techniques in lumbar interbody arthrodesis have shown a continued evolution of procedural approach and instrumentation. minimally invasive spine surgery aims to reduce approach related morbidity, while producing clinical outcomes comparable to its open predecessors. one important example of this is the development of minimally invasive techniques for lumbar interbody fusion, including transforaminal lumbar interbody fusion (tlif). the mi - tlif technique, has displayed comparable outcomes to open tlif, while adding the benefits of less approach - related morbidity, decreased intraoperative blood loss, and shorter hospital stays. however, critics of the technique have noted that the mi - tlif has longer operative times and exposes patients to increased fluoroscopic radiation. over the past decade mi - tlif has been shown to have a number of benefits, especially with regard to peri - operative outcomes. ultimately, comparing the known literature of a traditional, open tlif approach to published reports on mi - tlif will identify the unique risks and benefits associated with each. this understanding may help guide improved clinical decision making for patients presenting with lumbar degenerative disk disease. in this paper, we evaluate the literature to examine the efficacy of mi - tlif compared to its open counterpart. in addition, key studies discussing the risks and benefits of mi - tlif were included to more thoroughly explore the nature of the technique and its application. in this paper, the authors have used the pubmed / medline search engines to search for relevant reports addressing the topic of transforaminal lumbar interbody fusion., a few historical reports have been added for completeness. included in this search was the following key phrases : minimally invasive, further, although articles were first identified by abstract, only full text manuscripts were used to compile this review of the topic. further, inclusion criteria were based on the study 's contribution in terms of original data, technical variations, and contrasts between open and minimally invasive versions of the procedure ideally completed at the same institution. in total, 14 articles were selected on the aforementioned basis. all contributed to the established body of the literature pertaining to lumbar arthrodesis techniques, particularly different variants of tlif. six of the 14 articles were prospective studies, while the remaining 8 were retrospective (table 1). after failed conservative management for a minimum of 6 months, surgery becomes the next therapeutic option for patients presenting with degenerative disc disease (ddd), radiculopathy with spinal instability, and/or grade 1 spondylolisthesis. initially patients are assessed through radiological investigations including x - ray (ap, lateral, flexion, and extention), and noncontrast lumbosacral mri. length of hospitalization is determined by postoperative pain control and functional dependence, with patients of advanced age or medical comorbidities often requiring longer postoperative recovery. however, a majority of patients are admitted the day of surgery and discharged within 2472 hours after operation. under general anesthesia, the patient is prepped and draped in standard fashion, and a fluoroscopic c - arm is positioned in the sterile field. under fluoroscopic guidance the appropriate level is marked and a 3 cm incision is made 4.5 cm of off midline. a k - wire is targeted to the bony complex at the surgical level and serial dilators are consecutively passed to split the muscle fibers. a working channel is placed, the dilators are removed, and the channel is secured appropriately for adequate visualization of the medial portion of the facet and inferior lamina. a curette is used to detach the ligamentum flavum from the inferior edge of the lamina, and a kerrison is used to perform the hemilaminectomy. the unilateral facet can be removed using an osteotome or high - speed drill. following adequate exposure of the disc space curved and angled curettes and a disc scraper are then used to prepare the end plate. an appropriately sized interbody spacer is inserted into the disc space, and a half sponge of bmp is packed into the disc space. after removal of the working channel, a jamshidi needle is localized to the unilateral pedicle either above or below the discectomy level, and positioning is checked using fluoroscopic imaging. a k - wire driver is used to insert a guide wire into the superficial portion of the pedicle. a sextant percutaneous screw system (medtronic inc ; memphis, tn) is used to pass a cannulated pedicle screw over the k - wire and into the pedicle under fluoroscopic guidance. this is repeated at all desired pedicles on either side. the sextant holding sleeves are mated, the percutaneous rod holder and guide are attached, and a small skin incision is made to pass the rod percutaneously through the screw head. after correct positioning of the rod is confirmed with fluoroscopy, the screw head is tightened, the rod holder is released, and the holding sleeve is removed. skin closure is accomplished in the standard fashion. for a full detailed description see lawton., see figures 1 and 2 for illustrative cases from patients treated with the mi - tlif procedure. as noted, our review included 14 articles. follow - up times ranged across all articles from 6 months to 42 months. the mean follow - up was 20 months, with a mean patient cohort of 52 patients. within seven of the articles that directly compared outcomes of open tlif with mi - tlif, mean duration of mi - tlif surgery was 220 minutes, compared to 218 minutes for its open counterpart. furthermore, blood loss was found to be on average 282 ml in mi - tlif cases, while open tlif resulted in 693 ml of blood loss. the length of stay for mi - tlif was found to be 5.6 days, while open tlif had patients in the hospital for an average of 8.1 days (see table 2). though the literature displayed possible benefit of mi - tlif relative to its open counterpart, both procedures are associated with possible complications. major sources of complications shared by mi - tlif and open tlif are allograft malposition, pedicle screw malposition, and infection. some minor complications found in both open and mi studies were hematoma, anemia, and cerebrospinal fluid leakage. in both lumbar arthrodesis techniques, placement of a k - wire is necessary, and this k - wire is held in place to formally place the expandable retractor moving the k - wire could result in entrance to the vertebral canal and possible damage to the nerve roots or cauda equina, which had the potential to occur in either tlif technique. complications more likely to be found in the open tlif approach include infections and muscular trauma as a result of the increased exposure and soft tissue dissection. in addition, increased exposure has been shown to be potentially associated with 23.5% of reported complications being infectious in nature, within the open tlif studies. open tlif may have a slightly lower rate of neurological complications, for neurological deficits were a considerably lower proportion of total complications, 11.8%, when compared to mi - tlif 's 20.7%. however, there were a greater variety of unique complications to open tlif, as shown by 23.4% of complications coming in the form of dural tears, ileus, and atelectasis among others. please refer to table 3 for further analysis. in the mi - tlif literature reviewed, many authors discussed the challenging learning curve associated with mi - tlif, which makes certain complications, particularly those related to instrumentation more likely. endoscopic visualization of the spinal structure limits the field of view for the surgeon, making identification of already unfamiliar landmarks even more difficult. though visualization techniques have improved over time, percutaneous fixation systems do not have the ability to reposition three dimensionally. tubular dilator retractors can result in poor decompression while resulting in higher rates of neurological injuries. of all complications presented in the mi - tlif comparative literature, approximately 1 in 5 were related to neurological complications (table 4).. wrote of possible inexperience leading to inappropriate placement of transpedicular screws, and inadequate preparation of intervertebral cage and fusion site which can lead to further instrumentation related complications. the operative surgeon additionally must be familiar with 3d lumbar anatomy and be able to carefully interpret 2d radiographic images to make a mental reconstruction. this is a unique skill and one that is not as critical with a traditional, open approach. the surgeon must be able to read anterior - posterior and lateral imaging in order to accurately insert percutaneous pedicle screws, thereby allowing for possible misinterpretation leading to complications. screw misplacement and cage migration or subsidence accounted for 44.8% of complications reported in mi - tlif comparative studies. mi - tlif itself presents with increased risk to the surgeon related to increased radiation exposure due to lengthened intraoperative fluoroscopy times. though many may claim that a surgeon 's experience level with minimally invasive procedures will dictate their fluoroscopy times, some studies found no significant difference as experience increased. very few studies reported the duration and radiation exposure resulting from x - ray and fluoroscopy. authors who did report this data found that mi - tlif had greater duration of radiation exposure for patients undergoing the procedure [3, 5, 7 ]. due to the relative recent adoption of mi - tlif use, the long - term effects of increased radiation exposure have not been evaluated. the development of 2d computer assisted fluoroscopy systems as well as the o - arm is a modern means to decrease this exposure risk. following data collection and the literature review, it is clear that there is a paucity of data comparing mi - tlif and open tlif. to our knowledge, there remains no high - class studies that directly compare these two techniques. however, smaller studies, both prospective and retrospective in nature, have shown promise in regards to novel mi techniques for tlif. scheufler. compared percutaneous transforaminal lumbar interbody fixation (ptlif) with mini - open transforaminal lumbar interbody fixation (otlif) while utilizing the wiltse method. they found at 8 month and 16 month follow - up, overall clinical outcome did not differ between the two techniques. however, in terms of pain following the operation, ptlif resulted in significantly lower levels of pain (p < 0.01). though the study showed no decreased advantages due to the percutaneous approach, a longer prospective study would be needed to further discern the success and functionality of each multilevel fusion. in a study examining 42 patients with mean follow - up time of 29 months, dhall. the authors found that mean estimated blood loss for mini - open (194 ml) was significantly lower (p < 0.01) than the open - group (505 ml). the length of stay was decreased for mini - open patients by on average, 2.5 days (p < 0.01). however, there were complications of neurologic nature in 2 patients, while 2 other patients required further revision. all 42 patients displayed fusion, and the authors felt that the mini - open technique was a possible substitute to open tlif. performed one of the earlier studies (2001 - 2002) on 49 patients who had mi - tlif. majority of patients in the study either had degenerative disc disease with herniated nucleus pulposus (hnp) or spondylolisthesis. mean operative times were approximately 240 minutes, approximate blood loss was 140 ml, and hospital stays averaged 1.9 days. complications were limited to four patients, two of which required screw repositioning while two others developed radiculopathy following the procedure. vaps changed on average from 7.2 to 2.1 while odi changed from 46 to 14 from preoperative assessment to final follow - up. ultimately, all patients in the study had fusion on follow - up imaging. the author believed that mi - tlif is at least equivalent if not a marked improvement over its open counterpart. a variation of the accepted microendoscopic discectomy was completed by isaacs and colleagues, which was termed metlif. metlif resulted in less blood loss, shorter hospital stays, and decreased postoperative narcotic administration. there were no associated procedural complications associated with the multicenter study. ultimately, this new variation showed promise. schizas. examined their institutional experience executing both mitlif and open midline transforaminal lumbar interbody fusion. the study found that length of surgery, postoperative pain, analgesia requirements, and vas / odi scores were not significantly different between the mi and open procedures. however, they did find that the mi - tlif did result in significantly less blood loss and a shorter hospital stay. complications found in the mi - tlif group, three pseudorthrosis, may have likely been due to the surgeon 's gradual adjustment to the novel instrumentation and visualization techniques associated (table 5). lumbar arthrodesis is an effective method for treating spinal pathology such as spondylolisthesis, ddd, and spinal instability. as minimally invasive spine procedures have emerged, variants such as minimally invasive discectomy and minimally invasive cervical foraminotomies have allowed for reduced complications related to tissue trauma, while reducing blood loss and shortening recovery time [4, 8, 19, 20 ]. however, no procedure comes without inherent risks. due to mi - tlif being a novel procedure for some surgeons, it takes increasingly longer for them to become effective in carrying it out. compared safety and effectiveness of mi - tlif and open tlif, showing similar long - term outcomes over the course of the 37.5-month follow - up period. assigning 63 patients to the open arm and 76 patients to the minimally invasive arm of the study, the authors matched by prior lumbar surgery, diagnosis, and levels at they found significant improvement in mean estimated blood loss (p <.0001) for mi (163.0 ml) versus the open tlif (366.8 ml). the study found improvements (p = 0.02) in mean duration of hospitalization in mi - tlif (3 days) relative to their open counterparts (4.2 days). in addition, rates of neurological deficit were significantly higher (p = 0.02) in the minimally invasive arm of the study (10.2%) compared to the open cohort (1.6%). operative times, mean change in vas scores, patient satisfaction, all significantly favored the open tlif procedure. the authors hypothesized that the neurological deficits and other factors in favor of open tlif could have occurred as a result of the surgical learning curve. once the procedure is mastered, its application can positively impact patient care in numerous ways. but, the fundamental advantage of mi - tlif comes from its decrease in tissue trauma and overall exposure of the patient. a prospective cohort study was carried out by shunwu. with 62 patients that had undergone single level tlif by a single surgeon in a single hospital. one cohort of 32 patients underwent mi - tlif with the tubular retractor system, while the remaining patients underwent open tlif. serum creatine kinase levels, a measure of soft - tissue trauma, was measured on the third postoperative day. also, time to ambulation and number of transfusions were also measured in the study. shunwu and colleagues found that mi - tlif resulted in significantly lower serum creatine kinase levels were found, while patients needed less transfusions and were able to walk earlier than their open counterparts. when comparing the two approaches, this study displayed that mi - tlif still proposes significant quantifiable benefit in terms of decreased soft tissue trauma. as a minimally invasive procedure, mi - tlif can be utilized to treat particular pathologies, while maintaining the same high levels of clinical success as the open tlif, even with over two years of follow - up. park and foley contributed an article to the literature that described mi - tlif in 40 consecutive patients who were diagnosed with spondylolisthesis. their percutaneous approach resulted in reduction of spondylolisthesis in all cases, with an average follow - up time of 35 months. the average odi decreased from 55 to 16, while the vas scores decreased from 65 to 8 in leg and 52 to 15 in back. this was yet another proof of mi - tlif being a possible replacement to open tlif in patients with degenerative or isthmic spondylolisthesis. in a prospective study that contrasted clinical and imaging outcomes for mi - tlif and open tlif procedures, peng. found that mi - tlif had equivalent long - term outcomes with open tlif. the patient cohort had 29 patients in each arm of the study, and 48 of 58 patients were women. the study examined, fluoroscopic times and found that mi - tlif had significantly (p < 0.05) longer (105.5 seconds) compared to open (35.2 seconds). thus, it is clear that the mi - tlif cases ran significantly longer overall. then, the authors discussed the significantly less blood loss, less morphine, and short hospitalization utilized for patients in the mi - tlif cohort. yet, open tlif and mi - tlif both were very similar in providing significant benefit to patients when rated by odi, nass, and vas, all at follow - ups of six months and two years. in addition, there was no significant difference between open and mi - tlif in terms of fusion rates, both which were approximately 80%. peng and colleagues presented data that was supportive of mi - tlif in terms of pain, hospitalization, and recovery, while at the same time retaining the high - fusion rate associated with open tlif at two year follow - up. aside from particular pathologies that would benefit from mi - tlif, there are certain populations that could benefit from the decreased tissue disruption and decreased blood loss. in elderly patients, lee. completed a retrospective review of 27 consecutive cases and found a low complication rate and beneficial outcomes for patients over the age of 65. the average age of patients in the study was approximately 70 years, and each underwent a mini - open tlif. they were then followed up for three years, displaying fusion rates of nearly 80%, similar to that seen in other studies. however, 44% of patients displayed adjacent segment degeneration, which was statistically significant in terms of its relation to sacral tilt following the procedure (p = 0.006). two patients experienced minor complications in the perioperative period, one being a drug eruption and the other a urinary tract infection. overall, the authors strongly felt that mini - open tlif is a low - risk, beneficial option for the elderly. though the studies presented displayed heterogeneous patient populations with different indications for lumbar arthrodesis, there were many patterns seen across studies. aside from possible complications such as screw displacement and neurological deficit, which were often related to a steep learning curve, mi - tlif displayed no significant disadvantages when compared to open tlif or other standard lumbar fusion techniques. the risks of blood loss, narcotic administration, pseudorthrosis, and infection all are equivalent if not decreased when utilizing mi - tlif as a possible technique. various postoperative recovery and mi - tlif and open tlif are quite similar in absolute indications and often present with similar complications, thus a randomized clinical trial would be beneficial in further elucidating the risks and benefits associated with each. as other variations emerge for mi - tlif, such as metlif, there is still need for an overall meta - analysis of all available data, comparing minimally invasive technique to traditional, open procedures. | this paper reviews the current published data regarding open transforaminal lumbar interbody fusion (tlif) in relation to minimally invasive transforaminal lumbar interbody fusion (mi - tlif). introduction. mi - tlif, a modern method for lumbar interbody arthrodesis, has allowed for a minimally invasive method to treat degenerative spinal pathologies. currently, there is limited literature that compares tlif directly to mi - tlif. thus, we seek to discuss the current literature on these techniques. methods. using a pubmed search, we reviewed recent publications of open and mi - tlif, dating from 2002 to 2012. we discussed these studies and their findings in this paper, focusing on patient - reported outcomes as well as complications. results. data found in 14 articles of the literature was analyzed. using these reports, we found mean follow - up was 20 months. the mean patient study size was 52. seven of the articles directly compared outcomes of open tlif with mi - tlif, such as mean duration of surgery, length of post - operative stay, blood loss, and complications. conclusion. although high - class data comparing these two techniques is lacking, the current evidence supports mi - tlif with outcomes comparable to that of the traditional, open technique. further prospective, randomized studies will help to further our understanding of this minimally invasive technique. |
the most recent network technologies are enabling a variety of new applications thanks to the provision of increased bandwidth and better management of quality of service. nevertheless, telemedical services involving multimedia data are still lacking behind, due to the concern of the end users, that is, clinicians and also patients, about the low quality provided. wireless and mobile telemedicine underpins applications such as the transmission of video data from an ambulance, the rapid retrieval and remote display of video data stored in hospital databases, remote (first - level) diagnosis in rural areas, for example, robotic teleultrasonography and telesurgery. one of the key challenges is the ability to stream medical video over wireless channels. although wireless multimedia telemedicine services have been proposed before in [15 ], the application of these technologies in real scenarios has been constrained by the unacceptably poor quality of the medical multimedia data arising from the limited bandwidth. it would be desirable to store approximately 30 seconds of dynamic heart images per patient (i.e., three sections of the heart and 10 seconds for each section). even if frames are digitized with 512 512 pixels with 8 bits each and the frame rate is 25 hz, the size of the uncompressed digital video sequence would be (512 512 8 3 10 25) bits medical video compression techniques are thus required. for telemedical applications, such techniques must offer high fidelity in order to avoid the loss of vital diagnostic information. to achieve this, lossless compression techniques are often considered, but have the disadvantage of low - compression rates. therefore, when transmission is over band - limited and error - prone channels, a compromise must be made between compression fidelity and protection and resilience to channel errors and packet loss. it has been estimated that lossy compression ratios of 1 : 5 to 1 : 29 do not result in a lowering of diagnostic accuracy. furthermore, even in situations where the final diagnosis must be carried out on an image that has been reversibly compressed, irreversible compression can still play a critical role where quick access over a bandlimited channel is required. however, we can consider three types of lossless compression : information lossless compression, perceptually lossless compression, and diagnostically lossless compression. the first one is limited by the entropy (mean information) of the source ; the second is such that losses are not perceived by the human eye ; the latter is such that the diagnosis made on the basis of the image / video sequence is not affected by compression. in this paper, we focus on ultrasound video, although some considerations are still valid for different type of sources. we propose to exploit all the available context information (type and goal of ongoing / stored examination, status of the patient, transmission scenario) to design an appropriate transmission system for diagnostically lossless ultrasound video transmission over wimax systems. for instance, coronary heart disease can be diagnosed by measuring and scoring regional motion of the heart wall in ultrasound images of the left ventricle of the heart. such information can be taken into account as context, and regions of interest (roi) can be defined accordingly in each specific session. multiple regions of interest can be defined, and compression / transmission strategies can be tailored to such context information. we propose a global scheme for error - resilient transmission of ultrasound and ecg data designed based on the characteristics of the specific scenario. for this purpose we invoke a wide range of recent advancements in video compression, error resilient video coding, and transmission technologies, including specific tools of the h.264 video coding standard such as flexible macroblock ordering (fmo) for roi identification. two different unequal error protection methodologies, providing higher protection to the most diagnostically relevant data, are proposed and the whole transmission system is specifically designed for the aforementioned scenario. the performance is evaluated over a realistic wimax network, by considering real measurements. the paper is structured as follows. section 2 provides an overview of the state of the art in wireless telemedicine systems for diagnostic video transmission and also focuses on the main advancements in the relevant enabling technologies. following the problem statement and description of the proposed approach in section 3, section 4 details the system implementation and validation results. ieee 802.16/wimax (worldwide interoperability for microwave access) [911 ] is one of the most promising technologies for broadband wireless access, both for fixed and mobile use, currently being deployed in many countries worldwide. 2001 wimax has evolved from 802.16 to 802.16d for fixed wireless access, and to the new ieee 802.16e standard with mobility support [9, 10 ]. mobile wimax adds significant enhancements, including improvement of nlos coverage by utilizing advanced antenna diversity schemes and hybrid automatic repeat request (harq) ; the adoption of dense subchannelization, thus increasing system gain and improving indoor penetration ; the use of adaptive antenna system (aas) and multiple input multiple output (mimo) technologies to improve coverage ; the introduction of a downlink subchannelization scheme, enabling better coverage and capacity tradeoff. this brings potential benefits in terms of coverage, power consumption, self - installation, frequency reuse, and bandwidth efficiency. the 802.16e standard encompasses five quality of service classes for different types of traffic / applications. in particular, for medical applications in emergency areas it is important to have an easy setup of the infrastructure. at the same time, qos is critical in medical applications, thus proper prioritization and scheduling policies should be adopted in order to enable reliable and high - quality transmission of possibly critical medical data. in resource allocation is used to prioritize different type of connections (e.g., an emergency connection must have higher priority than a followup connection) over a ieee 802.16e network. an admission control scheme is used to reserve radio resources for higher priority connections and avoid congestion. three types of connections in the network are considered : connections from ambulances, clinics, and followup patients. wimax has dramatically improved with respect to previous systems in terms of features which are critical for medical applications. high end - to - end quality ; robustness and reliability : the system can not break down under stress and the connection can not be lost ; security : transmission of medical data should be secure and privacy of medical data must be preserved, medical data or patient identification can not be disclosed indiscriminately ; the fact that different health care providers have different access rights has to be considered. however, the baseline wimax scheme lacks error protection beyond phy / mac and unequal error protection is not considered at phy / mac, hence video sequences can be largely affected by errors and packet losses. in order to improve the video quality, in addition, if unequal error protection is not available, the video quality will degrade significantly when a mobile subscriber station (mss) experiences shadowing fading, temporal fading or interference. the idea of unequal error protection is to apply more robust channel coding to more important video content. therefore, the mss can at least decode some important video frames, for example, i frames and diagnostically important content. for this reason we propose in this paper to adopt an unequal loss protection strategy at the application layer, to improve packet error resilience for ultrasound video sequences transmitted over a wimax system. the advantages of an unequal loss protection at the application layer are mainly the availability of detailed source information at this layer (no need to pass such information through the osi protocol stack) and standard compatibility (phy / mac layers are standardized in wimax). teleultrasound systems for remote diagnosis have been proposed in the last ten years [1, 2, 4, 5 ] given the need to allow teleconsultation when the access of the medical specialist to the sonographer is not possible. more challenging scenarios include ultrasound guided remote telesurgery and wireless robotic teleultrasonography. in, the quality of received real - time medical video sequences after transmission was just acceptable for a first diagnosis, since the available wireless technologies (2.5 g, 3 g) did not allow sufficient bandwidth for good quality video transmission. recent studies reported by the authors in [13, 14 ] show the improvements achievable through the exploitation of appropriate rate - control strategies and cross - layer design over wlan/3 g systems. some projects and demonstrations the goal of the european ist project weird was the realisation of ieee 802.16/wimax - based testbeds, including novel applications running on top of a wimax - based end - to - end architecture. broadband access for medical personnel requiring high - resolution medical information in nomadic emergency camps and high - resolution video and data streaming from medical instruments were considered. the project highlighted the main challenges that wimax still has to face in e - health applications. the goal of the mobile healthcare services project in taiwan is to support emergency medical assistance and patient care services wherever it is required outside of a medical facility. with the assistance of high - bandwidth wireless communications (wimax), healthcare personnel in the field will be able to connect to critical medical resources, exchange important files, and arrange treatment, saving crucial minutes in the early treatment of patients. in australia, with the help of intel australia and airspan networks, the organizers of the australian grand prix deployed a wimax network to improve communication flow between the on - site trauma unit and medical specialists at the alfred hospital three kilometers away. auto racing events require a medical team capable of attending to the steady stream of injuries incurred by the drivers, mechanics, and other personnel throughout the competition. the trackside trauma facility was provided with high - speed wireless connection, linking the on - site medical staff with their counterparts in a hospital three kilometers away. the wimax network eliminated the need for the 20-minute trips previously required to manually transport radiology images, test results, and other medical information. furthermore, wireless web cameras installed at the remote site allowed medical staff in the field to run real - time video consultations and patient reviews with their colleagues in the hospital. the project focused on medical images and ambient video, not on medical video sequences. the goal of most of the aforementioned projects is / was to demonstrate the transmission of medical data over a standard network, with no effort to tailor the characteristics of the transmission system to the specificity of the transmitted data. one of the first works addressing the need of taking the specific characteristics of the medical application into account in the design of the transmission system was, presenting the design of a mobile teletrauma system using 3 g networks. the importance of considering a specific cross - layer strategy designed with the goal of maximizing the diagnostic quality of the received information was first identified in and then also addressed in [13, 14, 21, 22 ]. a recent work in this direction is also, where adaptive transmission of medical images and video is addressed using scalable coding and context - aware wireless medical networks. the authors propose a wavelet - based scalable coding scheme and context information is addressed here as the information on the patient state (normal / urgent). this work focused on teledermatology, mri, and ambient video (no ultrasound). since most networks deal with a limited amount of bandwidth, scaling techniques are introduced to send less data over the network with as little inconvenience as possible for the user. one of these techniques is region - of - interest coding (region of interest (roi)). roi divides an image into multiple parts, the most important part typically being the one the user is observing, called the roi. roi coding can be used to encode objects of interest with a higher quality, whereas the remainder of the image can be regarded as background information and can be encoded more coarsely. the advantage of this method is that the image parts that the viewer is looking at can be transmitted with a higher quality. the result is that the overall viewing experience remains satisfactory, while the transmission can be performed at lower bitrates. this can be realized by the use of slices (e.g., if slice - group-0 is transmitted first, by placing the roi in slice - group-0, it should arrive first at decoder side). when network congestion occurs, the probability of having a frame that contains at least something the viewer most likely wants to see, is higher with roi coded imagery than without roi. the roi can be defined by the user (e.g., clinician) by means of a mouse click, by making use of an eye tracking device or can be predicted, based on content recognition algorithms. medical video sequences typically consist of an area which is critical for the diagnosis and a surrounding area which provides the context, but is not critical for the purpose of the diagnosis. roi coding appears thus as a natural methodology for medical video and roi definition can be performed according to contextual information, either automatically or by the clinician. the image compression standard jpeg2000 allows both the definition of a roi of regular shape and defined by the user through a mask. the mpeg4 standard proposed for the first time the concept of objects independently manipulated in a video sequence. in the more recent h.264 standard [26, 27 ] there are several different models to implement roi - coding, all making use of slice groups. indeed, the concept of roi is not often exploited in the design of compression and transmission strategies. reference presents an implementation of multiple region - of - interest models in h.264/avc. reference presents a cost - distortion optimized unequal error protection for object - based video communications, with the goal of optimizing the global distortion of each image by adapting the transmission power to provide a different protection to shape and texture information. this approach is probably the most similar to our one, although the authors focused on power adaptation (physical layer), while we propose unequal loss protection at the application layer. furthermore, the authors did not consider medical video and did not exploit relevant context information. they exploited the object tool in the mpeg-4 standard, while we rely on the fmo tool in the h.264 standard for the identification of regions of interest. reference presents a region - based rate control and bit allocation for wireless video transmission. reference exploits the concept of roi and contextual information for context - aware multi - lead ecg compression based on image codecs. the following section describes a useful way to implement rois in the h.264 video coding standard. the h.264 standard [26, 32 ] includes three profiles (baseline, main, and extended), each having a different set of functionalities. the baseline profile was designed primarily for low - cost applications with reduced computational power. the most relevant functionalities in the baseline profile are fmo and arbitrary slice ordering (aso). both techniques are used for manipulating the decoding order of the macroblocks in the picture. a slice contains at least one macroblock and it can include all the macroblocks in the video frame. using fmo, groups of macroblocks consisting of one or more slices, known as slice groups, are formed according to a specific strategy. the fmo mode, in conjunction with advanced error concealment methods applied at the decoder, maintains the visual impact of the losses at a low level even at loss rates up to 10%. apart from predefined patterns, fully flexible macroblock ordering (explicit mode) is also allowed, where the macroblock classification can be changed dynamically throughout the entire video sequence based on the video content. the idea behind fmo is that if a slice gets corrupted, and the macroblocks within this slice are dispersed across the frame, it will be easier to conceal the lost macroblocks than in the case they are contiguous. however, according to our experience, in the case of medical video error concealment is not necessarily beneficial, since it may hide important irregularities present in the original video. for this reason, in this paper we consider fmo as a means to perform roi implementation in h.264 and not with the purpose of error concealment. the standard includes seven modes to map macroblocks (mbs) to a slice group and we will consider in the following the explicit mode (type 6), allowing the user to associate each of the macroblocks to a slice group independently. the fmo tool has already been used by a few authors for the purpose of roi definition and unequal error protection. in the authors propose a transcoding scheme to perform unequal error protection based on the information available at the output of the entropy coder. unequal error protection is performed here in the transform domain and context / content information is not considered for the unequal error protection strategy : the most relevant macroblocks in each frame are selected solely based on the distortion introduced at the decoder if the macroblock is lost. in a novel technique to represent rois using fmo is proposed, together with a rate control technique to improve the picture quality in roi. in the importance of every macroblock is calculated based on its influence on the current frame and future frames and macroblocks with the highest impact factor are grouped together in a separate slice group using the flexible macroblock ordering feature of h.264/avc. the authors suggest that their results could underpin the design of an unequal error protection strategy. due to the characteristics of video coding methodologies and standards [27, 36 ], it has been shown that joint source and channel coding / decoding techniques (jscc / d) are beneficial to wireless video transmission. although source coding and channel coding are usually treated separately, jscc / d techniques allow improvements in end - to - end video quality through the joint design of source coding on one side and channel coding and modulation strategies on the other. more in general, the different layers of the standard transmission protocol stack can be jointly designed, according to a paradigm that becomes to be known as cross - layer design. despite the demonstrable advantages in end - to - end video quality, there are few studies addressing the use of the cross - layer and jscc / d approach for mobile telemedical applications [13, 14, 20, 21 ]. examples of cross - layer methodologies include : rate control [38, 39 ], to adapt the source coding rate to the network and channel conditions ; rate - distortion optimized streaming of packetized media ; unequal error protection. since video packets may contribute differently to the overall video quality, unequal error protection (uep) is a natural way of protecting transmitted video data. the idea is to allocate more resources to the parts of the video sequence that have a greater impact on video quality, while spending less resources on parts that are less significant. in, an unequal error protection scheme based on rate - compatible punctured convolutional codes, a priority encoding transmission scheme is proposed to allow a user to set different priorities of error protection for different segments of the video stream. this scheme is suitable for mpeg video, in which there is a decreasing importance among i, p, and b frames. in general, error protection can come from various sources such as forward error correction (fec), retransmission, and transmission power adaptation. in the authors proposed an uep scheme for mpeg4 video where different packet partitions were protected with different channel codes. the mpeg4 data partitioning tool was exploited and a criterion was proposed in order to avoid passing the otherwise necessary side information about partition lengths. unequal error protection is also possible taking modulation into account, for example, through appropriate bit allocation over the different subcarriers in multicarrier modulation. an uep scheme based on different priorities of mpeg4 objects is presented in where shape and texture data are transmitted over different service channels. in this paper, unequal error protection is performed at the application layer through erasure codes ; on one side uep at the application layer keeps compatibility with the wimax standard, since mac / phy layers do not require modifications ; on the other side, the use of erasure codes allows the recovery of lost packets at the application layer, where the use of bit - error correction codes would be useless, since lower layer protocols remove packets with erroneous bits, unless mac - lite /udp - lite protocols are used to allow packets with erroneous bits in the payload to reach the application layer. ultrasound scanners produce conical images where the actual image acquired by the probe sensor is a fan - shaped window over a black background including patient data and in some cases the associated ecg waveform (see figures 2(a) and 2(b)). the fan - shaped window is the diagnostically useful area and it typically occupies 50%60% of the area of the full image. the actual size of this fan area in a given ultrasound clip depends on the machine and its settings. after detection of three key points, this area can be modeled as the sector of a circle centered in (a, b) and with radius c, that is, with equation (1)(xa)2+(yb)2=c2. the automatic detection of the fan area is not trivial, as the position and size of it varies in different frames and from clip to clip. although a fan - shaped mask can be detected for each frame, we assume the fan area is uniform across all frames. we therefore construct a fan - shaped mask by finding the union of the individual masks identified. it is possible to adopt a similar procedure for clip - to - clip variations, by identifying a universal mask. with the purpose of a context - aware design of the compression and transmission scheme, we identify three rois in each ultrasound video sequence (see figure 2(a)). diagnostically most important area identified by the clinician (see, e.g., figure 2(a)) ; fan - shaped sector (see figure 2(a)) ; black background with patient data and in some cases the associated ecg waveform. in the following, we will also consider roi 2 and roi 3 jointly processed, as in figure 2(b). in particular, roi 1 is selected by the medical specialist according to context information such as type of examination and a priori knowledge on the disease to diagnose. information in the background is typically text data, for example, about the patient, the instrument used in the examination, and the section of the organ visualized. the associated ecg wave can also be displayed in the background area, with the ecg sample corresponding to the visualized image highlighted with a bar in the waveform. this information can be extracted prior transmission and both text and the ecg waveform can be separately compressed. when dicom standard is used, such information can easily be separated from the rest of the image. we do not extract such information from the background prior to transmission and we transmit roi 3 as a separate roi or in the same transmission class as roi 2. in the first option, data and ecg waveform are separately encoded. when there is no requirement for high resolution for the diagnosis of a specific disease, ecg waveform is typically sampled at 360 hz with a resolution of 11 bits per sample. in some cases the information from different (up to eight) channels obtained from different leads is needed. the waveform of a single channel occupies 360 samples / s 11 bits / sample = 3960 bits / s 4 kbits / s without compression (for eight channels12 leads the rate is about 32 kbps). a channel can be compressed with acceptable quality down to 400 bits / s = 50 bytes / s (see, e.g., and references cited therein). when such information is removed and separately encoded, and application layer fec is adopted, we propose we embed such information in the padding bits needed to have a regular code structure (see section 3.4). note that synchronization between ecg data and ultrasound images is important, in order for the specialist to correlate the visualized image with the corresponding wave in the ecg signal. for instance, it is essential for a specialist to synchronize the measurement of the diameter of vessels to the r - wave spikes in the ecg trace, to eliminate the effects of periodic changes in diameter caused by the normal changes in blood flow with every heartbeat. we assume that we compress our medical video sequences according to the h.264 video coding standard, with the aid of the flexible macroblock ordering (fmo) tool for encoding separately the different rois. we also assume that each roi r in a frame f is composed of if, r slices. we assume that different quantization parameters are adopted for each m - th slice for roi r, r = 1,2, 3, in frame f, that is, quantization parameters are f, r, m. the transmission channel is characterized by a set of parameters (such as packet loss rate, loss burst length, etc.) as specified in section 3.3. for the sake of generality, we denote the service class associated to mth slice for roi r in frame f as f, r, m, with r = 1,2, 3 in our case. a service class can be intended as a qos class provided by the underlying network or as a level of protection provided by (possibly unequal) forward error correction. the corresponding probability of packet loss and loss burst length (in packets) are denoted as (f, r, m) and (f, r, m). the total transmission time per frame can be calculated as (2)tf, tot=r=1rtot m=1if, r[bf, r, m(f, r, m)r(f, r, m) ], where bf, r, m represents the encoding bits for slice m in roi r and rtot is the number of rois in the frame. in our case note that we do not consider here automatic retransmission request (arq) techniques and processing time for fec is neglected. in the example results reported in section 4, we consider the case where we keep quantization parameters constant in a session and we select different service classes for different rois through a group of pictures gop - by - gop unequal error protection scheme at the application layer. in this case f : quantization parameter for frame f ; f, r : service class for roi r in frame f ; (f, r) : corresponding probability of packet loss ; l(f, r) : corresponding mean burst length. the total transmission time per frame can be calculated as (3)tf, tot=r=1rtot[bf, r(f)r(f, r) ] and per gop : (4)tgop, tot=f=1ftottf, tot. the gilbert two - state channel model has been widely used in the literature to represent packet loss patterns in wireless fading channels. two states are considered to represent good and bad channel states in terms of packet errors. such a model, depicted in figure 3, is completely specified by two parameters : the probability of packet loss pe and the mean burst length lb. by denoting with s0 the packet error free state and s1 the packet error state, the channel state transition probability matrix p has elements pi, j such that, (5)pi, j = p[s(k)=sj s(k1)=si ] ; i, j{0,1 } representing the transition probability from state si at tk1 to state sj at tk. (6)q = p[s(k)=s0 s(k1)=s1],p = p[s(k)=s1 s(k1)=s0 ]. the transition matrix is given by (7)p=(1ppq1q) and it is (8)pe = p[s(k)=s1]=pp+q, lb=1q. the use of reed - solomon (rs) codes is described first, the global uep strategy adopted follows. we consider the use of reed - solomon (rs) codes for application - layer fec. when fec is used at the application layer, it is necessary to apply erasure codes across video packets ; the wimax mac layer discards the whole mac frame in the event of an error, that is, the erroneous frame at the receiving mac is never passed on to the higher layer. therefore, if rs coding is applied within a single packet at the application layer, the erroneous packet will not be available for error detection or correction at the application layer. similar to, we apply rs coding across packets using an interleaver, that is, k slices each of length lm bits are buffered at a matrix interleaver. the first symbol from each of the k slices is sent through an (n, k) rs coder resulting in n k parity symbols, each of which forms the first symbol of the n k parity packets. note that the symbol size in bits depends on the selected value of n, that is, c = log 2(n + 1). this is repeated for the whole slice, resulting in n k parity packets each of length lmax generated by the rs encoder. note that actually the slice lengths lm are not exactly identical and padding bits are needed to obtained equally long packets of length lmax. each video or parity packet is transmitted via rtp / udp / ip and an 802.16e mac frame ; if this frame is discarded at the receiving 802.16e mac layer due to channel errors, this results in a symbol erasure at the rs decoder in the application layer. the rs decoder at the application layer can correct up to n k packet losses out of n packets over which the rs coding was applied. first, the interlacing operation requires that k slices are accumulated to begin the rs(n, k) coding operation. second, once k packets are available, generating the redundant packets by applying the rs code involves data processing delay. due to the high hardware speeds currently available and the possibility to perform encoding in parallel ; the latter delay is very limited and we can only consider the time delay involved in having to wait for k packets. note that, since the rs code is systematic, it is not necessary to buffer packets to form rs codewords, but the information symbols can be transmitted directly if a local copy is kept to form the parity check symbols. these computed parity check symbols can then be sent immediately after the information symbols, eliminating interlacing delay at the transmitter. the total interlacing delay would then be the delay at the receiving end alone. every data block has its own block sequence number, which is useful at the receiver side, since it provides the rs decoder with the position of the lost block. the rs decoder can then recover up to (n k) lost blocks with this position information instead of recovering (n k)/2 lost blocks without the position information. the residual loss probability in case of independent packet losses is (9)pl=1ni = t+1ni(ni)pei(1pe)ni, where t = n k when erasures are considered and information on the position of the erasures is available. for the adopted gilbert - model the probability of packet error and burst length after fec can be calculated following the analysis in. if at least k out of n packets are correctly received, the underlying video information can be correctly decoded. this provides resiliency against a maximum packet loss rate of p = (n k)/n when considering that even fec packets may be affected by loss. thus, based on averaged packet loss rate (pe) measurements such as that provided by rtcp feedback, it is possible to dynamically adjust the redundancy amount h = n k as h = pek/(1 pe). when a decoding failure happens, there are (n i) < k correctly - received packets including both video and parity packets possibly. we utilize these video packets if there is any for the video decoding ; on average, (k / n)(k i) packets out of (n i) correctly - received packets should be video packets. we propose to provide a high protection to the most significant roi for the purpose of diagnosis (roi 1) and a lower protection to roi 2 and the background. patient data / ecg can either be transmitted as data and compressed ecg in padding bits of roi 1 and thus strongly protected, or transmitted in roi 2. we propose rs coding is performed gop by gop ; an rs block will include data from no more than one gop. for the selection of the rs block size, the erasure correction capability of the code and the slice size have to be defined first. the selection of slice size lf, r, m, when slices are not separated in smaller packets at lower layers, is linked to the network and channel characteristics. in the following we will assume that each roi is encoded in one slice of length lf, r. lmax, where rows are made of symbols from slices and padding bits to reach the length lmax, and columns, with data in groups of bits (rs symbol), represent the rs data blocks. lmax is a value which is fixed gop by gop depending on the size of the slices in the gop. the block is simply built by arranging the slices (+ padding bits) as the rows of the rs block until either the suggested value of k rows is reached or the slices belonging to a gop are terminated. after rs coding the structure has size n lmax due to the presence of n k parity packets. note that, with the assumptions above, the mac pdu size is (10)lpdu = lmac.header+lcrc+lrohc.header+lmax. the selection of the coding rate k / n depends on the characteristics of the channel / the network of the video sequence and of context information and the rate could be adapted dynamically gop - by - gop in order to adapt to the conditions of the network. instead of considering models for the impact of losses in the different regions on the global distortion, as typically done, in this case we give priority to context information for taking decisions on the protection rate, the relative importance of the region of interest with respect to the background is different for different types of examinations and we propose that this weight is provided by the clinician and considered for the selection of the protection rate of the different rois. after application layer unequal error protection, the total number of bits per frame is (11)bf, tot=r=1rtot[bf, r(f)rc(f, r) ] and per gop (12)bgop, tot=f=1ftotbf, tot, where the factor 0 < < 1 takes into account the overhead due to padding bits in the rs code matrix organization, and ftot is the number of frames in the gop. it is common practice in video transmission to conceal the effect of errors at the receiver side by, for instance, interpolating from neighbouring data in time and space. in the medical field, this practice may not be desirable when a medical doctor is performing a diagnosis. such concealment practice could be misleading since in this case the specialist can not factor into his or her decision an awareness of missing and potentially important data. for this reason, we propose that concealment is applied seamlessly only in roi2 and roi3 in order to smooth the not diagnostically important rois. although concealment is applied in roi1, we propose to inform the specialist that a specific mb has been concealed by highlighting concealed mbs in the portion of the video frame belonging to roi1. it is in fact important that the specialist can assess his / her confidence on the diagnosis. the ultrasound video clips used in our experiments are cardiac ultrasonography sequences, partly collected from a hospital and partly from public databases. the acquired medical video sequence is encoded according to the h.264 standard with the parameters reported in table 2. groups of slices are organized with the aid of the flexible macroblock ordering tool, in order to have separate groups of slices for different rois. information about the shape of the different rois is stored in the picture parameter set. the encoded image stream is then encoded through rs codes and delivered via rtp / udp / ip. we assume robust header compression (rohc) is adopted to reduce the overhead due to packetization headers and that rtp / udp / ip headers are compressed via rohc to three bytes. the main radio access network parameters of the reference testbed are shown in table 3. measurement conditions and measurement results from are reported for convenience in tables 4 and 5, respectively. we consider a vehicular environment, in order to simulate ultrasound video transmission to / from an ambulance. this is the case where immediate access to ultrasound examinations located in the hospital database is needed, and where the examination is performed in an ambulance through a portable ultrasonographer and the relevant video stream is transmitted in real time to the specialist in the hospital. in particular the gilbert channel model parameters are selected according to the measurements in table 5. the measurements only report the maximum packet - loss burst length. by assuming a geometric distribution for the burst length, we estimate the mean packet - loss burst length as lb = 5 ; in this case according to the geometric distribution the probability of having a packet - loss burst length higher than the measured maximum value is of the order of 10. note that measurements are done by transmitting a number of packets of the order of 10. we compare the following strategies for application layer (unequal) error protection (see table 5). no application layer protection ; in this case all the available bitrate is used for representing the video sequence. application layer equal error protection (eep) : in this case a higher protection is uniformly provided to the bitstream, resulting in a higher robustness in bad channel / network conditions, but in a reduced global quality when channel / network conditions are good. application layer roi - based unequal error protection : similar as in the case above, this scheme results in a higher robustness in bad channel / network conditions, but in a reduced global quality when channel / network conditions are good. in this case, however, the redundancy is exploited to protect the most important information from the point of view of the diagnosis and an improved quality in terms of probability to perform a correct diagnosis is expected also when channel conditions are bad. application layer roi - based and prediction - based unequal error protection ; this scheme results in a higher robustness in bad channel / network conditions, but in an even more reduced global quality when channel / network conditions are good. in this case, the redundancy is exploited to protect the most important roi from the point of view of the diagnosis and the most important information for motion compensation prediction (i frames). an improved quality in terms of probability to perform a correct diagnosis is expected also when channel conditions are bad. in medical applications, the target of the optimization of the transmission system should not be the minimization of distortion in terms of mean square error (or equivalently the maximization of the peak signal - to - noise ratio, psnr), but the maximization of the probability of performing a correct diagnosis based on the received video sequence. although not designed for this purpose, according to preliminary studies [13, 52 ] the structural similarity metric (ssim) better meets this criterion and for this reason we consider in this paper ssim in addition to the well - known psnr. although the performance assessment of such a scheme should be done through subjective metrics, results are presented in terms of the aforementioned well known objective metrics in order to allow easy comparison with results obtained by other authors and to give an indication of the local distortion achieved in different rois. we consider local distortion as in the following : (13)mseroir=1nri=1nr(xiyi)2,psnrroir=10 log 102552mseroir, where nr is the number of pixels in roi ; r, xi and yi represent the luminance of pixel i in the original and in the corrupted frame, respectively. we then consider a slightly modified version of the ssim metric in. the ssim index, as shown in (14), can be written as the product of three independent contributions, representing the luminance information, the contrast information, and the structural information. with x and y indicating image signals in the reference and received image, (14)ssim(x, y)=l(x, y)c(x, y)s(x, y), where the luminance comparison is represented by the term (15)l(x, y)=2xy+c1x2+y2+c1 and for the contrast comparison (16)c(x, y)=2xy+c2x2+y2+c1. the structural comparison term s(x, y) is (17)s(x, y)=xy+c3xy+c3. in the expressions above, we use a 11 11 circular - symmetric gaussian weighting function wi, with standard deviation of 1.5 samples, normalized to sum to unity : iwi = 1. the statistics are thus defined as (18)x=1ni=1nwixi,x2=1n1i=1nwi(xix)2,xy=1n1i=1nwi(xix)(yiy). we then define the mssim metric for roir as (19)mssimroir(xr, yr)=1mj=1mssim(xr, j, yr, j), where m is the number of local windows in roi r and j is the local window index. numerical results obtained in the conditions described above, and summarized in table 6, are reported in table 7. note that the quality of the diagnostically important region of interest is lower than the quality of the background in the unprotected case, due to the different complexity and the use of the same quantization parameter for the different rois. a uniform protection scheme at the application layer (eep) increases the total quality of the sequence, but it fails in sensibly increasing the quality of the most important roi for the diagnosis. we highlight here that the schemes where fec is applied at the application layer are compared with an uncoded scheme where a higher bitrate is adopted in source encoding, in order to allow a fair comparison. both the uep schemes manage to improve the quality of roi 1, at the expense of a slight decrease in quality in the remaining part of the images. the scheme uep 1 provides a slightly higher quality for roi 1, both in terms of psnr and ssim. scheme uep 2 provides an improvement of about 1 db in psnr with a decrease in quality with respect to scheme uep 1 of only 0.2 dbs in roi 1 and it can be preferable in some scenarios as also confirmed by subjective tests. the sequence corresponding to image [e ] is the one selected by the medical specialist involved in the study as the one best keeping diagnostic quality. we have proposed in this paper a context - aware transmission strategy for diagnostic - quality ultrasound video transmission over wimax systems. context, in terms of regions of interest (roi) in a specific session, is taken into account for the identification of multiple regions of interest, and compression / transmission strategies are tailored to such context information. we have presented a methodology based on h.264 medical video compression and fmo for roi identification. two different unequal error protection methodologies, providing higher protection to the most diagnostically relevant data, are compared. results show that the proposed scheme allows an improvement for the diagnostic region of interest of about 3 dbs in psnr and 0.31 in ssim with respect to the case where such an approach is not adopted, still obtaining a small improvement in quality in the rest of the image (0.81.6 in psnr for uep 1 and uep 2, resp.). | the most recent network technologies are enabling a variety of new applications, thanks to the provision of increased bandwidth and better management of quality of service. nevertheless, telemedical services involving multimedia data are still lagging behind, due to the concern of the end users, that is, clinicians and also patients, about the low quality provided. indeed, emerging network technologies should be appropriately exploited by designing the transmission strategy focusing on quality provision for end users. stemming from this principle, we propose here a context - aware transmission strategy for medical video transmission over wimax systems. context, in terms of regions of interest (roi) in a specific session, is taken into account for the identification of multiple regions of interest, and compression / transmission strategies are tailored to such context information. we present a methodology based on h.264 medical video compression and flexible macroblock ordering (fmo) for roi identification. two different unequal error protection methodologies, providing higher protection to the most diagnostically relevant data, are presented. |
the case we present here is about a 22-year - old female patient referred to the department of conservative dentistry and endodontics with the chief complaint of pain in the left lower back tooth for the past 2 days. her medical history was noncontributory and she gave a dental history of root canal treatment was done in that tooth before 2 months in a dental clinic. on clinical examination, it was found access opening was done in a grossly decayed left mandibular second molar. the treatment was continued by complete caries excauation and canal exploration after rubber dam isolation. on microscopic exploration of the floor of the pulp chamber a canal re is an extra third root in mandibular molars present on the lingual side, which is shorter in length than other two roots. however, here, it is seen on buccal side called as rp. on careful exploration on mesial root, presence of three canals after working length estimation done [figure 1 ], all the canals were enlarged until 20 size k file and biomechanical preparation was completed by protaper rotary systems. all three mesial canals were enlarged until f1, distal canal until f3 and the extra third root on buccal side until f2 with copious irrigation of saline simultaneously. then the master cones checkup were done as shown in figure 2. then the final irrigation was done with 17% ethylenediaminetetraacetic acid, 3% sodium hypochlorite and by saline. canals were dried with paper points and obturated with gutta - percha cones of respective sizes with kerr sealer. figure 4 shows the post obturation magnified view of floor of the pulp chamber showing the five canals with rp in middle. working length determination post obturation radiograph microscopic image of floor of pulp chamber showing type b rp the mandibular molars usually have two roots with two canals in mesial roots and one canal in distal root. it is more common that mandibular molars having four canals with two in mesial and distal root viz. the first molar. however, recent studies shows there are more chance for a 3 canal in mesial and distal root as middle mesial and middle distal, respectively. this presence in second molar is very rare but, recently few studies of ryan. and jain reported with cases of five or six canals in mandibular first molars only. the isthmus connecting the mesiobuccal and lingual or distobuccal and lingual is debrided and left uninstrumented in most of the cases. however, on careful exploration of this isthmus a third canal with a separate path of exit can be seen in many of the cases as reported by deepalakshmi. in their four cases. here, in this case of 37 with three mesial canals, all three files and gp points went until full working length independently without any fusion at the apex to exit as one. it seems that in all three canals, there will be isthmus communicating them in between laterally as it travel, these lateral connections were debrided completely with 3% sodium hypochlorite and sealed by the root canal sealer. re is an extra third root in mandibular molar, which is shorter than the mesial and distal roots ; seen in distolingual corner like a distolingual canal in the floor of the chamber. the rp occurrence rate is about 0%, 2%, and 5% in mandibular first, second and third molars respectively. carlsen and alexandersen described two different types of rp : type a : rp in which the cervical part is located on the mesial root complex ; type b : rp in which the cervical part is located centrally, between the mesial and distal root complexes, an additional cusp was present on the buccal side. the complex root canal anatomy stresses the operator to observe the canal morphology with care without underestimating it and missing the extra root and roots canals, which can occur in any cases as a rare finding. thus, the success of endodontics lies in the careful exploration of the canals and debridement of it. | mandibular first molar commonly has two roots, the mesial and the distal root. surprisingly in some mandibular first molar has an unusual extra third root in between mesial and distal root. this extra root is the most commonly seen in lingual side and is called as radix entomolaris. if this extra root is seen on the buccal side, it is termed as radix paramolaris (rp), which is a very rare phenomenon. recently, various case report studies has shown the presence of third canal in the mesial and distal roots of mandibular molars. they are named as middle mesial and middle distal canal, respectively. here, we present a case report of mandibular second molar showing three canals in the mesial root and a rp. |
stabilizer muscles of the trunk in the human body refer to the deep muscles that contribute to stabilization. stabilizer muscles in the waist region include the diaphragm, pelvic floor muscles, transversus abdominis, internal abdominal oblique, and multifidus1. among these stabilizer muscles, trunk stabilizer muscles are activated before the body initiates motion to stabilize the vertebral column3,4,5. when there is back pain, pain prevents some movements of the lumbar spine and pelvis, and such a phenomenon weakens the stabilizer muscles, which can lead to chronic back pain6. it has been confirmed that the contraction of stabilizer muscles is reduced and the order of muscle recruitment is reversed in patients with back pain7. therefore, physical therapy in patients with back pain focuses on rehabilitation of the stabilizer muscles to reduce pain, and improve muscle endurance and muscle strength8,9,10,11. these exercises are based on six principles : concentration, control, centering, flowing movement, precision, and breathing14. among these basic principles, breathing applies to all ranges of motion during all exercises, and breathing promotes activation of the trunk stabilizer muscles14. even though study of effects of pilates breathing on trunk muscle activation during the trunk flex in a previous study was existed15, there was not studied another movement of trunk (for example, extension) or function associated movement. it is necessary to investigate the actual effects of breathing on the trunk stabilizer muscles in various trunk movements. therefore, the purpose of the present study was to investigate the effects of pilates breathing on trunk muscle activities during trunk flexion and extension exercise. additionally, the relationship between pilates breathing and lifting, which is associated with back pain, was evaluated. participants did not have a history of trunk surgeries, existing back pain, or prior pilates experience, and they did not have any problems performing exercises. all participants received verbal and written information about the study and signed a consent form. surface electromyography (emg) (telemyo dts telemetry, noraxon, az, usa) was used to measure the trunk muscle activities. the sampling rate was set to 1,000 hz, band pass filter was set to 20450 hz, and notch filter was set to 60 hz. activities of the trunk flexors such as the rectus abdominis and trunk deep stabilizers such as the transversus abdominis, internal abdominal oblique and trunk extensor such as erector spinae, multifidus were measured. electrodes were placed on the muscles of the right side of the participants body, and the locations of the electrodes were shaved and cleaned with alcohol before the measurements were taken to reduce impedance. the electrode for the rectus abdominis was placed on upper belly of the muscle below the xiphoid process, 3 cm to the right of the medial line16. the electrode for the erector spinae was placed on the muscle, 3 cm to the right of the third lumbar spinous process. the electrode for the transversus abdominis / internal abdominal oblique was placed 2 cm below and medial of the right anterior superior iliac spine17. the electrode for the multifidus was placed 2 cm to the right of the fifth lumbar spinous process18. emg data were collected during 3 pilates based therapeutic exercises at baseline and after the breathing training. head and chest curl - ups and chest - head lifts were selected for this study, as they are representative motions for trunk flexion and extension. head and chest curl - ups in supine hook - lying position with both hands behind the head and maintain the chin - in position was performed flex the upper trunk in the sagital plane until scapula inferior angle. chest - head lifts in prone position with both hands behind the head and maintain the chin - in position was performed extend the upper trunk in the sagital plane until xiphoid process. additionally, lifting tasks, which are daily activities directly related to back pain, were also performed. lifting task was lifed 10-kg weight dumbbell placed in the middle of both knee with knee exed approximately 30 in 30 cm apart stance (indicated by oor markings) and keep straight arms and maintain neutral pelvic position that extend the only hip and trunk in the sagital plane until uplight the trunk. sufficient exercise training without breathing control was provided to participants by pilates instructor before the test so that the three exercises could be performed accurately. participants were allowed 30 minutes of rest after the training before proceeding to the experiment. each exercise was performed five times, and emg data were recorded middle three times. to prevent muscle fatigue, 2 minutes of rest were allowed in between each exercise. using a metronome, each exercise was performed for 6 seconds (3 seconds of contraction and 3 seconds of relaxation). five seconds of maximum isometric contraction was performed three times. after discarding 1 second from both the beginning and end, participants were allowed to rest for more than 5 minutes after the measurement was obtained. during the pre - measurement, participants were instructed to perform the exercises without restricting their breathing and by using their usual breathing methods. after the pre - measurement, participants underwent pilates breathing training, which was guided by a pilates instructor. a 60-minute pilates breathing training session was performed three times per week for 2 weeks. pilates breathing training session was consists of only breathing control in the supine position. changes in the muscle activities after the experiment (% mvic) were investigated by performing the paired t - test. the significance level was set to<0.05, and the kolmogorov - smirnov test was performed to test for normality. as shown in table 1table 1.comparison of muscle activities (mvic%)exercisemusclespre - restpost - testcurl - upra38.84 21.1746.83 18.62tra / io45.07 35.1568.34 40.64es5.52 4.845.84 3.48mf6.34 4.778.95 4.48chest - head liftra13.17 19.1922.64 31.46tra / io12.84 12.9740.36 25.20es39.72 20.7954.49 15.59mf45.69 14.2654.40 15.27lifting taskra6.13 4.16.61 4.59tra / io24.21 22.7239.22 25.75es47.69 17.2853.86 15.31mf50.17 12.6462.59 asterisks indicate a significant difference between pre - post test (p<0.05, p<0.01, p<0.001). ra : rectus abdominis, tra / io : transversus abdominis / internal oblique, es : erector spinae, mf : multifidus, all muscle activities increased after pilates breathing. statistically significant improvements were observed in the stabilizer muscles transversus abdominis / internal abdominal oblique and multifidus. asterisks indicate a significant difference between pre - post test (p<0.05, p<0.01, p<0.001). ra : rectus abdominis, tra / io : transversus abdominis / internal oblique, es : erector spinae, mf : multifidus the recovery of spinal stability can be achieved through breath training alone20, 21. pilates exercise also emphasizes the importance of appropriate breathing for stabilizing the trunk22. besides stabilizing the trunk, stabilizer muscles such as the transversus abdominis, internal abdominal oblique, and multifidus also contribute to voluntary respiration. the transversus abdominis, especially, along with the diaphragm, is activated during exhalation, and it experiences the largest change in thickness during maximal exhalation23. additionally, the transversus abdominis, along with diaphragm movement, can induce isometric contraction of the trunk stabilizer muscles and activation of the pelvic floor muscles by increasing abdominal pressure. therefore, incorporating abdominal respiration during trunk stabilization exercises can produce better results24, 25. since activities of the trunk stabilizer muscles can be regulated by one s breath, breathing our study s results showed that performing pilates breathing during curl - ups, chest - head lifts, and lifting tasks significantly increased activities of the transversus abdominis / internal abdominal oblique and multifidus. reported that activities of the transversus abdominis or internal abdominal oblique significantly increase when pilates breathing is incorporated, which coincides with our study s yoon. also reported that slow exhalations during curl - ups selectively increase activities of the transversus abdominis and internal abdominal oblique26. the present study differs from previous studies since an intervention period was allocated for breathing training. in addition, previous studies were limited to curl - ups and trunk flexions, whereas our study measured activities of all the flexion, extension, and stabilizer muscles of the trunk. the effects of pilates breathing on trunk muscles during lifting tasks were also investigated. isometric contraction of the trunk muscles can reduce the risk of back pain during lifting tasks30. increased activities of the trunk muscles with pilates breathing indicate that pilates breath training during lifting tasks can reduce the risk of back injuries. patients with acute back pain can not easily move their spine due to severe pain. trunk movements are also restricted in patients immediately after surgery due to severe pain and possible side effects. pilates breathing can induce muscle activation even without actual body movements. therefore, effectiveness of pilates breathing trainings in patients with acute back pain or in those whose trunk movements are restricted due to a recent back surgery or surgical procedure is suggest future studies. the current study s findings show immediate results after the breath training intervention. therefore, a further study in which participants are followed up for a specific period is necessary to determine the long - term effects of breath training. our study s results show that pilates breathing increases activities of the transversus abdominis / internal abdominal oblique and multifidus. therefore, the use of pilates breathing can more effectively increase activities of the muscles during trunk - stabilizing exercises. moreover, performing pilates breathing during lifting tasks can prevent potential injuries by increasing muscle activities. lastly, pilates breathing can be an effective method for patients with acute back pain or those who have undergone back surgery and have restricted trunk movement. | [purpose ] to investigate the effects of pilates breathing on trunk muscle activation. [subjects and methods ] twenty - eight healthy female adults were selected for this study. participants trunk muscle activations were measured while they performed curl - ups, chest - head lifts, and lifting tasks. pilates breathing trainings were performed for 60 minutes per each session, 3 times per week for 2 weeks. post - training muscle activations were measured by the same methods used for the pre - training muscle activations. [results ] all trunk muscles measured in this study had increased activities after pilates breathing trainings. all activities of the transversus abdominis / internal abdominal oblique, and multifidus significantly increased. [conclusion ] pilates breathing increased activities of the trunk stabilizer muscles. activation of the trunk muscle indicates that practicing pilates breathing while performing lifting tasks will reduce the risk of trunk injuries. |
metabolic encephalopathies are among one of the common causes of admission to hospital in the elderly age group. depending on the severity and rate of development of hypercalcemia, patient can present with mild drowsiness to coma. in more than 90% of cases, hyperparathyroidism is the cause ; however, in an emergency setting, malignancy related hypercalcemia is the commonest cause. milk alkali syndrome (mas) can cause acute, subacute or chronic hypercalcemia, which has been reported as early as in 1923, though the term mas was coined in 1949. though it became uncommon after some time, recently it has witnessed resurgence after increased general awareness about osteoporosis and overuse of calcium carbonate and vitamin d. injection teriparatide is known to cause transient hypercalcemia up to 6 hours of its administration ; severe hypercalcemia causing encephalopathy is not known. here, we report a case of hypercalcemic encephalopathy caused by mas in which a permissive role was played by injection teriparatide and other factors. an 82-year - old male presented to emergency with history of sudden onset with gradual deterioration of mental sensorium of 8 days duration. other history elicited from attendants revealed that the patient suffered from low impact fracture of third and fourth lumbar vertebrae 3 months back for which he was evaluated and found to have osteoporosis (l1l4 vertebrae : t - score 3.1, z - score 1.1 ; femur neck : t - score 3.0, z - score 0.7). during evaluation, he was detected to have prostatic carcinoma for which he was being treated with depot preparation of injection treptorelin acetate [gonadotropin - releasing hormone (gnrh) analogue ] 11.25 mg once in 3 months. he was prescribed oral calcium carbonate 500 mg thrice daily and cholecalciferol 60,000 u monthly. however, the patient was consuming 56 tablets of calcium carbonate (each tablet containing 500 mg elemental calcium) and his diet consisted of milk (1.5 l / day) and plenty of curd. he was started on injection teriparatide 20 g daily 10 days back in view of severe osteoporosis. at this time, his serum calcium (9.9 mg / dl), phosphorus (3.8 mg / dl) and 25(oh) vitamin d (84.4 ng / ml) levels were normal. he had limited physical activity in view of his age and disability. on examination, he had normal vital parameters with normal systemic examination except drowsiness and disorientation. investigations revealed hypercalcemia (serum calcium 14.3 mg / dl), normal phosphate levels (3.1 mg / dl), normal alkaline phosphatase (174 u / l), acute kidney injury as evidenced by raised serum creatinine (2.3 mg / dl as compared to basal creatinine levels of 0.9 mg / dl), metabolic alkalosis (ph 7.56, hco 39 meq / l, paco2 47 mm of hg) and hypokalemia (serum potassium 2.3 meq / l). his serum parathyroid hormone (pth) level was 7 pg / ml (10 - 74 pg / ml). he was euthyroid [t3 1.2 ng/ ml, t4 9.45 g / dl, thyroid stimulating hormone (tsh) 4.5 iu / ml ] and eucortisolemic (cortisol basal 8.3 g / dl, post acth cortisol 24 g / dl). he had low serum testosterone levels (0.1 ng / ml) consistent with gnrh therapy. in view of acute hypercalcemia, hypokalemia, metabolic alkalosis, acute kidney injury and history of consumption of 34 g of calcium carbonate daily, he was diagnosed as a case of hypercalcemic encephalopathy due to mas which was probably precipitated by concomitant use of injection teriparatide and immobilization. his drugs (calcium carbonate and teriparatide) were stopped and he was started on intravenous forced diuresis with 3 l of normal saline and 40 mg thrice daily injection of frusemide, with monitoring of cardiovascular status. he was also given injection zoledronic acid 5 mg in 100 ml normal saline over 30 min. his clinical and biochemical parameters normalized in 72 hours (serum calcium 8.6 mg / dl, ph 7.38, potassium 4.6 meq / l and creatinine 0.7 mg / dl). he was discharged after 6 days of hospital stay on vitamin d sachet (60,000 u) once monthly. hypercalcemia has various causes, and in an emergency setting, primary hyperparathyroidism, malignancy associated hypercalcemia and mas constitute most of the cases. our patient was diagnosed with mas on the basis of presence of acute hypercalcemia, hypokalemia, metabolic alkalosis, acute kidney injury and history of consumption of 34 g daily calcium carbonate, with normal phosphate and low pth levels. milk alkali therapy for treatment of peptic ulcer disease was developed by sippy in 1910. it constituted bed rest of 4 weeks duration and multiple doses of milk and cream with alkali. because of paucity of other treatment options, milk alkali treatment rapidly became standard for treatment of peptic ulcer disease. burnett for the first time named this syndrome of hypercalcemia, hyperphosphatemia, metabolic alkalosis and renal dysfunction as mas. in pathogenesis of mas, old age, pre - existing renal dysfunction, and ingestion of large dose of calcium carbonate are all known to play a role. initially, incidence of mas was reported to be 1015% which subsequently reduced on account of availability of effective alternate therapies for peptic ulcer disease. but the last two decades have seen resurgence of a modern form of mas, where calcium carbonate constitutes the source of calcium and alkali, because many doctors are prescribing calcium supplements due to increase awareness about osteoporosis. modern mas differs from classical mas in absence of male preponderance and presence of near - normal phosphate levels. in the present case secondly, at the age of 82, his estimated glomerular filtration rate will be compromised (~53 ml / min by cockcroft and gault equation). however, in our patient with compromised renal function, excessive calcium intake with vitamin d may have predisposed him to the development of hypercalcemia. finally, on initiation of injection teriparatide, he developed sudden worsening of sensorium and severe symptomatic hypercalcemia, along with deterioration in renal function and the biochemical features suggestive of mas. it was initially suspected that with the use of teriparatide, a synthetic analogue of pth, the incidence of hypercalcemia would increase. however, mostly transient and mild hypercalcemia is observed but persistent and severe hypercalcemia has been reported. hence, our patient developed mas secondary to excessive intake of calcium where a permissive role was played by age, immobilization and teriparatide for the development of hypercalcemic encephalopathy. therefore, it is suggested that when teriparatide therapy is begun, particularly in elderly patients who have limited physical activity, daily total, dietary and supplemental, calcium should be maintained at 1000 mg or less, so as to keep the serum calcium below 10 mg / dl. | an 82-year - old male, a known case of severe osteoporosis with vertebral fracture and prostatic carcinoma, was treated with gonadotropin releasing hormone analogue, calcium carbonate, cholecalciferol sachet and injection teriparatide. his diet consisted of milk and curd. he developed altered behavior and generalized weakness, and on investigation, hypercalcemia, hypokalemia, and metabolic alkalosis with low parathyroid hormone levels were detected. injection teriparatide was stopped and he was managed with forced saline diuresis and injection zoledronic acid. he was diagnosed as a case of milk alkali syndrome in whom teriparatide and prolonged immobilization played a permissive role in the development of hypercalcemic encephalopathy. |
during the surgical removal of impacted lower third molar teeth, since the region is constricted and the visibility is insufficient and the position and bony structure of the teeth may be rigid, the patient is affected by trauma causing postoperative problems such as pain, edema, decreased function, and trismus. these challenges cause problems for surgeons and patients since they disturb the aesthetics and function. moreover, they may affect the activities of daily living and may result in a severe labor force loss. to prevent all these complications, the most commonly used drugs for the prevention of inflammation after extraction of impacted lower third molar teeth are non - steroidal anti - inflammatory drugs (nsaids). it inhibits cyclooxygenase and the lipoxygenase enzyme, thus preventing the formation of prostaglandins and leukotrienes that play an essential role in inflammation by diminishing active oxygen radicals and inhibiting migration and phagocytosis of leucocytes. in addition to its anti - inflammatory effects by these mechanisms, it also has analgesic and antipyretic effects. corticosteroids are another group of drugs that also suppress the production of prostaglandins and leukotrienes at initial phases and thereby inhibit inflammation. methylprednisolone, a type of corticosteroid, inhibits the development of macrophages in the inflammation zone, diminishing the number and proliferation of fibroblasts in connective tissue, and suppressing the immune system. methylprednisolone, by stabilizing cellular and organelle membranes, reduces kinin and bradykinin formation and blocks histamine and histamine - like substances intracellularly. various routes and times of administration (e.g., oral, intravenous, and intramuscular ; preoperative and perioperative) have recently been proposed because of limited benefits when the therapy was applied postoperatively. although there are many studies in the literature about the effectiveness of corticosteroids and nsaids on reducing complications after the surgical extraction of impacted third molar teeth, there is still no consensus on the type of drug administration. in light of these data, the aim of the present study was to compare the effects of administration of pre - emptive intravenous (iv) tenoxicam (tilcotil roche - istanbul, turkey) (an nsaid) and iv methylprednisolone (prednol l mustafa nevzat istanbul, turkey) (a corticosteroid) in postoperative control of pain, edema, and trismus of patients following the extraction of impacted third molars. this study was a placebo - controlled, randomized, double - blind, clinical trial. a total of 60 adult patients admitted to our oral and maxillofacial surgery clinic due to impacted third molar teeth, without any known systemic diseases, were included in the study. informed consent was obtained from all patients. to standardize the 60 teeth with an orthodontic indication of surgical extraction in the study, their mesio - angular or vertical positions were recorded. patients who are exposed to severe trauma due to bony retention during surgery were excluded, as were patients who used an anti - inflammatory or analgesic agent during the study or within 15 days prior to the beginning of the study. the ethics committee at dicle university faculty of medicine approved the study, which has been performed in accordance with the ethics standards of the 2008 declaration of helsinki. the patients were randomized into 3 groups according to the drug administered before the operation ; iv 20 mg tenoxicam was administered to group a (n=20), iv 80 mg methylprednisolone sodium succinate was injected in group b (n=20), and iv isotonic sodium chloride was administered to group c patients (n=20) 1 hour before the operation. prior to surgery, the patients received extra - oral antisepsis with a solution of 10% povidone - iodine. local anesthesia was performed with lidocaine 2% and epinephrine 1:100 000. using standard methods, after the surgery, all patients were informed about the local hemostatic measures, feeding, and cleaning. during the postoperative period, prophylaxis amoxicillin 3500 mg, paracetamol 3500 mg as pain - killer, and chlorhexidine gluconate 2x1 times as oral antiseptic were prescribed to all patients. postoperative swelling was assessed by an ultrasound (toshiba ssh 1401) and a 7.5 mhz transducer. in the evaluation of edema, the soft - tissue measurements were performed in dicle university hospital, radiology department, division of ultrasound. the preoperative and postoperative 48 hour ultrasound imaging of all patients were performed from the same point while the teeth were on centric occlusion. to assure that the ultrasound images were taken from the same point, the preoperative measured point was marked with a pen. to achieve maximum standardization, all measurements were performed on the same machine by the same doctor, with minimum pressure on the skin (figure 1). after the radiological evaluation of all patients, they were asked to open the mouth as much as possible and limited mouth opening (trismus) was assessed by determining maximal unassisted mouth opening, measured with a simple calliper between the upper and lower central incisors. the measurement process was repeated 3 times and the mean of these 3 measurements was assessed each time. trismus was determined by the difference between the preoperative and postoperative period. in the evaluation of postoperative pain, visual analogue scale (vas) forms were given to the patients. to indicate the intensity of pain, the following categorization was used : 0 = no pain ; 2 = mild pain ; 4 = moderate pain ; 6 = severe pain ; 8= very severe pain ; and 10 = unbearable pain. the patients were asked to mark the pain level hourly during waking hours starting from the first hour of the operation. by use of these forms, the severities of pain were evaluated for the first 24 hours (figure 2). data analysis was performed using the statistical package for social sciences (spss for windows, version 18.0 ; spss inc., chicago, il, usa). in the determination of differences between the groups, one - way anova (variance analysis) test was used. for the multiple comparisons among groups in variance analysis, dunnett test was used to evaluate the comparison of significant differences between study groups and the control group. postoperative swelling was assessed by an ultrasound (toshiba ssh 1401) and a 7.5 mhz transducer. in the evaluation of edema, the soft - tissue measurements were performed in dicle university hospital, radiology department, division of ultrasound. the preoperative and postoperative 48 hour ultrasound imaging of all patients were performed from the same point while the teeth were on centric occlusion. to assure that the ultrasound images were taken from the same point, the preoperative measured point was marked with a pen. to achieve maximum standardization, all measurements were performed on the same machine by the same doctor, with minimum pressure on the skin (figure 1). after the radiological evaluation of all patients, they were asked to open the mouth as much as possible and limited mouth opening (trismus) was assessed by determining maximal unassisted mouth opening, measured with a simple calliper between the upper and lower central incisors. the measurement process was repeated 3 times and the mean of these 3 measurements was assessed each time. in the evaluation of postoperative pain, visual analogue scale (vas) forms were given to the patients. to indicate the intensity of pain, the following categorization was used : 0 = no pain ; 2 = mild pain ; 4 = moderate pain ; 6 = severe pain ; 8= very severe pain ; and 10 = unbearable pain. the patients were asked to mark the pain level hourly during waking hours starting from the first hour of the operation. by use of these forms, the severities of pain were evaluated for the first 24 hours (figure 2). data analysis was performed using the statistical package for social sciences (spss for windows, version 18.0 ; spss inc., chicago, il, usa). in the determination of differences between the groups, one - way anova (variance analysis) test was used. for the multiple comparisons among groups in variance analysis, dunnett test was used to evaluate the comparison of significant differences between study groups and the control group. the study included a total of 60 patients (41 females and 19 males) aged 1840 years (table 1). the duration of surgery of groups a, b, and c were 26.100.95, 25.700.98, and 23.800.79 minutes, respectively. no significant difference was found between the groups regarding the duration of surgery (f=1.798, p>0.05) (table 2). in evaluation of edema, postoperative edema (subcutaneous + masseter muscle) ratios of groups were 38%, 57%, and 150% in group a, group b, and control groups, respectively (p<0.05). there was no statistically significant difference between methyl prednisolone and tenoxicam groups regarding the edema (table 3). the postoperative trismus ratios of groups were 5.25%, 2.95%, and 13.90% in group a, group b, and the control group, respectively. although the trismus ratios were significantly better in both study groups than in the control group, the methyl prednisolone patients had statistically significantly superior results as regards the trismus compared with the tenoxicam patients (f=110.3, p<0.001) (table 4). mean pain scores of groups a, b, and c were 35.305.64, 48.207.22, and 81.404.79, respectively. in the evaluation of pain scores, the tenoxicam and methylprednisolone groups had similar mean pain scores, which were statistically significantly lower than in the control group (f=15.89, p<0.001) (table 4). in the follow - up period after single - dose administrations of iv tenoxicam or methylprednisolone, no postoperative complications, including gastrointestinal problems, hemorrhage or delay in wound healing, were observed in any of the patients. although there are many reports in the literature about use of drugs in prevention of postoperative pain, edema, and trismus after impacted third molar teeth extraction, to the best of our knowledge this is the first study comparing the effects of 2 very commonly used drugs moreover, even though this procedure is a very common oral surgical procedure, there is still no consensus about the optimal type, time, method of administration, and dose of drugs to use in prevention of complications. in this respect a gradual facial swelling occurs in response to tissue trauma in the third molar region, with peak swelling 48 h after surgery. there are various methods used in the evaluation of edema after oral surgeries, including visual analogue scale, measuring with silk suture, or by the aid of plethysmography. this effect may be explained by the long duration of action and high anti - inflammatory potencies of both drugs. each drug may be preferred for the purpose of reducing edema after impacted third molar teeth extraction. limited mouth opening is another unwanted effect commonly reported after oral surgeries. in investigations, inter - insical distance measurements before and after the operation is commonly used for the evaluation of trismus, as in our study. trismus impedes eating and talking and reduces the quality of daily life of patients ; in this sense, decreased trismus means decreased discomfort as well as increased life - quality for the patients. therefore, methyl prednisolone may be preferred to tenoxicam for patients with a lesser trismus. in evaluation of postoperative pain, visual analogue scale or number of analgesic tablets consumed after surgery are the methods commonly used. in this study, since patients were prescribed paracetamol 3500 mg routinely after surgery, number of analgesics consumed was not recorded. in pain control, in fact, this is an interesting finding because the exact contribution of corticosteroids in the control of pain is not yet fully clarified, but nsaids are very potent pain - killers. this finding may also be associated with or affected by the use of paracetamol 3500 mg routinely. there is no need to combine corticosteroids (with their adverse effects) with nsaids, since combining corticosteroids with a relatively safe drug (e.g., paracetamol) is enough to achieve the same pain - relief. supporting this data, joshi. compared the effect of preoperative ibuprofen, diclofenac, paracetamol with codeine, and placebo tablets on postoperative dental pain and found no significant difference between the different therapeutic groups. corticosteroids and nsaids are 2 large groups of drugs that are widely studied for the prevention of postoperative inflammatory complications and to diminish unwanted effects such as pain or trismus [1316 ]. nsaids have been reported to be effective in pain relief, while corticosteroids are effective in diminishing edema. this being the case, some investigators preferred to combine these 2 groups of drugs. studied the combined usage of ibuprofen and methylprednisolone in the prevention of pain and edema after third molar teeth extraction and revealed that combined usage of these 2 drugs decreased the edema by 56% and pain by 67% compared with the control group. however, in the present study we did not observe any significant difference between tenoxicam and methylprednisolone groups regarding pain relief. corticosteroids have been used widely in diminishing complications after third molar teeth extraction, but with different dosages and types.. gave 18 mg oral methylprednisolone 1 night before and 20 mg iv methylprednisolone during surgery and reported that this combination decreased the postoperative edema by 42% within the first 24 h and there was no trismus observed in these patients and no need for other analgesic preparations. investigated the effects of pre - emptive 8-mg oral dexamethasone on pain, trismus and edema of impacted third molar surgery patients and reported that this drug significantly decreased postoperative pain and edema but did not have any effects on trismus. more recently, in a study comparing dexamethasone 8 mg and methylprednisolone 40 mg in control of pain, swelling, and trismus following the impacted third molar teeth extraction, 8-mg dexamethasone has been determined to be better in control of swelling and trismus, with no difference in pain control between drugs. another study comparing the effects of weight - dependent methylprednisolone (4080 mg) or a placebo orally 1 h prior to surgery, determined that a single preoperative weight - dependent administration of methylprednisolone is a safe and effective method for diminishing postoperative discomfort, pain intensity, and total intake of analgesics after wisdom tooth extractions. there are also some studies in the literature comparing the effects of corticosteroids and nsaids on postoperative complications. sisk and bonnington compared the effects of methylprednisolone and flurbiprofen on postoperative pain, edema, and trismus after impacted third molar teeth surgery on 60 patients aged 1635 years. they determined that flurbiprofen was more effective in pain control while methylprednisolone was more effective in control of edema. compared 50-mg diclofenac with 50-mg diclofenac and 40-mg methylprednisolone combination and reported that the pain scores were diminished in the combined group but there was no significant difference between the groups in terms of trismus. compared the administration of diclofenac potassium alone and in combination with dexamethasone and found that the combination therapy was more effective in controlling pain, swelling, and trismus following third molar surgery. the doses of drugs examined in this study were based on previous reports of the maximum effective dosages without any adverse effects. kumara and zacharias gave 40 mg tenoxicam to impacted third molar teeth surgery patients orally to the first group 1 night before the operation and iv to the second group during the operation and reported that the methods were equally effective in healthy young patients. it has been reported that there was no significant difference between 20 mg or 40 mg iv tenoxicam after oral surgery in terms of pain relief. in light of these data, the recommended dosages range from 40 mg to 125 mg ; however, 80 mg was chosen as it was one of the most commonly used dosages. in a recent study, short - term outcomes of third molar operations (swelling, trismus, and pain) has been determined to differ depending on patient characteristics, including age, sex, and body mass index. moreover, in that study surgery characteristics such as operating time and tooth sectioning were also associated with postoperative variables. although the ages were similar, we did not calculate the body mass indices of patients. however, the duration of surgery was recorded and evaluated for each group to determine possible confounding factors that could influence our results. there was no statistically significant difference between groups in the duration of surgery, showing that the procedures, performed by the same surgical team, were as standardized as possible. in conclusion, the results of this study indicate that although methylprednisolone and tenoxicam are both effective in diminishing complications of impacted third molar teeth extraction, preoperative administration of 80 mg methylprednisolone achieves better control of trismus than tenoxicam, without any differences in edema and pain control. in light of these data, we conclude that methylprednisolone may be a better alternative than tenoxicam in the prevention of complications associated with impacted third molar teeth extraction. | backgroundthe aim of the present study was to compare the effects of preemptive intravenous tenoxicam and methylprednisolone administrations on extraction of impacted third molars.material/methodsthis was a placebo - controlled, randomized, double - blind, clinical trial. a total of 60 adult patients ages 1840 years with the complaints of impacted third molar teeth were included in the study.resultsthe postoperative swelling ratios (p<0.05) and pain scores (p<0.05) were significantly better in both study groups than in the control group and there was no statistically significant difference between methylprednisolone and tenoxicam groups with regards to the edema and pain relief.conclusionspreoperative administration of 80 mg methylprednisolone achieves better control of trismus than tenoxicam without any significant differences in edema and pain control in impacted third molar teeth extraction. |
the practice of clinical laboratory medicine requires that test results be assessed to determine if the patient is normal or is subject to some pathological condition. thus it s imperative that laboratories report test results along with reference intervals, in the past, typically called normal ranges. establishing reference intervals has always been a challenge as significant differences may exist in disease frequencies ; biological variation in analytes among ethnic groups, genders and ages ; specimen collection techniques ; test performance ; test interpretation ; and other factors. physicians rely on the availability of appropriate and reliable reference intervals to accurately interpret laboratory test results combined with data collected during medical interview and clinical examination. although health professionals understand the importance of reference intervals, many laboratories still do not have comprehensive data, especially ranges that are specific for their typical patient populations. there continues to be significant gaps in the available reference intervals as frequently intervals cited in the literature were obtained using older methodologies and instrumentation and cover a limited range of age groups or a relatively small number of samples. ferruccio ceriotti, a former chair of the ifcc committee for reference intervals and decision limits (c - ridl), noted that the theory of reference values was developed more than 30 years ago, but its application in most clinical laboratories is still incomplete today. and that this is due to the fact that obtaining a good reference interval is a very demanding activity, in terms of time, money and knowledge (1). cerriotti also noted that the time, effort and money required to establish reference intervals are large and clinical laboratories are disinclined to modify reference intervals as this is a demanding task also requiring education of clinicians and patients. large multicentre studies are needed to make real progress in this field and bridge the large gap now existing between a very nice theory (ifcc and clsi documents) and a very poor practice. between 1987 1991, the ifcc published a series of 6 seminal papers providing detailed guidance for establishing reference intervals (2 - 7). the clinical and laboratory standards institute (clsi, previously nccls) first published the c28 guideline (defining, esishing, and verifying reference intervals in the clinical laboratory) in 1995 and the current edition of the guideline, c28-a3, in 2008(8) the c28-a3 is a joint clsi / ifcc document and incorporates the series of ifcc papers. direct approach in which reference individuals are vetted to ensure that they are healthy and discourages the indirect approach in which existing data is used to establish ranges by retroactively identifying acceptable reference populations. friedberg. noted that a college of american pathologists (cap) survey of 500 laboratories in 2001 found that 390 (78%) adopted manufacturers values for reference intervals (9). in their own survey of 163 laboratories, of those laboratories, about one half tested between 21 50 samples and one quarter tested more than 100 samples. it is not clear how many of these labs followed an accepted guideline such as c28. we report here reference intervals established for hong kong (chinese) blood donors for twenty - five routine clinical chemistry analytes. all tests were performed following the manufacturer s package inserts for abbott reagents on the abbott architect c16000 analyzer (abbott diagnostics, chicago, il, usa). calibrator traceability and measurement uncertainty information for all assays were provided by the manufacturer (i.e., traceability to reference materials and/or methods as per iso 17511). they represented routine clinical chemistry assays commonly performed at the pamela youde nethersole eastern hospital (pyneh). the purpose of this work was to develop reference intervals for these key analytes that reflected the hospital s patient population after the architect became the test of record analytical system. hong kong blood donors, representing the local chinese population, who passed the routine red cross blood donation screening criteria, were invited to participate in the reference interval study. the intent was to collect samples from about 300 female and 300 male donors. donors signed a consent form allowing an extra blood sample to be drawn at the time of donation at the hong kong red cross blood transfusion service. the consent form read : the chemical pathology laboratory of pamela youde nethersole eastern hospital has recently changed its analyzers and has to re - establish the reference range for reporting purposes. blood samples from the normal population have to be collected in sufficiently large numbers for testing by the new analyzers in order to establish the reference ranges and your help by contributing a few cc or our blood would be highly appreciated. five milliliters of blood were collected into beckton - dickinson serum separator gel separator tubes (sst ii). the blood samples were first collected in the empty sampling pouch of the blood bag and then drawn into the gel tube through a luer adaptor. tubes were labeled with the red cross identification number and the donor information included date of birth, gender, ethnicity (chinese or non - chinese) and the date of blood collection. the samples were collected over a period of twelve days, with 39 89 donors recruited per day. a messenger received the samples from the red cross at about 4 pm and delivered them to pyneh in a specimen transportation box with an ice pack on the same day, arriving at the laboratory of pyneh at about 5 pm. a total of 687 samples, 378 male and 309 female, were collected, with ages ranging between 16 62, and were labeled with pyneh barcode labels after receipt. the samples were centrifuged, decapped, and assayed on the architect c16000 on the same day as sample collection. any excess samples were aliquoted and stored at 50 c. the data was analyzed using the rhoads ep evaluator (david g. rhoads associates, south burlington, vermont) software program. the reference intervals represent the central 95% range of the data set from a reference population consisting of apparently healthy individuals. it is intended to estimate intervals : (1) for endogenous analytes and not exogenous analytes (e.g., drugs), (2) for the patient population for which the intervals apply, (3) for a cohort of at least 120 reference specimens. the nonparametric method was chosen to calculate the reference intervals as it makes no assumptions about the shape of the population distribution. although over 300 samples for both genders were available for each analyte, the age range was large and intervals can vary with age and lifestyle and the nonparametric approach is a conservative method. the confidence interval is a measure of the precision of the interval and indicates that for a repeat study using a different reference population, 90% of the values should fall within the ci. the confidence ratio is the ratio of the average ci width to the reference interval width. a value of 0.10 or less is desirable and values over 0.30 are indicated in table 2. the confidence ratio is dependent on the sample size and improves with a larger number of samples. all tests were performed following the manufacturer s package inserts for abbott reagents on the abbott architect c16000 analyzer (abbott diagnostics, chicago, il, usa). calibrator traceability and measurement uncertainty information for all assays were provided by the manufacturer (i.e., traceability to reference materials and/or methods as per iso 17511). they represented routine clinical chemistry assays commonly performed at the pamela youde nethersole eastern hospital (pyneh). the purpose of this work was to develop reference intervals for these key analytes that reflected the hospital s patient population after the architect became the test of record analytical system. hong kong blood donors, representing the local chinese population, who passed the routine red cross blood donation screening criteria, were invited to participate in the reference interval study. donors signed a consent form allowing an extra blood sample to be drawn at the time of donation at the hong kong red cross blood transfusion service. the consent form read : the chemical pathology laboratory of pamela youde nethersole eastern hospital has recently changed its analyzers and has to re - establish the reference range for reporting purposes. blood samples from the normal population have to be collected in sufficiently large numbers for testing by the new analyzers in order to establish the reference ranges and your help by contributing a few cc or our blood would be highly appreciated. five milliliters of blood were collected into beckton - dickinson serum separator gel separator tubes (sst ii). the blood samples were first collected in the empty sampling pouch of the blood bag and then drawn into the gel tube through a luer adaptor. tubes were labeled with the red cross identification number and the donor information included date of birth, gender, ethnicity (chinese or non - chinese) and the date of blood collection. the samples were collected over a period of twelve days, with 39 89 donors recruited per day. a messenger received the samples from the red cross at about 4 pm and delivered them to pyneh in a specimen transportation box with an ice pack on the same day, arriving at the laboratory of pyneh at about 5 pm. a total of 687 samples, 378 male and 309 female, were collected, with ages ranging between 16 62, and were labeled with pyneh barcode labels after receipt. the samples were centrifuged, decapped, and assayed on the architect c16000 on the same day as sample collection. the data was analyzed using the rhoads ep evaluator (david g. rhoads associates, south burlington, vermont) software program. the reference intervals represent the central 95% range of the data set from a reference population consisting of apparently healthy individuals. it is intended to estimate intervals : (1) for endogenous analytes and not exogenous analytes (e.g., drugs), (2) for the patient population for which the intervals apply, (3) for a cohort of at least 120 reference specimens. the nonparametric method was chosen to calculate the reference intervals as it makes no assumptions about the shape of the population distribution. although over 300 samples for both genders were available for each analyte, the age range was large and intervals can vary with age and lifestyle and the nonparametric approach is a conservative method. the confidence interval is a measure of the precision of the interval and indicates that for a repeat study using a different reference population, 90% of the values should fall within the ci. the confidence ratio is the ratio of the average ci width to the reference interval width. a value of 0.10 or less is desirable and values over 0.30 are indicated in table 2. the confidence ratio is dependent on the sample size and improves with a larger number of samples. we have chosen to use gender specific intervals or the combined gender intervals as indicated in the table. the decision to adopt combined gender reference intervals is based on practical clinical considerations. using albumin as an example g / l, the female interval is 37.6 48.6 g / l, and the combined interval is 37.9 48.9 g / l. while there may be a statistically significant difference between the male and female intervals, they are both so close to the combined interval that it is deemed to not to be a clinically significant difference. using the electrolytes, na, k, and cl, as another example, the tight physiological control of these key analytes predicts that minimal gender difference would be observed. the observed data confirm that assumption, intervals differing by 1 (one) mmol / l or less and the gender combined intervals are therefore adopted. other analytes predictably demonstrate gender specific differences that are great enough to warrant maintaining separate gender intervals. examples of these analytes include ck and creatinine for which the difference in muscle mass between males and females results in both statistically and clinically significant differences. a third category of analytes are those for which reference intervals are typically used in clinical practice. this applies to cholesterol, d - hdl, and triglycerides for which the ncep clinical cutoffs for assessing cardiovascular disease risk are used (10) although the standard ncep medical decision levels are used when reporting the results for these lipid assays, it is nevertheless instructive to know the distribution of these lipids in the local population. while the need for up to date, relevant reference intervals is undeniable, the task of generating them is daunting. pertinent issues have been well reviewed by horn and pesce, who recognize the clsi c28 guideline as a good source document (11). c28 recommends the direct method, either a priori or a posteriori, meaning that the reference individuals are pre - selected and judged to be normal, or otherwise meet the desired criteria for inclusion in the study. the indirect method, in which existing data is used to determine reference intervals, variously known as data mining or the hoffman approach, can be useful as an alternative, although debate about it s limitations continues in the current literature (12,13). an interesting paper by seccombe describes application of the indirect approach for five analytes and reports intervals for them that are remarkably similar to intervals previously established using the direct approach (14). limitations of this technique include the need for very large numbers of observations from multiple laboratories that are all using similar instrumentation and assay methods, i.e., an analytically standardized peer group. while the indirect approach may be used successfully, as determined by intervals that are essentially equivalent to those based on the direct approach, the validity of the indirect intervals may be questioned unless the intervals from a proper direct approach study are available for comparison. several recent pediatric reference interval studies using the direct approach and following c28 have been published, two of them having used the architect c8000 analyzers and abbott reagents, as in the work reported here (15 - 17). we were able to cooperate with the local red cross to secure a large number of samples from healthy males and females representative of the indigenous chinese ethnic population in hong kong. we used statistical analysis based on a well recognized guideline for determining reference intervals, clsi / ifcc c28, to develop reference intervals for twenty - five common clinical chemistry analytes. we ve adopted the intervals for result reporting purposes and believe that they are appropriate for the patient population served by the pamela youde nethersole eastern hospital in hong kong. another reference interval study for the hong kong population has recently been reported (18). about half the number of samples (335 vs. 687) and consisted of 138 males and 197 females. the reference intervals were drawn from the staff of the queen elizabeth hospital, hong kong, and their families, relatives, and friends. subjects were excluded if glucose, cholesterol, and triglycerides were elevated and fasting specimens were collected. the intervals were general similar to those reported here except for phosphorus and alkaline phosphatase. another recent asian study included samples from cities in japan, korea, taiwan, indonesia, and hong kong.19 a total of 580 individuals, 279 males and 301 females, were tested, including 46 men and 74 women from hong kong ranging in age from 20 62. twenty two clinical chemistry analytes (electrolytes, metabolites, enzymes, proteins) were tested using an hitachi 7170 analyzer and a variety of different different methodologies. because the data was pooled, it s difficult to directly compare the intervals from this work with those reported here for the hong kong population. unexpectedly large between - city differences were noted for ld, total protein, globulin, na, k, cl, bun, and phosphorus. this work was not intended to define reference intervals to demonstrate any differences in analyte intervals between samples from the six asian cities included. there are limitations of this work, hardly surprising given the inherent difficulties in conducting a full scale reference interval study. blood donors underwent a screening process that included a limited medical history and cursory physical exam (e.g., blood pressure, hemoglobin, temperature) and had to meet certain predetermined criteria. it s reasonable to assume that all of the subjects were healthy, at least healthy enough to donate a unit of blood. normal in the sense of being free of pathological conditions that could affect the values of various analytes measured in this study. more extensive vetting of the subjects for inclusion in the study would have been preferable, but the approach taken was practical. the age range of the subjects was large, 16 62 years of age. it s quite possible that the reference intervals for some the analytes study vary by age. combining the data for this wide age range and using only gender as a partitioning factor however, to apply age as another partitioning factor, it would have been necessary to collect many more reference specimens, theoretically, a minimum of 120 for each age category and gender. the protocol did not strictly follow the clsi / ifcc c28 guideline, which would have been a daunting task, but attempted to conform to c28 as much as practical. the work reported here allowed the pamela youde nethersole eastern hospital to generate current reference intervals for the typical local patient population for a large number of routine analytes using a new analytical system (instrumentation and reagents). these intervals were more appropriate than literature citations or the intervals listed in manufacturer s package inserts. while the ranges reported here are by no means definitive, they represent a reasonably good start. a future study, or even a series of studies, using the same protocol should be manageable and the results could be used to assess the appropriateness of the original intervals. if appropriate, additional data might be pooled with the current numbers, allowing for a more statistically robust estimate of the intervals and potentially allowing for the application of more partitioning factors. | backgrounddefining reference intervals is a major challenge because of the difficulty in recruiting volunteers to participate and testing samples from a significant number of healthy reference individuals. historical literature citation intervals are often suboptimal because they re be based on obsolete methods and/or only a small number of poorly defined reference samples.methodsblood donors in hong kong gave permission for additional blood to be collected for reference interval testing. the samples were tested for twenty - five routine analytes on the abbott architect clinical chemistry system. results were analyzed using the rhoads ep evaluator software program, which is based on the clsi / ifcc c28-a guideline, and defines the reference interval as the 95% central range.resultsmethod specific reference intervals were established for twenty - five common clinical chemistry analytes for a chinese ethnic population. the intervals were defined for each gender separately and for genders combined. gender specific or combined gender intervals were adapted as appropriate for each analyte.conclusiona large number of healthy, apparently normal blood donors from a local ethnic population were tested to provide current reference intervals for a new clinical chemistry system. intervals were determined following an accepted international guideline. laboratories using the same or similar methodologies may adapt these intervals if deemed validated and deemed suitable for their patient population. laboratories using different methodologies may be able to successfully adapt the intervals for their facilities using the reference interval transference technique based on a method comparison study. |
symptomatic irreversible pulpitis is a pulpal state characterized by mild / severe pain that lingers after removal of a stimulus. in such cases, pulpectomy / extraction is required to alleviate the symptoms and prevent apical periodontitis. however, as the severity of pulpal inflammation can not be measured quantitatively, it must be based on clinical findings rather than histological diagnosis ; moreover, there is poor correlation between the two. in addition, pulps of teeth clinically diagnosed with irreversible pulpitis were shown to have the potential to heal after pulpotomy with appropriate biomaterials such as mineral trioxide aggregate (mta) and calcium enriched mixture (cem) cement.[37 ] a systematic review revealed that to reduce the risk of carious pulp exposure, partial caries removal (indirect pulp therapy [ipt ]) can result in long - term success compared with complete caries removal in the deep lesion. a clinical trial demonstrated high survival rate of permanent teeth when deep carious lesions were managed without exposing the pulp by placing an indirect pulp cap. these results demonstrated that the teeth pulps remained symptomless and maintained their healing potential and defensive capacity after treatment. elimination of the bulk of the infected dentin and sealing the remaining carious lesion from oral fluids with appropriate materials is an accepted treatment for badly decayed teeth. in addition, performance of ipt is simpler / more economical than rct. cem cement has been introduced as a water - based and tooth - colored endodontic biomaterial. after mixing with liquid, the ph of cem increases to > 10. it is assumed that in a high ph environment the calcium ions that are released from cem react with endogenous phosphates to form hydroxyapatite ; this would explain the favorable sealing ability / biocompatibility of the cement. an in vitro study revealed that the anti - bacterial properties of cem are similar to calcium hydroxide. cem has been employed as a root - end filling material, as well as direct pulp capping agent, pulpotomy agent, apical plug, in the repair of furcal perforations, management of root resorption, and regenerative endodontics. recently, an interesting report demonstrated favorable treatment outcomes of direct pulp capping with cem for management of a permanent mature molar with irreversible pulpitis and associated apical periodontitis. the following report discusses the first case of successful management of a symptomatic permanent tooth with irreversible pulpitis and associated apical periodontitis using ipt with cem, followed by a sandwich glass - ionomer composite restoration. a 12-year - old female with a symptomatic first left lower molar was observed at a private clinic. diagnostic tests were performed ; the involved tooth responded with mild pain to percussion and severe lingering pain to endo - frost cold spray (roeko ; coltene whaledent, langenau, germany). radiographic examination showed a large carious lesion associated with apical periodontitis [figure 1 ]. the adjacent / opposing teeth were asymptomatic and responded normally to all diagnostic tests. based on the clinical and radiographic examinations, the diagnosis of irreversible pulpitis with apical periodontitis was made. thereafter, ipt of the tooth was decided ; an informed consent was obtained from the patient 's legal guardian. intraoral pre - operative radiograph showing well - defined radiolucency of mesial and distal roots of symptomatic first lower left molar the patient was instructed to rinse her mouth with 0.2% chlorhexidine. the tooth was anesthetized with 2% lidocaine with 1:80,000 epinephrine (daroupakhsh, tehran, iran) and then isolated with rubber dam. the bulk of soft carious dentin was carefully removed by tungsten - carbide round burs (komet, lemgo, germany) at low speed ; medium and large spoon excavators (ash, london, uk) were also used. after that, the remaining carious dentin in pulpal floor was covered by 1.5 mm layer of cem cement (bioniquedent, tehran, iran). the remaining tooth cavity was then restored / sealed with glass - ionomer cement and composite resin sandwich technique [figure 2 ]. post - operative orthopantomogram (opg) showing threelayers ofrestorationconsistofindirectpulptherapy / calcium enriched mixture, glass ionomer and composite resin. left : higher magnification of treated first lower left molar revealing well defined periapical radiolucency of both roots the patient was re - examined clinically after 1 and 7 days. at the 1-day follow - up the patient complained of sensitivity to cold which had significantly decreased 1 week post - operatively. patient was recalled at 1 year for clinical / radiographic follow - up. clinical examination with cold test showed a vital tooth which was functional, asymptomatic, with normal physiologic mobility, normal probing depths, and a satisfactory coronal restoration. radiographic examinations showed normal periodontium, and evidence of periapical healing [figures 3 and 4 ]. six - month follow - up radiograph showing healing process of apical pathology one - year follow - up radiograph showing complete periapical healing this study conducted ipt with cem cement for a pulp with sign / symptoms of irreversible pulpits along with apical periodontitis. though, apical periodontitis seemed evident on the radiograph, the pulp was still vital. the general consensus in cases where irreversibly inflamed pulp has been diagnosed, is that the pulp no longer has the potential to heal and will not survive with any form of treatment. however, it should be considered that irreversible pulpitis is a clinical term for the classification of pulpal diseases ; when treating the deep carious lesion clinicians have to diagnose irreversible pulpitis on the basis of indirect diagnostic methods. there is little evidence to correlate the clinical signs / symptoms with histological feature of the diseased pulp. therefore, dentists are trained to diagnose irreversible pulpitis when patient complains of pain lasting for a few minutes to several hours, pain exacerbating with hot / cold fluids, and radiating pain ; as well as tender to percussion. however, there is no information to indicate which symptom is an indication that the pulp no longer can repair itself. conversely, recent reports have demonstrated that teeth with irreversible pulpitis showed favorable treatment outcomes with pulpotomy using cem / mta as pulp cappings.[357 ] though the diseased pulps in these cases were inflamed, they were still vital ; that is they still maintained a blood supply and therefore the most important factor for healing. in the light of recent high - level evidences, the inflammatory process should be re - examined to recognize its possible or probable positive effect on pulpal healing. our clinical / radiographic follow - ups also showed favorable treatment outcomes for ipt / cem. these results reveal that severe local inflammation in pulpal tissue may heal and be repaired if the irritant is removed and the pulp is well protected from further irritants. if further studies support this, there may be a need for reclassification of dental pulp diseases. a growing body of evidence has demonstrated that ipt is successful for the management of deep caries lesion in primary molars as well as two - stage technique in immature permanent teeth ; however, some recent studies have shown similar results in mature permanent teeth. ipt reduces the risk of carious pulp exposure, diminishes the substrate for microorganisms, prevents lesion development and promotes a physiological reaction in the pulp - dentin complex. several studies have demonstrated that once cariogenic bacteria are isolated from their nutritional supply by an adequate coronal seal, they either perish or become inactive. furthermore, once a seal is applied to carious dentin, it becomes dry / hard, arresting the carious process after ipt with a one - visit method. accordingly, the second appointment may not be needed, if the coronal restoration preserves the seal of cavity. our 1-year clinical follow - up showed that the one - visit ipt coronal filling was satisfactory, and therefore a second appointment was not scheduled. several dental materials have been suggested for capping the remaining carious lesion in ipt, that is calcium hydroxide, polycarboxylate cement combined with tannin - fluoride preparation, zinc - oxide eugenol cements, resin - bonded composite, glass ionomer cements and copper phosphate cement. the ideal capping material should be anti - bacterial, non - toxic, dimensionally stable, biocompatible, and create a hermetic seal as well as be able to induce regeneration of the pulp. considerable data has shown favorable properties of cem cement namely ; anti - bacterial properties, sealing ability, dimensional stability and biocompatibility when compared with other gold standard materials such as calcium hydroxide or mta. the favorable results in this case study may be partly attributed to the patient 's age. a young patient 's tooth may have slightly open apices and the dental pulp may respond more positively to pulp capping than a mature pulp. based on our clinical and radiographic observations, we can conclude that despite apparent pulpal and periradicular inflammation from the deep caries lesion, a conservative ipt can generate pulpal repair and periradicular healing in a mature permanent molar tooth. in addition, ipt / cem of deep caries lesion in young patients is an easier, more practical and valuable treatment plan than complete caries removal which may result in pulp exposure and root canal treatment. however, well - conducted clinical trials on this proposed technique will be required for more grounding clinical evidence. | dental pulp has the ability of repair / regeneration. indirect pulp therapy (ipt) is recommended for pulp preservation in asymptomatic teeth with extremely deep caries as well as teeth with clinical symptoms of reversible pulpitis. in this case study, we performed ipt with calcium enriched mixture (cem) cement on a symptomatic permanent molar. after clinical / radiographic examinations the tooth was diagnosed with irreversible pulpitis and associated apical periodontitis. ipt involved partial caries removal, the placement of cem cement pulp cap and overlying adhesive permanent restoration. at the 1 week follow - up, patient 's spontaneous symptoms had resolved. one - year follow - up demonstrated pulp vitality, clinical function, as well as the absence of pain / tenderness to percussion / palpation / cold sensitivity tests ; periapical radiograph showed a healing periradicular lesion with newly formed bone, that is normal pulp with normal periodontium. these favorable results indicate that ipt / cem may be a good treatment option in comparison to endodontic treatment in young patients. ipt of deep - caries lesion is an easier, more practical and valuable treatment plan than complete caries removal. |
the child who chronically breathes through the mouth might also have difficulties with functional capacity, compromised respiratory system, damaging lung ventilation with lower thoracic expansibility and, consequently, may develop a weakness of the respiratory muscles.1 2 3 4 5 currently researchers and clinicians are increasingly seeking methods of instrumental evaluation, especially with the goal to gather data complementary to clinical evaluations because it is necessary to evaluate the breathing muscles and consequences of those respiratory changes in the mouth breathing child.6 because the literature points to a physiologic relationship between mouth breathing standard and respiratory muscle strength and because there are few studies that evaluate the respiratory muscle strength in mouth breathers, the present study had the objective to develop a review to investigate studies that used the methods of evaluation of muscle strength in mouth breathers. the review was made in order to gather the clinical evidence available in the literature to answer the clinical question. first, the research question was formulated, considering the goal of the review in this study : what are the evaluation methods of respiratory muscle strength in mouth breathers ? next, a systematic search was made in medline databases via pubmed, latin american literature in the health sciences (lilacs), and scientific electronic library online (scielo), between november 2011 and october 2012, and included papers and dissertations covering the evaluation methods of rms in inspiration and expiration during mouth breathing defining the eligibility criteria. the keywords used for search followed the description of decs / mesh terms, being : muscle strength, mouth breathing, evaluation studies. the combinations between those words were made in each database mentioned above, using the boolean operator and, without language restriction (table 1). levels of evidence.7 studies with the major strength of evidence are in the first position in the classification. as for the level of scientific evidence, studies with the major strength of evidence are in the first position in the classification with a score of 1 and the lower strength with a score of 8.7 due to the lack of studies with evidence level 1, 2, 3, and 6, publications with evidence level 4 and 5 were selected for this review (table 1). the ones selected according to the inclusion criteria had their methodologic quality evaluated by the following classification : random allocation, blind subjects, blind therapists, control groups, statistical analysis, and statistical comparison between the selected study groups (table 2). the selected studies had their methodologic quality evaluated by two independent reviewers (r.a. and d.c.) and the differences were discussed with a third reviewer (h.j.). currently, there are still few systematic reviews with meta - analysis available in physiotherapy and other health fields. the reasons that hamper the execution of this kind of study include the use of different research protocols and variations in methodologic quality.8 in principle, all studies were identified by the electronic search in the computer screen. then, the studies were subsequently analyzed and the publications that addressed the evaluation methods of respiratory muscle strength in mouth breathers were included in the revision (one case control study and two case studies). among the 13 publications initially selected in databases, 10 were excluded. among the excluded ones, eight papers had no relation with the main subject according to their title and abstract and two were repeated (fig. 1). search and selection of studies for revision according to prisma (preferred reporting items for systematic reviews and meta - analyses).24 abbreviation : lilacs, latin american literature in the health sciences. to have a better presentation of results, the following were considered in the selected publications : author / year, country, sample, age average in years, methods, and equipments for evaluation of the respiratory muscle strength and results (table 3). abbreviations : mb, mouth breathing ; nb, nasal breathing ; pemax, maximum expiratory pressure ; pimax, maximum inspiratory pressure ; rb, respiratory biofeedback. the three included studies had as population children between 6 and 13 years of age with and without mouth breathing.9 10 11 the studies evaluated the respiratory muscle strength in mouth breathing children through the measurements of maximum static respiratory pressures (inspiratory and expiratory) by a manovacuometer. those studies included 144 children of both sexes : 82 with mouth breathing and 62 with nasal breathing (control group). a control group was included only in the study of okuro.9 of them, 32 children were evaluated pre- and post - adenotonsillectomy because they had enlarged tonsils, 20 were evaluated pre- and posttreatment using biofeedback, and 92 had postural assessment and exercise (table 3). regarding the methodologic quality, all studies had the inclusion criteria and statistical analysis, but no one had random allocation and blind subjects. from the three publications, only the okuro study had blind therapists and a control group.9 the three studies made a statistical comparison between the mouth breathers subgroups (table 2). one fact is that the authors were unanimous with relation to the manovacuometry method as a means to evaluate the breathing pressures pre- and posttreatment and pre- and postsurgery used to evaluate respiratory muscle strength in children with mouth breathing. the studies were not analyzed for efficacy of both method and equipment used in mouth breathing children (table 3). currently, the evaluation methods of respiratory muscle strength are becoming more important because the three studies included were made from 2007, which evaluated the breathing muscles in mouth breathers in order to detect or not the improvement before and after some intervention. this clinical importance of evaluating the breathing muscles was proposed in previous publications.12 13 it is observed that brazil was predominant in the three included studies because they were the only one that focused in the theme of this revision, due to the search by keywords and the inclusion criteria. this discovery may be explained because the manovacuometer equipment is made in brazil and is standardized and certified by the national institute of metrology, standardization and industrial quality, besides its commercialization, acquisition, and maintenance being more easily accomplished in brazilian territory. from this, the brazilian scientists started doing several researches about mouth breathing, respiratory muscle strength, posture, and their relationships. however, there is still a lack of studies correlating mouth breathing and respiratory muscle strength. some studies14 15 16 17 found in previous searches focused on changes of body posture or on association with the respiratory function in individuals with mouth breathing, given that musculoskeletal changes are the more easily identified signals. it was identified as a prevalent variation between 20 and 32 children with mouth breathing (911), and this compromises the reproducibility of those findings for the general population because of the reduced number of individuals present on studies. in the publications selected for this review, the age of analyzed subjects was between 7 and 13 years, and according to the world health organization,18 the age group from 7 to 12 years old is defined as prepubertal and was established because this period is considered of transition in breathing system development and marks the end of rapid growth and structural changes of the periphery breathing units.19 there was clinical homogeneity among the studies favoring the reliable evaluation of breathing pressures in the population set by the authors by manovacuometry. this measurement technique used by the included studies is widely found in the literature because there is a consensus in relation to the ideal method of manovacuometry to evaluate this maximum respiratory pressures (pimax and pemax).12 these pressures are measured in the mouth using a manovacuometer. the higher values exclude significantly clinical weakness of the breathing muscles.12 measurements are useful for differentiation between a neuromuscular weakness of abdominal muscles and a specific weakness of the diaphragm or others inspiratory muscles.20 21 22 furthermore, it is necessary to evaluate the respiratory muscles and the consequences of this respiratory change in the mouth breathing child.6 the child that chronically breathes through the mouth may develop changes in the respiratory system,1 2 impairing lung ventilation,22 demanding less strength from the breathing muscles, which would lead to muscle weakness and lower chest expansion, sagging, and abdominal protrusion.3 23 moreover, it is possible to say that when there is a treatment with attention focused on respiratory muscle strength, there is significant increase in pemax and pimax values, improving this strength and all respiratory mechanics in the mouth breathing. given the few studies found for this review, it is noted that the lack of the evaluation of the respiratory muscle strength interferes in the diagnosis of some of this strength deficit, as well as in the definition of the treatment and in the revaluation process of respiratory strength to evidence if this treatment is effective or not. in clinical practice, the delay to detect the diagnosis of mouth breathing and the unimportance of this clinical condition may lead to few scientific studies focused on respiratory muscle strength of the mouth breather being performed. thus, it is expected that further studies aimed at the methods used to evaluate respiratory muscle strength in mouth breathing will be performed, as studies correlating results of manovacuometry with data collected from other instruments for this evaluation, because there are many gaps to be filled in correlation studies of diagnoses in mouth breathing. despite manovacuometry being used in clinical practice to evaluate the respiratory muscle strength in individuals with and without mouth breathing, it was found in this review that there is not yet enough evidence to support the recommendation of this technique. the low methodological rigor of the available studies hinders a truthful and reliable result to support or refute the use of manovacuometry. finally, it was observed that there are few studies evaluating respiratory muscle strength in mouth breathing people, through manovacuometry, suggesting that new research needs to be performed. | introduction the child who chronically breathes through the mouth may develop a weakness of the respiratory muscles. researchers and clinical are seeking for methods of instrumental evaluation to gather complementary data to clinical evaluations. with this in mind, it is important to evaluate breathing muscles in the child with mouth breathing. objective to develop a review to investigate studies that used evaluation methods of respiratory muscle strength in mouth breathers. data synthesis the authors were unanimous in relation to manovacuometry method as a way to evaluate respiratory pressures in mouth breathing children. two of them performed with an analog manovacuometer and the other one, digital. the studies were not evaluated with regard to the method efficacy neither the used instruments. conclusion there are few studies evaluating respiratory muscle strength in mouth breathing people through manovacuometry and the low methodological rigor of the analyzed studies hindered a reliable result to support or refuse the use of this technique. |
as discussed by both citron and nerem and also bergman and nerem, the medical device industry evolved in the second half of the 20 century. first, there was kidney dialysis, a technology accredited to the pioneering work of a dutch physician, willem kolff. beyond the treatment of chronic kidney failure, kolff 's technologies formed the foundation to the membrane oxygenator. in turn, this led to cardiopulmonary bypass being performed safely for extended periods of time. there followed the development of prosthetic heart valves with the first implant being performed in 1952. clinically - practical electrical stimulation therapies for cardiac rhythm disorders began to emerge in the 1950s. in 1958, c. walton lillehei collaborated with earl bakken, an electrical engineer, in the use of the world 's first transistorized battery - powered cardiac pacemaker that was externally powered. however, a few years later, in 1960, william chardack implanted the first pacemaker that was completely internal, i.e. within the body. this was possible because of the mercury / zinc battery that had been designed by wilson greatbach, an engineer who passed away in 2011. this industry grew in the last 50 years to become a major industry with $ 200 billion in annual sales, with a workforce of 300,000, and an industry that invests seven percent of revenues back into r&d. bioengineering in universities has developed in parallel to the development of the medical device / implant industry. back in the 1950s and 1960s, bioengineering involved the application of the traditional engineering disciplines to problems in medicine and biology. however, this all began to change in the 1970s with the establishment of academic departments, called either biomedical engineering or bioengineering. this development grew slowly until the 1990s when there was an acceleration in the formation of such academic units. this was due to many factors ; chief among these was the impact of the biological revolution and the significant investments made by the whitaker foundation. worldwide, there are now more than 100 departments of this type, and the field of biomedical engineering / bioengineering is now recognized as being its own academic discipline, an engineering discipline based on the science of biology. cardiovascular research and orthopaedic research have been two major areas of focus in the evolving discipline of bioengineering. each section focuses on a certain area of cardiovascular research in which this author has been personally involved. an important research area in the historical development of bioengineering is the role of hemodynamics in the disease atherosclerosis, particularly in the early atherogenic stage of the disease. in the 1960s, there was already evidence that the pattern of atherogenesis appeared to correlate with the pattern of blood flow. as studies developed, it became clear that there was greater predilection of the disease in low - shear stress regions. the vascular endothelium, which was in contact with the flowing blood and the associated shear stress environment, became a focus on the effects of the hemodynamic environment on vascular endothelial biology. these studies included the effects of flow, the effects of cyclic stress and in some cases even pressure ; however, the major focus was on the study of the influence of flow and the associated shear stress environment. much of this research over the years has been done in vitro with cultured vascular endothelial cells exposed to a variety of flow environments either using a parallel - plate flow chamber or a cone - plate device. the flow environments studied have included steady laminar flow, a purely oscillating flow, and pulsatile type flows, either with a reversing or a non - reversing waveform. although a key indicator of the influence of flow is the change in morphology, as illustrated in figure 1, and the reorganization of the cytoskeletal network, there are also changes in gene expression and protein expression. for a flow environment where there is a non - zero mean flow component, vascular endothelial cells elongate and align their major axis parallel to the direction of flow. there is reorganization of f - actin, such that the fibers also are aligned with the flow ; and if the actin cytoskeleton is disrupted, then the vascular endothelial cells do not elongate and align. this thus indicates that the change in morphology is due to a reorganization of the f - actin. interestingly, there is no morphological change (no elongation nor alignment of the vascular endothelial cells) for a purely oscillatory flow. with respect to gene expression, obviously, much has been learned about vascular endothelial biology from these in vitro studies and later confirmed with in vivo studies using a variety of animal models. interestingly, some life scientists studying atherosclerosis in the 1970s did not believe that the physical forces associated with the hemodynamic environment could have any influence at all on cell behavior and the cellular processes involved in the initiation of the disease. in fact, it now is clear that the function of a cell is determined by the signals associated with the microenvironment in which the cell resides. this symphony of signals, what i call nature 's orchestra, is made up of the soluble molecules to which the cell is exposed, the other cells which are in contact, the substrate to which it is adhered (extracellular matrix and/or some type of synthetic material), and the mechanical environment in which the cell resides, i.e. the physical forces to which it is exposed. one may speculate that the reason much was learned about vascular endothelial function from in vitro studies was because these monolayer studies mimicked the fact that the vascular endothelium is a monolayer in vivo. as the environment in cell culture is not physiologic, there have been efforts to engineer a more physiologic in vitro environment. this has included using a co - culture of vascular endothelial cells with vascular smooth muscle cells. it is now clear that smooth muscle cells in a three - dimensional environment have different characteristics than such cells in a two - dimensional environment. even with the advances made, much due to the involvement of engineers in the study of vascular biology, there is more to be done. this includes continuing to develop in vitro models that are more physiologic and better at simulating in vivo environments. it should also be noted that, although there were many engineers who learned biology to be leaders in this field, there were also life scientists who became more like engineers in their approach in the study of vascular biology. thus, the bioengineering community that emerged from studies in this area has been very much an interdisciplinary one, and this provided the foundation for the emergence of bioengineering as a discipline in its own right, a discipline based on the science of biology, a discipline in which biology and engineering are very much integrated. heart valves engineering is another area, in which engineers have been involved and which has fostered the growth of bioengineering. initially, much of the engineering effort was focused on the development of improved prosthetic valves to be used as defective valve replacements. this certainly was important to the emerging medical device / implant industry in the 1970s and 1980s, and it was an area in which university researchers became very active. a key issue was to optimize the fluid mechanical characteristics of such heart valves implants. engineers have begun to make a contribution to the area of heart valves engineering in another way, in order to learn more about the basic biology of heart valves, a tissue that resides in an extremely dynamic mechanical environment. our knowledge about the biology of blood vessels has increased considerably over the last few decades ; this was largely driven by studies aimed at achieving a better understanding of the biology and pathobiology associated with atherosclerosis. although there were clinical problems associated with heart valves, there seemingly was not satisfactory motivation to study the basic biology of heart valves. several bioengineering laboratories are participating in this, and the area of heart valves has became an important part of bioengineering research. one example is the heart valve team from georgia tech and emory university school of medicine in atlanta, georgia. this team includes the laboratories of professors hanjoong jo and ajit yoganathan, and robert m. nerem. an issue being investigated is whether there is any difference between endothelial cells on the different sides of the aortic valve leaflets, i.e. the ventricularis side as compared to the fibrosa side. whereas the hemodynamic environment on the ventricularis side may be characterized as a unidirectional, time varying laminar flow, on the fibrosa side it is a reversing pulsatile flow (figure 2). if so, are these differences due to the very different hemodynamic environments on each side, or alternatively due to fundamental differences between the endothelial cells ? is the difference between the endothelial cells on the two sides genetic or is it environmental ? we found over 700 genes downregulated and over 300 genes upregulated by oscillatory flow as compared to steady laminar flow. however, no significant difference has been found when fibrosa side and ventricularis side endothelial cells are exposed to the same shear stress conditions. there is no side - dependency and no apparent difference other than differences in their respective hemodynamic environment. also, mirna array analysis yielded 30 shear - sensitive mirnas and three side - specific mirnas. moreover, mirna validation confirmed four of 17 shear - sensitive and one of three side - dependent mirnas. although there is clearly much more to do, we are slowly beginning to better understand the biology of heart valves. the above is only an example ; however, the biology of heart valves including their biomechanical properties has become a major topic with several sessions at virtually every bioengineering conference. it thus has become very much a part of the evolution of bioengineering as a field and the intertwining of this field with cardiovascular research. tissue engineering is another research area where bioengineering has had a significant involvement, and thus has been intertwined with the growth of this new engineering discipline. it was only in 1987 that the term tissue engineering was created ; and it was in 1988 that a conference called tissue engineering was first held at lake tahoe, california. the focus was on fabricating replacement tissues and organs outside of the body using cells and scaffolds for later implantation into the body. however, a much broader interdisciplinary effort evolved in the 1990s as stem cells received more interest. furthermore, the field of tissue engineering broadened into what now is called regenerative medicine and includes, in addition to replacement, also repair and regeneration. chief among these are the development of a small - diameter blood vessel substitute for the use in coronary bypass surgery, repair of a damaged myocardial wall following a heart attack and the development of a valvular substitute for use in defective heart valve replacement. the tissue engineering of a heart valve substitute is in fact what has stimulated much of the interest in the biology of heart valves. furthermore, the pediatric population is one of the main targeted patient populations. a young child that has a heart valve with a congenital defect if one had a replacement valve made of living cells that would grow over time as the child grows, then only a single surgery would be needed. to date there has been some success with the two major efforts of the laboratories of john mayer in boston sacks. and simon hoerstrup in zurich, switzerland, with both laboratories having engineers as part of their teams. the successful tissue engineering of a heart valve requires a combination of the right cells, a scaffold to provide the initial architecture, and the signals necessary to drive the process. although none of the current efforts has progressed to human studies yet, large animal experiments have been conducted. furthermore, from this one can see that there is a real role for engineering. the tissue engineering of a small - diameter blood vessel substitute in many ways may be viewed as one of the field 's holy grails. this is because there are many patients who need the coronary bypass procedure but do not have native vessels available for use. here again success depends on the right combination of cells, a scaffold either biologic or a synthetic material, and the necessary signals. in this area some progress has been made, and at least three efforts have been able to move into clinical trials. first is the work of shinoka and colleagues, who reported in 2001 the first clinical use of a tissue - engineered blood vessel (tevb), based on an autologous cell - seeded biodegradable scaffold, to repair cardiac defects in the low - pressure circulation of children. although the cells initially were taken from excised tissue and cultured in vitro, later studies were performed with cells isolated from the bone marrow and then directly seeded into the scaffold in the operating room. shinoka moved to yale university, where he received fda clearance to start a clinical trial. this was based on the 1999 report of the use of a tubular synthetic, biodegradable scaffold composed of polyglycolic acid and seeded with smooth muscle cells as a vascular graft. in a recently published study by by niklason 's startup company, humacyte perhaps the most important achievement in that study was to produce a small diameter human tebv with a burst pressure in excess of 3000 mmhg. these vessels, which were produced in 10 weeks in a pulsating bioreactor and decellularized, showed better compliance than that of a human saphenous vein, but still considerably less than that of human arteries. the third promising approach has as its foundation in the research conducted in 1990s by the laboratory of dr. the innovation here was to create sheets from the matrix secreted by cells, with these self - assembled sheets then rolled into the many distinct layers that compose a natural blood vessel. this method was used to construct the first tissue - engineered human blood vessel that was truly biological and that displayed physiological mechanical properties. in 2000, cytograft tissue engineering, inc. was founded with the purpose of developing this new technology and bringing it to the clinic. the company aimed to simplify the complex in vitro model that was developed in auger 's laboratory, while retaining its many biological advantages. this was done by eliminating the medial layer of smcs. the argument for eliminating this layer however, by eliminating the medial layer, the mechanical properties of this tebv substitute were significantly altered. preliminary results from initial human trials of this type of graft were reported a few years ago. this trial involved the use of the graft as an arterio - venous (av) shunt for hemodialysis access. biomechanical testing indicated an average burst pressure in excess of 3000 mmhg. furthermore, production of the graft proved to be reproducible. cytograft is focusing its clinical trials in europe and asia, and the phase iii clinical studies for hemodialysis access employs both autologous and allogeneic grafts. of interest is that the new allogeneic model has the potential of reducing the overall production time to 3 weeks. furthermore, it allows 1000 of grafts to be fabricated from a single master cell line. the more recent approaches of niklason and her co - workers, l'heureux and the cytograft team involve creating a biological scaffold made up of extracellular matrix components. still, a major issue is cells ' source. if one is to use an autologous cell approach, then the concept of a tebv off - the - shelf availability is not possible to implement. on the other hand, the use of an allogeneic cell approach could lead to off - the - shelf availability. however, one must again emphasize that, if allogeneic endothelial cells are to be used, then one must incorporate some type of immune - suppressive strategy. alternatively one could possibly recruit endothelial cells from the patient into a non - endothelialized tebv which has been implanted. for all the progress in the last decade, it still appears that we are a few years away from having a tebv substitute achieving fda approval. another important area is that of the heart itself and the use of a cell - based therapy for myocardial repair. although there have been a variety of studies, they have not been particularily encouraging. this is because there has been only modest improvements in left ventricular function no matter what cell type is used. also, cell engraftment has been poor, and it is very unlikely that significant repair / regeneration actually occurred. the fact that the use of different cell types leads to very similar results suggests that it is a paracrine effect, not one of cell replacement. this is an area where engineers can contribute with one example being the work of simpson. a critical issue in tissue engineering and regenerative medicine is that of cell source. because of this, the field of stem cell technology has taken on an important role in the field of tissue engineering and regenerative medicine. engineers are also contributing to the advances being made in this field. here again there have been studies of the role of physical forces, in this case in the modulation of stem cell behavior. one example is the use of flow and the associated shear stress to influence the differentiation of mouse embryonic stem cells to endothelial cells. there is an emerging tissue engineering and regenerative medicine industry. in the translation of the benchtop research to patient therapies there also is a recognition that there is a need for the further development of bioprocessing systems for stem cell biomanufacturing. these systems will need to provide for the scaleup in cell numbers needed for a patient therapy and also the systematic assessment of the cell population required to provide for quality control and thus regulatory approval. over the past half century, bioengineering has emerged as an engineering discipline in its own right. at the same time, there have been major advances in our understanding of cardiovascular disease and in the development of therapeutic approaches. as described here, there has been an intertwining of these advances ; and although one might argue that cardiovascular research would have advanced without the involvement of bioengineers, at the same time engineers have made major contributions. the participation and contribution of bioengineers to cardiovascular research has been illustrated here using three specific areas : hemodynamics and atherosclerosis, heart valve engineering, and tissue engineering. these are areas in which the biomechanical aspects of the problem proved to be important. this was true in terms of the role of flow and the associated shear stress in the development of atherosclerosis, the fluid mechanic characteristics of prosthetic heart valves and more recently side - specific differences in the function of valvular endothelial cells, and in the development of innovative therapies using tissue engineering and regenerative medicine approaches. there have been other contributions by bioengineers that have led to advancements in our understanding of the cardiovascular system and disease processes. this includes imaging, which engineers have contributed to the advancement of technologies that range from ultrasound to computerized tomography to magnetic resonance imaging to position emission tomography. engineers also have contributed to our understanding of the electrophysiological characteristics of the heart and to cardiac rhythm therapies. after all, it was only over half century ago that critical contributions of engineers led to the implantable pacemaker and to the establishment of the medical device industry. the story told here, however, has been based on biomechanics and its role in various aspects of the cardiovascular system. furthermore, it may be argued that it was individuals with an engineering background that recognized the importance of biomechanics in biology and in physiology. as noted earlier, the mechanical environment to which cells are exposed is part of the symphony of signals that orchestrates function, both normal and pathological. the result is that biomechanics has not only contributed to our understanding of the cardiovascular system, but it also has contributed to the establishment of the discipline of bioengineering. looking into the future, there will be the continuing involvement of bioengineers including biomechanicians in cardiovascular research, and with this there will be continuing advances in our understanding of the cardiovascular system, including the basic biology and pathobiological aspects of disease as well as the development of new therapeutic approaches. | the development of the modern era of bioengineering and the advances in our understanding of the cardiovascular system have been intertwined over the past one - half century. this is true of bioengineering as an area for research in universities. bioengineering is ultimately the beginning of a new engineering discipline, as well as a new discipline in the medical device industry. |
he reported chronic non - productive cough, weakness for the preceding three weeks, loss of appetite, and weight loss of about ten kilograms in six months. the interview and the medical history documentation indicated that he was suffering from diabetes type 2, paroxysmal atrial fibrillation, hypertension, chronic renal failure grade 3, and from generalised atherosclerosis. at age 45 it was located in segment 6 of the right lung, with diameter of 4.5 cm and lysis in the centre. in addition, the ct revealed in the enlarged lower pulmonary vein and in left atrium of the heart an hourglass - shaped structure with dimensions 5.4 1.6 cm. the results of laboratory tests of serum revealed the elevation of d - dimers (791 ng / ml) and c - reactive protein concentration (37 mg / l). in bronchofiberoscopy the additional longitudinal echo ranging from right pulmonary vein ostia to the mouth of mitral valve was recorded b) the loss of contrast in the left atrium and thickening of the pulmonary vein. after cardiothoracic consultation the patient was qualified for surgery. in the first stage sternotomy with extracorporeal circulation was performed. we incised the left heart atrium and revealed a pale pink tumour, which we removed completely from the atrium and from the final section of the right lower pulmonary vein. in intraoperative cytology of the tumour imprints the pathologist found non - small cell lung carcinoma cells. we decided to perform a right - sided thoracotomy in the second stage with right lower lung lobectomy and mediastinal lymphadenectomy. 2c the incised right pulmonary vein is shown with its wall infiltration forming the beginning section of the cardiovascular part of the tumour. a) intracardiac part of the tumour just before the resection and (b) just after the resection. c) right lower lobe the pulmonary vein is cut and the infiltration of the vein wall is shown. in the final examination of the tumour the pathologist diagnosed mixed heterogeneous lung cancer (squamous cell and non - small cell endocrine) in stage iiia. there are very few cases of lung cancer extension via the pulmonary vein into the left atrium of the heart [2, 3 ]. they can be diagnosed by use of transoesophageal echocardiograms, magnetic resonance imaging, and ct scan [1, 3 ]. most of those tumours show rapid growth and comprise a large mass in the lung parenchyma. resection of the intra - atrial mass seems to be a life - threatening procedure because the patient may die from cardiac inflow obstruction and sudden cardiac arrest or massive tumour embolism involving the major organs [2, 4, 5 ]. the possibility of systemic tumour embolisation should be considered in patients with large, central tumours and particularly those that abut the pulmonary veins [4, 5 ]. computed tomography with contrast allows the detection of the spread of lung cancer through the vessels into the heart cavity. malignant lung tumours penetrating into the cavity of the heart can be, in some cases, radically removed. in this case, the tumour did not infiltrate the left atrium of the heart but only grew into its light through the lower pulmonary vein (iiia t3n1m0 stage). the rapid progression of the tumour in the presented case (the floating part of the tumour in the atrium grew 1 cm in a week, as seen by comparing ultrasound examinations of the heart) rapid multidisciplinary collaboration of the two centres enabled fast and efficient qualification procedure. | computed tomography is performed in every patient before lung tumour resection. the presented case realises how important it is to perform this study with contrast. in a 75-year - old male we detected a tumour ingrowing from the right lung through the right lower pulmonary vein into the left atrium of the heart. the patient was qualified for primary sternotomy with extracorporeal circulation and resection of the intracardiac part of the tumour. in the second stage, right - sided thoracotomy was performed, and right lower lung lobectomy was done. mixed heterogeneous lung cancer was diagnosed (squamous cell and non - small cell endocrine) in stage iiia. the perioperative period was uncomplicated. the patient, due to renal failure, was not eligible for adjuvant chemotherapy. if the patient were qualified for lobectomy based directly on computed tomography without contrast, there would have been a high risk of perioperative death due to embolic incidents and heart failure. effective multidisciplinary collaboration allowed us to avoid this sort of complication. |
a total of 24 male sprague - dawley rats aged 10 weeks were obtained from a specific pathogen - free colony at samtako co. (osan, republic of korea) and used after one week of quarantine and acclimation. the institutional animal care and use committee of chonnam national university approved the protocols for the animal study, and the animals were cared for in accordance with the guidelines for animal experiments of chonnam national university. ach was purchased from sigma - aldrich co. (st. louis, mo, usa). the test chemical was diluted to the appropriate concentration with corn oil (sigma - aldrich, st. louis, mo, usa) and was administered orally to rats for 7 days. healthy males were assigned randomly into four experimental groups of 6 rats each : three treatment groups receiving 3, 10, and 30 mg / kg / day ach and a vehicle control group. the doses selected were on the basis of toxicity studies described earlier. all animals were observed daily for any clinical signs of toxicity and body weights were measured on days 0, 1, 2, and 7 of the test. at the scheduled termination day (day 7 of the study), all male rats were euthanized by carbon dioxide and exsanguination from the aorta. the absolute weights of the prostates, seminal vesicles, testes and epididymides were measured and their weights relative to body weight calculated. the left testis was homogenized and sonicated with 12 ml distilled water for sperm head count. the sperm suspension was put into a hemacytometer (neubauer, germany) and the number of homogenization - resistant sperm heads was counted under a light microscope (leica, germany). for motility measurements, the sperm was obtained from the left cauda epididymis, placed in hanks ' balanced salt solution (ph 7.2) containing 5 mg / ml bovine serum albumin (sigma chemical co., st. louis, mo, usa) and maintained at 37. motility was observed using a microscope with a stage warmer. sperm morphology was also examined using optical microscopy of the sperm smears (sperm suspension containing 1% eosin y) collected from the left cauda epididymis. these sections were stained with hematoxylin - eosin for histopathologic examination and then examined microscopically. the weighed frozen right epididymis was homogenized in a glass - teflon homogenizer with 50 mm phosphate buffer (ph 7.4) to obtain 1:9 (w / v) whole homogenate. the homogenates were then centrifuged at 11,000 g for 15 min at 4 to discard any cell debris, and the supernatant was used for the measurement of concentration of malondialdehyde (mda), reduced glutathione (gsh) content and activities of catalase and glutathione - peroxidase (gpx). the concentration of mda was assayed by monitoring thiobarbituric acid reactive substance formation by the method of berton.. total protein concentrations were determined by the bradford protein assay (bio - rad), using bovine serum albumin as a standard. results are expressed as meansd, and all statistical comparisons were made by one - way anova followed by tukey - kramer multiple comparison test. a total of 24 male sprague - dawley rats aged 10 weeks were obtained from a specific pathogen - free colony at samtako co. (osan, republic of korea) and used after one week of quarantine and acclimation. the institutional animal care and use committee of chonnam national university approved the protocols for the animal study, and the animals were cared for in accordance with the guidelines for animal experiments of chonnam national university. the test chemical was diluted to the appropriate concentration with corn oil (sigma - aldrich, st. louis, mo, usa) and was administered orally to rats for 7 days. healthy males were assigned randomly into four experimental groups of 6 rats each : three treatment groups receiving 3, 10, and 30 mg / kg / day ach and a vehicle control group. all animals were observed daily for any clinical signs of toxicity and body weights were measured on days 0, 1, 2, and 7 of the test. at the scheduled termination day (day 7 of the study), all male rats were euthanized by carbon dioxide and exsanguination from the aorta. the absolute weights of the prostates, seminal vesicles, testes and epididymides were measured and their weights relative to body weight calculated. the left testis was homogenized and sonicated with 12 ml distilled water for sperm head count. the sperm suspension was put into a hemacytometer (neubauer, germany) and the number of homogenization - resistant sperm heads was counted under a light microscope (leica, germany). for motility measurements, the sperm was obtained from the left cauda epididymis, placed in hanks ' balanced salt solution (ph 7.2) containing 5 mg / ml bovine serum albumin (sigma chemical co., st. louis, mo, usa) and maintained at 37. motility was observed using a microscope with a stage warmer. sperm morphology was also examined using optical microscopy of the sperm smears (sperm suspension containing 1% eosin y) collected from the left cauda epididymis. these sections were stained with hematoxylin - eosin for histopathologic examination and then examined microscopically. the weighed frozen right epididymis was homogenized in a glass - teflon homogenizer with 50 mm phosphate buffer (ph 7.4) to obtain 1:9 (w / v) whole homogenate. the homogenates were then centrifuged at 11,000 g for 15 min at 4 to discard any cell debris, and the supernatant was used for the measurement of concentration of malondialdehyde (mda), reduced glutathione (gsh) content and activities of catalase and glutathione - peroxidase (gpx). the concentration of mda was assayed by monitoring thiobarbituric acid reactive substance formation by the method of berton.. total protein concentrations were determined by the bradford protein assay (bio - rad), using bovine serum albumin as a standard. results are expressed as meansd, and all statistical comparisons were made by one - way anova followed by tukey - kramer multiple comparison test. there was no treatment - related mortality and body weight change in any of the animals treated with ach during the study period (data not shown). however, the rats treated with ach showed treatment - related occurrence of clinical signs such as nasal discharge, soft feces, and decreased locomotor activity in a dose - dependent manner (table 1). although the difference was not statistically significant between the groups, cystic pustule of the caput epididymis was found in all animals of the high dose group (data not shown). as shown in table 2, the rats treated with 30 mg / kg / day of ach showed a statistically significant increase in the relative weights of both epididymides when compared to the control group. spermatic granuloma (n=6), cell debris in the ducts (n=6), epithelial cell vacuolization (n=6), and oligospermia (n=6) were observed in caput epididymis of the 30 mg / kg group (figure 1). spermatic granuloma (n=1), cell debris in the ducts (n=2), epithelial cell vacuolization (n=4), and oligospermia (n=1) were also found in the 10 mg / kg group. although the difference was not statistically significant between the groups, the incidence of histopathological lesions observed in the 10 and 30 mg / kg groups was higher than the control group. as shown in table 4, no treatment effects were detected in terms of the number of testicular sperm heads and morphology of epididymal sperm. in contrast, the sperm motility of epididymis in the 10 and 30 mg / kg groups significantly decreased in a dose - dependent manner when compared with that of the control group. the concentration of mda, an end product of lipid peroxidation, in the 10 and 30mg / kg groups were significantly increased in a dose - dependent manner when compared with the control (table 5). however, the concentration of gsh were significantly decreased in the 30 mg / kg group compared to the control group. the catalase and gpx activities in all of ach treatment groups were also significantly decreased when compared with the control group. the toxicity of ach has been extensively studied in short and long term animal studies over the past several decades. the present study was undertaken in an attempt to evaluate the potential effects of ach on sperm and epididymal function after 7-day repeated oral administration in rats. treatment - related clinical signs such as nasal discharge, soft feces and decreased locomotor activity were observed in this study. these findings may be attributed to mucus and gastrointestinal irritation effects of ach because ach is a chemical that acts as an eye and nasal irritant. on the other hand, there were no significant differences in the body weights between the groups. a dose - dependent increase in the relative weights of epididymis these results are similar to the finding of kawaguchi., who showed that the relative epididymis weight increased when ach was administered at 20 mg / kg / day for 19 days to rats. at the scheduled necropsy, the cystic pustule of the epididymis was observed in all cases in the high dose group. the increased incidence of the cystic pustule was considered to be a treatment - related effect, since this finding is uncommon in normal control rats and was consistent with the significantly increased weight of the epididymis. cooper. also reported that a single dose ach at 50 mg / kg / day produces large retention cysts in the ductuli efferentes and proximal caput of the epididymides in rats. characteristic histopathologic findings observed in the present study included spermatic granuloma, cell debris in the ducts, epithelial cell vacuolization, and oligospermia in proximal caput epididymis. and jelks and miller also demonstrated that a single oral dose of ach at 25 mg / kg / day results in a white pustule, sperm granuloma formation, sloughed epithelial cells in the lumen, and epithelial vacuolization and disruption in the rat epididymis. recently, kawaguchi. reported that oral administration of ach to rats for 18 days resulted in cell debris in the tubules of the caput epididymis and sperm granulomas in the tubules of the cauda epididymis at 20 mg / kg / day. the results of the above studies and the present study clearly show that ach has adverse effects on epididymal histology in rats. in the present study, although there were no obvious differences in the incidence of abnormal sperm in caudal epididymis and sperm head count in testis among the groups, epididymal sperm motility in the high dose group was significantly decreased in a dose - dependent manner. it was previously reported that evaluation of epididymal sperm motility seems to be a potential indicator of male reproductive toxicity. hoyt. demonstrated that when rats received 5 mg / kg / day for 2 weeks follow by a 2-week withdrawal period, there was a decrease in the percentage of motile sperm. the 8-day repeated oral dose of 10 mg / kg / day to rats resulted in reductions in the mean percentage of motile sperm, curvilinear velocity, straight - line velocity, lateral head displacement, and linearity. ours and the results of previous studies strongly indicate that ach has adverse effects on sperm function in rats. ros production and oxidative stress plays a role in sperm functions during maturation and capacitation, because their plasma membranes contain high content of polyunsaturated fatty acids and their cytoplasm contains low concentrations of scavenging enzymes. ros are considered to damage spermatozoa through lipid peroxidation, resulting in altered sperm functions. in the present study, the animals treated with ach showed decreased activities of antioxidant enzymes such as catalase and gpx, and gsh concentration in a dose - related manner, while they increased mda concentration in the epididymis. an increase in the lipid peroxidation indicated that ach induced oxidative stress in the epididymis by decreasing the activities of antioxidant enzymes, thereby leading to an excessive generation of ros. therefore, these results suggest that the administration of ach elicits depletion of the antioxidant defense system in the epididymis, indicating that oxidative damage plays a role in sperm and epididymal toxicities. it was reported that oxidative damage and ros production cause sperm pathology such as atp depletion. the inhibitory effect of ach on metabolic activity of sperm in vitro is thought to occur via ach oxidation within the sperm to form 3-chlorolctaldehyde, which then inhibits gapdh. gapdh, a glycolytic enzyme, is known as a major target protein in oxidative stress and is an important key enzyme to generation of atp which is necessary for sperm motility and male fertility. in addition, sperm are believed to contain a more susceptible isoform of this enzyme. it has been demonstrated that ach directly affects spermatozoa by the depletion of atp levels and the inhibition of gapdh activity in rat sperm and epididymis. therefore, we can consider that oxidative damage induced by ach administration can cause an inhibition of gapdh activity in rat sperm and epididymis, followed by sperm atp depletion. these results from previous studies were consistent with the decrease of sperm motility observed in our study. overall, it can be concluded that the 7-day repeated oral dose of ach to rats elicits oxidative damage at 3 mg / kg / day and spermatotoxicity in the epididymis at 10 mg / kg / day, and that the adverse effects of ach on sperm functions and epididymal histology may be at least partially due to the induction of lipid peroxidation and decrease of antioxidant activities in rats. | this study was conducted to investigate the potential effects of -chlorohydrin (ach) on epididymal function and antioxidant system in male rats. the test chemical was administered to male rats by gavage at doses of 0, 3, 10, and 30 mg / kg / day for 7 days. twenty - four male rats were randomly assigned to four experimental groups, with six rats in each group. spermatotoxicity was assessed by measurement of reproductive organ weight, testicular sperm head count, epididymal sperm motility and morphology, histopathologic examination, and oxidative damage analysis in rats. at 30 mg / kg / day, an increase in the incidence of clinical signs, epididymis weight, and gross necropsy findings of the epididymis, a decrease in the sperm motility, and an increased incidence of histopathological changes of the epididymis were observed in a dose - dependent manner. at 10 mg / kg / day, an increased incidence of clinical signs and histopathological changes and decreased sperm motility were observed. in the oxidative damage analysis, an increase in the malondialdehyde concentration and a decrease in the glutathione content and glutathione peroxidase and catalase activities in the epididymal tissue were detected at 3 mg / kg / day. the results show that graded doses of ach elicit depletion of the antioxidant defense system and that the spermatotoxicity of ach may be due to the induction of oxidative stress. |
this disease presents a slightly higher predominance in males, and most patients are diagnosed within the first fourth decades of life.1 although up to one third of dermoid cysts are found at birth, most of them are diagnosed in the second and third decades.2 almost 7% of all dermoids are located in the head and neck.3 they are frequently found in the region of the lateral part of the eyebrow, in the periorbital region, and in the midline nasal region. in the neck they usually occur in the submental region, above the hyoid and always in the midline.3 this tumor accounts for fewer than 5% of all intracranial masses.2 tumors arising from the sinonasal region usually present late as their symptoms are often banal and may be overlooked by patients and their clinicians. the recent onset of unilateral nasal symptoms, without improvement with medical therapy, and orbital and neurologic symptoms should be investigated with imaging studies. computed tomography (ct) and magnetic resonance imaging (mri) are used to characterize tumors of this region.4 endoscopic sinus surgery is a well - established technique for the treatment of sinus diseases, including chronic sinusitis and nasal neoplasms. endoscopic sinus surgery to approach nasal neoplasms is limited, as the tumor can be too extensive.5 in this report, we discuss diagnosis, differential diagnosis, and possible approaches to lesions arising from the pterygopalatine fossa. a 23-year - old man was admitted to a tertiary care center after a car accident, in which he sustained a pelvic / femoral fracture. there was no evidence of any associated injury, and a conversion to tracheostomy after long - term intubation the patient was referred for brain mri ; a heterogeneous ovoid lesion hyperintense in t2-weighted imaging in the pterygopalatine fossa was discovered. as a matter of fact, this lesion was indenting the middle fossa and was closely related to the maxillary artery, sphenopalatine ganglia, and maxillary posterior wall. the patient was referred to the otolaryngology department of clinics hospital university of so paulo. prior to surgery, laboratory workup, including a complete blood count with differentials and determination of liver enzymes and electrolytes, showed no abnormal findings. brain ct showed a heterogeneous lobulated lesion in the left masticator space (infratemporal region), 3 3, 5 2 cm wide, medial to lateral pterygoid muscle, with erosion of the inferior wall of foramen rotundum (fig. we repeated brain mri, which showed a lobulated extra - axial formation in the left masticator space, between lateral and medial pterygoid muscles, with fat heterogeneous tissue, hypointense in t2-weighted image, gadolinium - enhanced in t1-weighted image (fig. there were small hyperintense lesions in t1-weighted images in the suprasellar cistern and in the sylvian fissure, suggesting previous dermoid rupture (fig. computed tomography showing a heterogeneous lobulated lesion in the infratemporal region. magnetic resonance imaging (mri) of the lesion with heterogenous content in the inferior region. benign and malignant tumors can arise from any of the structures within the infratemporal fossa and parapharyngeal space.6 dermoid inclusion cysts are benign tumors and are mainly unilocular and expand slowly, enlarging over years or decades, by the accumulation of cutaneous products. they may show lipid content, derived from sebaceous secretions, and secretions of apocrine sweat glands and hair.2 the causes of dermoids include failure of surface ectoderm to separate from underlying structures and sequestration of surface ectoderm at lines of epithelial fusion during embryonic development. most congenital dermoid cysts probably arise due to an embryologic accident during early stages of development, between the third and fifth weeks of gestation.2 3 clinical examination of the fossa pterygoid is difficult because it is deep lying and not easily accessible. lesions are often discovered only at a late stage either because there are no clinical signs or, if they do occur, they are so common as to be overlooked.1 6 7 in our case, the diagnosis was incidental. tumors that arise in pterygopalatine fossa are usually asymptomatic. ct and mri provide precise imaging information and may be necessary for making the diagnosis. in addition, imaging data can help to differentiate infection from tumor lesions and primary tumors from secondary tumors.1 7 although they are slow - growing, dermoid inclusions cysts produce pressure changes on surrounding structures that are visible at radiograph study. in a case series, yu evaluated 86 patients with tumoral lesions of pterygopalatine and infratemporal spaces.1 most of the lesions (81%) originated in oral and maxillofacial regions other than pterygopalatine and infratemporal spaces and extended to this region. only one case of teratoma was found. the most frequent diseases reported among the other cases were squamous cells carcinoma, adenoid cystic carcinoma, inflammatory disease, and hemangioma. differential diagnosis in cases of tumoral lesions in pterygoid fossa should be performed (table 1).1 8 9 10 11 12 13 treatment of dermoid cysts consists of complete surgical excision of the lesion, avoiding recurrence.6 rupture of dermoid cysts can produce severe chemical meningitis, usually attributed to the irritating effects of the cholesterol in the cellular debris.2 in our case, brain mri showed signs of previous rupture, which may account for the patient 's epilepsy. uppal excised a dermoid cyst in the infratemporal fossa, extending inferiorly to the parapharyngeal space, using a lateral approach to that region.6 in our case, we performed a minimally invasive treatment, using an endoscopic transnasal transpterygoid approach with a wide access to the lesion. it was possible to proceed with complete excision of the tumor, without intraoperative complications. the clinical diagnosis and management of these lesions can be challenging because of the relative inaccessibility of the region, which contains the maxillary artery, the maxillary nerve, and the pterygopalatine ganglia, with its branches. surgical advantages of the endoscopic endonasal approach in comparison with traditional transcranial approaches include a more direct midline exposure, decreased brain parenchyma injury, and lack of neurovascular structure manipulation. from the patient 's perspective, decreased surgery time, decreased length of stay, increased patient comfort, and lack of external incision are advantages of the endoscopic endonasal approach. although rare, dermoid cysts should be considered in the differential diagnosis of expansive lesions in the pterygopalatine fossa and can be excised by a transnasal transpterygoid endoscopic approach. | objective to describe a case of dermoid cyst arising from the pterygopalatine fossa and review the literature. methods we report a case of a 23-year - old man who suffered a car accident 2 years before otolaryngologic attendance. he had one episode of generalized tonic - clonic seizure and developed a reduction of visual acuity of the left side after the accident. neurologic investigation was performed and magnetic resonance imaging revealed an incidental finding of a heterogeneous ovoid lesion in the pterygopalatine fossa, hyperintense on t2-weighted imaging. results endoscopic sinus surgery with transpterygoid approach was performed. the ovoid lesion was noted in the pterygopalatine fossa. puncture for intraoperative evaluation showed a transparent thick fluid. surprisingly, hair and sebaceous glands were found inside the cyst capsule. the cyst was excised completely. histologic examination revealed a dermoid cyst. the patient currently has no evidence of recurrence at 1 year postoperatively. conclusion this unique case is a rare report of a dermoid cyst incidentally diagnosed. an endoscopic transnasal transpterygoid approach may be performed to treat successfully this kind of lesion. although rare, it should be considered in the differential diagnosis of expansive lesions in the pterygopalatine fossa, including schwannoma, angiofibroma, esthesioneuroblastoma, osteochondroma, cholesterol granuloma, hemangioma, lymphoma, and osteoma. |
aquagenic syringeal acrokeratoderma (asa) is an acquired or hereditary keratoderma of unknown etiology. drug - induced cases of asa of the palms have been reported, associated with rofecoxib, celecoxib, aspirin, and in one case, tobramycin used for cystic fibrosis. several pathogenic mechanisms have been proposed, including structural or functional defects of the horny layer during adolescence, primary disease of the sweat ducts, increased sodium concentration in the skin, thereby increasing the water - retention capacity of the horny layer or a reaction to drugs. we present a case of a woman who used spironolactone for polycystic ovary syndrome (pcos) and developed asa 2 weeks later, after withdrawing the drug. a 21-year - old woman presented to our outpatient clinic with complaints of palmar eruption. she noted wrinkling and edema of the skin on the palms after soaking in water for 5 - 10 min, 3 weeks earlier. the wrinkling and edema were accompanied by sensations of tightness, tingling, and pruritus. the condition became better 10 - 15 minutes after exposure to water had ceased, but did not completely resolve. these findings regressed gradually after drying the hands, but were not completely resolved. on dermatological examination, whitish, keratodermic, macerated plaques, and dilated punctae were observed over the volar surface of the hands [figure 1 ]. after soaking the hands in water for 5 min, the lesions became more translucent, noticeable, and symptomatic [figure 2 ]. before soaking the hands in water, whitish keratodermic macerated plaques and dilated punctae were observed after soaking the hands in water for 5 min, the lesions became more translucent, noticeable, and symptomatic from her medical history, we learned that she used spironolactone for pcos up to 2 months earlier. she denied any concomitant hyperhidrosis, a personal or family history of cystic fibrosis, abnormal scalp hair, or atopic diathesis. her cousin had similar lesions years ago, but her symptoms were completely resolved with treatment. the disease had been treated at another center with 19% aluminum hydrochloride cream with no improvement. the laboratory findings, including sweat chloride concentrations were within normal limits, except for very small elevation of prolactin levels. we wanted to perform genetic testing to define cystic fibrosis transmembrane conductance regulator (cftr) mutations, but she would not accept any further tests. the patient was treated with topical pomade containing 10% urea and salicylic acid twice a day and topical 10% urea lotion (excipial lipo ; orva). asa has been described in the literature under various names for similar conditions, patients with flat - topped, pitted, or translucent papules, with pebbly or white, prominent eccrine pores that are macerated in appearance and that appear on the hands and feet, exacerbated by water immersion. the disease was first reported as a hereditary papulotranslucent acrokeratoderma in 1973 by onwukwe. they reported a condition that appeared soon after puberty, demonstrated an autosomal dominant mode of inheritance and associated with fine - textured scalp hair and an atopic diathesis. afterwards, in 1974, aquagenic wrinkling was reported by elliot in a letter to the editor, describing an anecdotal observation in children with cystic fibrosis. subsequently, several reports presented by several names, including transient reactive papulotranslucent acrokeratoderma, asa, aquagenic keratoderma, transient aquagenic palmar hyperwrinkling, and early aquagenic wrinkling. histopathological changes include orthohyperkeratosis with increased thickness and abnormal staining of the stratum corneum, dilated acrosyringia, and dermal eccrine ducts with hyperplasia of eccrine glands, clear cell changes and vacuolations, and increased capillaries around and adjacent to the eccrine glands. although asa is often related to cystic fibrosis, researchers have reported that it is not only specific for cystic fibrosis ; it is also seen in different conditions, such as marasmus and nephrotic syndrome. drug - induced cases of asa of the palms have been reported in single case reports, associated with rofecoxib, celecoxib, aspirin, and, in one patient, with tobramycin used for cystic fibrosis. the proposed mechanisms for the cases using rofecoxib, celecoxib, and aspirin involve an increase in the sodium retention of epidermal cells. in these cases, the proposed mechanism is cyclooxygenase-2 (cox-2) inhibition in epidermal cells, which may cause increased sodium reabsorption in a mechanism similar to the effect of cox-2 inhibitors on kidney cells. adrenal steroids have recently been shown to play an important role in the regulation of renal cox-2 expression. according to zhang., blockade of mineralocorticoid receptors with spironolactone leads to upregulation of renal cortical cox-2 expression. in our patient, who was using spironolactone for pcos for approximately 2 months withdrawing the drug may have caused increased sodium retention of epidermal cells. during treatment, 20% aluminum chloride solution, botulinum toxin injections, antihistamines, pomade containing 5% salicylic acid, a mixture of mometasone furoate and petroleum jelly, and a cream containing 20% urea were used for treatment. our patient had not improved with 19% aluminum hydrochloride cream, perhaps because she had not suffered from palmar hyperhidrosis. withdrawing spironolactone may have caused increased sodium retention of epidermal cells, thereby inducing asa. | aquagenic syringeal acrokeratoderma is a rare, transient, and usually bilaterally symmetric, palmoplantar keratoderma. patients complain of tingling and pain in the hands starting a few minutes after exposure to water and lasting for 20 - 30 minutes after removal. clinically, there is marked wrinkling with edematous white papules on the palms or, less often, the soles. we present the case of a 21-year - old woman who used spironolactone for polycystic ovary syndrome and had similar clinical features 2 weeks later, after withdrawing the drug. |
scorpions are fascinating animals. their venom with various potentialities has made them look both scary and fascinating. the venom of some species of scorpion has a remarkable impact on religious attitudes, concepts, and views of different societies in the east and west. also, they are believed by a few traditional groups in some parts of the world. ancient egyptians believed that scorpions were created through decomposed corpses, crocodiles, and lizards. according to an algerian legend, therefore, they built the palace using totally smooth and flattened stones to be impenetrable by scorpions. one day, a basket of grapes with a scorpion hidden in it was brought to the prince. such legends describe to some extent the story of how the human is stung by scorpions. relatives of an authority in ahvaz jundishapur university of medical sciences (ajums) took some vegetables for him. his spouse was stung by a scorpion while cleaning them, and lost her life some days later. treatment of those stung by venomous animals and their safety in ancient iran has contributed to the formation of various stories and legends. mithridates vi was a cautious man and because he was afraid of being poisoned by his solders, he was accustomed to use some poison in his food every day. the king was coincidentally defeated by the romans ; then, he decided to commit suicide. however, whatever poison he ate he did not succeed, so he ordered one of his soldiers to stab him. it seems that getting accustomed to a little and then gradually more amounts of poison has been common in ancient iran. on this occasion, mithridates vi is known as the first immunologist in iran. mithradatism is the practice of protecting oneself against a poison by gradually self - administrating nonlethal amounts for the body to become accustomed to the poison. later, galen made an antidote and named it therikos mithridatus. in countries such as sudan, scorpions are put in sesame oil to make medicine for those stung by scorpions. scorpion sting is one of the main health problems in developing, tropical, and subtropical countries causing immense clinical consequences and sometimes death. the status of scorpion sting varies in different regions and countries due to lifestyle, economic social status, residential status, way of providing health services, and species present in every geographical area. scorpion sting is one of the main medical concerns of some african, middle eastern, eastern, and central american countries. a total of 300,000 cases of scorpion sting are reported annually in mexico. in 1995, 7000 cases of scorpion stings occurred in brazil, and 1% were fatal despite receiving antivenom serum. more than 20 species of scorpion exist in the arabian peninsula and their stings result in death in some cases, and 86 species of scorpions has been found in india. iran, due to its climate and weather, is very rich in arthropods especially scorpions. iran is one of the countries with a vast variety of scorpions, especially dangerous species. reports of scorpion stings are obviously notable in the old iranian sources. to treat stings, different drugs under the name of antidotes, applying medicines, and ointments these varieties of medicines prove that scorpion sting has always been a main medical concern in the previous historical eras. in a manuscript dated 1908 ad, cities such as tehran, ghazvin, ghom, kashan, tafresh, khoramshahr, ahvaz, soleymanieh, korestan, kerman, bampour, shoushtar, and shahroud also, in recent decades, cases of scorpion stings and related mortalities are being reported annually in iran. thus, it can be said that arthropods are the most dangerous venomous animals in iran. scorpions have been long known in iran due to their stings ; therefore, there is considerable need for the prevention or treatment of their stings. in the traditional sources and texts, ways of treatment, and getting rid of and destroying scorpions have been discussed in detail. at present, the main way for treating scorpion sting (of any species) in the world and iran is the use of antivenom serum, which has been under question in the treatment of some cases. due to the significance of scorpion sting in the world and iran in particular, study of this matter along with the related therapeutic methods especially in the light of ancient and traditional medicine of iran is of vital importance. therefore, our aim is to find applied methods proposed in the traditional medicine to manage and handle the problem, at least in parts of the world. this descriptive review was done by searching keywords such as traditional medicine, scorpion sting, and treatment of scorpion sting through relevant websites, specialized journals, articles, and books. all the articles and sources covering detailed and technical description of scorpion sting and its treatment, traditional medicine, prevention and control of dangerous species were studied ; and the rest were excluded. articles not related directly to the study and those studying other aspects of scorpions such as the biochemical aspect of venom, were omitted. all articles and reference books published in the past 30 years in the persian scientific journals were studied and finally different points of view of various authors about prevention, control, and treatment of scorpion sting in the traditional medicine were offered. out of 85 sources, 47 were selected and analyzed based on their potentiality and usefulness. scorpions have always been hated by human being because they have almost always been associated with sin and criminality. fear of mad dogs, scorpions, and other venomous animals was very common in the middle ages and various books were written on preventing or treating related diseases. the same therapeutic perspective is present in the iranian literature. in some countries, people often carry an image of a scorpion with them to guard themselves against scorpion sting. in iran, images of scorpions have been discovered carved on silver objects. one of these texts deals with the point that if one scorpion is placed in fire, it stings and kills itself. scorpions have attracted attention due to their venomous, painful, and sometimes deadly stings. like other nations, when mithras, iranian god of the sun scarified a sacred cow for its blood to fertilize the world, a mischievous demon sent a scorpion to sting the cow s testicles for destroying its source of life. the ancient iranians used to hold the women s celebrations on sepandrmazgn, the 5 day of march. they called it good news (mozd). on this day, in addition to giving presents to women, a ceremony was conducted to prevent scorpion sting. killing animals, vermin (or destroying kharfaster) was very common in the ancient iran so that it was considered as one of the main oblations. scorpions were also involved in the fate of wars in iran. in the late sassanid period, kashan was so famous and thriving that it sent an army of warriors headed by shirzad to fight with the arabs. the army of abu musa ashari, the arabian commander was encountered with persistence and tenacity. abu musa ashari, who was disappointed to not capture kashan, distributed pitchers of scorpions throughout the city. the arabs soon after killed the people, looted properties and animals, and took the soldiers captive. a number of western travellers wrote things about scorpions in their itineraries that often did not match reality. sir thomas herbert, in one of his journeys in 1626 described scorpions of kashan : scorpions are small snakes with long fingers. these reports from the ancient iran remained in the religious and historical texts prove the archaism of this issue in iran. due to the existence of these animals all over iran, which provides them with a desirable climate for survival, therefore, the iranians should have gathered comprehensive and complete information to control and treat scorpion sting. despite damage brought to the scientific and cultural body of iran during wars in different periods, sources available and accessible to the iranian in the past illuminate like a gem in the ancient scientific world. by contrast, iran has been a cradle of civilization for centuries and has presented precious scientists to the world of medicine. in the modern era, studies on scorpions were started more seriously with the onset of great wars from the 20 century. medical importance of scorpions was of more concern with the beginning of the imposed wars against iran. kamali highlighted cases of scorpion stings in the province of khuzestan, especially in towns and villages. radmanesh reported sting rates of 41%, 45%, and 13% for androctonus crassicauda, mesobuthus eupeus, and hemiscorpius lepturus species, respectively in khuzestan. he also stated a sporadic rate for stings by buthotus saulcyi and apistobuthus petriygocerus scorpions. he (radmanesh) said a rate of 1015% sting by this most dangerous and venomous scorpion in khuzestan. zayerzadeh in a study points out that about 40% of the total referring to stings ward > 70% of the stings in ahvaz are pertained to mesobuthus eupeus species. he believes that the classic six as (anesthetic, antibiotic, antitetanic, anitvenom, anti - inflammatory, and analgesic) method is not applicable for scorpion sting in the province of khuzestan. according to radmanesh, 10% of the scorpion stings in khuzestan are from hemiscorpius lepturus. dehghani reported that 30%, 62%, and 8% of the stings in kashan are related to black, yellow, and indistinguishable scorpions, respectively. afzali and pezeshki studied cases of acute renal failure induced by the gadim scorpion in children in abuzar hospital of ahvaz in 1994. they concluded that renal failure in the gadim - stung patients is always due to hemoglobin urine. according to dehghani and fathi, 42,500 cases of scorpion stings occur annually in the country. this is because a few cases of scorpion stings take place in the remote villages and are not recorded. except for khuzestan, which is always at the top, the placing of some provinces change a little in the annual statistics. bigdeli reported the rate of scorpion stings for 100,000 people of khuzestan province from 1998 to 2000 as 709 cases, 706 cases, and 733 cases, respectively. the cities of ramhormoz and masjedesoleiman, along with rural regions of this province, had the highest rates of scorpion stings. based on these studies, scorpion stings have been reported from all over the country, but the province of khuzestan is first in terms of occurrences and related complications. one of the main concerns in managing scorpion sting is recognition of stings of dangerous species. by knowing complications of stings of different species, we can find specific therapeutic methods for each. stings by hemiscorpius lepturus leave severe complications that have been mentioned in the iranian old medicine. ibn sina (avicenna), the well - known persian physician, about 1000 years ago described a scorpion that can undoubtedly be attributed to the h. lepturus species based on the clinical signs given in the canon of medicine. venomous scorpions (dragging its tail) own segmentary bodies with a tapered and sharp tail. their poison is very strong and is usually very common in askar mokaram of khuzestan. if one is stung by them, one does not feel anything immediately. the day after or on the person feels the pain. the person gets depressed, their complexion gets pale and may take jaundice. after being stung, the tongue is inflamed, site of sting is infected, urinates blood and sometimes dies. do nt neglect if the pain is little because sting of this scorpion is highly venomous and fateful. what is inferred from these clinical manifestations from 1000 years ago is that scorpion sting dates back to centuries ago in iran and only h. lepturus species is accompanied with such clinical signs. poison of this species has cytotoxic and hemolytic effects (fig. 1). many are stung by this species in iran especially in provinces of khuzestan and hormozgan, which is followed by severe clinical signs particularly in children. h. lepturus is an exception among the medical species in terms of clinical manifestations. severe skin inflammation and hemolysis are of clinical complications attributed to this type of scorpion. gadim s clinical manifestations reported by ramanesh are the same as that stated by the persian ancient physicians such as avicenna, which reveal their current or previous attention in that area. avicenna described morphology, habitat, absence of pain at the beginning of the sting, and all the other clinical complications of the above - mentioned scorpion. his knowledge was not only applicable in his time, but also useful for us today. in another part of avicenna s description about the clinical manifestation of scorpion sting, he states that the individual feels a sharp and sudden pain at the site of the sting like a needle penetrating into the body. the individual then begins to sweat ; the lips become convulsant and cool ; the hairs become dry ; the male sexual organ seems longer and more erect than usual ; the sting feels fiery hot ; the individual s complexion is altered totally ; a kind of sticky liquid comes to the lips and sticks to them ; and sticky tears are coagulated in the corners of the eyes. these clinical signs indicate that this persian scientist was talking about scorpion sting with neurotoxic venoms, which have been widely discussed in detail by iranian and other scientists in the modern era (fig. 2). studies on the effect of venoms of these scorpions on laboratory animals demonstrate how avicenna precisely explained the situation 1000 years earlier. this proves that avicenna reported clinical manifestations of those stung by scorpions based on the type of venom. most of the scorpions in iran and other parts of the world possess neurotoxic poisons ; therefore unrestrained knowledge of avicenna could be useful for the researchers and physicians in tropical and semitropical regions. according to another reference, each patient is treated individually in traditional medicine because their temperament is a mixture of four humors and the balance is different from one person to another. at present, all researchers agree with this principle and believe that treatment of scorpion sting varies in different victims based on their individual and genetic characteristics. of course, in age, sex, and weight groups, clinical complications of certain species are similar. in other words, there are fixed basics that should be considered in the treatment. therefore, due to the problem of scorpion sting in iran especially in some regions, experienced local physicians familiar with traditional medicine and environmental factors are needed. this improves quality of treatment and offers a general applicable therapeutic protocol for each patient. in iranian traditional medicine, various first aid methods have been used as the primary and general basic prior to manage scorpion sting. one of the suggestions by abu - hsab in this regard is as follows : before doing anything, site of the sting must be sucked just like the way mentioned in the chapter on absorbing poison. further, as zink reported to save the patient, bloodletting can be done at the site of the sting to let the blood along with the poison out of the body or else the site can be sucked under certain circumstances. also, the site can be cupped to let the poison out with the blood. furthermore : the poison must be drawn out of the body and then medication should be done. as stated in these texts, ways of neutralizing the poison or drawing it out of the body have been common since long ago. some of the ancient first - aid practices are still used in the present time. methods of drawing the poison out of the body are nowadays practical and recommended in the modern medicine with few changes. drawing the poison out of body by lancetting or by sucking is still ordinary as a primary action in most of the scorpion - prone areas. this method is relatively dominant in aran and bidgol, a scorpion - prone area in the province of isfahan. therapeutic effects of these methods depend on the precision in drawing the poison out of the victim s body. application of hot steam and warming the sting site is one of the methods of treatment recommended in traditional medicine. in other references, burning the site of the sting or putting hot olive oil and zeft resin on the site have been stated as well. this method for the treatment of stings has been known as one of the most effective ways in this regard. nowadays, in the countries separated from the former soviet union, burning the site of the sting by venomous animals such as the black spider by a piece of metal or burning matches is used as a way for treatment. for instance, in ramhormoz in the province of khuzestan, hot sands are used to burn the site of the sting. using this method, scorpion poison made of organic compounds and protein mostly is slightly burned along with the surrounding tissue. cutaneous leishmaniasis is treated by the local healers in abuzeidabad in aran and bidgol but scars remain because of burning. this method is very invasive and can jeopardize the patient s life if necessary therapeutic equipment can not be used in emergencies. in traditional iranian sources, especially in the canon of medicine, different medicines such as assa foetida resin to neutralize the poison have been mentioned to treat scorpion sting and decrease complications especially the pain created. if garlic is taken with walnut and is followed with some wine, it is more effective in lessening the pain. after eating the garlic and drinking the wine, they should cover in a warm place so as to sweat. the important point is sweating in order to drive the obscene material out of the body. oral drugs in the treatment of scorpion sting include bitter lettuce juice, desert chicory juice, beer, and whatever pacifying the temperature especially in the case of severe inflammation. elsewhere, it has been stated that a mixture of garlic along with warm wine and bottle grass, angedan resin, anacyclus pyrethrum, and opoponax galbanum is outstandingly useful in treatment. all drugs common in modern medicine owe debt to the traditional medicine common in some countries. nowadays, researchers investigate through different countries, regions, and even tribes of latin american and african indians to get acquainted with usage of herbal medicines. there is a concise and complete list of medicinal drugs in the iranian traditional texts. traditional medicine is now practiced in india, pakistan, and bangladesh especially among muslims. richness of traditional medicine in the indian subcontinent is mostly due to the immigration of iranian physicians in different centuries. of course, there are discussions about some of the drug cocktails that have been mingled with superstitions. to get rid of scorpions, different methods have been suggested in iranian traditional medicine. in the canon of medicine, it says that a scorpion dies if a radish leaf or a piece of lead is put on it. provide styrax officinalis, orpiment (arsenic natural sulfur), sheep droppings, and sheep fat in equal quantities ; let the fat be melted and put in front of the scorpion s hole : the scorpion will leave the hole. if a slice of radish is put in front of scorpion s hole, it never dares come out of it. if smoke of a killed scorpion is directed towards a scorpion hole, the scorpion dies inside. smoke of sulfur, orpiment, fat, and sheep droppings frighten scorpions. among cases advised in the traditional medicine texts, some are precisely effective in getting rid of or killing scorpions. smoke produced by burning different materials is today used for keeping away arthropods from people, which is most common among rural populations. orpiment or arsenic sulfur (as2s3) is an old poison used now for pests. severity of poisoning depends on factors such as health status, age of victim, site of sting, scorpion species, size of scorpion, and degree of the scorpion s stimulation. scorpion species is of vital importance because each species has different fractions and organic compounds in its poison, resulting in different mechanisms in the body of humans or animals. thus, color of scorpions conveys different species, which has been insisted on in the canon of medicine. scorpions appear in nine different colors : white scorpion, yellow scorpion, gray scorpion, grayish black scorpion, green scorpion, golden scorpion in which tip of the tail and forks is black, vinaceous scorpion with needle - pricking sting and great pain, and dingy scorpion that its sting results in excess laughing and delirium. in total, 52 species of scorpions have been identified in iran, with a few subspecies for some. all of the scorpions have various ecotypes for each climate, which include nearly all the colors described by avicenna in the canon of medicine. identification of scorpions and their morphology has received special attention in the traditional and ancient scientific sources of iran. differences between male and female scorpions are given as follows : male scorpion is skinny and slim, while the female one is big in size ; sting of the male sex is thick and that of the female is thin. about scorpion sting, it has been mentioned that some scorpions have double stings which remain as two bites. site of the bites is cold and the whole body is warm accompanied sometimes with cold sweat. having two distinct stings in the telson is not morphologically matched with a specific species today. it is notable that there are two glands of poison in the telson that separately carry the poison to the back of the sting. therefore, at the end of the sting, a unique duct of poison is formed containing poison of both glands. there is no flying scorpion among various species today and based on the sources available, no species of scorpions have ever been winged. however, in some old texts, flying scorpions have been mentioned. also, according to laymen in regions rich in scorpions such as kashan, flying scorpion has been reported. according to the canon of medicine, flying scorpions are bigger than the usual ones and they use their wings to fly safely from one place to another. in the sand dunes of desert regions especially near kashan, movement of black scorpion a. crassicauda has been watched in strong winds by the author. these scorpions in strong winds and storms move easily and safely from sand dunes due to their hard and firm exterior skeleton. when winds blow, a great deal of sand is brought to human residential sites. this matter is confirmed by lay observations in some regions of the country that scorpions are more common when the wind blows. usually, these arachnids rest 10 cm or more deep under layers of sand to shelter from the severe heat of the desert. the same source says that there are scorpions with a six - segment tail that becomes very dangerous and deadly when the sheri star (after august 15) rises. the first part about the tail has been explained well. in the morphology of scorpions the telson as a separate segment, the ancient report about the tail and deadliness of scorpion in august comes true. however, the reports about scorpions with less than six - segment tails is not in reality believable. one of them says that if a scorpion is surrounded by fire, the scorpion stings itself. these statements by ordinary people originate from the observation that when a circle of fire with a noticeable height is made around the scorpion, the animal is invaded by the fire from all sides. therefore, it tries to keep the fire away from itself with the only defensive tool it possesses the sting. then, it stings repeatedly different parts of its body to get rid of the fire burning the skin. prior to being killed by its poison, organic materials and proteins in the scorpion s body are coagulated and denatured. if a human is surrounded by a circle of fire, no reaction nevertheless, if part of the body of an adult in particular is stung by an insect like a bee, we beat on the site immediately. therefore, it is not stinging itself, but it is trying to keep itself away from danger. scorpions live in varied habitants such as indoors, outdoors, suburbs of villages and cities, and most regions of iran. therefore, due to their contact with people, various cases of scorpion stings and mortalities have been reported in iran in the ancient medical texts and after islam. thus, more studies and investigations in the iranian traditional medicine can open new horizons in the treatment, control, and prevention of scorpion stings. scorpion sting and its venom have been regarded as a major medical and health problem, with a long history in iran. by considering and scrutinizing the other side of these works, it can be claimed that dangers of scorpion, the beliefs, the realities, and analyzing customs has played a significant role in forming the role of this creature, which was occasionally realistic and in some cases mixed with superstitions. | due to the medical and therapeutic importance of scorpions in iranian traditional medicine, this review was conducted on the treatment of scorpion sting as performed by traditional healers in order to realize complications, clinical manifestations, diversities, and deficiencies in the prevention, control, and treatment as mentioned in the pertained literatures. this study tried to make known and investigate attitudes of the iranian national and traditional medicine towards controlling these venomous animals. keywords and articles were searched through relevant sites on the internet. we investigated different journals and references for the iranian traditional medicine. based on the articles and books found, we tried to find suitable solutions to problems from the viewpoint of traditional medicine. scorpion sting dates back to ancient iran and has been widely reflected in the resources of iranian traditional medicine. the traditional medicine offers various guidelines that can be beneficial in this respect. new attitude towards scorpion sting with regard to traditional medicine resources can enhance control and prevention of scorpion stings. consequently, this attitude leads authorities and researchers to a decreased level of scorpion stings or related consequences. |
insulator proteins confer activity to insulators sequences, a class of functional elements with important roles in the regulation of chromosomal organization. in this study, we investigated the origin of the seven known proteins associated with insulator activity in d. melanogaster. to find potential orthologs of these proteins in other organisms, we followed two independent strategies. on the one hand, we performed blast searches and analyzed a large number of candidate sequences in phylogenetic experiments (table 1 ; figs. s1s4). on the other hand, we combined hmm searches and mcl clustering to examine which of the candidates share orthology with a known drosophila insulator (table s4). with both methods we found robust evidence that the known drosophila insulators (except ctcf) are restricted to arthropods and have been acquired successively during arthropod evolution (fig. 2 ;, we did not find evidence for the existence of known drosophila insulator proteins outside the arthropods by analyzing seven genomes from four protostome phyla (annelida, mollusca, platyhelminthes, nematoda). it is unlikely that a lack of sensitivity is responsible for this result as we find ctcf orthologs in the annelid capitella spi, in the mollusc lottia gigantea, and in the nematode trichinella spiralis, faithfully replicating the results of previous work with an independent method (heger. the detection of ctcf in all 19 analyzed arthropod genomes, including so far unknown orthologs (e.g., from the myriapod strigamia maritima and the strepsipteran m. moldrzyki), further confirms the specificity and sensitivity of our approach. second, the zf proteins ctcf and su(hw) are consistently present in arthropods from which genome sequences are available plus in some additional orders with a significant number of ests. in both cases, the presence of orthologous sequences in the three subgroups of arthropods indicates that the common ancestor of arthropods already had these proteins. such an assumption is well supported for the ctcf protein that has been found in all bilaterians (heger. the origin of the su(hw) protein is less clear. as it is absent in nematodes (table s4 ; heger. 2009), it could have evolved in the ancestor of arthropods or in the common ancestor of arthropods and a closely related ecdysozoan sister group, for example, tardigrades or onychophorans. third, in contrast to ctcf and su(hw), the proteins zw5 and beaf-32 are restricted to a remarkably limited subset of arthropods. we obtained with both methods only few beaf-32 candidates outside the drosophila genus (64 and 95, respectively), and none of them could be placed into the drosophila beaf-32 orthology group. although the domain composition of zw5 issued a much higher number of candidates (28,431) across the 26 genomes (table s4), we could only recover zw5 orthologs in drosophila and g. morsitans, another brachyceran fly. a zw5 ortholog could not be found in nematoceran dipteres and other arthropods despite the existence of several sequenced genomes and large amounts of ests. these results are based on the most comprehensive study undertaken so far and provide consistent evidence that zw5 and beaf-32 likely emerged in or close to the common ancestor of the genus drosophila. finally, our results with respect to the btb domain containing insulators suggest that at least some of them have evolved at intermediate stages when compared with the ubiquitous proteins ctcf / su(hw) and the restricted the mapping of cp190, for example, shows that it is present in all insects with a sequenced genome and in three crustacean orders. the inability to detect this protein in a myriapod and two mite genomes and in a large amount of ests from chelicerates suggests that cp190 evolved in the ancestor of pancrustacea. gaga factor, on the other hand, formed a highly supported cluster containing exclusively sequences from diptera and hymenoptera in phylogenetic experiments (fig. s2). the hmm - based approach confirmed this result, but assigned in addition sequences from the hemipteran rhodnius prolixus to the gaga factor orthology group (table s4). in all further insect, crustacean, and chelicerate genomes, orthologs were not detectable, indicating that this protein likely emerged in the common ancestor of hemiptera, hymenoptera, and diptera. importantly, these findings imply that gaga factors do not exist outside the arthropod phylum. to elucidate the conflict of these findings with the proposed existence of vertebrate gaga factors (matharu. 2010 ; kumar 2011), we performed phylogenetic analysis with two sets of candidates, acquired by blast and hmm searches. however, neither the four proposed vertebrate gaga factors nor our additional candidates were placed to the insect orthology group (figs. although our study provides evidence for a consecutive gain of insulator proteins in arthropods, it also suggests that insulator evolution is dynamic in terms of losses. although it is difficult to prove the absence of a gene in potentially inaccurate genome assemblies, our results indicate that inference of at least some secondary losses is reasonable. the two best examples are the repetitive loss of gaga factor in coleoptera / strepsiptera and in lepidoptera and the loss of su(hw) in lepidoptera, each supported by the analysis of two genomes with both methods (fig. we were not able to discover an expected ortholog in a single genome (table s4). this may be a consequence of incomplete genome assemblies, but could alternatively reflect a dynamic nature of insulator evolution in arthropods. we did not observe a loss of the more ancient ctcf insulator in any arthropod genome. this is compatible with the idea that ctcf function is needed for fundamental processes in the bilateria (heger. 2012) and might have been supplemented and modified by additional components in the arthropod lineage. although our results have been established by two fairly independent methods, we can not formally prove the absence of orthologous proteins from certain clades. with growing databases and dependent on the sensitivity of homology detection tools, the exact placement of the origin of some proteins may still change. however, irrespective of uncertainties in the exact time of gain and loss, our observations reveal a consistent model for the evolution of the known d. melanogaster chromatin insulators through a series of successive acquisitions. it has been confirmed repeatedly that insulator proteins colocalize and interact with each other (gerasimova. 2007 ; bartkuhn. 2009 ; negre. 2010 ; van bortle. 2012), thereby creating a network of dependencies that is thought to contribute to cell - specific differences in nuclear organization and gene expression (yang and corces 2011). moreover, there is recent evidence that each d. melanogaster insulator subclass, determined by the presence of su(hw), ctcf, beaf, or gaf, shares the cp190 protein and possibly also mod(mdg4) (van bortle. our findings point out that the mechanisms, interactions, and components of insulator complexes must be considerably different from drosophila not only in the great majority of arthropods (that share two of the seven factors), but also in other bilaterian phyla that only have ctcf in common. according to our findings, the presence of different insulator systems and the complex interactions between their components seen in d. melanogaster today are a result of ongoing evolution and diversification. these processes started more than 600 million years ago in the ancestor of bilaterians (www.timetree.org) with ctcf, the oldest known chromatin insulator of multicellular animals (heger. 2012). at or close to the root of arthropods, these two basal systems experienced subsequent additions and modifications, with the gain of beaf-32 in the genus drosophila, 60 million years ago, being the most recent acquisition. to what extent the successive acquisition of new insulator genes is involved in adaptive processes, shall be the topic of future investigations. although we can describe in detail an expansion of insulator proteins only in the drosophila history, millions of arthropod species trace back to the same common ancestor that was equipped with the ctcf and su(hw) insulators. it is therefore possible that other arthropods simultaneously increased their initial repertoire of insulator proteins during the past 600 million years, giving rise to a plethora of unexplored territories. thus, the expansion of insulator mechanisms that happened in d. melanogaster history might in fact be a general characteristic of arthropods and other animals. the description of non - ctcf insulators in echinoderms exemplifies that such an expansion could indeed apply to a greater variety of animals (yajima. if so, this characteristic could provide a mechanism to modulate an ancient insulator system and fine - tune gene expression in a lineage - specific way. with the known drosophila insulator proteins as query (dctcf, gi:21356747 ; su(hw), gi:33860216 ; cp190, gi:23171337 ; gaf, gi:83287912 ; mod(mdg4), gi:158030328 ; zw5, gi:45549097 ; beaf, gi:17647187), standard blastx, blastp, or tblastn (altschul. 1997) searches were conducted in publicly available sequence databases at ncbi (http://blast.ncbi.nlm.nih.gov/). to minimize the chance of missing an ortholog, we performed parallel searches in different databases (nucleotide, est, and protein) and subdivided a search into smaller entities if a given taxonomic range reported > 500 hits below threshold (= download restriction at the ncbi blast web interface). we set the threshold for btb domain protein candidates to 10 because in preliminary experiments this value effectively incorporated btb domain proteins distinct from btb domain containing insulators, for example, tramtrack or broad complex. collected nucleotide sequences were translated to the appropriate reading frame using emboss (rice. we obtained from the initial dataset a collection of unique sequences using the emboss tool all other candidates from the initial dataset were passed to the silix clustering algorithm (miele. 2011) as partial sequences (command line parameter -p)., we took all reference sequences plus candidates that clustered to one of the references. for the zw5, su(hw), and ctcf proteins, the described drosophila orthologs served as positive control for cluster identification. similarly, we included the known drosophila orthologs of the btb - domain proteins gaga factor, cp190, and mod(mdg4) to specify these clusters. outgroup for the analysis of zf insulators was the widely distributed zf transcription factor yy1. for btb domain insulators, we used as outgroup a set of lola - like orthologs from diverse arthropods. phylogenetic trees resulting from the alignments were computed under the maximum likelihood criterion using parallel raxml version 7.2.6 (stamatakis 2006) with 100 bootstrap resamplings. as optimal models of sequence evolution we used the wag+ or dcmut++f model (zf domain proteins), the jtt++f model (btb domain proteins), and the wag++f model (vertebrate gafs) as selected by prottest3 (darriba. likelihood trees were visualized and arranged with figtree (http://tree.bio.ed.ac.uk/software/figtree/) and then graphically edited with adobe illustrator software. to verify our results with an independent method, we combined a search on the basis of insulator - specific hidden markov models (http://hmmer.org/) with the markov cluster algorithm (van dongen 2000). we downloaded from various sites (table s3) genome and, if available, proteome and unplaced reads data of 19 different arthropod and seven outgroup species. the 26 selected species represent maximal diversity while avoiding over - representation of well - sampled groups such as dipterans. as subsequent comparisons relied on protein sequences, we translated the 26 genomes and the respective unplaced reads data into all six reading frames (> 90 nucleotides) and combined the resulting open reading frames of each species with the corresponding protein set as offered by the sequencing center. to obtain specific hmmer profiles, we selected with attention to maximal diversity and length for each insulator protein 815 previously verified orthologs as representatives of a cluster. we then calculated and manually refined multiple alignments of these sequences using the mafft einsi algorithm (katoh. 2005) and derived a representative full length hmmer profile for each insulator protein. all 26 orf sets were scanned with the six custom made hmmer profiles and all sequences below the default inclusion threshold of hmmsearch (e - value 500 hits below threshold (= download restriction at the ncbi blast web interface). we set the threshold for btb domain protein candidates to 10 because in preliminary experiments this value effectively incorporated btb domain proteins distinct from btb domain containing insulators, for example, tramtrack or broad complex. collected nucleotide sequences were translated to the appropriate reading frame using emboss (rice. we obtained from the initial dataset a collection of unique sequences using the emboss tool all other candidates from the initial dataset were passed to the silix clustering algorithm (miele. 2011) as partial sequences (command line parameter -p)., we took all reference sequences plus candidates that clustered to one of the references. for the zw5, su(hw), and ctcf proteins, the described drosophila orthologs served as positive control for cluster identification. similarly, we included the known drosophila orthologs of the btb - domain proteins gaga factor, cp190, and mod(mdg4) to specify these clusters. outgroup for the analysis of zf insulators was the widely distributed zf transcription factor yy1. for btb domain insulators, we used as outgroup a set of lola - like orthologs from diverse arthropods. phylogenetic trees resulting from the alignments were computed under the maximum likelihood criterion using parallel raxml version 7.2.6 (stamatakis 2006) with 100 bootstrap resamplings. as optimal models of sequence evolution we used the wag+ or dcmut++f model (zf domain proteins), the jtt++f model (btb domain proteins), and the wag++f model (vertebrate gafs) as selected by prottest3 (darriba. likelihood trees were visualized and arranged with figtree (http://tree.bio.ed.ac.uk/software/figtree/) and then graphically edited with adobe illustrator software. to verify our results with an independent method, we combined a search on the basis of insulator - specific hidden markov models (http://hmmer.org/) with the markov cluster algorithm (van dongen 2000). we downloaded from various sites (table s3) genome and, if available, proteome and unplaced reads data of 19 different arthropod and seven outgroup species. the 26 selected species represent maximal diversity while avoiding over - representation of well - sampled groups such as dipterans. as subsequent comparisons relied on protein sequences, we translated the 26 genomes and the respective unplaced reads data into all six reading frames (> 90 nucleotides) and combined the resulting open reading frames of each species with the corresponding protein set as offered by the sequencing center. to obtain specific hmmer profiles, we selected with attention to maximal diversity and length for each insulator protein 815 previously verified orthologs as representatives of a cluster. all 26 orf sets were scanned with the six custom made hmmer profiles and all sequences below the default inclusion threshold of hmmsearch (e - value < 0.01 ; 39,573 unique sequences) were fed to a dedicated orthomcl pipeline (li. we used the recommended inflation parameter 1.5 for the mcl step. before clustering, we removed duplicates and supplemented each of the 26 sequence collections with reference insulator sequences of that species (verified by phylogenetic experiments) to facilitate cluster recognition. zw5, and beaf, we detected a single orthology group whereas su(hw) (2), cp190 (4), and gaf (3) orthologs split to more than one group. like described above, we constructed two multiple sequence alignments from representative members of the gaga cluster (full length and btb domain only) and uploaded these alignments to hhpred (http://toolkit.tuebingen.mpg.de/hhpred) for highly sensitive profile profile searches. we searched with default parameters in the proteomes of h. sapiens and mus musculus, the only deuterostome datasets available. after removing duplicates, we analyzed the remaining 45 candidates with phylogenetic methods (see above). additional supporting information may be found in the online version of this article at the publisher s website : figure s1. download location of 26 genomes and proteomes used for a hmm / orthomcl - based search for insulator proteins. | alteration of regulatory dna elements or their binding proteins may have drastic consequences for morphological evolution. chromatin insulators are one example of such proteins and play a fundamental role in organizing gene expression. while a single insulator protein, ctcf (ccctc - binding factor), is known in vertebrates, drosophila melanogaster utilizes six additional factors. we studied the evolution of these proteins and show here that in contrast to the bilaterian - wide distribution of ctcf all other d. melanogaster insulators are restricted to arthropods. the full set is present exclusively in the genus drosophila whereas only two insulators, su(hw) and ctcf, existed at the base of the arthropod clade and all additional factors have been acquired successively at later stages. secondary loss of factors in some lineages further led to the presence of different insulator subsets in arthropods. thus, the evolution of insulator proteins within arthropods is an ongoing and dynamic process that reshapes and supplements the ancient ctcf - based system common to bilaterians. expansion of insulator systems may therefore be a general strategy to increase an organism s gene regulatory repertoire and its potential for morphological plasticity. |
antimicrobial resistance can also lead to a delay in the administration of appropriate drugs, and the drugs required to treat resistant pathogens can be toxic (2). antimicrobial resistance has become a worldwide problem with the exception of a few countries in northern europe (3). however, even in sweden, where resistant bacteria are rare, increases of ciprofloxacin- resistant escherichia coli and imipenem - resistant pseudomonas aeruginosa have been reported (4). the prevalence of resistant bacteria varies significantly in different countries as it is influenced by the amount of antimicrobial agents used. the major reasons for performing surveillance are to determine the size of the problem, to see whether resistance is increasing or not, to detect any previously unknown types of resistance, and to determine whether any particular type of resistance is spreading or is associated with an outbreak (1). analysis of routine susceptibility data has some limitations, which include its inaccuracy, but it does not require much resource. longitudinal studies of resistance trends are considered most beneficial for the detection of subtle changes in resistance (5). a previous nationwide surveillance in korea showed increasing resistance rates of enterococcus faecium to vancomycin, enterobacteriaceae to fluoroquinolones, the 3rd generation cephalosporins, cephamycins and fluoroquinolones, and p. aeruginosa and acinetobacters to carbapenems (6). it is hoped that the korean national health insurance program, which in 2001 abolished over - the - counter sales of antimicrobial agents and started to scrutinize proper use of antimicrobial agents at hospitals, has had some impact upon reducing resistant bacteria. the aim of the surveillance in 2001was : to determine any changing trends in the above - mentioned serious resistances in particular, besides the common resistances at korean nationwide surveillance of antimicrobial resistance (konsar) hospitals in different locations, and to determine possible emergence of new resistances. routine susceptibility test data for major aerobic pathogenic bacteria in 2001 were collected from 31 hospitals located in 8 cities / provinces in korea. however, the data obtained from 30 hospitals were analyzed, excluding one hospital with poor performance versus the who / cdc quality control program. as did in the previous study (6), less than 20 isolates of a species from a hospital main methods of susceptibility testing used were, the nccls disk diffusion test (7) by 21 hospitals and commercial broth microdilution systems, i.e., vitek (biomerieux, marcy l'etoile, france) or microscan (dade microscan inc., west sacramento, ca, u.s.a.) by 9 hospitals. methicillin - resistant staphylococci were tested using oxacillin, and penicillin g - nonsusceptible pneumococci were screened mainly by the oxacillin - disk method (7). hospitals were divided into three groups according to location and bed capacity (1,000 beds countrywide, < 1,000 beds in seoul, and < 1,000 beds in non - seoul). resistance rates did not include intermediates. the mean resistance rate in each hospital group was calculated from the mean resistance rates at each hospital, to minimize the influence of a large number of isolates at some hospitals (8). the resistance rates of some important organisms to certain antimicrobial agents were compared to those of previous years. however, the statistical significances of changes in resistance were not determined, because this depends on the statistical method used (8), and because the purpose of surveillance includes the detection of outbreaks of resistant infections (9). routine susceptibility test data for major aerobic pathogenic bacteria in 2001 were collected from 31 hospitals located in 8 cities / provinces in korea. however, the data obtained from 30 hospitals were analyzed, excluding one hospital with poor performance versus the who / cdc quality control program. as did in the previous study (6), less than 20 isolates of a species from a hospital main methods of susceptibility testing used were, the nccls disk diffusion test (7) by 21 hospitals and commercial broth microdilution systems, i.e., vitek (biomerieux, marcy l'etoile, france) or microscan (dade microscan inc., west sacramento, ca, u.s.a.) by 9 hospitals. methicillin - resistant staphylococci were tested using oxacillin, and penicillin g - nonsusceptible pneumococci were screened mainly by the oxacillin - disk method (7). hospitals were divided into three groups according to location and bed capacity (1,000 beds countrywide, < 1,000 beds in seoul, and < 1,000 beds in non - seoul). the mean resistance rate in each hospital group was calculated from the mean resistance rates at each hospital, to minimize the influence of a large number of isolates at some hospitals (8). the resistance rates of some important organisms to certain antimicrobial agents were compared to those of previous years. however, the statistical significances of changes in resistance were not determined, because this depends on the statistical method used (8), and because the purpose of surveillance includes the detection of outbreaks of resistant infections (9). the susceptibility data analyzed were of 169,032 isolates, which consisted of 79,167 (46.8%) isolates of gram - positive cocci, and 89,865 (53.2%) isolates of gram - negative bacilli. proportions of the organisms were : staphylococcus aureus 24.1%, coagulase - negative staphylococci 11.1%, enterococcus faecalis 6.0%, e. faecium 3.8%, pneumococci 1.8%, e. coli 12.0%, klebsiella pneumoniae 7.0%, enterobacter cloacae 3.8%, serratia marcescens 3.4%, non - typhoidal salmonellae 0.6%, acinetobacters 9.4%, p. aeruginosa 16.5%, and haemophilus influenzae 0.4%. those of s. aureus to oxacillin, erythromycin, and gentamicin were 70%, but the rates of coagulase - negative staphylococci to these antimicrobial agents were somewhat lower. the resistance rates of s. aureus to cotrimoxazole were lower than that of coagulase - negative staphylococci (12% vs. 42%), but were higher to ciprofloxacin (64% vs. 34%) and to tetracycline (63% vs. 43%). the rate of penicillin - nonsusceptible pneumococci was 72%, and that of ciprofloxacin - resistance was 6%. the resistance rates of e. faecium were much higher than those of e. faecalis to ampicillin (88% vs. 2%), and to ciprofloxacin (88% vs. 43%), but lower to tetracycline (23% vs. 81%). the vancomycin resistance rates of e. faecium and e. faecalis were 16% and 1%, respectively. resistance rate of e. coli to ampicillin was 75%, and to both piperacillin and cotrimoxazole 50%. resistance rates to other antimicrobial agents were : 9% to ceftazidime, 11% to cefoxitin, 30% to gentamicin, and 28% to fluoroquinolone. twenty - seven per cent and 20% of k. pneumoniae were resistant to ceftazidime and cefoxitin, respectively, and 12% were resistant to amikacin. of the e. cloacae isolates, 40% were resistant to cefotaxime and ceftazidime, and 40% were to tobramycin. imipenem resistance rates of e. cloacae and s. marcescens were 0.4% and 0.6%, respectively. the resistance rates of s. marcescens to cefoperazone - sulbactam and amikacin, at 15% and 22%, respectively, were the highest among enterobacteriaceae species analyzed. the resistance rates of p. aeruginosa to ceftazidime and cefotaxime were 21% and 57%, respectively. the resistance rates to imipenem and amikacin 17% and 26%, respectively. of the acinetobacters, 65% were resistant to piperacillin, cefotaxime, ceftazidime, and aztreonam. cefepime resistance rate of 52% was the highest among gram - negative bacilli. also, 61% of the isolates were resistant to aminoglycosides, fluoroquinolone and cotrimoxazole. the imipenem resistance rate of all isolates was 6%. among the 990 isolates of non - typhoidal salmonellae, 35% were resistant to ampicillin, and 4% to cotrimoxazole, but none were resistant to fluoroquinolone. in the case of h. influenzae, some hospitals tested ampicillin susceptibility, while others tested -lactamase production, and still others tested both. among the 699 isolates of h. influenzae, 64% were ampicillin resistant and 66% were -lactamase producers. during the period 1997 - 2001, the resistance rates of s. aureus to oxacillin and erythromycin remained similar (fig. penicillin - nonsusceptible pneumococci declined slightly from 75% to 72%, whereas ampicillin - resistant e. faecium increased steadily from 70% to 88%. vancomycin - resistant e. faecium isolates increased at all hospital groups : from 2% to 16% at large hospital group, from 2% to 19% at the seoul medium hospital group, and from 7% to 13% at the non - seoul medium hospital group (fig. cefoxitin - resistant k. pneumoniae increased from 16% to 20%, and ampicillin - resistant h. influenzae from 58% to 64% (fig. the amikacin resistance rate of p. aeruginosa declined slightly from 33% to 26%, while that of acinetobacters gradually increased from 50% to 61%. 4), while the rates to imipenem remained to be 5% to 6% (fig. 3). ceftazidime- and imipenem - resistant p. aeruginosa were present in all hospital groups (fig. the surveillance of resistance is an important part of modern clinical microbiology (5), but surveillance methods are considered often inappropriate (1). the surveillance, which is based on collecting data from each laboratory has many problems due to the different methods used for species identification and for susceptibility testing. more accurate results can be obtained if isolates are collected and tested at a central laboratory (5), but this method is very expensive. current konsar program consists of two different forms of surveillance : one is the analysis of routine susceptibility test data, which were determined by individual participating hospitals, i.e., this study, and the other is the determination of the prevalence of certain resistances in isolates collected from participants, e.g., the study on metallo--lactamase - producing strains (10). both the necessity and the limitations of surveillance were discussed in a previous study (6). it is difficult to distinguish between community- and nosocomially - acquired pathogens, although their resistance rates differ markedly. however, some organisms, such as methicillin - resistant staphylococci, vancomycin - resistant enterococci, p. aeruginosa and acinetobacters are typical nosocomial pathogens, while non - typhoidal salmonellae are mostly community - acquired pathogens. the high proportion of s. aureus (24.1%) among the isolates tested in the present study indicates its continued importance. methicillin - resistant s. aureus (mrsa) is one of the typical nosocomial pathogens (11). the proportion of mrsa and methicillin - resistant coagulase - negative staphylococci, 70% and 69%, respectively, were similar to those reported for 1999 and 2000 (fig. surveillance in 1998 - 2000 in kinki, japan, showed the proportion of mrsa was approximately 60% (12). the more active drug against mrsa remained to be cotrimoxazole except for vancomycin, but the rate of 12% was much higher one than the 1% found in japan. two isolates of true vancomycin - resistant s. aureus were reported in the united states in 2002 (13, 14). the penicillin - nonsusceptible rate of pneumococci decreased slightly from 78% in 1999 to 72% in 2001. a reduction in penicillin - nonsusceptible isolates may be the result of the abolition of over - the - counter sales of common antimicrobial agents in 2001 in japan, the proportion of penicillin - nonsusceptible pneumococci, by the broth microdilution method, was 65% in 1998 and 81% in 2000 (12). we should know that the proportions were based on breakpoint for the treatment of meningitis (7). therefore, pneumonia caused by penicillin - intermediate isolates may respond to penicillin therapy (15).. the ampicillin - resistant rate of e. faecalis, 1%, was slightly lower than the 2% found in 2000, though still higher than the < 1% found at the coordinating laboratory of the konsar program. it was considered that ampicillin - resistant e. faecalis was due to misidentification of the species (1). these indicate the need to retest the species when an e. faecalis isolate shows resistance to ampicillin. vancomycin resistance rate of e. faecium in 2001 remained similar to those in 2000 at large- and medium - hospital groups in seoul. although the rate at the non - seoul medium - hospital group declined from 24% in 2000 to 13% in 2001 (fig. 2), vancomycin - resistant e. faecium seemed to be established in all hospital groups, as was at the coordinating hospital (16). it is a concern that once a resistance gene has became accumulated to an environment, it is impossible to eliminate them (17). the ampicillin resistance rate of e. coli did increase further and that of h. influenzae increased only slightly. ampicillin had been a commonly used drug because it was available without prescription, but currently no antimicrobial agent can be bought without a prescription since 2001. it is interesting to see the future surveillance could show a downward trend again. in the present study, 64% of the h. influenzae isolates the proportion of -lactamase - producing isolates in the united states was 36%, while that was only 3% in japan. in a european study (18), the prevalence of -lactamase producers varied from 8.1% to 34.8% depending on countries. it is interesting that in japan 39.6% of the isolates were -lactamase - negative ampicillin - resistant (blnar) strains (19). the resistance rates of non - typhoidal salmonellae, i.e., 35% to ampicillin, 4% to cotrimoxazole and none to ciprofloxacin, were similar to those reported in 2000 (6). when empirical antimicrobial treatment of non - typhoidal salmonellae infection is required (20), cotrimoxazole or ciprofloxacin should be appropriate first - line drugs in korea. in the present study, further study is required to determine whether extended - spectrum -lactamase (esbl)-producing isolates are present. in a korean hospital, esbl - producing non - typhoidal salmonellae were detected as early as 1995 (21). fluoroquinolones are increasingly used, as they are one of the three major broad - spectrum classes of antimicrobial agents (22). fluoroquinolone resistance rate of e. coli gradually rose from 24% in 1997 to 28% in 2001. the rate of acinetobacters, 65% was only slightly lower than that of the 70% in 2000 (6). expanded - spectrum cephalosporins are ineffective for the treatment of infections caused by esbl - producing isolates of e. coli and k. pneumoniae, although they may be inhibited by low concentrations of these drugs (23). it was reported that bacteremic patients with esbl - producing k. pneumoniae had higher initial treatment failure rates than those with non - esbl - producing isolates (24). also, the cefoxitin resistance rate 20% of k. pneumoniae suggests plasmid - mediated ampc -lactamase production (25). therefore, ceftazidime resistance rates can not reflect the proportion of esbl producers, although high prevalence of esbl - producing k. pneumoniae has been reported in korea (26). cephamycins, such as cefoxitin, are active against esbl - producing isolates, but plasmid - mediated ampc -lactamase - producing isolates are resistant not only to all cephalosporins but also to cephamycins. carbapenems are very useful drugs as they are stable to hydrolysis even to esbl- and ampc -lactamases (27). although vim-2 metallo--lactamase - producing s. marcescens and e. cloacae have been reported (28, 29), the imipenem resistance rates of e. cloacae and s. marcescens were low (0.4% and 0.6%, respectively), suggesting that carbapenem - resistant enterobacteriaceae are rare. the imipenem resistance rates of p. aeruginosa to ceftazidime and imipenem remained similar in all hospital groups (fig. about 9% of imipenemresistant p. aeruginosa were due to the production of acquired metallo--lactamases (30). 29% of acinetobacters isolated in 1996 - 1998 were resistant to imipenem, though the hospital did not use imipenem at that time (31). in our present study, however, the imipenem resistance rate rose to 13% in 2002 at the coordinating laboratory. another study showed that approximately 11.4% and 14.2% of imipenem - nonsusceptible isolates possessed the vim-2 or the imp-1 metallo--lactamase gene (10). given the increase in the prevalence of resistant bacteria, the empirical selection of antimicrobial agents becomes increasingly difficult and rapid microbiological testing increasingly crucial. as was found in the previous study, some laboratories tested susceptibility to too few antimicrobial classes (data not shown). diagnostic bacteriology results can not be obtained immediately, but clinical microbiologists should remember that early results only can aid the appropriate management of patients, and consequently reduce emergence and the spread of resistance. it was reported that early preliminary microbiology results of blood stream infection had a greater impact on antimicrobial management than susceptibility data (32). in conclusion, methicillin - resistant staphylococci, penicillin - nonsusceptible pneumococci, ampicillin - resistant e. faecium and h. influenzae, and expanded - spectrum cephalosporin - resistant gram - negative bacilli remain prevalent. vancomycin - resistant e. faecium, fluoroquinolone - resistant gram - negative bacilli, and imipenem - resistant p. aeruginosa are increasing at all groups of hospitals. given this increasing resistance, not only rapid and accurate routine susceptibility testing, but also the nationwide resistance surveillance become even more important for optimal patient management, the control of nosocomial infection, and eventually to conserve newer antimicrobial agents. | the 5th year konsar surveillance in 2001 was based on routine test data at 30 participating hospitals. it was of particular interest to find a trend in the resistances of enterococci to vancomycin, of enterobacteriaceae to the 3rd generation cephalosporin and fluoroquinolone, and of pseudomonas aeruginosa and acinetobacters to carbapenem. resistance rates of gram - positive cocci were : 70% of staphylococcus aureus to oxacillin ; 88% and 16% of enterococcus faecium to ampicillin and vancomycin, respectively. seventy - two percent of pneumococci were nonsusceptible to penicillin. the resistance rates of enterobacteriaceae were : escherichia coli, 28% to fluoroquinolone ; klebsiella pneumoniae, 27% to ceftazidime, and 20% to cefoxitin ; and enterobacter cloacae, 40% to cefotaxime and ceftazidime. the resistance rates of p. aeruginosa were 21% to ceftazidime, 17% to imipenem, and those of the acinetobacters were 61% to ceftazidime, aminoglycosides, fluoroquinolone and cotrimoxazole. thirty - five percent of non - typhoidal salmonellae were ampicillin resistant, and 66% of haemophilus influenzae were -lactamase producers. notable changes over the 1997 - 2001 period were : increases in vancomycin - resistant e. faecium, and amikacin- and fluoroquinolone - resistant acinetobacters. with the increasing prevalence of resistant bacteria, nationwide surveillance has become more important for optimal patient management, for the control of nosocomial infection, and for the conservation of the newer antimicrobial agents. |
the etiology of kienbock 's disease, also known as (osteonecrosis of the lunate, remains controversial. it commonly occurs in patients twenty to fourty years old and presents with pain and stiffness in the dorsomedial aspect of the wrist. several risk factors have been established to help explain its etiology : acute or repetitive trauma, variation in blood supply to the lunate, differences in the anatomy and shape of the lunate bone, and venous congestion (1, 2). abnormal biomechanics at the radiocarpal joint between the distal radius and ulna has also been implicated in kienbock 's disease (3, 4). ulnar variance describes the length relationship between the articular surfaces of the radius and ulna at the radiocarpal joint. positive ulnar variance indicates that the ulna is longer than the radius, while negative ulnar variance indicates that the ulna is shorter at the wrist joint. in neutral ulnar variance 80% of the axial load at the wrist is transmitted through the distal radius. as ulnar variance decreases to more negative values, the load transmission across the distal radiocarpal joint increases, subsequently exposing the lunate to abnormally higher pressures and potentially increasing the risk of kienbock 's disease (3, 4). kienbock 's disease is also associated with systemic lupus erythematosus (sle) (5 - 8), antiphospholipid antibody syndrome (9), sickle cell anemia (10), and crohn 's enteritis (11). multiple hereditary osteochondromata (12), carpal coalition (13, 14) and congenital shortening of the ulna in langer - giedion syndrome (15), are other anatomic abnormalities that have been reported with kienbock 's disease. rheumatic diseases, including scleroderma (16 - 18), rheumatoid arthritis (19), gout (20, 21) and dermatomyositis (22) have been published in association with kienbock 's, but there have been no identifiable cases in patients with juvenile idiopathic arthritis (jia). this report presents a case of osteonecrosis of the lunate in a patient with jia and no prior history of trauma. furthermore, a literature review is done to illustrate the proposed etiologies of kienbock 's disease and its association with other rheumatologic conditions. a 20-year - old right - handed female with known rheumatoid factor negative polyarticular jia presented to the clinic because of pain and limited range of motion in the left wrist. she was diagnosed with jia at the age of nine after a two - month history of pain and swelling in both hands and knees. during the course of her illness several other joints progressively became involved. during the first year of treatment, she was prescribed nonsteroidal anti - inflammatory medication and low - dose prednisolone. to help control her symptoms she required diseasemodifying antirheumatic drugs. her medications included methotrexate 20 mg weekly, folinic acid 2.5 mg weekly, and etanercept 25 mg twice a week. three weeks prior to presentation, the patient experienced a severe flare of her arthritis due to non - compliance with her medication. this lead to persistent left wrist pain and limited rangeof- motion. on examination she demonstrated synovial thickening of her left wrist with no palpable effusion. magnetic resonance imaging of her left wrist showed mild synovial thickening and erosive arthropathy throughout the carpus, radiocarpal, and carpometacarpal joints. in addition, there is possible sclerosis and edema, since she had collapse of the lunate with mixed signal intensity. plain radiographs revealed negative ulnar variance, sclerosis, and loss of lunate height (fig. the imaging was compatible with stage 4 osteonecrosis of the lunate (23). the patient was managed non - operatively and at two - year follow - up was asymptomatic with no concomitant worsening of lunate osteonecrosis on radiograph. a. coronal t1-weighted fast - spin - echo magnetic resonance image showing collapse of the lunate with mixed signal intensity suggesting sclerosis and edema consistent with kienbock 's disease (arrow). b. antero - posterior plain x - ray of the left wrist demonstrates sclerosis and slight loss of height involving the lunate compatible with kienbock 's disease (arrow). the current literature indicates that most cases of kienbock 's disease develop without a history of trauma and posit that e extraosseous and intraosseous blood supply to the lunate have a role in the disease process. the extraosseous blood supply is formed by a series of dorsal and volar vascular arches (1, 24). the intraosseous blood supply is made up of braches entering the volar and dorsal poles however the composite of this vasculature is variable (1, 25 - 27) ; branches demonstrate different anastomosis patterns inside the lunate, or the lunate may be supplied by only one dorsal or volar branch or by both a dorsal and volar arterial supply the lunate without any anastomosis (26). when an anastomosis does exist, it can be characterized as a y, x, or i pattern, depending on the amount of vessels supplying each pole (24, 27). depending on the intraosseous vascular anatomy of the lunate, certain lunate bones are predisposed to kienbock 's disease. this concept was highlighted in a case report of kienbock 's disease associated with sickle cell anemia (10). the osteonecrosis was thought to have developed from an at - risk lunate - single volar arterial supply - along with significant vascular sickling and stasis. pathogenesis of osteonecrosis of the lunate venous congestion has also been attributed to the pathogenesis of kienbock 's disease (2). during surgery, jensen measured increased pressure inside the lunate compared with the radial styloid and capitate. he concluded that the higher pressure was caused by venous congestion leading to osteonecrosis of the lunate. negative ulnar variance is also implicated in the pathogenesis of kienbock 's disease (3, 4, 28). the altered relationship between the ulna and radius at the distal radioulnar joint modifies the biomechanics at the radiolunate joint and increases strains on the lunate. this postulation is still controversial because several studies, including a meta - analysis, have shown that negative ulnar variance is not a risk factor for developing kienbock 's disease, (29, 30). on the contrary, ledoux. performed a finite - element analysis on cadaveric lunate bones and found that the progression of a fracture of the lunate was present with negative ulnar variance, a high lunate uncovering index, which is the amount of lunate outside the lunate fossa of the radius compared to the amount of lunate articulating with the lunate fossa, and angulated trabeculae (31). this suggests that given the circumstance, the lunate can be at risk for developing osteonecrosis due to abnormal stresses. further cases have also reportedpatients with conditions that may have caused altered stresses on the lunate. in particular, two cases have been reported involving carpal coalition (13, 14). it was postulated in these cases that carpal coalition caused a progressively increasing stress on the lunate, which in turn led to kienbock 's disease. schuind. reported a case of kienbock 's disease associated with congenital shortening of the ulna as seen in langer - giedion syndrome (15). it was suggested that kienbock 's disease developed from microfractures sustained by an abnormal stress distribution (15). multiple hereditary osteochondromata in the forearm was also found in association with kienbock 's disease and was attributed to an excess load on the lunate by negative ulnar variance, but with no carpal slip (12). systemic lupus erythematosus has been associated with avascular necrosis of bone. in 1977, urman presented several cases of patients with sle and osteonecrosis of the carpal bones, including a case report of a patient with sle and kienbock 's disease (8). the patient also had a history of raynaud 's phenomenon and was taking highdose corticosteroids. in sle patients treated with corticosteroids and who developed osteonecrosis, there was one patient who developed kienbock 's disease (5). this patient was treated with large doses of corticosteroids compared to those who did not develop kienbock 's disease. mok. reported a case of bilateral kienbock 's disease in a patient with sle (6). the patient described in this case report was never treated with corticosteroids and the etiology of kienbock 's disease was thought to be due to a vasculopathy caused by either vasculitis or antiphospholipid antibodies, even though this patient tested negative for lupus anticoagulant and anticardiolipin antibodies. more recently, taniguchi. described two cases of kienbock 's disease in sle after taking high doses of steroids (7). one of the cases was of a patient who developed bilateral osteonecrosis of the lunate. it is apparent that the cases of kienbock 's disease in patients with sle were either attributed to high dose corticosteroid use or to the disease itself. more over, patients with crohn 's disease who use corticosteroids to control disease symptoms are known to develop kienbock 's disease and osteonecrosis of the hip (11). the same scenario also occurred in a patient with dermatomyositis taking high doses of corticosteroids for sixteen months (22). scleroderma associated with kienbock 's disease was first reported by agus. in a patient with bilateral osteonecrosis of the lunate (16). this patient had severe raynaud 's phenomenon and was never treated with corticosteroids. the contributing factors were hypothesized to be vasculopathy, raynaud 's phenomenon, and a lunate consisting of a single nutrient vessel. ribbans also reported a case of kienbock 's disease in a patient with scleroderma and severe raynaud 's phenomenon (18). the vasculopathy, scleroderma, and repeated use of the patient 's affected wrist predisposed this patient to kienbock 's disease. matsumoto. reported three cases of kienbock 's disease in three patients with scleroderma, two without a history of steroid use and one who only used low dose steroids prior to the diagnosis of osteonecrosis (17). all three patients also had limited skin involvement, but had severe raynaud 's phenomenon. they reported that scleroderma related vascular disease was likely the cause of the circulatory impairment leading to osteonecrosis. mok. reported a patient with rheumatoid arthritis who was found to have osteonecrosis of the lunate. they believed that the kienbock 's disease occurred in this patient due to an increase in intra - articular pressure within the wrist compartment, causing impedance of venous return, vascular insufficiency to the lunate, and subsequent osteonecrosis (19). in addition to the various risk factors mentioned above, kienbock 's disease b is prevalent in specific patient populations. rooker. found an increased prevalence of kienbock 's disease in a group of patients with cerebral palsy (9.4%) (32). this was thought to be related to an abnormally flexed posture of a spastic wrist, which was present in all patients with kienbock 's disease in this study and could impede blood flow.. also found an increased prevalence of kienbock 's disease in patients with cerebral palsy (2.7%) (33). they believed that the high muscle tone across the wrist in an ulnar negative wrist caused an increased pressure on the lunate. this cause is consistent with repeated microtrauma leading to vascular compromise and ultimately osteonecrosis. while the association between kienbock 's disease and several of the reported cases could be coincidental, having a rheumatologic comorbidity such as jia could represent a risk factor for kienbock 's disease, as seen in the presented case report. the wrist is the second most common site of growth abnormality in patients with jia (34). several changes occur at the wrist, including narrowing of the intercarpal spaces, premature ossification of the carpal bones, and early fusion of the ulnar epiphysis leading to a shorter ulna (negative ulnar variance) (35). the wrist ultimately becomes displaced ulnarly and volarly leading to a dislocation of the wrist and bayonet deformity. therefore, it is possible that the abnormalities in the wrist associated with jia could lead to abnormal stresses and or pressures in the wrist that led to kienbock 's disease. furthermore, the erosive changes in other carpal bones may lead to a change on the normal force patterns in the patient 's carpus. seven years prior to the onset of kienbock 's disease, our patient was also treated with low - dose corticosteroids which could have also disrupted circulation and led to the development of lunate osteonecrosis. several theories attempt to explain its pathogenesis, which suggests that it may be multifactorial. many risk factors have also been identified ; steroid treatment, a predisposing rheumatologic disease, a variation in lunate blood supply, and possibly negative ulnar variance. it also suggests that kienbock 's disease could be a possible cause of wrist pain and stiffness in patients with jia. | kienbock 's disease or osteonecrosis of the lunate is an uncommon cause of wrist pain.. though there have been several reports of cases in patients with various rheumatologic diseases, the precise etiology has currently not been established. we report a case of kienbock 's disease that occurred in a patient with juvenile idiopathic arthritis. to our knowledge, this is the first case report with an association between these two conditions. |
pharmacopuncture refers to be in the limelight acupuncture therapy that treat diseases by using techniques such as warming (chunghwa), alcohol extraction or pressing to extract drugs from one or more oriental medicines and then injecting them into specific acupoints related to the disease and adjusting meridian functions. cathami semen is the seed of the safflower and is composed of 20%-30% fatty oil (acids), whose main constituents are glycerides, linoleic acid, and oleic acid, as well as serotonin, serotonin conjugate, and serotobenin. carthamus tinctorious l. contains safflower - yellow cathamin, which reportedly extends blood vessels, excites the uterus, and reduces blood cholesterol. in oriental medicine, carthamus tinctorious l. has warmness, which can affect detoxification, pain relief and blood circulation. carthmi - flos herbal acupuncture (cf), a representative meridian pharmacopuncture medicine, is an oriental medicine made by extracting and refining oil from cathami semen. cf was prepared in a sterile laborotary at the korean pharmacopuncture institute (kpi). after the seeds had been cleaned, ground and peeled, they were screw pressed on a prepared mat. water soluble carthmi - flos herbal acupuncture (wcf) was prepared by homogenizing cf, water for injection (wfi) and an emulsifier. pharmacopuncture with carthamus tinctorious l. is a representative pharmacopuncture method whose efficacy was demonstrated by many studies. various studies on the effects of pharmacopuncture with carthamus tinctorious l. in treating painful diseases, such as anti - pain effects in an arthritis model on experimental animals, pain - relief effects in degenerative knee arthritis patients, and effects in reducing backache according to the oswestry disability index, were conducted, as were studies on immune - function and blood - vessel effects, including its anti - carcinogenic activity and its effect on suppressing blood clots induced by endotoxin. other studies addressed hematological diseases (the treatment of alopecia areata by using pharmacopuncture with carthamus tinctorious l.), and gynecological diseases (the treatment of oligomenorrhea with pharmacopuncture and carthamus tinctorious l.). thus, the effects of pharmacopuncture combined with carthamus tinctorious l. have been demonstrated in a variety of different fields. studied the safety of pharmacopuncture with carthamus tinctorious l. against skin irritation and eye irritation, and an. verified its safety through acute and subacute toxicity experiments. because pharmacopuncture with carthamus tinctorious l. is expected to show gradual diversification of clinical use as an oriental medicine, studies on toxicity, dose, and side effects must be pursued because pharmacopuncture inserts foreign substances into the human body. in this study, single intramuscular- dose toxicity tests were performed on rats to verify the safety and to identify any side effects of wcf prepared using carthamus tinctorious l. preliminary study was performed at biotoxtech by non - good laboratory practices (glp). these tests were performed by biotoxtech according to glp and the standards of the ministry of food and drug safety as requested by the kpi. wcf was developed to increase the treatment effects by increasing absorptivity and to reduce the side effects by reducing residue of existing cf. wcf was prepared in three steps : in step 1, cf, wfi and primary emulsifier were mixed together and homogenized for 30 minutes by using a high - speed homogenizer. emulsifier was added in step 2 and homogenized at 30 - 80. in step 3, the temperature was reduced, and the mixture was homogenized to yield wcf. the rats, 24 males and 24 females, were 5-weeks old with body weights of 111.8 - 135.8 g and 107.4 - 127.9 g, respectively. general symptoms and body weights were observed to verify that no problems existed with the animals. during the grace period, individual subjects were marked, and at the end of the grace period, 20 males and 20 females closest to the standard body weight were selected and randomly divided into 4 groups of 5 male rats and 5 female rats so that the mean weights of the four groups were uniform. solid feed (teklad certified irradiated global 18% protein rodent diet 2918c) and filtered tap water (cheongju tap water filtered using a filter water sterilizer and irradiated with ultraviolet rays) were freely available. the breeding environment was as follows : a temperature of 20.0- 22.8, relative humidity of 48.5%-65.8%, 10- to 15-hour ventilation, 12-hour / day lighting cycle, and illuminesence of 150 - 300 lux. the clinical dose of wcf was 1.0 ml per application, as no fatalities had been observed at doses up to this value in preliminary tests (biotoxtech study no. : thus, under consultation with the test s requester, 1.0-ml/ animal was configured as the high dose. medium and low doses were 0.5- and 0.1-ml / animal ; the same amount of saline solution, 1.0-ml / animal, was injected into the control group. (table 1) injection was performed using a disposable syringe (1 ml, 26 g). single doses were injected into the left femoral muscle for the low - dose and the medium - dose groups. for the control and the high - dose groups, single injections, 0.5 ml / site, were done into the left and the right femoral muscles. on the day of injection (day 0), clinical symptoms and fatalities were observed at 30 minutes and at 1, 2, 4 and 6 hours after injection. from day 1 to day 14, general symptoms were observed once a day. body weights were measured (cp3202s scale) on days of injection (before injection) and on days 3, 7 and 14. for the hematology test, blood was collected from the abdominal aorta on day 15 after applying anesthesia by isoflurane. hematological analyses were carried out by placing 1 ml of collected blood into a tube containing ethylenediaminetetraacetic acid, which was then analyzed using a blood corpuscle analyzer (advia 120, siemens, germany). for the coagulation test, about 2 ml of collected blood was placed in a tube containing 3.2% sodium citrate and centrifuged for 10 minutes at 3,000 rpm, after which the blood plasma was collected. measurement was done using a coagulation time analyzer (coapresta 2000, sekisui, japan). the prothrombin time and the activated partial thromboplastin time (aptt) were measured. after completing the hematology tests, for the clinical chemistry test, we centrifuged the remaining blood for 10 minutes at 3,000 rpm and collected the blood serum. a clinical chemistry analyzer (7180, hitachi, japan) and an electrolyte analyzer (avl9181, roche, germany) were used. visual inspection of all body organs and tissues was performed on all animals after necropsy. body organs and tissues were extracted and fixed in 10% neutral buffered formalin, and histopathological observations were carried out. body - weight, hematology and clinical chemistry results were tested using sas (version 9.3, sas institute inc. bartlett tests were performed for homo - scedasticity (significance level : 0.05). for homo - scedasticity, one - way analysis of variance tests were performed to yield the significance (significance level : 0.05), and multiple dunnett s t - tests were carried out (significance level : 0.05 on both sides and 0.01). for hetero - scedasticity, significance (significance level : 0.05) was checked using the kruskal - wallis test. accordingly, computation of the ld50 was impossible ; however, a single intramuscular dose of wcf at 1.0 ml / animal caused no mortalities. (table 2) no abnormal clinical symptoms (type of toxicity symptom, time of expression, time of recovery, etc.) were observed on the day of injection (day 0) at 30 minutes and at 1, 2, 4 and 6 hours after injection, as was the case for days 1 to 14 after injection. all groups showed a continued increase in body weight, but the differences among the groups were not significant. tables 3, 4, 5) hematological analyses on the ratsblood showed wcf to have no effect. however, aptt (sec) mean of the g3 female group showed a statistically significant change (p < 0.01),but it was a miscellaneous change with no dose dependence, clinical meaning or toxicological meaning. clinical chemistry analyses showed no significant differences for any of the measured items between the normal saline and the wcf groups. in no male or female experimental group were visual abnormalities observed. (tables 6, 7, 8) on the histopathological tests, one female in the highdose group showed infiltration of mononuclear cells and a multi - nucleated giant cell around eosinophilic materials caused by wcf in an area similar to a cross section of muscular fiber, and 8 eosinophilic materials were clustered at a local part. wcf is prepared by homogenizing a mixture of cf, wfi and emulsifier and increases the treatment efficacy while maintaining the characteristics of existing cf. the effects of cf have been demonstrated by various studies ; i.e., cf can be used for pain relief, is effective for a variety of different pains, increases regional cerebral blood flow significantly, and can be used in a variety of different fields with different effects such as anti - carcinogenic [7, 12 ] and anti - oxidative effects. in this experiment, an intramuscular - dose toxicity test of wcf was performed by biotoxtech to evaluate the safety of wcf manufactured by the kpi according to the glp. no mortalities occurred in any group. also, observations taken after injection of wcf showed no abnormalities in the clinical symptoms of the four groups. hematologically, a change in the aptt (sec) mean value of the g3 female group was observed, but had no clinical or toxicological meaning. finally, histopathological observation showed that wcf caused a change in one female in the high - dose group. visual examination showed the subject to have suffered infiltration of mononuclear cell and to have had a multi - nucleated giant cell around eosinophilic material. studies on the side effects and the safety of cf are continuously being conducted. according to, pain, flare and edema after 4 days, side effects, such as flare and edema, mostly disappeared. they were thought to have occurred due to a mixture of highly - toxic drugs in the oil press during cf during preparation. suppuration and pain appeared in the surgical parts in 1 patient who received cf therapy of 0.1 ml/1 time / week for 8 months and 1 patient who received cf therapy of 0.1 ml/1 - 3 times / week for 3 months. grade - minimal unlike in, the side effects were deemed to be due to an accumulation of cf in the human body from long - term therapy. the safety of cf was reported in based on a toxicity test of cf, but this was only a short - term study on acute and subacute toxicity. chronic toxicity tests on cf must be carried out in the future. although histological abnormalities caused by wcf were found in a subject of the high - dose group in this experiment, according to, no histological abnormalities were observed in sprague - dawley rats treated with cf 3 times a week for 1 - 2 weeks. however, the group with 4 weeks of therapy showed histological ambiguity regarding the boundary of muscular tissues, observation of flares, and induction of inflammations. a histological study with a greater number of subjects and a longer period is deemed necessary to examine long - term or high - dose effects of cf or wcf. the safety of cf was demonstrated, but its side effects were presented in several studies. thus, the toxicity and the safety of wcf must be investigated, and long - term histological studies based on large numbers of subjects are deemed necessary. the results of single intramuscular - dose toxicity tests with wcf on 4 groups of sprague - dawley rats, 3 experimental groups (0.1 ml / animal, 0.5 ml / animal and 1.0 ml / animal) and 1 control group (1.0 ml / animal of saline solution), are as follows : in all groups no mortalities occurred. there was no abnormalities in clinical symptoms, body - weight, hematology tests, clinical chemistry tests, necropsy, histopathological tests. in 1.0 ml / animal dose of wcf, this study do not show wcf s safety of human body. to examine the safety of wcf, we need to carry out testing to check whether or not the toxicity would have negative effects on human body. | objectives : this experiment was conducted to examine the toxicity of water soluble carthmi - flos herbal acupuncture (wcf) by administering a single intramuscular dose of wcf in 6-week - old, male and female sprague - dawley rats and to find the lethality dose for wcf.methods:the experiment was conducted at biotoxtech according to good laboratory practices under a request by the korean pharmacopuncture institute. this experiment was performed based on the testing standards of toxicity test standards for drugs by the ministry of food and drug safety. subjects were divided into 4 groups : 1 control group in which normal saline was administered and 3 test groups in which 0.1, 0.5 or 1.0 ml of wcf was administered ; a single intramuscular dose was injected into 5 males and 5 females in each group. general symptoms and body weights were observed / measured for 14 days after injection. at the end of the observation period, hematological and clinical chemistry tests were performed, followed by necropsy and histopathological examinations of the injected sections.results:no mortalities were observed in any group. also, symptoms, body weight, hematology, clinical chemistry and necropsy were not affected. however, histopathological examination of the injected part in one female in the 1.0-ml group showed infiltration of mononuclear cells and a multi - nucleated giant cell around eosinophilic material.conclusion:administration of single intramuscular doses of wcf in 3 groups of rats showed that the approximate lethal dose of wcf for all rats was in excess of 1.0 ml, as no mortalities were observed for injections up to and including 1.0 ml. |
stress may be broadly defined as the constellation of physiological and behavioral responses to any challenge that overwhelms, or is perceived to overwhelm, selective homeostatic systems of the individual (selye, 1980 ; day, 2005). a hallmark feature of stress entails activation of the hypothalamic - pituitary - adrenal (hpa) axis. this neuroendocrine cascade is initiated when visceromotor neurons in the paraventricular nucleus of the hypothalamus (pvh) stimulate the release of pituitary adrenocorticotropic hormone (acth) into the bloodstream, which, in turn, activates glucocorticoid (gc ; cortisol in humans, corticosterone in rodents) secretion from the adrenal gland (antoni, 1986). gcs are the end - products of hpa axis activation, and facilitate catabolic processes throughout the body during stress by increasing energy metabolism and utilization. gcs also have activating effects on cardiovascular output, and inhibit non - essential processes, such as immune and reproductive functions. finally, hpa axis activation during stress alters cognitive and emotional processes relevant for behavioral adaptation (e.g., shors., 1992 ; mcintyre., 2003). despite the critical role that stress plays for adaptive coping and survival of the individual, it is widely implicated in the onset of psychiatric disease, most notably depression and post - traumatic stress disorder (kessler, 1997 ; yehuda, 2002). initial studies revealed that patients hospitalized for major depressive illness commonly manifested hypercortisolemia and hpa axis insensitivity to gc receptor agonist treatment (i.e., dexamethasone supression test ; carroll., 1976). a wealth of research implicates elevated gcs in compromised brain function, disruptions in the neural circuits imparting negative feedback control over the hpa axis, and further endangerment of brain regions targeted by gcs (for reviews, see sapolsky., 1986 ; conrad, 2008). since the neural substrates providing restraining influences over the stress axis are also regions that play important roles in cognition and emotion, elevated gc levels and hpa axis dysregulation may be key steps in producing the disordered thought and affect that characterize stress - related mental illnesses. animal models of repeated stress (e.g., chronic variable stress, chronic intermittent stress, chronic social defeat stress) have proven useful for modeling hpa axis hyperactivity and depression - like behaviors, and would appear to provide the appropriate setting for teasing apart the role of the hpa axis in the pathogenesis of depression. however, progress has been hampered by the fact that the neural circuitry and mechanisms accounting for limbic forebrain control over the hpa axis have proven difficult to unravel. while a number of these candidate regions have been implicated in hpa axis inhibition during emotional stress (herman., 2003 ; radley and sawchenko, 2011), none of these cell groups provide any appreciable direct innervation of the pvh. combined pathway tracing and immediate - early gene mapping studies have helped to identify a number of candidate cell groups that could serve as disynaptic relays to interface between forebrain regulators and the pvh. the picture that emerges is one involving a complex network of higher - order structures interconnected in a parallel and multisynaptic manner with the pvh (cullinan., 1993 ; roland and sawchenko, 1993 ; van de kar and blair, 1999 ; herman., 2003). here we highlight recent advances in our research suggesting an entirely different organization for limbic forebrain control over the stress axis : one involving convergence onto a circumscribed cluster of gabaergic neurons within the anterior bed nuclei of the stria terminalis (abst), that, in turn, directly inhibits the pvh and hpa activation. this model has several implications for neural circuits and mechanisms underlying hpa axis control and gc - dependent negative feedback. an unforeseen but not incidental feature is that this model helps to clarify the sequelae of chronic stress - induced hpa axis hyperactivity, whereby structural reorganization within limbic forebrain cell groups (i.e., synapse loss / gain) throughout the network leads to an attrition of hpa axis control. over the years, attempts to organize stressors into a taxonomical framework have resulted in two major groupings, physiological (a.k.a., systemic), and emotional (a.k.a., neurogenic, psychogenic) (fortier, 1951 ; allen., 1971). more recent immediate - early gene mapping as a generic index of cellular activation in stress - related circuits has helped to provide a considerable degree of face validity for these distinctions (cullinan., 1995 ; li and sawchenko, 1998 ; dayas., 2001). physiological stressors are generally considered to involve more targeted challenges that overwhelm selective homeostatic systems, such as hemorrhage, hypoxia, or immunogenic stimuli. emotional stressors require interpretation by exteroceptive sensory modalities and integration with distinct cognitive (comparison with past experience) and affective information processing systems in the brain (herman and cullinan, 1997 ; sawchenko., 2000 ; whereas each class of stressor enlists brainstem and hypothalamic effectors for activation of the sympathoadrenal and hpa axis output, emotional stressors manifest widespread activation in the limbic forebrain, and correspond to a broad array of behavioral changes (e.g., vigilance, fear, anxiety) that help to facilitate adaptive coping as required by the specific environmental demand (cullinan., 1995 ; campeau., 1997 ; li and sawchenko, 1998 ; dayas., 2001 functional and lesion studies implicate a network of limbic forebrain cell groups in the inhibitory control of hpa activation during emotional stress (cullinan., 1995 ; herman and cullinan, 1997 ; akana., 2001 ; noteworthy examples of regions implicated in hpa axis inhibition are the septum (feldman and conforti, 1980b), posterior paraventricular nucleus of the thalamus (pvtp ; jaferi and bhatnagar, 2006), ventral subiculum (vsub, the region issuing extrinsic projections of hippocampal formation involved in stress regulation ; herman., 1995), and mpfc (diorio., 1993). these cell groups are conspicuously lacking in any direct innervation of hpa effector neurons within the pvh, instead issuing projections throughout numerous basal forebrain and hypothalamic structures (sesack., 1989 ; cullinan., 1993 ; herman., 2003). many of these regions (notably, mpfc and hippocampal outputs) give rise to predominantly excitatory projections, utilizing the neurotransmitter glutamate (malthe - sorenssen., 1980 ; walaas and fonnum, 1980 ; ottersen., 1995), implicating a hitherto unknown, gabaergic relay. previous work has identified candidate gabaergic cell groups (i.e., preoptic area, abst, posterior bst, dorsomedial hypothalamic nucleus, pvh - surround regions) between vsub and pvh (cullinan., 1993), laying a foundation for understanding how controls over the axis may be organized. nonetheless, whether influences from vsub, and other hpa - inhibitory cell groups, are mediated via several disynaptic relays arranged in parallel to each other, and which relays are capable of integrating inhibitory signals from the limbic forebrain during emotional stress, has remained elusive. our starting point into this problem was to first address the nature of mpfc involvement in acute emotional stress - induced hpa activation, and we have shown that distinct subregions of mpfc differentially modulate the stress axis (radley., 2006a). these studies were inspired from the idea that a variety of other functions subserved by mpfc are differentiated in a dorsal - to - ventral manner (morgan and ledoux, 1995 ; heidbreder and groenewegen, 2003). indeed, previous reports in the stress literature tended to treat the mpfc as a homogeneous structure, and discrepancies remained concerning the nature of mpfc 's influence (excitatory or inhibitory) on hpa output (sullivan and gratton, 1999 ; akana., 2005). through a series of experiments employing discrete excitotoxin lesions in cortical subfields of mpfc, we found that lesions of dorsal mpfc (encompassing prelimbic cortex, pl, and portions of dorsal anterior cingulate cortex, acd) enhanced, whereas ventral mpfc (infralimbic cortex, il) lesions inhibited hpa activation in response to acute restraint stress (radley. furthermore, dorsal mpfc lesions resulted in a prolonged elevation of plasma corticosterone after the cessation of restraint, which is consistent with its role as a target site for gc negative feedback under normal conditions (diorio., 1993) (figure 1). top : darkfield photomicrographs showing corticotropin - releasing factor (crf) mrna expression in the paraventricular nucleus of the hypothalamus (pvh) as a function treatment condition. restraint stress results in a marked increase in crf mrna expression in the pvh dorsal medial parvicellular subdivision of intact animals, which is enhanced in prelimbic (pl)-lesioned rats. no such enhancement of stress - induced crf mrna expression was seen in infralimbic (il)-lesioned animals. bottom : stress exposure also significantly increases plasma corticosterone (cort) levels in sham - lesioned animals. pl lesions result in a prolonged increase in stress - induced plasma cort, while il - lesioned animals show a more rapid recovery to pre - stress levels. differs significantly from basal (0 min) values from within each group, p < 0.05 ; differs significantly from sham - lesioned animals for a given time point, p < 0.01. follow - up work has shown that a discrete cluster of gabaergic neurons in abst forms the missing link in a circuit conveying hpa - inhibitory influences of pl during emotional stress (radley., 2009). first, functional neuroanatomical experiments assayed for sources of gabaergic input to pvh whose sensitivity (i.e., as measured with fos activation) to an acute stress or (restraint) was diminished by dorsal mpfc lesions. of the stress - sensitive, gabaergic, pvh - projecting regions analyzed, a circumscribed region in the abst (corresponding to the dorsomedial and fusiform subdivisions of dong., 2001) was exclusive in showing a decrement in fos activation following pl lesions (radley., 2009). by contrast, il lesions were noted to attenuate fos activation in pvh - projecting neurons in the same region, albeit in a subpopulation of non - gabaergic neurons (figure 2). top : brightfield photomicrograph showing stress - induced fos immunoreactivity (black nuclei) and fluoro - gold (fg ; brown cytoplasm) in anterior bed nuclei of the stria terminalis (abst). retrogradely - labeled cells are concentrated in fusiform (fu) and dorsomedial (dm) subnuclei of abst following tracer injections centered in the pvh. inset : coronal section showing the approximate location of abst corresponding to the region comprising the relevant subdivisions (red box). middle left : following restraint stress, cells doubly - labeled for fos and fluoro - gold (arrows) are abundant in sham - lesioned animals. middle right : concurrent labeling for fos (brown) with glutamic acid decarboxylase (gad67) mrna (black grains) showing comparable increases in doubly - labeled cells (arrows) in the sham - lesioned group following restraint stress. bottom : mean + sem number of neurons co - labeled for fos and fluoro - gold, and for fos and gad67 mrna, in abst of treatment groups. whereas both pl and il lesions reliably diminished stress - induced activation of pvh - projecting neurons in abst, only pl lesions resulted in a decrease in the activation of gabaergic neurons in this subregion, implicating different relays for prefrontal modulation of the stress axis. differs significantly from sham - lesioned control animals, p < 0.05. differs significantly from sham - lesioned stressed animals, p < 0.05. portions of these data have been derived from radley. data on il lesion effects on stress - induced abst activation are previously unpublished. in a second series of experiments, functional ablation of gabaergic neurons in abst recapitulated the effects of pl lesions on acute stress - induced hpa activation (radley. these studies were performed by focally administering an immunotoxin in abst that preferentially ablates gabaergic, while sparing non - gabaergic, neurons (radley., 2009). ablation of gabaergic cell groups in abst enhanced activation of pvh and hormonal indices of hpa axis output in response to acute restraint. previous reports that indiscriminate lesions to abst attenuate stress - induced hpa output (choi., 2007), whereas stimulation of abst may either facilitate or inhibit hpa activity (dunn, 1987), are consistent with the idea that distinct hpa - regulatory influences arise from neurochemically heterogeneous subpopulations. thus, opposing influences of the dorsal and ventral mpfc may commingle within the same region of abst onto separate populations of pvh - projecting gabaergic and non - gabaergic neurons, respectively, to modulate emotional stress - induced hpa output (figure 3). two disynaptic pathways from medial prefrontal cortex (mpfc) are proposed to account for the differential modulation of emotional stress - induced hpa output. whereas evidence highlighted in the text [see also, radley. (2009) ] supports gabaergic neurons in the anterior bed nuclei of the stria terminalis (abst) as interceding for the hpa - inhibitory influences of prelimbic cortex (pl ; red lines), the pathway from infralimbic cortex (il) is suggested, and remains to be verified with functional studies. ac, anterior commissure ; acth, adrenocorticotropic hormone ; ant. pit., anterior pituitary gland ; cc, corpus callosum ; crf, corticotropin - releasing factor ; ot, optic tract ; pvh, paraventricular nucleus of the hypothalamus. subsequent anatomical pathway tracing studies have that pl is the cortical subfield that provides the source of hpa - inhibitory influences that emanate from the mpfc (radley. whereas the subcortical projections of dorsal and ventral mpfc are considered to be highly divergent (e.g., vertes, 2004), their projections to abst distribute in a topographically graded, increasing dorsal - to - ventral manner (radley., 2009). anterograde tracer injections centered in the most dorsal aspect of mpfc (acd) fail to label any projections to abst, more ventrally placed injections label progressively more inputs, with pl providing a moderate innervation of abst, and il providing the densest input. acute restraint stress increases activation of abst - projecting neurons throughout pl and il, and most prominently in the medial - to - rostral aspect of pl (radley and sawchenko, unpublished observations). finally, dual tracing experiments show that pl projections overlap extensively, and make appositions with, pvh - projecting cell groups in abst (radley and sawchenko, 2011). insight into the broader organization of hpa axis control has been gleaned from examination of a second limbic forebrain region implicated in the inhibitory regulation of the neuroendocrine stress response. the hippocampal formation (hf) is similar to mpfc from the standpoint that its extrinsic projections are excitatory, it avoids direct innervation of pvh proper, and is capable of inhibiting emotional - stress induced hpa output (swanson and cowan, 1977 ; walaas and fonnum, 1980 ; canteras and swanson, 1992 ; cullinan., 1993). previous studies have shown that hpa - inhibitory influences of hf are localized to vsub (herman., 1995 ; herman and mueller, 2006), implicating a disynaptic gabaergic relay to pvh. vsub contains the bulk of the extrinsic projections of hf that innervate candidate pvh - projecting gabaergic cell groups, such as various subregions of the bst and hypothalamus, and preoptic area (cullinan., 1993). nonetheless, attempts to define the precise relays subserving vsub influences on the stress axis, or its relation to other relays involved in hpa axis inhibition, had not been previously clarified. we have found that vsub influences on the hpa axis are also interceded for by abst, similar in nature to pl (radley and sawchenko, 2011). first, gabaergic pvh - afferent cell groups in abst showed a diminished functional activation in animals bearing excitotoxin lesions of vsub. in these experiments, vsub lesions were also noted to increase multiple indices of acute restraint - induced hpa activation, as previously reported (herman., 1995). although hf is not typically regarded as one of the more stress - responsive regions in the limbic forebrain (li and sawchenko, 1998), vsub, particularly its abst - projecting neurons, does in fact display a moderate degree of engagement following acute restraint stress (radley and sawchenko, 2011). finally, animals bearing dual tracer deposits show that vsub projections overlap extensively, and make appositions with, stress - sensitive pvh - projecting cell groups in abst. collectively, these studies highlight a neural circuitry from vsub abst (gaba) pvh, with each node in the pathway showing functional activation in response to acute restraint stress, and lesions of vsub resulting in corresponding alterations in output (i.e., decreased abst, increased pvh / hpa activation). a key feature of abst, in addition to its role as a site of convergence, is that it appears to integrate limbic cortical influences (radley and sawchenko, 2011). for instance, animals bearing excitotoxin lesions of both pl and vsub were found to exhibit more exaggerated central indices of stress - induced hpa responses as compared to lesions of either alone. furthermore, ablation of gabaergic cell groups in abst produced a greater enhancement of hormonal indices of hpa activation in response to acute restraint, as compared to animals with vsub lesions alone. given that dual lesions of pl and vsub, or separately, by disruption of their interceding inhibitory relay, result in a greater overall effect on stress - induced hpa output than lesions of either, implicates abst as a key integrator of stress - inhibitory influences emanating from the limbic cortex. indeed, our examination of the projections of pl and vsub reveal extensive overlap in their terminal innervation of pvh - projecting neurons within abst, with evidence of some convergence onto single neurons (figure 4). top : overlap of retrograde tracer injections in pvh (fluoro - gold, fg ; cyan), and anterograde tracers in pl (bda ; green) and vsub (flouroruby, fr ; red) was evaluated in fluorescence preparations using confocal microscopy. instances of bda- (arrows) and fr - labeled (arrowhead) terminals were found to make appositions onto single pvh - projecting neurons in abst, by analysis of single optical planes containing fluorescence labeling for all three markers. bottom row : after a single stress exposure, numerous instances of fos - labeled nuclei are evident in pvh - projecting neurons containing appositions from bda- (left) and fr - labeled (right) terminals. the elucidation of this network should help to address some of the lingering questions concerning the central organization of hpa control. first is the generality of abst as a site of convergence and integration of additional forebrain limbic influences on emotional stress - induced hpa output (figure 5). none of the other forebrain cell groups implicated in the inhibition of emotional stress - induced hpa activity (i.e., septum, posterior paraventricular thalamic nucleus) provides any substantial direct innervation of pvh, although each projects to abst (shin., 2008). thus, abst gabaergic neurons are poised to receive and integrate these along with prefrontal and hippocampal influences. the amygdala is generally considered to exert an excitatory influence on hpa axis activation (prewitt and herman, 1997 ; sullivan., 2004), however, the circuits and mechanisms accounting for this are poorly understood. both cea and mea issue a massive gabaergic input into gabaergic regions of abst (sun and cassell, 1993 ; tsubouchi., 2007), particularly the aforementioned pvh - projecting population. this suggests that excitatory effects on hpa output may be mediated via disinhibition of this modulatory pathway. the basolateral amygdala (bla), which consists predominantly of pyramidal - like glutamatergic neurons, is also implicated in stimulating emotional stress - induced hpa activation (bhatnagar., one likely scenario is for bla to access the pvh via the bla - to - cea pathway widely implicated as the direction of information flow for the genesis of fear - related autonomic and behavioral responses (pitkanen., 1997 ; ledoux, 2000), then proceeding via a cea (gaba) abst (gaba) pvh pathway. proposed role of anterior bed nuclei of the stria terminalis (abst) as an integrator of limbic forebrain influences on emotional stress - induced hpa output. previous work of ours supports the pathways highlighted in red, with abst providing an important source of gabaergic innervation of pvh, and relaying limbic cortical influences. other forebrain cell groups known to influence hpa output (highlighted in black), notably via gc receptor - mediated negative feedback, also project to abst, whose integrated output targets pvh directly. like ventral subiculum (vsub) and prelimbic cortex (pl), these regions do not provide any appreciable innervation of pvh, but do issue projections to the abst. acth, adrenocorticotropic hormone ; amyg, amygdala ; cc, corpus callosum ; crf, corticotropin - releasing factor ; glu, glutamate ; hf, hippocampal formation ; ls, lateral septum ; ot, optic tract ; pit., pituitary gland ; pvh, paraventricular nucleus of the hypothalamus ; pvt, paraventricular thalamic nucleus. the model as proposed may also help to advance our understanding of the circuits and mechanisms accounting for gc receptor - mediated negative feedback. a number of cell groups implicated in inhibiting emotional stress - induced hpa activation (i.e., pl, hf, pvtp, lateral septum) are also capable of imparting gc receptor - mediated negative feedback on the axis (feldman and conforti, 1980a ; jacobson and sapolsky, 1991 ; diorio., 1993 ; jaferi and bhatnagar, 2006). this raises the possibility that abst may integrate steroid - dependent feedback information from the limbic forebrain for conveyance to the pvh. evidence increasingly suggests that gc negative feedback in the limbic forebrain may be mediated via an endocannabinoid signaling mechanism. for example, hill and colleagues (2011) recently reported that gc receptor activation in mpfc neurons mobilizes the release of endocannabinoids and increases excitatory outflow from principal neurons via the presynaptic inhibition of gaba release from local interneurons (hill., 2011). understanding of a broader circuitry for imparting inhibitory influences over the stress axis should allow for assessment of whether gc negative feedback is restricted to upstream mediators, or whether abst can intercede for these influences as a proximate source of steroid - mediated feedback, and, the generality of endocannabinoid signaling in relaying gc - dependent feedback in other components of the network. finally, if the proposed framework is inhibitory in nature, this helps to clarify a fundamentally important question of what drives the initial activation of pvh and hpa output during stress. as previously noted, il appears to exert an excitatory influence on hpa output via a distinct realy in abst, and may comprise one of the upstream components for an activating network. the idea that a non - gabaergic subpopulation of abst neurons relays excitatory influences from il to the pvh is consistent with evidence that indiscriminate excitotoxin lesions in abst reliably attenuate acute emotional stress - induced hpa output (choi., 2007). one proposal from choi and colleagues (2007) is that the non - neuroendocrine crf - expressing subpopulation within abst (corresponding to the fusiform subdivision of dong., 2001) may provide a source of excitatory input into pvh, and future studies will help to clarify this relationship further. at least with regard to physiological stress, hpa output appears to be mediated predominantly via medullary aminergic inputs to pvh, as ablation of this pathway completely blocks central and peripheral indices of hpa activation under exposure to these challenges (ritter. by contrast, this pathway does not mediate hpa activation during acute emotional stress (ritter., 2003 ; schiltz and sawchenko, 2007), and evidence for an equivalent activating system under this category of challenges remains elusive. chronic stress induces profound structural and synaptic changes in a variety of limbic forebrain regions. mpfc (acd, pl, and il) and ca3 hippocampal neurons show regressive alterations in apical dendritic and synapse morphology (watanabe., 1992 ; magarinos and mcewen, 1995a ; cook and wellman, 2004 ; radley., 2004, 2006b ; stewart., 2005 ; hajszan., 2009), whereas amygdala (bla) neurons show increases in these indices (vyas., 2002, 2006). these changes are paralleled by reductions in gray matter volume and functional impairments in mpfc and hf of depressed individuals (sheline., stress - induced structural plasticity is dependent on elevated gcs and excitatory glutamatergic signaling (liu. such structural alterations have been linked with disruptions in learning and memory (luine., 1994 ; stewart., 2005 ; liston., 2006 ; dias - ferreira. conrad, 2010), and increases in anxiety - like behaviors (e.g., elevated plus maze performance ; mitra., 2005). hpa axis hyperactivity (i.e., sensitization, facilitation) is also widely documented to result from chronic stress (ottenweller., 1989 ; dallman., 1992 ; willner, 1997 ; bhatnagar and dallman, 1998 ; figueiredo., 2003a ; weinberg., 2010), although its relation to structural plasticity in the limbic forebrain remains to be thoroughly examined. much of the previous literature relevant to studying the effects chronic stress on structural plasticity entailed examination of dendritic branching patterns. this is likely due to the fact that stress and gcs produce robust changes on neurons that are readily manifest at the morphological level, and that changes in dendritic branching patterns (i.e., complexity, length, branch numbers) generally were thought to correlate with changes in synaptic connectivity. nonetheless, increasing attention has been given to more detailed analyses of synaptic alterations in neural circuits following chronic stress, given their critical role as junctional points of connectivity that mediate information flow between neurons. dendritic spines represent sites of postsynaptic contact for the majority of excitatory synaptic input in cortical structures. spines are highly motile and dynamic structures that exhibit a wide degree of morphological diversity, with alterations in shape and number providing a cellular correlate for learning capacity, learning, and memory (bailey and kandel, 1993 ; sorra and harris, 2000 ; kasai., 2003 ; wilbrecht., for instance, long and thin spines tend to be regarded as immature, and are more abundant during development, whereas mushroom - shaped spines (large diameter head, small diameter neck) represent stronger, more well - established excitatory synapses. a number of studies suggest that chronic stress leads to a net loss of excitatory synapses in pl neurons (radley. notably, dendritic spines in the distal portions of the apical dendritic tree appear to be most profoundly impacted by chronic stress, inclusive of retraction of distal processes and decreases in spine density (figure 6) (radley., 2006b ; liu and aghajanian, 2008). we conducted a high - throughput analysis of over 17,000 dendritic spine morphologies in pl and acd pyramidal neurons (radley., 2008a), and found that chronic stress resulted in an overall decrease in apical dendritic spine density, manifested by a loss of mushroom - shaped spines, and an increased frequency of long and thin spines. another recent report employing two - photon microscopic in vivo imaging of spines in has provided the most compelling evidence to date for the capacity of gcs to mediate stress - induced spine alterations in the cortex (i.e., primary motor, secondary motor, somatosensory ; liston and gan, 2011). whereas acute gc exposure increased the rate of spine turnover (elimination and formation), prolonged gc exposure selectively increased the elimination of spines, particularly ones that were older and more stable. these, and other studies (michelsen., 2007 ; liu and aghajanian, 2008), support the idea that chronic stress, via increases in gc levels, may selectively target the mature, stable population of excitatory synapses throughout cortical structures. summary of effects of chronic stress on structural plasticity in mpfc pyramidal neurons. in these studies (radley., 2004, 2006b, 2008a), high resolution analyses were performed in digitally reconstructed dendritic segments from fluorescent dye - injected pyramidal neurons in dorsal anterior cingulate (acd) and prelimbic (pl) areas. an atlas plate (lower left) depicts the approximate region within mpfc that neurons were filled for morphologic analyses. arrows highlight the fact that dendritic atrophy and spine / excitatory synapse loss is most prominent on distal apical dendrites (right). spine morphologic analyses reveal fewer spines with mature (stubby, mushroom - shaped), and a greater number with immature (long and thin) phenotype. chronic stress has also been shown to decrease synapse and spine density in hippocampal neurons (sousa., 2000 ; sandi., 2003 ; stewart., 2005 ; hajszan., in one of the more rigorous demonstrations of this phenomenon, one study employed electron microscopy (em) and stereological 3-d reconstructions in, finding that chronic stress induced significant decreases in dendritic spine density and synapse number, and was reversible following a stress - free recovery period (sandi., 2003). in another study employing em, magarinos and colleagues (1997) reported ultrastructural differences in presynaptic terminals of synapses in the mossy fiber pathway in ca3 neurons following chronic stress, indicative of an up - regulation of presynaptic activity and release of glutamate. taken together, decreases in density and in overall numbers of postsynaptic excitatory contacts may help to limit the extent of excitotoxic damage that would otherwise result from prolonged activation of glutamatergic synapses under chronic stress. a number of studies have begun to identify the cellular mechanisms underlying chronic stress - induced spine synapse loss (for reviews, see arnsten, 2009 ; duman and voleti, 2012). for example, reduced expression of certain neurotrophic / growth factors (notably, brain - derived neurotrophic factor) in the hippocampus, and more recently in mpfc, may contribute to dendritic spine synaptic compromise in these regions (liu., 2012 ; alterations in protein kinase c signaling have also been shown to underlie dendritic spine loss in mpfc (hains., 2009). finally, the mammalian target of rapamycin (mtor) signaling pathway has recently been implicated in synaptic deficits that result from excessive glutamatergic stimulation, such as that which ensues under chronic stress (magarinos and mcewen, 1995b ; li., these studies highlight potentially important cellular mechanisms for investigating their role in the circuit alterations underlying neuroendocrine adjustments following chronic stress. from the network perspective, large - scale decreases and destabilization of the excitatory synapse population in mpfc and hf could uncouple excitatory afferent input from excitatory outflow in pl and/or hf, resulting in their diminished influence over pvh - projecting gabaergic neurons in abst. concurrent increases in bla neuronal dendritic branching and spine densities could also drive disinhibition of pvh - projecting gabaergic neurons, via increasing activation in the extrinsic gabaergic projections from cea. one challenge concerns whether changes throughout the entire network are necessary for hpa axis hyperactivity following chronic stress, or whether this phenotype is regulated by a distinct pathway or mechanism. as many of the stress - related changes in hf have been demonstrated more dorsally in ca3, and to some extent in dg and ca1 (e.g., sousa., 2000 ; snyder., 2011) it is unclear whether vsub serves as a way station, or whether stress effects within vsub proper (or ventral hippocampus) account for alterations in excitatory outflow to abst. another issue concerns the fact that little is known about how chronic stress impacts other cell groups implicated in the stress - inhibitory network, such as pvtp and lateral septum. the fact that gcs appear capable to exert widespread effects throughout the cortex, inclusive of sensorimotor regions (liston and gan, 2011), poses additional challenges in teasing apart neural circuits that underlie stress - related behavioral and physiological alterations. it has been previously established that the hpa axis response to emotional stress involves a network of limbic forebrain afferents that exert their effects on the pvh via multisynaptic and parallel pathways. recent evidence lends support for at least two limbic cortical regions, mpfc and hf, that impart their inhibitory influences over the stress axis by converging on a discrete target, the abst, that in turn inhibits the pvh and hpa activity. importantly, gabaergic neurons in the abst exhibit the capacity to integrate the inhibitory prefrontal and hippocampal influences that they impart on the stress axis. there are a number of hypotheses that derive from this model that should help to inform future work. one idea is that abst serves as a neural hub for receiving and integrating stress - modulatory influences from other limbic forebrain regions (i.e., pvtp, septum, amygdala). another implication is that this network, notably via gabaergic relays in abst, may serve to integrate gc receptor - mediated negative feedback signals from some, if not all of, these limbic forebrain regions via a presynaptic endocannabinoid signaling mechanism. as this network is inhibitory, this may help to inform the search for hpa - activating networks ; i.e., something akin to the medullary aminergic inputs to pvh that are known to drive hpa output in response to physiological stressors. chronic stress - induced neuroplasticity throughout the limbic cortex, or within key regions, may lead to an attrition of their excitatory influence on the pvh - projecting gabaergic cell population in abst, producing hpa axis hyperactivity. a key feature of this hypothesis is that regressive changes are evident in regions that normally serve to inhibit hpa axis activation during emotionally stressful experiences, and hypertrophic changes (i.e., increased branching and synapse number) are evident in regions that contribute an excitatory influence on hpa output. whereas considerable gains have been made in understanding the cellular mechanisms underlying dendritic spine dynamics, much of this work has not been applied to enhance our knowledge of how chronic stress leads to long - term changes in neuroendocrine function. moreover, examination of why stress and gcs have bidirectional effects on excitatory synapse plasticity in bla, relative to hf and mpfc, may shed light on what are likely to be categorically distinct effects on the regulation of gene expression in these cell types that are imparted by gcs. finally, susceptibility to stress - related psychiatric illnesses depends on a number of factors (e.g., genetics, early - life experiences, previous stress exposure) that may help to explain why some individuals go on to develop stress - related disorders while others do not. the extent to which structural plasticity in limbic cortical regions is predictive of adaptation or failure of stress / hpa control systems is of fundamental importance for informing the issue of how these more complex hereditary and environmental factors may tip the balance between stress resilience and pathology. the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. | a network of interconnected cell groups in the limbic forebrain regulates hypothalamic - pituitary - adrenal (hpa) axis activation during emotionally stressful experiences, and disruption of these systems is broadly implicated in the onset of psychiatric illnesses. a significant challenge has been to unravel the circuitry and mechanisms providing for regulation of hpa output, as these limbic forebrain regions do not provide any direct innervation of hpa effector cell groups in the paraventricular hypothalamus (pvh). recent evidence will be highlighted that endorses a discrete region within the bed nuclei of the stria terminalis serving as a neural hub for integrating and relaying hpa - inhibitory influences to the pvh during emotional stress, whereas the prevailing view has involved a more complex organization of mulitple cell groups arranged in parallel between the forebrain and pvh. a hypothesis will be advanced that accounts for the capacity of this network to constrain the magnitude and/or duration of hpa axis output in response to emotionally stressful experiences, and for how chronic stress - induced synaptic reorganization in key cell groups may lead to an attrition of these influences, resulting in hpa axis hyperactivity. |
type 2 diabetes mellitus (t2 dm) and obesity are increasingly assuming importance as public health problems in our country, especially among the north indians. both conditions have a complex etiopathogenesis and epidemiology. it is well - known that across the globe indians are genetically and environmentally predisposed to develop metabolic syndrome (ms), diabetes and cardiovascular disease (cvd) at younger age and lower indices of body fat. worldwide increased body iron store (bis) has been emerging as putative risk factor for development of insulin resistance and cvd. basic, clinical, and epidemiological studies have generated enough scientific evidence to suggest that iron overload is proinflammatory and proatherosclerotic. measurement of bis involves use of many variables, of which serum ferritin (sf) has been found to be a reliable tool, providing that confounding effects by inflammatory, hepatic, or neoplastic diseases are excluded. body circumference measures, such as waist circumference (wc) represent adiposity and these have been undisputedly associated with, insulin resistance syndrome and t2 dm, thus wc may be significantly associated with bis as reflected by sf concentrations. this study was carried out to study the relationship of bis with t2 dm and obesity in north indian population. the basic idea was to evaluate the utility of sf as an additional marker of ms ; and to identify the subgroup of individuals at risk for iron - related tissue damage, diabetes and cvd. this was a cross - sectional observational study carried out on a randomized sample of patients presenting to our medical outpatient department for a period of 10 months (february - november 2009). the study population was categorized into four groups consisting of : group a : normal healthy individuals (controls) ; group b : obese nondiabetic individuals ; group c : lean diabetic patients ; group d : obese diabetic patients. this study was restricted to men and postmenopausal women to eliminate the influence of pregnancy, hormones and reduce possible confounding by iron deficiency. the study was also restricted to persons between 40 and 65 years of age to eliminate age related bias in sf values. individuals having recent acute or chronic infectious, inflammatory or neoplastic condition ; or with laboratory evidence of inflammation (c - reactive protein > 0.5 mg / dl or white blood cell count > 11,000/ul, platelet count > 400,000/ul) were excluded. similarly, other exclusion criteria were patients on aspirin, iron, or hormonal therapy. smokers and individuals with alcohol consumption of > 20 g / day were also excluded. individuals who had anemia (hemoglobin 100 fl, or with history of overt blood loss were also refused enrollment. the height was measured to the nearest 0.1 cm, weight to the nearest 0.01 kg, and wc and hip circumference (hc) to the nearest 0.1 cm according to the standard guidelines and equipments. wc was measured in a horizontal plane at the level of the mid - point of the iliac crest and lower rib margin using nonstretchable flexible tape at the end of normal expiration, in the fasting state, with the subject standing erect and looking straight forward and observer sitting in front of the subject. the variables were computed as : waist - to - hip circumference ratio (whr), body mass index (bmi = weight/[height ]). since sf is an acute - phase reactant and does not accurately reflect the body 's iron stores in the presence of inflammation, serum c - reactive protein estimation was done to exclude patients with underlying inflammation. patients were considered to have diabetes if they met at least one of the following definitions : fasting plasma glucose > 126 mg / dl on two or more occasions, postprandial glucose > 200 mg / dl, or a random glucose > 200 mg / dl with symptoms, or patient currently on insulin or oral hypoglycemic agents. patients were considered to be obese if their bmi was 25 kg / m or more ; high wc was defined as larger than 90 cm in men and 80 cm in women. individuals were classified into the groups on the basis of their bmi (25 or more classified as obesity as recommended in the consensus statement for diagnosis of obesity, abdominal obesity and the ms for asian indians and recommendations for physical activity, medical and surgical management) and not on the basis of the wc since we found that even among the nonobese individuals very few individuals had wcs as per the norms established for asian indians, especially among the north indian women. these lower cut - offs for obesity were important in our study population as several investigators have shown that asian indians are more predisposed to develop insulin resistance and cardiovascular risk factors at lower levels of bmi when compared to other ethnic groups. asian indians have different body composition, elevated percentage body fat, and abdominal adiposity at lower or similar bmi levels when compared to white caucasians. in this study, we needed distinctly dissimilar groups to compare the iron stores, thus we decided on lower cut off of bmi as recommended by the consensus statement above. for the estimation of sf, overnight fasting blood samples were obtained, centrifuged at 1500 g for 30 min and frozen at 70 c. quantitative estimation of sf was carried out by using the enzyme immunoassay, org 5fe sf kits procured from the orgentec diagnostika gmbh, carl - zeiss - strae 49 55129 mainz - germany were used. we used the following available reference range of sf (as on the kits) for the age and sex matched population : male (20 - 50 years) : 34 - 310 ng / ml ; female (20 - 50 years) : 22 - 112 ng / ml ; male (51 - 90 years) : 4 - 665 ng / ml ; female (51 - 90 years) : 13 - 651 ng / ml. comparison of the mean sf values of each group was drawn against the control group using the t - test by means of computerized software available (statistical package for the social sciences, spss inc). comparison of the mean sf values of each group was drawn against the control group using the t - test by means of computerized software available (statistical package for the social sciences, spss inc). group a - d comprised of 41 healthy normal controls, 50 obese individuals, 51 nonobese diabetic individuals, and 55 obese diabetic individuals, respectively. as can be observed from tables 1 and 2, the groups are quite distinct as per the mean body mass indices. however as is obvious from the tables, the wc of the females even in the nonobese category is on the higher side. summary of the anthropometric and sf data of males summary of the anthropometric and sf data of females a wide variation was observed in the sf values among the individuals in each group. mean values of sf in all our groups were on the lower side of the reference range available. two individuals each (men) in the control group and the obese group (a and b) and three individual each from the diabetic groups (c and d) had sf in the upper normal range. comparison of the mean sf values of each group was drawn against the control group, that is, group a. when the groups b - d were compared to their sex matched control group a to examine the difference in their sf concentrations, no statistically significant difference was established. thus, our result showed that as supposed the sf was neither increased in the nondiabetic obese nor in the lean or the obese diabetic individuals. the data analysis is shown for the males and females in tables 1 and 2, respectively. iron has been linked to the pathophysiology of various diseases primarily because of its chemical property of getting reversibly oxidized and reduced. this unique property makes iron potentially hazardous because it leads to generation of powerful reactive oxidant free radicals. excess body iron can impose oxidative injury that is associated with several cardiovascular risk factors including dyslipidemia, insulin resistance, and inflammation. intake of dietary iron, especially highly bioavailable heme iron, was recently associated with greater risk of t2 dm and coronary heart disease. many elegant studies have concluded that bis are positively associated with the development of glucose intolerance, t2 dm, and gestational diabetes. a study from finland supports the theory that increased iron stores, even in the range not considered to be associated with hemochromatosis, contribute to the development of noninsulin dependent diabetes. in a turkish study within the diabetic subgroup sf was increased in those participants having poor glycemic control when compared to participants with good control. raj and rajan from india have published their data on 86 diabetic patients and found a positive correlation between sf, fasting blood sugar and hba1c. smotra and kudyar in a similar study concluded that increased sf levels are associated with increased serum insulin reflecting insulin resistance, poor glycemic control and complications of t2 dm. in recent years, increased iron stores have been found to predict the development of t2 dm while iron depletion was protective. most of these hypothesis were derived from the observations that dm is a common complication of hemochromatosis and other iron overload states ; 53 - 80% of patients with hemochromatosis develop diabetes. frequent blood donations, leading to decreased iron stores, have been demonstrated to reduce postprandial hyperinsulinemia in healthy volunteers and to constitute a protective factor for the development of t2 dm. the clinical impact of these intimate interactions depends on the genetic predisposition and the time frame in which this network of closely related signals acts. the mechanisms behind hyperferritnemia in ms may be ferritin gene over expression as a part of adaptive adipocyte response to iron - induced oxidative stress. sf was increased and adiponectin was decreased in t2 dm and in obese diabetic subjects. these findings demonstrate a causal role for iron as a risk factor for insulin resistance. in a population study by martinelli., hepcidin (key iron regulatory hormone) levels increased significantly and linearly with increasing number of ms, paralleling the trend of sf. the authors apprehended that due to the pleiotropic effects of hepcidin, insulin resistance may worsen and contribute to the cardiovascular complications of ms. a novel adipokine visfatin predominantly secreted by visceral adipose tissue has also been found to be significantly associated with parameters of iron metabolism, especially in participants with altered glucose tolerance. it has been postulated that elevated iron stores may induce diabetes by several mechanisms including oxidative injury to pancreatic beta cells, impaired hepatic and muscle insulin extraction, and impaired peripheral glucose disposal. systemic iron overload is not only linked in the causation and induction of diabetes, but also associated with the progression of the disease. iron induced damage has also been shown to modulate the development of chronic diabetes complications such as microangiopathy and atherosclerosis. serum ferritin levels are often elevated in ms (dysmetabolic hyperferritinemia) or associated with a true hepatic iron overload (dysmetabolic iron overload syndrome). however, only a few studies have reported the relationship between the sf concentrations and various indices of adiposity, including the visceral and subcutaneous fat area. in indians, central (abdominal) adiposity is a characteristic feature, which correlates directly with the metabolically active visceral adipose tissue. increased sf levels have been found in overweight and obese women with polycystic ovary syndrome and has been shown to improve with metformin therapy. elevated sf concentrations early in gestation are associated with an increased risk gestational diabetes. the association, at least in part, is mediated by the maternal fat mass and obesity. recently, the european prospective investigation into cancer and nutrition - potsdam study examined the association between biss and risk of type 2 diabetes through a case cohort study. they concluded that high ferritin levels are associated with higher risk of type 2 diabetes independently of established diabetes risk factors. have recently conducted a systematic meta - analysis where they analyzed the association of biss or dietary heme - iron intake with t2 dm risk and have suggested that increased ferritin levels and heme - iron intake are both associated with higher risk of t2 dm. indians in general have been found to have low bis when compared to other asians, partly probably due to the predominant vegetarian diet. it has been also shown in many studies that the mean sf concentration is significantly lower in asian men when compared with the native - americans. in india, few if any studies have examined the relationship of sf and insulin resistance syndrome or diabetes. our study highlights that there is no evidence of iron overload in the nondiabetic obese, lean diabetic and obese diabetic north indians. since our study does not favor iron overload in this population, we may assume that excess iron may not contribute to the pathogenesis of these diseases in indians. it has been observed that mean sfs increased across heme - iron intake quartiles but decreased across nonheme iron intake quartiles. luan. found no significant association between nonheme - iron intake and diabetes and lee. demonstrated that nonheme iron intake was negatively associated with diabetes the most important reason for low sf levels even in the obese and diabetic indians appears to be the predominant vegetarian diet and nonheme iron consumption in our patients. it has been established by various cross - country surveys that nonheme iron contributes about 90 - 95% of total daily iron in indian diets, in contrast to the predominant highly bio - available heme iron in the western diets. further indian diet is plagued by low iron content (largely cereal based) and poor absorption (due to dietary lack of ascorbic acid and alpha tocopherol). the data also suggest that the average iron density of an indian diet is not more than 8.5 mg/1000 kcal. iron and other micronutrient deficient diet coupled with occult gastrointestinal blood loss, dietary restrictions in obesity and diabetes, certain medications and unrecognized renal involvement all may contribute to poor iron stores in indians. other important reason is a very low prevalence of the c282y or h63d gene mutations responsible for iron overload in indians as compared to the caucasians. in a study on nonalcoholic liver disease from india, it was shown that in indians there was no significant association of iron overload with this component of ms. in fact in a small case - control study on indian coronary heart disease patients, the bis were found be lower as compared to controls. having said so, however it strongly needs to be emphasized that iron even if not in excess is still available as catalytic iron to mediate in free radical reactions and its role in inducing oxidative injury and contribution to metabolic abnormalities can not be completely ignored. our observation of normoferritnemia in obesity and diabetes is in sharp contrast to the earlier studies published from the west. further research is needed to elucidate the mechanism and significance of these findings in indians. it is also important that such similar studies are done at community level with larger sample size to impart good statistical power to the study. furthermore, large prospective studies may help to establish or negate a causal relationship between iron overload, ms and diabetes. nevertheless, this data reassures us that public health and individual strategies to supplement iron must continue, without apprehension of precipitating ms or diabetes. | introduction : evidence from various epidemiological and clinical studies suggests that iron overload is proinflammatory and proatherosclerotic. excess body iron has been positively associated with insulin resistance, type 2 diabetes and obesity.aim of the study : to study the relationship of body iron stores with type 2 diabetes and obesity in middle aged north indian population.materials and methods : the participant population consisted of four groups of randomly selected participants (between 40 and 65 years of age and postmenopausal women) ; group a : normal individuals (controls), group b : obese nondiabetic individuals, group c : lean diabetic patients, group d : obese diabetic patients. blood was examined for hematological, biochemical estimations, c - reactive protein, and serum ferritin (sf).observation and results : a total of 197 participants were enrolled. the mean sf levels (ng / ml) among males were : group a (n = 18) 148.56 119.90 ; group b (n = 25) 129.11 94.77 ; group c (n = 27) 127.96 109.65 and group d (n = 22) 148.36 104.94. the mean sf levels (ng / ml) among females were : group a (n = 23) 67.44 37.59 ; group b (n = 25) 59.62 43.56 ; group c (n = 24) 77.97 91.46 and group d (n = 33) 66.46 86.05. no statistical difference was found among the groups in both the sexes.conclusions:our observation is in sharp contrast to the earlier studies published from the west stressing that iron stores are increased in obesity and diabetes. we conclude that sf may not be a strong risk factor in the pathogenesis of obesity and diabetes in middle aged north indians. |
enema administration has been a common practice worldwide for several centuries for the treatment of chronic constipation and for preparation of patient for a diagnostic test or a surgery. it seldom causes so prolonged lower gastrointestinal bleed that requires massive blood transfusion and rarely requires colectomy. we report an unusual complication of herbal enema in the form of severe ileo - colitis with persistent massive lower gastrointestinal bleed immediately after administration of enema for the treatment of chronic constipation. the bleeding was refractory to conservative treatment and was managed with emergency total laparoscopic colectomy. a 57-year - old male patient presented with persistent massive bleeding per rectum for one month that developed immediately after administration of herbal enema by a religious quack for the treatment of chronic constipation. he was admitted at some other hospital for the same complaint and was managed conservatively with oral glucocorticoid, 5-aminosalicylate preparation, metronidazole, entofoam (hydrocortisone acetate) and sucralfate enema. he had history of 48 units of blood transfusions during the course of conservative management to raise the haemoglobin above 8 mg / dl, but the condition did not improve and the patient was referred to our centre for further management. on clinical examination, the patient was pale, had tachycardia (pulse, 136/min.) and hypotension (systolic blood pressure, 70 mmhg). colonoscopy revealed extensive ulcerations and friability of entire colon, more on left colon and ileal intubation also showed multiple ulcerations [figure 1 ]. cect of abdomen showed distended small and large bowel with no air - fluid level. endoscopic view : ulcerations and friability of colonic mucosa we gave the patient a trial of conservative management because the condition was not permissible to withstand a major abdominal surgery. conservative management did not work and once again patient developed massive lower gastrointestinal bleed bringing haemoglobin down to 4.9 g%. patient was planned for emergency laparoscopic total colectomy. on laparoscopy, there was evidence of mild ascites, thick oedematous inflamed and friable large bowel and distal 30 cm of ileum. large bowel was very friable and developed 3 small inadvertent perforations by bowel holding grasper during dissection and manipulation of colon [figure 2 ]. the patient developed hyponatremia and paralytic ileus in early postoperative period that were managed conservatively. oral liquids were started on postoperative day (pod) 3 and after that stoma started functioning. drains were removed on pod5 and patient was discharged on pod 7 with advice of restoration of bowel continuity after 8 - 10 weeks. majority of the population of india is seemingly unaware of the potential hazards associated with herbal medications and of the limited knowledge and diagnostic skill of those who are prescribing such type of treatments. contrary to the widespread belief that because it is natural it is safe, herbal therapy probably carries major risks and produces more serious side - effects than any other form of alternative medicine. the common natural ingredients used in enemas are aloe, coffee, garlic and milk thistle. other types of enemas include the ones made with mineral water, epsom salt, glycerin, vinegar, bark of the marula tree, fruit of the cucumis africanus, various wild herbs, industrial thinner, turpentine, undiluted dettol, ginger, pepper or soap. the injurious side - effects of herbal enema vary from mild abdominal discomfort and self - resolving haemorrhagic proctocolitis to severe colitis. most of the time, colitis responds conservatively and rarely require blood transfusion and colectomy. herbal enema induced massive lower gastrointestinal bleed is a life threatening clinical condition usually refractory to conservative management as in our case. nowadays, the indication for surgery is mainly limited to acute, uncontrollable, and recurrent forms of lower gastrointestinal bleed. there are few studies in literature that address the feasibility of laparoscopic colectomy in emergency conditions. marcello reported in their case - control study comparing laparoscopic total colectomy for acute colitis with a matched open colectomy group that laparoscopic total colectomy is feasible and leads to a faster recovery. the mortality rate from a subtotal colectomy in emergency setting is approximately 20% in most collected series, because of the reluctance of surgeons to perform a subtotal colectomy early and defer it till desperate circumstances arise. on the basis of surgical outcomes of this patient and literature support, we need for spreading awareness regarding the potentially disastrous adverse effects of herbal medications prescribed by quacks and advocate early intervention in such type of severe ileocolitis with massive rectal bleed. | various colonic side - effects of herbal enema have been reported in literature ranging from mild abdominal discomfort to self - limiting haemorrhagic colitis. it rarely requires blood transfusion or subtotal colectomy. we report a 57-year - old male patient developing severe ileo - colitis with persistent massive rectal bleeding immediately after herbal enema administration for the treatment of chronic constipation and was resistant to conservative management. patient was managed successfully with emergency total laparoscopic colectomy. post - operative recovery of the patient was excellent. |
attention - deficit / hyperactivity disorder (adhd) is a psychobiological disorder that is described by attention deficit, hyperactivity, and reduced impulse control. based on the diagnostic and statistical manual of mental disorders, fifth edition (dsm - v), adhd is a persistent pattern of inattention and/or hyperactivity and impulsivity that interferes with functioning or development. the inattention manifests behaviorally in adhd as wandering off task, lacking persistence, having difficulty sustaining focus and being disorganized, and is not due to defiance or lack of comprehension. hyperactivity refers to excessive motor activity (such as a child running about) when it is not appropriate, or excessive fidgeting, tapping, or talkativeness (1). based on dsm - v results, the 3 subtypes that are related to adhd include predominantly hyperactive, predominantly inattentive, and a combination of one or more of the aforementioned types. a child must demonstrate at least 6 of the 9 symptoms and adolescents must demonstrate at least 5 of the 9 symptoms in a cluster form before the 3 subtypes (1). past research illustrated that symptoms related to hyperactivity may decrease as the child grows older, while the child s inattentiveness remains relatively unchanged (2, 3). barkley defined the main characteristic of adhd as difficulty in behavioral inhibition (4). unintentional behavior possessed by the child diagnosed with adhd appeared to affect their academic achievement throughout their academic career. in addition, these children displayed problems with their social skills and difficulty communicating, due to the apparent frustrations with their academic performance (5). the dsm - v shows that the rate of adhd was greater in males than in females, with a ratio of nearly 2:1 in children and 1.6:1 in adults (1). attention deficit includes the combination of biologically determined reactivity of the nervous system and failure to self - regulate, and environmental disturbances (6, 7). students who can not centralize their thoughts are impatient and evoke negative responses from their environment, which can provoke a perception of social refusal. the continued experience of refusal at school mixed with the vulnerability of the child / adolescent nervous system is thought to produce negative perceptions, evoking compensatory behaviors, such as, aggressive comic behavior, feeling alone, and/or a depressive status. to decrease such symptoms, the creation and protection of a supportive and caring home situation would appear to be an important factor. students must have a feeling of being accepted, the ability to develop trusting relationships, and also a belief that there are methods that can help them. this situation can be quite difficult to achieve if the parents displays symptoms similar to their child. adhd indicatorsare not just limited to childhood, and the relative dynamics of this disorder may cause the condition to persevere. based on previous studies, the principal treatments of adhd have focused on the use of medication, behavioral, cognitive, cognitive - behavioral, and neural treatments (8 - 16). a review of the literature between 2000 and 2013 identified the multimodal treatment of adhd for the adhd child and/or adolescent. these combination treatments include self - control, self - regulation, cognitive, cognitive - behavioral, cognitive play, behavioral play, motor - perceptual rehearsal, functions management training, verbal self - education training, education of children by parents, positive therapy, training of parents, parental constructive program, and training of stress coping skills. these studies demonstrated that the mentioned treatments were more successful and had longer lasting effects on attention deficit, social skills, and behavioral problems in adhd than the singular medication treatment or behavioral therapy (17 - 28). in this meta - analysis study, the researcher used published journal articles and theses on the topic of the effectiveness of intervention / treatment programs on adhd from the scientific information database (sid), the iranian research institute for information science and technology (irandoc), the national library and the archives of the islamic republic of iran (nlai), and the united states national library of medicine (nlm) and national institutes of health (pubmed) websites during 2000 - 2013. database search was performed with the following keywords : treatment program and adhd, intervention program and adhd, and instruction. in the preliminary database search, 72 related articles were obtained from sid, 64 dissertations and abstracts from the irandoc website, 23 dissertations and articles from the nlai website, and 175 dissertations and articles from the pubmed website. all findings were screened for inclusion, based on the title and abstract, using the following restrictions regarding the treatment / intervention program on adhd. after reviewing the preliminary articles and theses on adhd, a total number of 17 articles were screened for possible inclusion (table 1). these articles had to meet each of the subsequent criteria : the article must have been an original investigated report (experimental, quasi - experimental research), included treatment of at least one independent variable, and the students must have been diagnosed with adhd. articles excluded if they did not meet the above criteria, and if the students intelligence quotient scale (iqs) were below 90, or if data for the adhd students could not be separated from data of the group without adhd. a total of 17 articles were screened for inclusion in this meta - analysis. in the present study, the researcher employed coding procedure (40). a coding structure and operational definitions were made to record quantitative information from each of the 17 studies. preparation training treatments were categorized as either content area preparation or self - regulation preparation. 2. direct training treatments included teacher - led, explicit, and systematic training. 3. behavioral treatments included treatments in which the employment of positive reinforcement was dependent on the successful completion of an assignment. practical treatments contained treatments in which the child was candidate for reiterative performance of an ability that was intended to be already within the student s repertoire, for example : the student would stay for an extra 20 minutes to read the task. textbook modification included any treatment that modified an original textbook to decrease its stage of difficulty. the final category included any intervention that did not fit in the above - mentioned categories such as cognitive treatment, cognitive - behavioral treatment, and short - term executive function training. moreover, the mean and standard deviations of pretests for the 17 adhd studies are illustrated in table 3. furthermore, mean and standard deviations of posttests for the 17 adhd studies are provided in table 4. abbreviations : adhd, attention deficit hyperactivity disorder ; cgi - s, clinical global impressions - severity ; csi-4, children symptom inventory-4 (screens for adhd and other emotional and behavioral problems) ; dsm - iv, diagnostic and statistical manual of mental disorders ; gaf, global assessment of functioning ; ssrs, social skill rating system ; wis - r, wechsler intelligence scale for children - revised. a test to measure adhd children. the researcher used continuous means as a part of unmatched group from comprehensive meta - analysis software. the results are separated into 3 parts : a) mean, b) standard deviation, and c) sample size for each group. to test treatment results of experimental and control groups which were different, the effect size on all samples of each group means, standard deviations, and cohen s d are given in table 5. abbreviations : ll, low limited ; sd, standard deviation ; se, standard error ; ul, upper limited. the results of the study illustrated that there is a difference between the mean scores of the experimental and control groups. the overall meta - analysis of these studies illustrated a statistical significance (figures 1 and 2). in addition, overall standardized differences in means ranged between 0.79% and 95%, confidence interval ranged between 0.57 and 1.08, and z - value was equal to -1.45 (d = mean d = -0.68 (look at below of equation 1 and equation 2)). furthermore, the q statistic for heterogeneity of 347.74 is statistically significant (p 0.80) included cognitive - behavioral therapy, pharmacological treatment, and combinational treatment. this study, like the meta - analysis by nigg. showed that treatment programs reduced symptoms of adhd in children and adolescents (42). the difference between overall mean change for treatment and control groups ranged from -0.08- to- 4.45. this showed that the groups receiving treatment programs illustrated significantly larger improvements in term of adhd symptoms. consistent evidence from both experimental and quasi - experimental studies suggests that treatment programs had desirable effects for the majority of adhd studies (43, 44). effective treatment programs for adhd students reduced problems in social skills and academic performance, and behavior disorders, which are the cause of the disruption of daily life functioning. these treatment programs are a necessity for children with adhd in order to overcome the challenges that accompany an adhd diagnosis over the course of their education. consequently, improvements in daily life functioning and decreasing of functional disorders are the important, socially suitable standards against which treatment results must be evaluated. the majority of studies regarding effective treatment programs on adhd illustrated that treatment programs are effective on adhd. in this meta - analysis, the treatment programs used included cognitive treatments, cognitive - behavioral treatments, behavioral treatments, pharmacological treatments, and combinational treatments. previous studies have demonstrated that students, who received treatment in the various therapy programs, had a higher potential of being mainstreamed into society and showing improvement in their academic and social skills in comparison to students who did not receive some types of therapy. based on the findings of this research, it is recommended that cognitive - behavioral treatment and pharmacological treatment be used for a higher rate of success in aiding with the adhd symptoms in children and adolescents. in this meta - analysis, the treatment programs used included cognitive treatments, cognitive - behavioral treatments, behavioral treatments, pharmacological treatments, and combinational treatments. previous studies have demonstrated that students, who received treatment in the various therapy programs, had a higher potential of being mainstreamed into society and showing improvement in their academic and social skills in comparison to students who did not receive some types of therapy. based on the findings of this research, it is recommended that cognitive - behavioral treatment and pharmacological treatment be used for a higher rate of success in aiding with the adhd symptoms in children and adolescents. | context : the aim of this study was to determine the experimental evidence of treatment / intervention programs for deficits in social skills, attention, and behavioral disorder in children and adolescents with attention deficit hyperactivity disorder (adhd).evidence acquisition : meta - analysis procedures were employed to investigate whether children and adolescents with adhd exhibit deficits in attention and social skills. a total of 17 empirical research studies published between 2000 and 2013 met our inclusion criteria. attention and social skills measures were categorized according to both modality and type of processing required.results:children with adhd exhibited deficits in multiple components of attention and social skills that were not related to language - learning disorders and weaknesses in general intellectual abilities. the overall percentage effect for attention and social skills in students with adhd was calculated (effect size = 0. 79, confidence interval = 0.57 - 1.08). this meta - analysis study showed that treatment programs reduced attention deficit and social skills in adhd children and adolescents.conclusions:the evidence of attention and social skills deficits in children with adhd supports recent studies in adhd deficits. further research is required to explain in detail the nature, severity, and specificity of the deficits in individuals with adhd. |
therapeutic proteins and peptides have potential to elicit immune responses [1, 2 ], resulting in anti - drug antibodies (adas) that can pose problems for both patient safety and product efficacy. clinical consequences can range from relatively mild to serious adverse events [35 ], such as anaphylaxis, cytokine release syndrome, and cross - reactive neutralization of endogenous proteins mediating critical functions. ada can affect drug efficacy and biodistribution and drug clearance, and complicate the interpretations of toxicity and pharmacokinetic (pk) and pharmacodynamic (pd) data [68 ]. during drug development immunogenicity is examined by risk - based approach along with specific strategies for developing fit - for - purpose bioanalytical approaches. enzyme - linked immunosorbent assays (elisa) and electrochemiluminescence (ecl) immunoassays are the most widely used platform for ada detection due to their high sensitivity and throughput. lower affinity ada can be detected by surface plasmon resonance, biolayer interferometry, or other platforms. typically, detection of ada is followed by assessments of the magnitude (titer) of the ada response and the in vitro neutralizing ability of ada, especially in late - stage clinical studies. additional characterization of ada such as immunoglobulin subclass or isotype determinations, domain - mapping, relative binding affinity, cross - reactivity with endogenous proteins, or complement activating ability of the ada may be driven by product - specific, indication - specific, or risk assessment - based objectives [9, 12, 13 ]. recommendations for ada assay development, method validation, and testing strategies have been published by the ligand - binding assay bioanalytical focus group (lbabfg) of american association of pharmaceutical scientists (aaps) [10, 12, 1416 ]. additionally, scientific publications on risk - based approaches to immunogenicity assessments [9, 12, 1719 ] and regulatory documents from the us food and drug administration (fda) and the european medicine agency (ema) are also available [2023 ]. together, these documents provide ample guidance for the application of appropriate ada detection methods in clinical studies. since the late 1990s, liquid chromatography coupled to mass spectrometry (lc / ms) has been a dominant tool for sensitive, accurate, and rapid analysis of small - molecule drugs, metabolites, and biomarkers. in recent years, lc / ms has emerged as a promising platform for quantitation of biotherapeutics and protein biomarkers in biological matrices [2527 ]. the vast majority of lc / ms - based protein quantifications are performed at peptide levels, mainly due to consideration of assay sensitivity. a typical procedure for lc / ms - based quantification includes enzyme digestion and quantification of the target proteins based on selected signature peptides derived from the target [29, 30 ]. recently, furlong. developed a universal peptide method to quantitate human antibody fc region - containing therapeutic protein candidates in nonclinical species. surrogate tryptic peptide vvsvltvlhqdwlngk for igg1, igg3, and igg4 and vvsvltvvhqdwlngk for igg2 were identified in the fc region of human immunoglobulins (igg), respectively. the method was shown to be capable of supporting bioanalysis of a diversity of human fc region - containing therapeutic protein candidates in plasma samples of all commonly used animal species, thus eliminating the need to develop unique peptide methods for each individual therapeutic candidate. with a similar approach, dongen. achieved a higher sensitivity of 4 ng / ml for a monoclonal ab drug, infliximab, using a different universal peptide (slslspgk) from the c - terminal of fc. used a stable isotope labeled common mab as internal standard for quantitation of therapeutic mab in preclinical samples. not only was the common whole ab internal standard able to correct for variations from the beginning of sample processing to ionization in the mass spectrometer but also it allowed rapid method development with flexible choice of a suitable surrogate peptide for new applications, such as different species or different mab. stable isotope labeled human monoclonal ab incorporating [c6,n4]-arginine and [c6,n2]-lysine is now commercially available and can be used for the universal peptide methods. lc / ms has also been reported to assess ada in the presence of excess protein therapeutic in support of clinical programs addressing the safety and tolerability of human growth hormone analogues. this methodology overcame drug tolerance issues, which are often associated with traditional ada detection [3537 ], by completely saturating available ada binding sites with the addition of excess therapeutic. drug - ada complexes were then isolated using protein g immobilized on magnetic beads, followed by elution and digestion. resultant peptide from the target therapeutic proteins was quantified by lc coupled with matrix - assisted laser desorption ms and the results were correlated to the binding capacity of total ada. in another application, lc / ms was used to evaluate neutralizing ab (nab) assay by simultaneously quantitating residual mab - drug, endogenous igg, and nab - positive control in bead eluates. in the study, the low levels of the residual drug and human igg in the bead eluates indicate that the bead efficiently removed the high concentration drug and serum components from the serum samples. meanwhile, the nab - positive control recovery (~42%) in the bead provided an acceptable detection limit for the cell - based assay. this novel application of lc / ms to immunogenicity assay development demonstrates the advantages of lc / ms in selectivity and multiplexing, which provides direct and fast measurements of multiple components. we describe here an immunocapture - lc / ms - based approach for simultaneous ada isotyping and semiquantitation in human plasma. biotinylated drug or anti - drug ab is used to capture adas or drug - ada complexes in plasma, respectively. the resulting ada - drug or ada - drug - ab complexes after washing, ada is released from the beads and subjected to trypsin digestion followed by lc / ms detection of specific universal peptides for each ada isotype. protein z (containing no human fc) was a proprietary experimental biotherapeutic of boehringer ingelheim pharmaceuticals (ridgefield, ct) and produced in - house. the mouse anti - protein z monoclonal ab (mab) was supplied in - house. human igg1, igg2, igg3, igg4, and igm as well as bovine serum albumin (bsa) were purchased from sigma aldrich (st. louis, mo), human ige was from mp biomedicals (solon, oh), human iga1 was from abcam (cambridge, ma), and human iga2 was from emd millipore (billerica, ma). internal standard peptides with stable labeled c - terminal [c6,n4]arg or [c6,n2]lys were synthesized at genscript (piscataway, nj). streptavidin magnetic beads (1 m dia.), tpck trypsin, and ez - link sulfo - nhs - lc biotinylation kits were obtained from thermo scientific (rockford, il). all other lab chemicals, reagents, and buffer solutions were obtained from sigma aldrich, thermo scientific or invitrogen (grand island, ny). human preexisting ada (pea) positive and negative plasma were obtained in - house. biotinylation of protein z and the mouse anti - protein z mab was performed using an ez - link sulfo - nhs - lc biotinylation kit. a 1 mg / ml solution of the drug or mab was combined with a 10-fold molar excess of biotin and allowed to react at room temperature for 60 minutes. a haba assay was used to determine the amount of biotin incorporation in the sample after desalting. typical biotin incorporation was approximately 2 biotins per drug and 47 biotins per mouse mab. the biotinylated drug and biotinylated mouse mab solutions were diluted to 0.1 mg / ml in water and stored at 80c prior to use. the magnetic beads (10 mg / ml) were transferred to a polypropylene tube and placed on a magnetic stand to remove supernatant and collect the beads. the beads were then washed with 10x volume of tris buffered saline containing 0.1% tween-20 (tbs - t) and resuspended in 2x volume of tbs - t to yield a final working bead concentration of 5 mg / ml. an aliquot of 95 l of human plasma sample and 5 l of 5.85 m acetic acid were pipetted into a 96 deep - well polypropylene plate. the plate was incubated with gentle mixing for 1 hr at room temperature. after adding 75 l aliquot of 0.1 mg / ml biotinylated protein z and 40 l of trizma base (1.5 m tris, ph 10) to each sample, a 40 l aliquot of freshly prepared 5 mg / ml magnetic beads and 475 l of tbs - t binding buffer were added to each sample and the plate was gently mixed for 1 hr at room temperature. the beads were then separated, washed three times with 300 l of tbs - t and once with 300 l of water, and eluted with 150 l of 0.1 m glycine (ph 2.0) on a kingfisher flex bead handler (thermo scientific, san jose, ca). the eluent was immediately neutralized with 45 l of 1 m tris - hcl (ph 8.0) followed by the addition of 10 l of 0.1% bsa. an aliquot of 144 l of human plasma sample and 6 l of 5 mg / ml protein z aqueous solution were pipetted into a 96-deep - well polypropylene plate. the plate was incubated at 37c with gentle mixing for 1 hr and then stored at 80c overnight. a 100 l aliquot of 0.1 mg / ml biotinylated mouse mab was added to each sample and the plate a 100 l aliquot of freshly prepared 5 mg / ml magnetic beads and 475 l of tbs - t binding buffer were added to each sample and the plate was gently mixed for 1 hr at room temperature. the beads were separated, washed three times with 300 l of tbs - t and once with 300 l of water, and then eluted with 150 l of 0.1 m glycine (ph 2.0) on a kingfisher flex bead handler. the eluent was immediately neutralized with 45 l of 1 m tris - hcl (ph 8.0) followed by addition of 10 l of 0.1% bsa. commercial stock solutions of the different immunoglobulins (igs) ranged from 1 to 4.18 mg / ml. a series of 5010,000 ng / ml spiking calibration standards were prepared in 0.1% bsa using the stock solutions and stored at 80c prior to use. pooled human blank plasma matrix calibration standards were prepared by adding 10 l of the spiking calibration standards to the magnetic bead eluent of the pooled human blank plasma. to the immunocapture eluent, matrix calibration standards, or neat ig pbs solution, 5 l of 0.1% rapigest in 100 five l of a stable labeled internal standard solution of the universal peptides (0.2 g / ml) and 5 l of 50 mm tcep in 100 mm ammonium bicarbonate were added and followed by incubation at room temperature for 20 min. after adding 5 l of 50 mm iodoacetamide in 100 mm ammonium bicarbonate, the plate was gently shaken for 20 min while protected from light. a 5 l aliquot of solution containing trypsin (0.2 mg / ml) and calcium chloride (0.2 m), prepared immediately before use, was added to each sample and the plate was incubated at 37c overnight with gentle mixing. the samples were mixed for 40 min at 37c and then centrifuged at 4400 rpm for 10 min prior to lc / ms analysis. eksigent ekspert microlc 200 coupled with ab sciex 6500 triple quadrupole mass spectrometer (ab sciex, framingham, ma) was used. chromatographic separation was performed using acquity uplc peptide beh c18 column (1 mm 50 mm, 1.7 m, 300) operated at 60c. mobile phases consisted of (a) 0.1% formic acid and (b) 0.1% formic acid in acetonitrile running at a flow rate of 60 l / min. for information - dependent acquisition (ida), the lc gradient was 5% to 50% b over 18 minutes. for ada isotyping and semiquantitation, the lc gradient was 12% to 17% b over 2.8 minutes and then to 47% b over 6.2 minutes. key instrument parameters were as follows : + 5000 v electrospray voltage, 65 nebulizer gas units, 30 axillary gas units, 375c ion source temperature, 10 collision gas units, and unit resolution on both q1 and q3. for identifying unique peptides for each ada isotype with ida, up to 4 multiple - reaction - monitoring (mrm) pairs were used for screening followed by enhanced product ion scan. for ada isotyping and semiquantitation, 13 mrm pairs of universal peptides despite its many advantages and potential, the use of lc / ms for protein quantitation is not as straightforward as for small - molecules and oftentimes demands comprehensive method development. plasma and serum are very complex matrices that contain several hundreds of thousands of proteins and protein isoforms in a wide concentration range. upon digestion these are all cleaved into multiple peptides, from which just one or a few have to be quantified. when no protein or peptide purification is employed, lc / ms sensitivity is significantly compromised due to matrix interference from the peptide - rich digest. for high sensitivity lc / ms applications immunopurification can be done either at the protein - level prior to digestion [40, 41 ] or at the peptide level after digestion. immunopurification of peptides requires anti - peptide ab for each peptide of interest. unlike proteins, small peptides are usually less or even not immunogenic, which presents significant challenges for anti - peptide ab production moreover, ada and/or drug - ada complex has to be pulled down prior to digestion and peptide pull - down. in consideration of these factors, we employed protein - level immunopurification for sample preparation. antibodies are secreted by plasma cells and come in different isotypes with genetic variations or differences in the constant regions of the heavy and light chains. in humans, there are five heavy chain isotypes (: iga ; : igd ; : igg ; : ige ; and : igm) and two light chain isotypes (and). in addition, igg has 4 subclasses (igg1, igg2, igg3, and igg4) and iga has 2 subclasses (iga1 and iga2). relative ab abundance (% total igs) in human varies significantly among isotypes / subclasses : igg1 (65%), igg2 (25%), igg3 (5%), igg4 (5%), iga1, iga2 (13% iga1 + iga2), ige (0.9910 except for peptide wyvdgvevhnak (igg3, which was 0.9858). the calibration linear ranges and correlation coefficients (r) are listed in table 4. representative calibration curve of igg1 in human plasma eluent after immunocapture is shown in figure 3. using the calibration curve, only 2 out of the 11 pea positive samples had ada levels (for igg1 only) above the lloq 0.5 g / ml. lots 3 and 11 had ada igg1 level of 0.660 and 0.680 g / ml, respectively. it is expected that ada igg1 levels in some of the 9 samples could be quantitated if the lc / ms assay sensitivity was further improved. obviously, in order to increase the assay lc / ms specificity, one has to further eliminate endogenous igs to minimize the background response. this was consistent with the fact that igg1 is the most dominant antibody in human plasma. the biggest caveat of the semiquantitation approach was that the calibration standards did not go through the immunocapture process whereas the study samples did. the recovery could be estimated using well characterized polyclonal human ada positive controls, which were not available for protein z. based on our experiences with immunocapture in similar experimental setting and those reported in literature, immunocapture recovery varied from project to project but usually falls within a 3050% range. if this also held true for protein z, the measured ada levels in the pea plasma would be around 3050% of actual concentrations. different from ecl assays, the ada levels measured by the immunocapture - lc / ms represent absolute amounts. this allows one to compare ada isotype levels between samples, studies, and different biotherapeutics. database of such information could be gradually built and provide valuable insight to better understand immunogenicity and immunology of biotherapeutics. as with traditional ecl drug - bridging assays, the immunocapture - lc / ms method could be hampered by drug tolerance issues when drug is present. this limitation may be overcome by using acid dissociation to break up the drug - ada complex and release ada. biotinylated drug is then added to the samples so that the biotinylated drug competes with the existing drug in forming (biotinylated)drug - ada complexes. if the amount of the biotinylated drug is much more than that of existing drug which is usually determined with a pk assay, the drug interference is greatly reduced and assay sensitivity is improved. in ecl drug - bridging assays, one binding domain of ada has to bind to biotinylated drug while the other binds to sulfotagged drug in order to form (biotinylated)drug - ada-(sulfotagged)drug complex and be detected. in the immunocapture - lc / ms assay, on the other hand, only one arm of ada needs to bind to biotinylated drug and the other can still bind to the unlabeled drug. therefore, drug interference is expected to be less in immunocapture - lc / ms assay platform. it should be noted that if drug contains the human ig fc region, it may also bind to the beads via nonspecific binding just like endogenous proteins and could contribute to interference in the lc / ms assay. on the other hand, biotinylated drug that binds to streptavidin beads will not be eluted out under the elution conditions due to very strong biotin - streptavidin interaction. the binding between streptavidin and biotin has long been regarded as the strongest, noncovalent, biological interaction known, with a dissociation constant kd in the order of 4 10 m. the bond forms very rapidly and is stable in wide ranges of ph and temperature [53, 54 ]. it was evident that the results from the immunocapture - lc / ms assay confirmed pea positive results in most of these samples and were in good agreement with the drug - bridging ecl assay. the second immunocapture approach was using a mouse anti - protein z mab to capture ada in human plasma. in this approach, all adas had to be first completely converted to drug - ada complexes by adding excessive protein z to the samples. biotinylated mouse mab was then added to capture the protein z - ada complexes along with free protein z. in the presence of mab, the drug - ada complexes and free drug were converted to drug - ada - mab complexes and drug - mab complexes, respectively. after adding streptavidin magnetic beads, the complexes were immobilized on the beads and were subsequently separated from plasma using a magnet. ada was then eluted from the beads, digested, and assayed by lc / ms following the same procedures with drug as the capture reagent described previously. the merit using anti - drug ab as the capture reagent lies on that drug no longer interferes with the assay. this offers a huge advantage when drug levels in the study samples are high enough such that drug tolerance becomes a concern in other types of assays. the most important element of this approach is that the capture ab should not compete with ada for the drug ; that is, the two should not share the same binding domain on the drug. to confirm this for the mouse mab, protein z was spiked at 5 ng / ml to the pooled pea negative and the 11 positive human plasma samples and its concentration was determined using an immunocapture - lc / ms pk assay. the pk assay was developed in our lab to support clinical studies. in the pk assay, protein z was captured using the mouse mab, and the resulting drug - mab complex was then immobilized on magnetic beads, separated from plasma, eluted out from beads, digested, and analyzed by lc / ms. a unique peptide from protein z was monitored by lc / ms and used to quantitate protein z. the immunocapture recovery was determined by comparing protein z concentrations in the pea positive human plasma with the pooled pea negative plasma. no difference in protein z concentration was observed between the pea positive and pea negative samples (data not shown), and protein z recovery was more than 82% with averaging 97%. it was evident that the mouse mab was indeed able to capture protein z regardless of whether it is in ada - protein z complexes or free form. however, one has to be cautious as human adas come in many different forms and some may bind to the same domain on the drug as the capture ab. therefore, it is recommended to run this test using ada positive samples from the study. similar to the first approach using drug as capture reagent, the 11 pea positive and 9 pea negative blank human plasma samples were used to evaluate immunocapture using the mouse mab as the capture reagent. besides using a different capture reagent, the only difference between the two approaches was that in the second approach there was an additional step to convert ada to ada - drug complexes. to ensure a complete conversion, 6 l of 5 mg / ml the amount of protein z added was overwhelmingly more than the pea level (680 ng / ml) estimated by the first approach. the same amount of protein z was also added to the pooled pea blank plasma used for preparation of calibration standards. tables 5 and 6 provide the lc / ms peak area ratio response of ada in the pea negative and positive samples, respectively. the lc / ms chromatograms of igg1, igm, and ige unique peptides from the blank human plasma and lloq samples are shown in figure 4. the lc / ms response for ada (igg1) from the 9 pea negative samples ranged from 0.0210 to 0.0815, with a mean of 0.0361 and sd of 0.0194. the calculated cut - point at 95% was 0.0680 for igg1. using the cut - point, 7 of the 11 pea positive samples were also ada positive with the immunocapture - lc / ms assay. these 7 plasma lots were also ada positive in the first approach using drug as capture reagent. plasma lot 2 was ada positive in the first approach but negative in the second approach. in both approaches, lc / ms response of plasma lot 2 was close to the respective cut - point, so it was not surprising to see the discrepancy between the two approaches. calculated cut - points for all other ada isotypes / subclasses are listed in table 5. based on cut - points, lot 7 was ada positive for igg2, lot 8 was ada positive for igg3, lot 11 was ada positive for igm, and lots 4, 5, 6, 9, and 11 were ada positive for iga1 + iga2. however, lot 7 for igg2, lot 11 for igm, and lots 6 and 11 for iga1 + a2 were considered false positive due to the presence of interference as discussed below. the ada must be bound to the drug first, and the resulting drug - ada complexes had to be bound to the mouse mab and survive the immunocapture procedure in order to be detected by lc / ms. endogenous components such as igs that cross - reacted with the mab could also interfere with the assay and give false positive results. although this potential interference was already accounted for in the cut - point determination, it was further assessed for the pea positive plasma samples without the addition of excessive protein z. the drug plasma samples were spiked with the mab and then processed with the immunocapture procedure followed by lc / ms analysis. the lc / ms responses from the drug samples of lots 6 (0.0731) and 11 (0.0974) were above the cut - point of 0.0680, suggesting possible interference. however, both responses were slightly above the cut - points and much less than those (0.1800 and 0.5690) from their respective + drug samples. therefore, lots 6 and 11 were still considered ada positive despite the presence of small interference. likewise, lot 4 was considered positive for iga1 + a2, as the above cut - point drug response (0.1380) was much less than + drug responses (0.2300). the remaining drug positive samples, lot 7 for igg2, lot 11 for igm, and lots 6 and 11 for iga1 + a2 gave similar responses as their respective overall, seven of the eleven pea positive plasma samples were positive for igg1, one was positive for igg3, and three were positive for iga1 + iga2 using the mab as ada capture reagent. calibration curves were established for each ada isotype in the same way as in the first approach. hloq was also set at 10 g / ml for all isotypes / subclasses. the lloq, calibration ranges, and r were all similar to those from the first approach using drug as capture reagent. using the calibration curve, ada (igg1) level in lot 3 and lot 11 plasma was determined to be 0.570 and 1.25 g / ml, respectively. these two plasma samples were also the only ones with ada (igg1) level above lloq in the first immunocapture approach. the ada (igg1) level from the first approach was 13.6% and 45.6%, respectively, compared with the second approach. given the two totally different immunocapture approaches and the limited sample size, the two sets of semiquantitative data were considered in good agreement with each other. besides igg1, ada levels for other ada isotypes / classes were all blq in these 11 pea positive samples. it should be noted that the anti - drug ab capture approach may not be used if the biotherapeutic proteins contain constant human fc regions. unlike using drug as capture reagent, anti - drug ab captures both free drug and drug - ada complexes and during the ada elution step drug is also eluted out from magnetic beads and thus interferes with lc / ms detection. for instance, humira (adalimumab), a tnf inhibiting anti - inflammatory drug and the first fully human monoclonal antibody drug approved by the fda, is an igg1 made by phage display technology with amino acid sequences only from the human germline, making it indistinguishable in structure and function from natural human igg1. based on in silicon digestion prediction, humira would yield the universal peptides of human igg1, gpsvfplapssk, and thus interfere with the universal peptide ada assay. in this case, unique peptide(s) from the drug instead of the ada peptides might be monitored by lc / ms and the results can be qualitatively correlated to ada, as neubert. reported. another option is to use the first immunocapture approach with biotinylated drug as the ada capture reagent. we demonstrated for the first time that immunocapture - lc / ms can be used for simultaneous ada isotyping and semiquantitation in human plasma. either biotinylated drug or biotinylated anti - drug ab could be used as the immunocapture reagent, each with its own merits and shortfalls. biotinylated drug can readily capture ada but drug interference could be an issue if drug levels in the samples are high. on the other hand, immunocapture using an anti - drug ab eliminates drug interference, providing that the ab is able to capture drug - ada complex in addition to free drug. with this method, unique peptides from each ada isotype / subclass were identified and monitored by lc / ms. similar to traditional drug - bridging elisa assay, cut - points at 95% were established. the assay was used for screening, isotyping, and semiquantitating preexisting adas in human plasma. endogenous ig interferences need to be reduced in order to improve the assay sensitivity and specificity, and human positive ada controls will be needed for more accurate ada quantitation. owing to lc / ms 's advantages such as high specificity, selectivity and reproducibility, wide dynamic range, and multiplexing capability, it is expected that, with further improvements, immunocapture - lc / ms will become an invaluable tool in immunogenicity assessment. it can be easily implemented in bioanalytical lab settings for routine ada isotyping and semiquantitation. as ada levels measured by immunocapture - lc / ms represent absolute amounts, one can compare ada isotype levels between samples, studies, and different biotherapeutics, providing that consistency in positive controls is achieved to determine recovery. database of such information could be gradually built and provide valuable insight to better understand immunogenicity and immunology of biotherapeutics. | therapeutic proteins and peptides have potential to elicit immune responses resulting in anti - drug antibodies that can pose problems for both patient safety and product efficacy. during drug development immunogenicity is usually examined by risk - based approach along with specific strategies for developing fit - for - purpose bioanalytical approaches. enzyme - linked immunosorbent assays and electrochemiluminescence immunoassays are the most widely used platform for ada detection due to their high sensitivity and throughput. during the past decade, lc / ms has emerged as a promising technology for quantitation of biotherapeutics and protein biomarkers in biological matrices, mainly owing to its high specificity, selectivity, multiplexing, and wide dynamic range. in fully taking these advantages, we describe here an immunocapture - lc / ms methodology for simultaneous isotyping and semiquantitation of ada in human plasma. briefly, ada and/or drug - ada complex is captured by biotinylated drug or anti - drug ab, immobilized on streptavidin magnetic beads, and separated from human plasma by a magnet. ada is then released from the beads and subjected to trypsin digestion followed by lc / ms detection of specific universal peptides for each ada isotype. the lc / ms data are analyzed using cut - point and calibration curve. the proof - of - concept of this methodology is demonstrated by detecting preexisting ada in human plasma. |
they act via mechanisms classified as central or peripheral ; though some act through both pathways. the antitussive effect of an ethanolic seed extract of picralima nitida (pne) has previously been established by the authors. however, the mode of activity of cough medicines could be attributed to at least five reasons : pharmacological, physiological, true placebo, psychological and non - specific action. plant / plant products exhibiting antitussive activity may not have one mode of expressing this pharmacological effect.3, 4 the purport of this study was to establish the possible mode of antitussive and expectorant activity of an ethanolic seed extract of p. nitida. this will be ascertained by ; evaluating muco - suppression effect, assessing mast cell stabilization effect, and establishing antibacterial, anxiolytic, antioxidant activity in various rodent models. the pods of p. nitida were collected from the knust botanical garden in kumasi, ghana, in february, 2013. the pods were opened, the seeds removed, air - dried, and milled into powder. the powder weighing four (4) kg was extracted by cold maceration with 70% ethanol over a period of 72 h. the resulting extract was then concentrated at a temperature of 40 c and under low pressure to a syrupy mass in a rotary evaporator (rotavapor r-210, buchi, switzerland). the syrupy mass obtained was then dried in a hot air oven (gallenkamp, uk) maintained at 40 c to obtain 0.532 kg (% yield : 13.3%) of a solid mass of p. nitida extract (pne). macau) ; ammonium chloride (philip harris, hyde - cheshire ; uk) ; phenol red and sodium chloride (bdh chemicals ltd, poole, england) ; ketotifen fumarate (novartis pharma ag, basle, switzerland) ; compound 48/80 and toluidine blue (sigma chemical co., st. louis, mo, usa) ; sodium hydroxide (avondale, england) ; acetic acid, diazepam (sigma aldrich inc., st. louis, mo, usa), caffeine (sigma aldrich inc., st. balb / c mice (2030 g) and a sprague dawley rat (130 g), obtained from the animal house of the department of pharmacology, knust, kumasi, ghana were used in this study. they were fed on standard rodent pellet diet (agricare ltd, tanoso, kumasi, ghana) and water ad libitum. the animals were kept in the experimental area of the departmental animal house at ambient conditions of light, temperature and humidity for seven (7) days prior to experimentation and during experimentation. the muco - suppressant effect of pne was determined using the ammonium chloride - induced tracheal phenol red secretion model ; described previously. v, (n = 5) and pre - treated, for 30 min, as follows : group i, two (2) ml / kg normal saline (p.o) ; group ii, 100 mg / kg sodium cromoglycate (i.p) ; groups iii v, 100, 300, and 500 mg / kg of pne (p.o) respectively. tracheal mucus secretion was then induced with five (5) mg / kg ammonium chloride per os. animals were then injected with 500 mg / kg phenol red, intraperitoneally, 30 min later. the trachea was excised from each mouse and cleared of adhering tissues, after sacrificing it by cervical dislocation (30 min after the phenol red injection). each excised trachea was washed in three (3) ml physiological saline ; sodium hydroxide (0.3 ml naoh, one (1) m) was then added to the washing to stabilize the ph of the lavage fluid. the absorbance of the mixture was then taken at a wavelength of 460 nm using a spectrophotometer (t90 + uv / vis spectrometer pg instruments ltd). a calibration curve for phenol red was made ; from which concentrations of phenol red secreted by mice tracheae were extrapolated. mast cell stabilizing effect was ascertained using compound 48/80-induced mesenteric mast cell degranulation as described previously. dawley rat intestinal mesenteries was excised into several pieces and put into five groups, g1g5 (n = 5), in petri dishes containing tyrode solution. the mesenteries were then subjected to the following treatment : g1, normal saline ; g2, 20 g / ml ketotifen fumarate ; g3g5, 100, 300, and 500 g / ml pne respectively. the petri dishes were then incubated at 37 c for 15 min after which one (1) ml of compound 48/80 solution (10 g / ml) was added and incubated at 37 c for ten (10) min. the mesenteric pieces were then fixed in 10 % buffered formalin and processed in xylene and acetone. they were then stained with 0.1 % toluidine blue and observed under a leica dm 750 microscope (leica microsystems cm5 gmbh, wetzlar germany) for both intact and degranulated cells. ten (10) test tubes (labeled t1t10) each containing 20 ml nutrient agar were stabilized. the molten agars were inoculated with 0.2 ml each of the following organisms : t1 and t2, staphylococcus aureus ; t3 and t4, streptococcus pneumonia ; t5 and t6, salmonella typhi ; t7 and t8, escherichia coli ; t9 and t10, klebsiella pneumonia. the seeded agars were poured into labeled sterile petri dishes (p1p10) and allowed to set. using a sterile cork borer number seven (7), various concentrations of pne were prepared and poured in the wells to three - quarter (3/4) full as follows : p1, p3, p5, p7 and p9 each received 0.05, 0.5, 5, 25 mg / ml pne, while p2, p4, p6, p8 and p10 received 0.1, 1, 10, 50 mg / ml pne, in four of the five wells created. the petri dishes were covered and left on the bench for 45 min in order to allow effective diffusion of the extract. they were then incubated at 37 c for 24 h, after which they were examined for zones of growth inhibition around the wells. amoxicillin (1 %) was used as a positive control in the test against s. aureus, s. pneumonia, e. coli and k. pneumonia. the apparatus was made of plexiglas and consisted of two open arms and two closed arms of dimensions 30 cm 5 cm 15 cm. these arms extend from a central square platform (5 cm 5 cm). the maze was elevated to a height of 60 cm above the floor and placed in a lit room. mice were divided into ten groups (n = 6) and received the following treatment : vehicle - control, pne (100, 300, 500 mg / kg), diazepam (0.1, 0.3, 1.0 mg / kg), and caffeine (3, 10, 30 mg / kg). the vehicle, pne, caffeine were orally administered to their respective animals ' an hour before the experiment, whiles diazepam was given intraperitoneally 30 min before the experiment. at the start of the experiment, animals were individually placed at the center of the maze, facing one of the enclosed arms and their behavior videotaped for 5 min with a digital camera placed 100 cm above the maze. behavioral parameters were scored from the videotapes as follows:1)number of closed and open arm entries(absolute value and percentage of the total number);2)time spent in exploring the open and closed arms of the maze absolute time and percentage of the total time of testing3)number of head - dips (absolute value and percentage of the total number)protruding the head over the ledge of either an open (unprotected) or closed (protected) arm and down toward the floor;4)number of stretch - attend postures (absolute value and percentage of the total number)the mouse stretches forward and retracts to original position from a closed (protected) or an open (unprotected) arm. number of closed and open arm entries(absolute value and percentage of the total number) ; time spent in exploring the open and closed arms of the maze absolute time and percentage of the total time of testing number of head - dips (absolute value and percentage of the total number)protruding the head over the ledge of either an open (unprotected) or closed (protected) arm and down toward the floor ; number of stretch - attend postures (absolute value and percentage of the total number)the mouse stretches forward and retracts to original position from a closed (protected) or an open (unprotected) arm. the behavior was tracked by jwatcher version 1.0 (university of california, los angeles, usa and macquarie university, sidney, australia ; available at http://www.jwatcher.ucla.edu/.) the open field method has previously been described. the test was conducted in clear plexiglas boxes (40 cm 40 cm 30 cm). the floor of this box was divided by red lines into 16 equal squares. for behavioral analysis, the arena of the open field was designated as;1)corner (one of the four corner squares);2)periphery (the squares along the walls) ; or3)center (the four inner squares). corner (one of the four corner squares) ; periphery (the squares along the walls) ; or center (the four inner squares). the animals were divided into ten groups (n = 6), and received either the extracts (100, 300, or 500 mg / kg, p.o.), the vehicle or the reference drug diazepam (0.1, 0.3 or 1 mg / kg, i.p.). thirty minutes after i.p and 1 h after oral administration, the animals were placed at the center of the open field and were allowed to explore for 5 min. this was recorded by a video camera which was suspended 100 cm above the arena. behavioral parameters for all the tests were scored from videotapes with the aid of the public domain software jwatcher version 1.0. the numbers of entries, as well as the duration of stay in individual zones were assessed. the antioxidant property of pne (0.130 mg / ml) was evaluated in 2, 2-diphenyl-1-picrylhydrazyl hydrate (dpph) free radical scavenging, linoleic acid lipid peroxidation, reducing power, and total antioxidant assays by standard procedures previously described by amoateng., 2011. statistical analyses were done by one - way analysis of variance (anova) with dunnett 's multiple comparison test (post hoc test) using graph - pad prism for windows version 6.0 (graph - pad software, san diego, ca, usa). some aspects of the open field and elevated plus maze were done with two - way analysis of variance followed by bonferroni 's post - hoc test. differences between means of treated groups and the control were regarded as statistically significant at p 0.05. sodium cromoglycate, and pne (100300 mg / kg) significantly (p 0.050.001) reduced tracheal phenol red secretion compared to the control (fig. treatments with ketotifen fumarate, and pne (100500 mg / kg) were also able to reduce significantly (p 0.050.001) inhibit mast cell degranulation induced by compound 48/80 (fig. 1b). significant (p 0.050.0001) antibacterial activity was observed e. coli, s. typhi, k. pneumonia, s. pneumonia, and s. aureus from 1050 mg / ml. the lowest effect was against s. typhi at 5 mg / ml with a zone of 13.0 0.00 mm whiles the highest response was observed against s. pneumonia at 50 mg / ml with a zone of 22.3 0.88 mm (table 1). pne (100, 300, 500 mg / kg) enhanced, dose - independently, activities in the open arm by increasing percentage of entry into open arms (p 0.0001 ; fig. 2f) and percentage time spent in open arms (p = 0.1278 ; fig. 2c). there was also reduction in risk assessment by decreasing both the percentage protected head dips (p = 0.0030 ; fig. 3c) and percentage protected stretch attend postures (p = 0.0860 ; fig. diazepam (0.11 mg / kg) also dose dependently and significantly increased percentage of open arm entries (p = 0.0005 ; fig. 2e) and percentage time spent in open arm (p = 0.0004 ; fig. diazepam also reduced risk assessment by decreasing both the percentage protected head dips (p = 0.0002 ; fig. 3b) and percentage protected stretch attend postures (p = 0.0192 ; fig. these effects confirmed the anxiolytic effects of both pne and diazepam. with regard to caffeine, it increased open arm avoidance by reducing percentage of open arm entries (p = 0.8997 ; fig. 2d) and percentage time spent in open arms (p = 0.0285 ; fig. also, caffeine increased both the percentage protected head dips (p = 0.7205 ; fig. 3a) and percentage protected stretch attend postures (p = 0.0247 ; fig. pne (100, 300, 500 mg / kg) dose - independently increased the percentage entry into central zone (p = 0.0201 ; fig. 4d) and percentage time spent in central zones (p = 0.0427 ; fig. the reference anxiolytic, diazepam (0.11 mg / kg) dose dependently increased the percentage entry into central zone (p = 0.0023 ; fig. 4c) and percentage time spent in central zones (p = 0.0603 ; fig. the reference antioxidant, n - propyl gallate (0.0010.1 mg / ml), and pne (0.130 mg / ml) exhibited dpph free radical scavenging effect (fig. 5a), and showed concentration dependent ability to inhibit the autoxidation of linoleic acid lipid peroxidation (fig. 5b). pne and n - propyl gallate also dose dependently reduced fe to fe in the reducing power assay (fig. pne (0.130 mg / ml) showed a concentration dependent increase in total antioxidant capacity expressed as ascorbic acid equivalent (aae) (fig. ec50 values for the reference antioxidants and pne are as shown in table 2. this study carried out on pne to determine muco - suppressant, antibacterial, mast cell stabilizing, anxiolytic, and antioxidant properties in a bid to establish its mode of activity in the management of cough evolved from previous work on pne, by the authors, which established antitussive, and expectorant properties of the extract. tracheal mucus secretion, mast cell degranulation, bacterial infection of the airways, psychogenic factors (e.g. anxiety) and oxidative stress could culminate into cough. sodium cromoglycate and pne significantly reduced tracheal phenol red secretion after ammonium chloride induction, indicating muco - suppressant activity. sodium cromoglycate is known to exhibit muco - suppressant effect by prevent the release of inflammatory mediators resulting from chronic inflammation of the airways. the importance of this result lies in the fact that mucus in the airways mechanically stimulates and increases rapidly the activity of rapidly adapting receptor (rar) ; a cough sensor. the presence of excess mucus moves the cough sensor response up the stimulus / response curve and gives the impression of sensitization. by pne 's ability to inhibit mucus secretion from the airway, stimulation of rar compound 48/80-induced mast cell degranulation is used to test mast cell stabilizing effects of potential therapeutic agents. ketotifen stabilizes mast cells by inhibiting the release of histamine, serotonin and other inflammatory mediators. mast cell stabilization could contribute to antitussive effect in that leukotriene and histamine (which can stimulate c - fibers) are not release. apart from such direct effect, leukotrienes and histamine can induce mucus secretion and bronchoconstriction which can in turn stimulate rar receptors. mast cell stabilization also reduces chronic inflammation of the airway which can lead to hypertrophy and hyperplasia of surface goblet (mucous) cells and sub - mucosal glands, which consequently increases mucus production. pne demonstrated antibacterial activity against two gram positive and three gram negative bacteria, indicating a possible remedy for cough arising from bacterial infection of the airway. increase in cough sensitivity seems to be the most likely reason why infection contributes to cough. treatment and/or prevention of airway infection could be an important way of reducing inflammation and hyper - secretion and hence cough sensitivity. it is worth noting that there could be a potential reduction in c - fiber stimulation as a result of anti - bacterial effect. bacterial infection is known to reduce the level of endopeptisase especially substance p which gets released from c - fiber. considering the fact that substance p can augment c - fiber activity and that of the rar through mucus secretion and bronchospasm it induces suppressing bacterial proliferation will enhance the level of endopeptidase, which in turn will reduce substance p mediated events. anxiety in particular has been found to be an independent risk factor for both productive and non - productive cough. this may reflect a somatic manifestation of a wide range of psychological problems, an increased awareness of physical symptoms in anxious individuals as well as difficulty in maintaining voluntarily cough suppression.23, 24 therapeutically, therefore, anxiolytics may help to relieve cough. psychogenic cough is generally regarded as a respiratory tic and may be precipitated by a variety of emotional stimuli. in an attempt to establish the fact that pne could be useful in managing anxiety - related cough, its anxiolytic effect was established using the elevated plus maze and the open field tests. the open - field test examines anxiety - related behavior characterized by avoidance of an open, brightly lit area by rodents. animals placed in a novel environment express anxiety and fear, by showing alteration in certain parameters, such as decreases in ambulation and exploration time in the center of the open field with an attendant increased peripheral movement. the elevated plus maze utilizes the fear of a novel, brightly lit open space and fear of balancing on a relatively narrow, raised platform. generally, anxiolytics increase the number of entries into and the time spent in the open arms of the epm. the fact that pne reduced the percentage protected head dips and stretch attend posture in the epm also confirm its ability to reduce fear related behavior. oxidative stress contributes to cough in that reactive oxygen species (ros) have the potential of stimulating c - fibers. in testing the antioxidant effect, dpph, 2, 2-diphenyl-1-picrylhydrazyl hydrate a stable radical having a characteristic violet color (and maximum absorption at 517 nm) and which accepts an electron or hydrogen in the presence of a suitable free radical scavenger (reducing agent) was used. from the ic50 values oxidants can cause damage by interacting with anti - proteases or other processes leading to the development of chronic lung damage. intake of fruit and vegetable, which are major sources of antioxidants, have been associated with higher lung function and reduced symptoms of cough with phlegm. it is to be noted that the mucolytic effect of acetylcysteine have been attributed to their antioxidant activities and that gives an indication of the fact that pne may also have mucolytic activity. there seems to be notable similarities between mucolytics and expectorants : mucolytics just like expectorants have been found to be effective in productive cough34, 35 also the model used in testing expectorant effects i.e. the tracheal phenol red secretion has been used in ascertaining mucolytic activity of various extracts. the antioxidant effect established in this work can then be said to contribute to the extracts ability to manage productive cough ; a property which is typical of expectorants. however this antioxidant effect could also account for pne 's antitussive activity since reactive oxygen species (ros) are known activators of c - fibers. the mast cell stabilization, muco - suppressant, and antioxidant effects were the most prominent effect of pne even though it shows significant antimicrobial and anxiolytic activity. preliminary phytochemical screening of pne indicated the presence of alkaloids, tannins, steroids, glycosides, anthraquinones, and terpenoids some of which would have contributed to its properties.37, 38, 39 a review of the medicinal uses, phytochemistry and pharmacology of p. nitida has indicated that its alkaloids (especially from the seed) have significant antimicrobial, analgesic, and anti - inflammatory properties while its coumestan glycosides have antimicrobial activity ; these which could make it useful as antitussive. alkaloids, phenolic compounds (tannins), glycosides, saponins, sterols, and terpenoids has been reported to have mast cell stabilizing effect.42, 43, 44 it is possible that the anxiolytic action could be mediated by synergistic action of these phytochemicals as seen in several studies. the lower dose of hie (i.e. 100 mg / kg) seemed to be more effective ; giving more consistent results than the higher doses (i.e. 300 and 500 mg / kg). this may not be surprising since the extract may have several other components, some of which may have antagonist effects to the agonist effect being elicited ; but would only be of significance at higher doses i.e. proportional to the dose administered. the ethanolic seed extract of p. nitida has demonstrated very significant mast cell stabilizing, mucus suppressant and antioxidant activity as well as substantial antibacterial and anxiolytic properties ; all of which could contribute to its antitussive and expectorant property. | it has been established that picralima nitida has antitussive effect. this study therefore aimed at determining the possible mode of antitussive and expectorant activity of an ethanolic seed extract of p. nitida (pne). the muco - suppressant, mast cell stabilization, and the anxiolytic effects of pne were ascertained using ammonium chloride - induced phenol red secretion in balb / c mice ; compound 48/80-induced mesenteric mast cell degranulation assay ; and the open field and the elevated plus maze models respectively. antibacterial potential was ascertained by the agar plate diffusion method and its antioxidant potential by the 2,2-diphenyl-1-picrylhydrazyl hydrate (dpph) free radical scavenging, linoleic acid lipid peroxidation, reducing power, and total antioxidant assays. data obtained was analyzed using one - way analysis of variance (anova) with dunnett 's multiple comparison post hoc test. pne (100500 mg / kg) reduced (p 0.050.001) tracheal phenol red secretion. the extract (100500 g / ml) also dose - dependently (p 0.050.0001) stabilized mast cells. pne (100500 mg / kg) increased open arm activities in the elevated plus maze (p 0.05) as well as central zone exploration (p 0.05) in the open field test. pne (1050 mg / ml) showed activity against staphylococcus aureus, streptococcus pneumonia, escherichia coli, klebsiella pneumonia, and salmonella typhi. by the assays, pne showed significant antioxidant effect. the ethanolic seed extract of p. nitida has demonstrated very significant mast cell stabilizing, mucus suppressant, and antioxidant activity as well as substantial antibacterial and anxiolytic properties ; all of which could contribute to its antitussive and expectorant property. |
biliary cast syndrome (bcs) is characterized by the presence of casts within the intra- or extrahepatic biliary system. retained lithogenic material in the bile duct may lead to obstruction of the biliary tract and also to cholangitis [1, 2 ]. bcs is an uncommon complication which is mostly described in orthotopic liver transplantation [1, 2, 3, 4 ]. bcs is characterized by the presence of casts causing obstruction in the bile duct tree. formation in this syndrome included exudative inflammation of the bile duct epithelium [6, 7 ], ischemic injury to the biliary endothelium, use of postoperative biliary drainage tubes and biliary infection. bcs has also rarely been reported in non - liver transplant patients [9, 10, 11, 12, 13, 14 ]. there is no recommended standard management for patients who develop cholangiopathy secondary to bcs, and each case should be managed individually. we describe a male long - term opium inhaler with bcs who underwent successful endoscopic removal of the cast by balloon enteroscopy - guided endoscopic retrograde cholangiopancreatography. a 52-year - old man, who was a known case of opium addiction, presented with the chief complaint of epigastric pain for 1 week prior to admission. routine laboratory evaluation revealed cholestatic liver enzyme elevation with alkaline phosphatase 1,810 iu / l (reference range 80306 iu / l), total bilirubin 0.5 mg / dl (reference range 0.11.2 mg / dl) with normal aminotransferases, high erythrocyte sedimentation rate (90 mm / s) and normal prothrombin time. serum levels of fasting blood sugar, blood nitrogen urea, creatinine, triglyceride, total and low - density lipoprotein cholesterol and uric acid as well as thyroid function tests were in the normal range. an abdominal ct scan demonstrated dilated intrahepatic and extrahepatic bile ducts (the common bile duct measured 11 mm). magnetic resonance cholangiopancreatography showed a distended gallbladder and a dilated bile duct with the conclusion of ampullary and periampullary lesion (fig. endoscopic retrograde cholangiopancreatography showed a linear filling defect in the intra- and extrahepatic duct (fig. after removal of the biliary cast he is receiving ursodeoxycholic acid (udca) 750 mg per day and does not report any problem 4 months after treatment. his last laboratory evaluation revealed a significant decrease in erythrocyte sedimentation rate (30 mm / s) and a mild decrease in alkaline phosphatase (1,423 iu / l). bcs is an obstructive cholangiopathy, defined by the presence of casts within the intra- or extrahepatic biliary tract due to several mechanisms [1, 2 ]. most of the previously reported cases of bcs were described in orthotopic liver transplantation [1, 2, 3, 4 ]. based on our knowledge and the literature, there are only five published report of bcs in a non - transplant situation [9, 10, 11, 12, 13, 14 ]. described bcs in a patient with traumatic head injury and prolonged intensive care unit stay. gleeson. described bcs in two patients, one with hepatic infarction due to antiphospholipid syndrome and the other with fasting, parenteral nutrition and infection. koo. described a case of bcs in an elderly man after head trauma and long - term bed - ridden state. those studies considered increased bile pigment load due to absence of gallbladder [9, 12 ], ischemic events [10, 11, 12, 13 ] and gallbladder hypocontractility [11, 14 ] as the leading causes for biliary cast formation. structurally, in the liver transplant patients the biliary cast is composed of high bilirubin content (1050%), bile acids (1015%), few cholesterol and protein (510%) or collagen from necrotic biliary epithelial cells. however, the chemical composition of biliary casts in the setting of non - liver transplant patients is unclear. there is no standard management for bcs patients, and a variety of therapeutic and surgical methods are used to treat bcs. as far as we know, this is the first reported case of bcs in an opium inhaler. in our case none of the previously reported risk factors, including liver transplantation, fasting, total parenteral nutrition, hemolysis, abdominal surgery, trauma or biliary infection, was detected. in long - term opium users, bile duct size and biliary pressure thus, we think that biliary dyskinesia due to long - term opium use was the predisposing factor for biliary cast formation in our case. the efficacy of udca use in the treatment of persistently elevated liver enzymes following endoscopic removal intervention was reported previously. the supposed mechanism by which udca acts in the treatment of bcs is dissolution of the cast in the small intrahepatic bile ducts, its hepatocytoprotective action and its effect on biliary acid secretion and immunomodulatory properties. generally, this case brings up the effect of opium use on bcs, and with attention to the high prevalence rate of opium consumption in iran, this subject should be considered in these patients. also, this case showed an effect of udca in the completion of treatment in bcs patients after cast removal. it seems that biliary dyskinesia due to long - term opium use can be another predisposing factor for biliary cast formation, and that administration of udca after removal of the biliary cast is an effective treatment for bcs. | biliary cast syndrome (bcs) is an uncommon complication which is mostly described in orthotopic liver transplantation. however, bcs has also been reported rarely in non - liver transplant patients. we describe a male long - term opium inhaler with bcs who underwent successful endoscopic cast removal by balloon enteroscopy - guided endoscopic retrograde cholangiopancreatography. a 52-year - old man, who was a known case of opium addiction, presented with the chief complaint of epigastric pain for 1 week prior to admission. routine laboratory evaluation revealed cholestatic liver enzyme elevation. a cholestatic pattern was seen in radiographic modalities. endoscopic retrograde cholangiopancreatography showed a linear filling defect in the intra- and extrahepatic duct. a long biliary cast was successfully removed using an extractor balloon. after removal of the biliary cast the patient is receiving ursodeoxycholic acid and does not report any problem 4 months after treatment. it seems that biliary dyskinesia due to long - term opium use can be a predisposing factor for biliary cast formation. |
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